Chapter 7
heredity,variation and breeding of
microbes
Heredity(inheritance) and variation is the most
intrinsical atrributes of all living organisms.
Let’s get married our
babies will be as beautiful
as me and as intelligent as
you……,
If they are as ugly as
me and as foolish as
you……,
The genotype(遗传型又称基因型 ) of an organism is the
genetic information stored in its genetic material,the
exact sequence of the bases or nucleotides in its DNA,
This sequence dictates what proteins or RNA molecules
can be produced by the organism,how the genes may be
regulated and the functioning of the protein once it is
synthesized,Not all proteins are made at the same time
and the full genotype of an organism cannot be
discovered unless the genome is sequenced,
genotype
The phenotype(表型 ) of an organism is the expression or
manifestation of that genotype as it can be observed or
measured; the manifestation of the genotype in the form of
characteristics which is observable and measurable,for
example,whether or not it produces a capsule or is resistant
to an antibiotic,The most common form of an organism is
often referred to as the wild-type,
Alterations to the genotype can therefore be seen as changes
in the phenotype,(遗传性的改变可通过表型改变来观察 )
phenotype
Any change to the sequence of bases (genotype) in
the DNA of an organism is a mutation or variation(突变或变异 ),The change may be a single-base change
called a point mutation or involve a much larger
sequence of DNA in which case it is called a multi-site
mutation,DNA may be deleted,inserted,inverted,
duplicated or substituted,(that means the change of
structure or amount),
Variation or
mutation
Modification(饰变 ) is the changing of phenotype
occurs only on the transcriptional and translational
levels but not involve the change of the genotype,
that is,the genetic material or the nucleotides,thus
it’s only a ornamental alteration.
Modification
Different nomenclatures/abbreviations are used
for describing genes and mutations in different
organisms,but in bacteria the following
convention is used:
Phenotype is described using a three-letter
abbreviation of roman letters with the first letter
capitalized,A superscript + or - is used to designate
the presence or absence of this phenotype;
For example,Phe- indicates the inability to
synthesize phenylalanine or Lac+ indicates
the ability to utilize lactose as a carbon
source,The superscripts 's' and 'r' may also
be used to designate sensitive or resistant.
Genotype is indicated by three lower case letters
(usually reflecting the phenotype) with a fourth
upper case letter to indicate specific genes
involved,all of which are italicized,for example,
pheA or lacZ.
Although,naturally,there may be a large number of
individuals of a species with slightly different phenotypes,
microbiologists and in particular microbial geneticists try
to work with organisms that are genetically identical,
Consequently,in the laboratory,we work with pure
cultures where microbes are descendent from the same
original parent to minimize the genetic variability,That
parent may often be referred to as the wild-type.
Section A
substantial basis for genetic variation
Part A
Three classical experiments
1,Transformation
2,Bacteriophage infection
3,Reconstruction of tobacco mosaic viruses
Our knoelege that DNA is the genetic material
has come from the experiments based on the fact
that some species of bacteria are capable of
naturally taking up DNA from their surrouding
medium,and incorprating it into their genomes,by
a process called transformation.
Fred Griffith in the late 1920s first showed that a
rough,non-capsulated,avirulent strain of
Streptococcus pneumo-niae could be converted into
a smooth,capsulated,virulent strain by the addition
of heat-killed smooth cells,
Oswald Avery and his group in a series of
experiments in the 1930s showed that the
material that carried this transforming
ability was DNA and not the proteins of the
cell.
transformation
Bacteriophage infection
转化试验
Transformation experiments
噬菌体感染试验
T4 bacteriophage are infectng E.coli
Part B
Genetic material form and site in the cell
1,Seven levels
① cell
② nucleolus
③ chromosome
④ nucleic acid
⑤ gene
⑥ genetic codon
⑦ nucleotide
2,Plasimid
① definition and features
② utilization in genetic
engineering
③ isolation and identification
④ classification
⑤ typical plasma
① Cell
Most DNA of prokaryotes and eukaryotes located in the
nucleolus （ eukayoote） or nucleoid（ prokayote）,The
number of the nucleolus or nucleoid varies for different
microbes and different cells of the same species.
some fungus are single-nucleolused such as Saccharomyces
cerevisiae(酿酒酵母 ),Aspergillus niger(黑曲霉 ) A.nidulans(构巢曲霉 ),Penicillium chrysogenum(产黄青霉 ),
While others are multi-nucleolused such as Neurospora
crasssa(粗糙脉孢霉 ) and A,oryzae (米曲霉 )
Algae and actinomyces,the hypha has more than one
nucleolus,while their spore has only one.
For bacterium,most bacillus has two nucleoids,and
coccus is single.
Genome
Mitochondrum
Cytoplasm gene
Choloroplast
coexisting organism
2μm plasmid(circle)
F plasmid
R factor
Col plasmid
prokaryote Ti plasmid
Ri plasmid
Mega plasmid
Degradative plasmid
eukaryote
Non-
nucleolus
chromosome
Types of genetic
material
② nuleolus
③ chromosome
A,The number of chromosome varies
greatly for different species.
B,Chromosomal set—— Heoloid and diploid
heploid has only one set of chromosome in
one cell and diploid has two same set of
chromosomes in one cell.
④ nucleic acid
A,Categories
B,Structure
C,length
⑤ gene
promoter
operon operator genes
structural genes
regulatory genes
Gene regulatory system
prokaryotes
exon
Gene
intron
eukaryotes
6,Genetic code(遗传密码 )
Specific nucleotide sequence in the DNA that determining
every amino acid,The information unit for genetic code is
condon (遗传密码的信息单位是密码子 )
A codon is composed by a triplet (三联体 )nucleotides.
A codon is described using a three uninterrupted nucleotide
sequence.
non-sense codon (UAA,UAG,UGA)
7,nucleotide
Functional unit for genetic—— gene
Information unit for genetic—— codon
The minimum unit for mutation and exchanging—
— nucleotide or base
AMP
TMP
GMP
CMP
2,Plasmid
2.1 definition：
prokaryote
free from the genome
independent replication（ independent
replicon独立性的复制子 ）
ccc DNA （ circular covalently closed DNA）
dsDNA
2.2 features：
Shape,Linear plasmid later be found in actinomyces such
as streptomyces coelicoler（天蓝色链霉菌） and the
prokaryote such as Borrelia hermsii（赫氏婢疏螺旋体）
Structure,twisted supercoil
Size,1.5~300kb,
Relative molecular weight,106~108,≈1% genome
Funtion,carrring non essential genes
Replicon
stringent replication control （ 1~2 plasmids）
relaxed replication control（ 10~15 plasmids even more）
Plasmid curing or elimination：
一些染料,丝裂霉素 C,UV，利福平，重金属离子,or
high tempreture
Integration （ episome附加体） F facfor
Utilization in genetic engineering
Plasmid has the following advantages in the manipulation of
genetic engineering,
1,Small volume facilitated the isolation of DNA and operation；
2,Circular—— keeping stability during the isolation
3,Replication origin independent of the genome
4,Multi-copy
5,Carring selective marks
Many cloning vectors are the modification of plasmid.
Isolation and identification of plasmid
Cell lysis
Removing of
RNA and
proteins
Seperation of
plasmid DNA and
chromosome DNA
identification
EM,
Agarose electrophoresis
PAGE
Density gradient centrifugation
Isolation
MW
measurement
Restrictive
endonulease
fragments
analysis
Kinds of plasmid
1.Conjugative plasmid
F plasmid (E.Coli)
pfdm and k plasmid (pseudomonas)
P plasmid (vibrio cholerae)
SCP plasmid (streptomyces)
2,antibiotic and
heavy metal ion
resistant plasmid
R plasmid (enterobacteria and
staphylococcus)
3,bacteriocin or antibiotic producing plasmid
enterobacteria
clostridium
streptomyces
4,Plasmid of physiological funtion
Utilize lactose,sugar,urea and nitrogen fixation
Decompose octane,camphor,naphthalene,水杨酸
Producing pigment
Symbiotic nitrogen fixing
5,toxin-producing plasmid
① exotoxin,endotoxin,K（ capsule） antigen—— E.coli
② burl-causing (致瘤 )—— Agrobacterium tumefaciens(根瘤土壤杆菌 )
③ tooth decay—— Streptococcus mutans(变异链球菌 )
④ 凝固酶,hemolysin,fibrinlysase溶纤维蛋白酶 和 enterotoxin—
— Staphylococcus aureus
① F plasmid (sex factor,fertility factor,F factor)
② R plasmid (resistance plasmid,R factor)
③ Col plasmid (colicin plasmid,colicinogenic factor,col factor)
④ Ti plasmid (tumor inducing plasmid诱癌质粒 )
⑤ Ri plasmid (root inducing plasmid)
⑥ megaplasmid (巨大质粒 )
⑦ degradative plasmid
typical
plasmids
Section B
gene mutation and breeding by induced
mutation
Part A gene mutation
a kind of variation
heritable changing
molecular structure and
amount of the genetic material
occur spontaneously or be induced
at a rather low frequency（ 10-6~10-9）
Definition
Wild type strain (野生型菌株,简称野生型 )is
isolated from the nature,wild type for short,
Mutant(突变株,突变体或突变型 ) derived from the
wild type via mutation and carries new traits.
A1 types of mutation
auxotroph
selectable mutant resistant mutant
conditional lethal mutant
morphological mutant
non-selectable mutant antigenic mutant
metabolite quantitative mutant
plus-mutant
minus-mutant
There are many genes in the cell whose gene product is
essential for cell growth such as proteins necessary for
DNA replication,It is therefore impossible to isolate
mutations in these genes,which totally inactivate the gene
function,as the cell would die,Conditional mutants can
be used in these circumstances,These are mutations which
are expressed only under certain circumstances,the most
common being high growth temperature,
Conditional
mutants
These temperature-sensitive mutants have a wild-
type phenotype at low temperatures but when
switched to a higher temperature the mutation is
manifested which allows the effect of the mutation to
be studied.
A2 mutation rate
definition
description
detection
A3 features of gene mutation
1,non-correspondent不对应性
2,rare 稀有性
3,independent独立性
4,inducible可诱变性
5,stable稳定性
6,reversible可逆性
7,Spontaneous 自发性
A4 mechanisms of gene mutation
transition
transversion
deletion
insertion
deletion
inserftion
translocation
inversion
substitution
frame-shift
mutation
point mutation
aberration
induced
mutation
Spontaneous
mutation
Gene
mutation
Replacement of a purine with another purine (or a
pyrimidine with a pyrimidine) is called a transition,
The change of a purine to a pyrimidine or vice versa
is a transversion,
Trransition and
transversion
The consequence of a point mutation on the
phenotype of the cell depends on how it alters the
protein sequence,The possibilities are,
same-sense,
mis-sense,
nonsense
frameshift mutations.
Consequence of a
point mutation
Point mutation - single base change
Deletion - loss of DNA
Insertion - addition of DNA
Inversion - DNA rearrangement
Duplication - repetition of a DNA sequence
Fig,1,Possible changes to DNA sequence that may result in mutation.
A mutation is any change in the nucleotide sequence
of the DNA in a cell,This change may be seen as an
alteration in the phenotype of the cell but it may be
silent if the change occurs in a non-essential part of a
protein sequence or in a non-coding part of the genome,
Mutations in essential genes such as those necessary for
DNA replication may be lethal and therefore never
isolated,
mutation
The changes in the DNA sequence range from single
base changes,normally called point mutations,to large
rearrangements of the genome,sometimes called multi-
site mutations,which involve short or long stretches of
DNA and may affect a number of genes,These multi-site
mutations may be deletions,insertions,inversions,
substitutions and duplications of sequence and occur as a
result of recombination(重组 ) or transposition(转座 ) in
the genome.
point mutation and
multi-site mutation
Point mutations may be the change of a purine for
another purine (A G) or a pyrimidine for another
pyrimidine (C T),called transitions,whereas the
changes of a purine to a pyrimidine and vice versa are
called transversions,The effect of a base change in the
genotype on the phenotype depends on the nature of the
mutation and where it occurs on the genome,There are
four types of point mutation illustrated in the following
figure and described below.
point mutation
1,Same-sense mutations are due to the redundancy( 冗余性 ) in the genetic code; the same amino acid is inserted
into the protein so no change in the phenotype is seen.
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Same sense mutation - same amino acid inserted
5 -AUG CCU UCA AGA UGU GGA CAA-3'
Met Pro Ser Arg Cys Gly Gin
Same-sense mutation
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Missense mutation - different amino acid inserted
5 -AUG CCU UCA GGA UGU GGG CAA-3'
Met Pro Ser G]y Cys Gly Gln
missense mutation
2,Missense mutations occur when a different amino acid is
inserted into the chain,The consequence of this change is
dependent on whether the alter ation is in an essential or non-
essential part of the protein,In the first case,protein function
may be altered or lost but in the latter no change to,the
phenotype may be observed.
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Nonsense,the creation of a termination codon - protein synthesis
stops
5-AUG CCU UCA AGA UGA GGG CAA-3
Met Pro Ser Arg STOP
nonsense mutation
3,Nonsense mutations result when the change in the base
sequence alters an amino acid-encoding codon to a STOP codon
(UAA,UAG,UGA),Protein synthesis terminates prematurely,
leading to the production of a truncated protein.
4,Frameshift mutations occur when one or two
nucleotides are deleted or inserted into the DNA
sequence,therefore altering the translational
reading frame and producing a completely
altered amino acid sequence from the point of
change.
frameshift mutation
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Frame shift mutation - from the point that a base is inserted or
deleted the amino acids are altered
Deletion or insertion of one or two bases
5'-AUG CCU UCA AGU GUG GGC AA-3'
Met Pro Ser Ser Val Gly etc.
Fig,The nature of point mutations as seen in the mRNA
sequence and their consequence on protein sequence.
frameshift mutation
Transposable element
It includes the following forms:
insertion sequence (插入序列,IS) prokaryotes
transposon(转座子,Tn) prokaryotes
transposable phage(转座噬菌体 ) Mu phage of E.coli
Transposable element(转座因子,TE) is also
known as moveable gene(可移动基因 ) and mobile
genetic element(可移动遗传因子 ) or jumping
gene(跳跃基因 )
The effect of a mutation can be reversed by a number of
means,The simplest is by reversion where a back
mutation (回复突变 )occurs to give back the original base
sequence (the wild-type),Selection for reversions is a good
test to indicate if the original mutation was a point
mutation as deletions do not revert easily.
reversion of
mutations
Another way of achieving a wild-type phenotype is by a
mechanism called suppression in which a second mutation
occurs at a different point in the genome,which
compensates for the effect of the first mutation,The second
mutation may be at a different site in the same gene in
which case it is called an intragenic suppressor or in a
completely different gene when it is termed an intergenic
suppressor.
第二节 微生物的基因突变
突变的机理
碱基的置换
移码突变
诱发突变的机理
辐射类诱变剂
碱基类似物诱变剂
引起 DNA碱基发生化学变化的诱变剂
自发突变的机理
Part B breeding by mutation
B1 breeding by spontaneous mutation
B2 breeding by induced mutation
Breeding by spontaneous mutation
1,Breeding in the production
Plus mutant (正突变株 ) is metabolite quantitative
mutant (产量突变株 )which has better productive
attributes than the original strain(原株 ).
2.Directional cultivation of better strains (定向培育优良菌株 )
Breeding by spontaneous mutation
2.Directional cultivation of better strains (定向培育优良菌株 )
based on the spontaneous mutation
under specific selective condition
constantly transplant
Time consuming and is a passive way of breeding,the
result is inpredictable,so is replaced by modern
breeding techniques.
BCG vaccine (卡介苗 )
B2 Breeding by induced mutation
1,treatment,mutagen (physical or chemical)
2,Microbe,evenly distributed or dispersed
3,Mutation rate,remarkably increased
4,Screening,by simple but efficient and rapid ways
5,For,mutant is utilized in later scienticific research and
production
6,Significance,nearly all strains used in the fermentation
industry are mutant famed by penicillium
chrysogenum(青霉素生产菌株,产黄青霉 )
1,Basic steps of breeding by induced breeding
die
unchange
survive minus
mutated a little
plus unsuitable
a lot
suitable for
productionoriginal strain
mu
tage
n
2.Principles of breeding by induced mutation
① select simple,easy and efficient mutagen
② use better original strain
③ treat the single-cell or single-spore suspension
④ use optimum mutating agent
⑤ take advantage of synergism of complex treatment
⑥ utilize and create the correlation of morphology,
physiology and yield
⑦ design high-efficient screening scheme
3,Means of screening
Although mutations occur spontaneously in
bacteria this is at a very low frequency so it is usually
desirable to use a mutagen such as UV light to
increase the chance of isolating the mutation of
interest,Identification of the presence of that
mutation depends on the nature of the gene of interest,
but generally these fall into three categories:
isolation of mutants
1,Mutants that are resistant to some toxic
compound such as antibiotics or to the
infection by a bacteriophage,These
mutations can be selected by growing in the
presence of the toxic agent,Only the
resistant mutant strains will grow.
2,Auxotrophic mutants that are unable to
synthesize a compound essential for growth such
as an amino acid or vitamin,These mutants
cannot be isolated directly but can be screened
for by replica plating(超连 ) as described below.
3,Mutants that are unable to use particular
substrates such as lactose or maltose for growth,
These may also be identified using replica plating.
Mutants resistant to toxic compounds
can be selected for by growth in the
presence of that agent,Mutations
leading to altered growth requirements
may be screened for by replica plating.
Colonies of bacteria are transferred,using
a sterile velvet pad,to two sets of media; one
on which the mutant will not grow and the
other on which it will,The mutant Colony is
identified by its lack of growth on the former
medium but purified from the latter.
1.Screening for metabolite quantitative mutant
2.Screening for resistant mutant
3.Screening for auxotroph mutant
琼脂块培养法
Gradient plate （梯度平板法）
4 steps for auxotroph mutant screening
① treating with mutagen
② eliminating the wild type
③ detecting the auxotroph
④ identification of the auxotroph
4 methods for the detection of the auxotroph
① layer plating method
② 限量补充培养法
③ 逐个检出法
④ replica plating影印培养法
营养缺陷型的鉴定 （ 生长谱法）
A 1 2 3 4 5 6 7
B 2 8 9 10 11 12 13
C 3 9 14 15 16 17 18
D 4 10 15 19 20 21 22
E 5 11 16 20 23 24 25
F 6 12 17 21 24 25 26
G 7 13 18 22 25 26 27
A
B
C
D
E
F
G
基础培养基
Layer plating method（夹层培养法及其结果）
This is a method used to screen a large number of colonies
for the presence of a particular mutation,Bacteria are spread
on to plates,containing medium on which both the parent and
the mutants will grow,at a dilution such that individual
colonies can be seen on the plate,After incubation,the
colonies are transferred to plates containing media that will
allow the detection of the presence of the mutation,using a
sterile velvet pad (丝绒布包裹的园柱章的园垫 ),
replica plating
The nature of that media depends on the mutation
required,In the case of auxotrophic mutations the
colonies may be replica plated on to plates with and
without a particular growth factor,Mutant strains that
are unable to synthesize that factor will not grow on the
minimal medium but will grow on the minimal medium
that does contain the growth factor,Having identified the
mutant colony by its lack of growth,it may then be picked
off and purified from the plate on which it has grown.
Putative
mutants
unable to
grow on
selective
medium
original plate
replica plate
nonselective
medium
selective
medium
replica plating for
the isolation of
mutant colonies
replica plating
Section C
Gene recombination and breeding by
hybridization
Part A gene recombination of prokaryote
① fragmental
② one direction from the donator to the recipient
③ unique and diverse mechanism ——
conjugation，
transformation
transduction
features
1,transformation
Definition
Microbes
Competence
Transforming principle
Process
transfection
Transformation is the result of the uptake of
free DNA from the surrounding medium and
the incorporation of that DNA into the genome,
In eukaryotes the ability of cells to take up
DNA is called transfection.
definition
转化,受体菌直接接受来自供体菌的 DNA片段，将其组合到基因组中，从而获得供体菌部分遗传性状的现象转化的必要条件
外部吸附
协助进入
不被降解
可以重组
Our knowlege that DNA is the genetic material
has come from the experiments based on the fact
that some species of bacteria are capable of
naturally taking up DNA from their surrounding
medium,and incorporating it into their genomes,
by a process called transformation.
Fred Griffith in the late 1920s first showed that a
rough,non-capsulated,avirulent strain of
Streptococcus pneumo-niae could be converted into
a smooth,capsulated,virulent strain by the addition
of heat-killed smooth cells,
Oswald Avery and his group in a series of
experiments in the 1930s showed that the
material that carried this transforming
ability was DNA and not the proteins of the
cell.
They were fortunate in their choice of
experimental microorganism as only a few species
are naturally able to take up DNA,including
Streptococcus,Bacillus,Neisseria,Haemophilus
and some species of archaebacteria,Uptake of
DNA by eukaryotic cells is usually referred to as
transfection.
microbes
The ability of a cell to take up DNA is dependent
on the cell being in a particular state called
competence,Competence is related to the presence
of receptors for DNA being present on the surface
of the cell,Other bacteria,such as ￡,cod,can be
induced to competence by chemical treatment
under cold conditions.
Depending on the species,DNA binding and
transfer into the cell may be of specific or non-
specific sequences,The DNA bound is normally
ds but in most cases it is made ss as it enters the
cell,Haemophilus influenzae takes up ds DNA,
The incoming DNA is integrated into the
chromosome by recombination.
Not all species within a genera will necessarily be
capable of transformation nor all cells within a
population,The ability of a bacterial cell to take up
DNA is associated with the development of a
competent state in which DNA receptors on the cell
surface and other transformation-specific proteins are
present,For example,in Neisseria gonorrhoeae,only
piliated cells are competent for transformation.
competence
Competence is generally dependent on growth
conditions,Competence may occur in only a few
cells in a culture (B,subtilis) or in nearly all cells
(S,pneumoniae) depending on the genera,
Competence may last for a few minutes (S,
pneumoniae) or for several hours (B,subtilis).
Competence can be induced in species that are
not naturally transformable,for example,￡,
coli,using chemical treatments such as ice-cold
Cad; treatment followed by a brief heat shock,
This competent state is not the same as natural
competence.
The nature of the DNA taken up is dependent on the
genera,Most bacteria will take up any DNA molecule,
but Haemophilus requires the presence of a 10 bp
specific sequence on the DNA,Haemophilus,a Gram-
negative bacterium,binds and takes up ds DNA in
contrast to the Gram-positive bacteria,Bacillus and
Streptococcus,which bind ds DNA but degrade one
strand as the other strand is imported into the cell,
Transforming
principle
The incoming linear DNA is protected from
degradation by competence-specific proteins and
is integrated into the genome by recombination
involving RecA protein,During artificially
induced competence in E,coli,circular plasmid
DNA are preferentially taken up and in this case
remain independent of the chromosome.
fate of DNA
Incomingsimple-
strandedDNA
Specific proteins
prevent DNA
degradation
homologousDNA
recipient chromosome
Transformation is probably an important
mechanism by which genes are transferred
naturally in the environment,Transformation
has also been used in genetic mapping as genes
that are close together are more likely to be taken
up on the same fragment of DNA and
incorporated together into the chromo some of a
recipient cell.
A mechanism by which DNA can be moved from one
cell to another is through the agency of a phage particle;
this is called transduction,Phage can pick up pieces of
chromosomal or plasmid DNA from one bacterium,
called the donor,and transfer it to a recipient bacteria
where it may be integrated into the recipient genome by
recombination,
transduction
Transduction has been found in a wide range of bacteria and
phage and is thought to play a significant role in the transfer of
genetic information in nature,Transduction is also an
important tool for geneticists as it allows the transfer of genes
between bacteria and the mapping of bacterial genomes; genes
that are close together will be transferred to a recipient cell
together,Many of the original uses of transducing phage have
been replaced by gene cloning,but they are still useful tools
today for the creation of bacterial strains and the analysis of
genomes.
roles of transduction
Generalized transduction is the transfer of any piece
of host DNA to a recipient by a phage particle,This is
due to the fact that some phage,at the point of phage
assembly,will accidentally package a phage-size
piece of host DNA into the phage head instead of the
phage genome,These transducing phage,on release
from the host,are capable of attaching to another cell
and injecting the DNA into this recipient,
普遍转导一、原核微生物 的基因重组
转导,以噬菌体为载体，将供体菌的部分
DNA传递给受体菌的过程普遍性转导局限性转导
The DNA,however,is not phage DNA and is
not capable of replicating,It will therefore only
survive if it is integrated into the host
chromosome by recombination.
Fig,1,Generalized transduction showing the transfer of
bio genes to a recipient.
Normal phage
replication and lysis No bio
+ transduction bio+ transduction
Frequently,especially if the donor and
recipient are closely related,the new DNA
will not make a significant difference to the
recipient,but if the donor and recipient
have different genotypes,a gene or genes
of the recipient cell may be converted to
that of the donor,
Fig 2 integration of transducing DNA into the host
chromosome by recombination
× ×
×
Transduced DNA
Chromosome
Recombination
cross-overs
Integration of incoming
DNA into chromosome
bio+
bio
bio+
Transduction is easy to carry out in the laboratory,A phage
preparation is added to a culture of the donor strain which is
allowed to incubate until complete lysis has been achieved,
The phage lysate containing a few transducing phage is then
used to infect a recipient culture of bacterium and this is
plated out on agar plates,Bacteria that receive the wild-type
phage will be lysed,but bacteria that receive transducing
phage will be able to grow as colonies,
The transfer of genes of interest may be
selected by using appropriate media,The
frequency at which a particular gene may be
transferred is about one cell in every 106 to 108,
Phage P22 in Salmonella typhimurium and
phage PI in E,coli are two systems frequently
used in the laboratory.
Specialized transduction is a rare occurrence
associated with a number of lysogenic phage that
integrate into the host chromosome,Normally when a
prophage is induced it excises from the chromosome in
a very precise fashion to form a complete phage
genome,Very rarely an excision event occurs which is
inaccurate; a piece of chromosome adjacent to the
phage is removed and a piece of the phage is left in the
chromosome,
specialized transduction
The excised phage is replicated normally,
packaged into phage heads and released to infect
another cell,The nature of the genes lost from the
phage particle is dependent on which end of the
genome is lost,In the example shown in Fig,3
where λ is integrated at its normal att site,on
excision,it may pick up gal or bio genes to form
λgal or λpbio,
Fig,3,Production of specialized transducing phage.
Theλgal phage have lost essential genes for
making mature phage so are incapable of
replicating and forming plaques in a new host
unless these functions are provided by a helper
λphage,The λbio phage are plaque-forming,
hence the 'p' designation,without any additional
functions being provided,as the genes that are
lost are not essential for phage replication.
The fate of the DNA in the recipient is various as shown
in Fig,4,The transducing phage may enter the lytic cycle
and produce many copies of the phage and the genes it
carries or it may integrate into the host chromosome,
Integration may be by site-specific recombination at the
att site to form a lysogen (Fig,4a) in which case there are
now two copies of the transduced gene(s) (partial diploid),
or recombination may occur between genes on the phage
and in the recipient chromosome (Fig,4b).
In contrast to generalized transduction,the particles
produced by specialized transduction are capable of
moving genes at a high frequency of transduction in a
specific manner,as all phage in a prepa ration carry the
same piece of DNA,Using a variety of genetic techniques
specialized transducing phage have been produced for
most areas of the ￡,coli chromosome which were used in
the past for the production of large amounts of DNA for a
particular gene,the transfer of genes between strains and
for the creation of partial diploid strains in order to study
gene function.
Fig,4,(a) formation of A lysogen;
Some of the fates of a specialized transducing phage on infections
of a recipient:
(b) replacement of recipient chromosomal DNA with
DNA carried on A by generalized recombination.
Transduction is the movement of host DNA
from one bacterial cell (donor) to another cell
(recipient) by a bacteriophage.
defective phage (缺陷噬菌体 )
summarization
Generalized transduction is the result of a rare
error during phage assembly,leading to the
packaging of a piece of host genome into the phage
head instead of the phage DNA,Such transducing
phage are capable of infecting new hosts and
injecting in the DNA but,as this DNA is not phage
DNA,it will be lost unless the DNA is integrated
into the host genome by recombination.
Generalized
transduction（普遍转导）summerization
This is a feature of some lysogenic phage which spend
some time integrated into sites in the host chromosome,At
induction,instead of the phage genome accurately excising
from the chromosome,it excises a piece of adjacent
chromosome as well,Phage particles containing these
genes will infect recipient cells where the whole phage may
integrate into the chromosome to form a lysogen,or the
chromosomal DNA may be integrated by recombination.
局限转导summarization
Special transduction has two categories:
① Low frequency transduction
② High frequency transduction
Donor,λdgal（转导噬菌体）
Recipient,E.coli K12gal-
Frequency,10-1~10-6
Transductant,gal+
Low frequency transduction
High frequency transduction
Based on F plasmid,E.coli can be divided into
four types:
① F+ strain
② F- strain
③ Hfr strain (high frequency recombination)
④ F- strain
研究细菌接合方法的基本原理
Integration and excision of F plasmids from the
chromosomes showing the production of F’
factors by aberrant excision.
Conjugation is the mechanism by which genetic
material is transferred between two bacterial cells
by plasmids,A cell containing a conjugative plasmid
(F+ fertility+) forms a mating pair with a cell that
does not contain a conjugative-plasmid (F-) by means
of an F-pilus on the surface of the cell,The pilus
contracts,pulling the two cells into contact,and the
DNA is transferred from the plasmid-containing cell,
the donor,to the recipient,The tra genes,carried
on the F plasmid,contain all the information for the
conjugative process.
Key notes——conjugation
The DNA that is transferred is single
stranded and is synthesized by rolling-circle
replication,The plasmid is nicked in one
strand at a site called oriT,origin of transfer,
The intact strand acts as a template for DNA
synthesis while the nicked strand is displaced
and transferred into the recipient cell,Once
inside the recipient cell,a complementary
strand of DNA is synthesized to form a new F
factor.
Key notes —— DNA transfer
F factors can integrate into specific sites in the E,
coli chromosome by homologous recombination between
insertion sequences found on the chromosome and the
plasmid,Even though integrated,the plasmid is still
capable of initiating the rolling-circle mechanism of
replication at oriT,followed by the transfer of single-
stranded DNA into a recipient,(continue)
Key notes integration and H fr strains
The whole chromosome therefore acts like a
large F plasmid,allowing the transfer of
chromosomal genes to another bacterium,Genes
closest to the plasmid are transferred first,In
the recipient,the donor chromosomal DNA is
integrated into the recipient chromosome by
recombination,Strains which have an F plasmid
integrated into the chromosome are called Hfr
(high-frequency recombination) strains.
Key notes
F plasmids integrated in the chromosome
normally excise themselves as precisely as they
entered to give an intact F plasmid,However,in
some cases,the excision is not precise and a
piece of the chromosome is inte grated into the
F plasmid,These are called F' factors.
Key notes F’ factors
一、原核微生物 的基因重组
接合,供体菌与受体菌的完整细胞之间直接接触而传递和重组 DNA的过程
F+和 F-的接合一、原核微生物 的基因重组
接合,Hfr菌株与 F-接合一、原核微生物 的基因重组
接合,F’转导
An important mechanism by which DNA can be
transferred between cells is conjugation,
mediated by some plasmids,which requires
direct contact between cells,Conjugative
plasmids,typified by the F plasmid of E,coli,
have the ability to transfer themselves
between cells and,in some cases,also to
transfer pieces of chromosomal DNA,
conjugation
F plasmids and their relatives carry a group of
genes called the tra genes which encode all the
proteins required to form a mating pair with another
bacterium that does not contain an F plasmid,DNA is
then transferred from the plasmid-containing cell,
called the donor or F+ cell,to the recipient,F- cell,A
map of the F plasmid of E.coli is shown in Fig,The F
plasmid also contains a number of insertion
sequences distributed throughout the molecule,
A simplified map of an F plasmid
The plasmid is large,95
kb in size,and normally
exists as one or two
copies in the cell,
Vegetative replication of
the plasmid at cell
division is by normal
semi-conservative,
bidirectional DNA
synthesis from oriV
followed by segregation
of the plasmid into the
daughter cells.
Cells containing F plasmids (F+) produce specialist F-pili on
their surface which are encoded by the tra genes,These are
proteinaceous,filamentous structures that attach to the
surface of F- cells to form a mating pair (Fig,a),They will not
form mating pairs with F+ due to a phenomenon called surface
exclusion which is mediated by a pair of tra-gene encoded
proteins in the outer membrane of F+ cells,The F-pilus
retracts on contact,pulling the two cells into close contact.
电镜照片显示两个细菌细胞的接合。
为了更易看见，性丝已经用与它特异结合的病毒标记。
A single-stranded copy of the F plasmid is
transferred into the recipient cell,It is produced by
rolling-circle replication,A single-strand nick is made
in the DNA at a site called oriT (origin of transfer)
which is located beside the tra genesThe 3' end of the
nicked DNA is used as a primer to start synthesis of a
new strand of DNA,using the intact strand as a
template by rolling-circle replication,The nicked
strand is displaced and transferred through a pore into
the recipient cell (Fig.b).
DNA transfer
Transfer of chromosomal genes to a recipient
by Hfr strains
Synthesis of a complementary strand of DNA
occurs in the recipient cell,The order of
transfer of the F plasmid is such that the last
genes to be transferred are tra genes,The
plasmid circularizes in the recipient to give an F+
cell and the mating-pair separates (Fig c).
Stages in conjugation,(a) a mating pair; (b)
DNA transfer;(c) separation
Integration and Hfr strains
As mentioned previously,the F plasmid has a
number of insertion sequences scatttered
about the molecule as does the E.cvoli
chromosome,Occasionally,recombination
between these homologous sequences leads th
the integration of the F plasmid into the
genome at specific sites,
Strains that have an F plasmid integrated into the
chromosome are called Hfr strains (standing for
high-frequency recombination for the reasons
described below),The F plasmid,once integrated,is
still capable of forming mating pairs and initiating
transfer
replication at oriT,However,in this case,the
chromosome plus plasmid acts like a large F
factor so F-plasmid DNA followed by the E,coli
chromosome is transferred into the recipient
(Fig,4),Chromosomal genes closest to the oriT
are transferred first,The order of transfer can
be clockwise or anticlockwise,depending on the
orientation in which the F plasmid is inserted.
The number of genes transferred depends on how
long the mating pair is held together but,in theory,
it takes 100 min to transfer the whole E,coli
chromosome,The last genes to be transferred are
the tra genes,In practice,the mating pairs break
at random and,normally,only part of the DNA is
transferred,The incoming single-stranded,
chromosomal DNA cannot recircularize to form a
plasmid or a second chromosome,However,it can
integrate into the recipient chromosome by
homologous recombination,thus allowing the
transfer of genes from donor to recipient.
This is why the strains are named high-frequency
recombination strains as they allow the transfer of
chromosomal genes from one strain to another at
high frequency,Hfr strains are used in genetic
mapping for determining the position of genes on the
chromosome of ￡,coli as the order of genes can be
determined by the time at which they enter a
recipient during mating.
F factors excise themselves from the chromosome by a
process which is the reverse of integration,Normally
this is an accurate process but occasionally an error is
made and the plasmid picks up a piece of the adjacent
chromosome to form an F' factor,These F' factors
can be used to transfer chromosomal genes into a
recipient cell and are used by bacterial geneticists
particularly for creating partial diploids of genes in a
cell for investigating dominance/reces sive
relationships between alleles of the same gene.
F’ factors
Protoplast fussion(原生质体融合 )
Definition
Microbes
Stages
通过人为的方法，使遗传性状不同的两个细胞的原生质体进行融合，借以获得兼有双亲遗传性状的稳重组子的过程，称为原生质体融合。由此法获得的重组子称为融合子（ fusant）。
Bacteria Actinomycetes
Yeast,mold,mushroom,
Plant cell
Animal cell（ without the cell wall）
Parent strain selecting （ placed in isotonic solution）
Remove of
the cell wall centrifugation
Addition of PEG to
facilitate the fusion
Diluting with
isotonic
solution
Plating on the
minimum medium
Replica
plating
Plating on
the selective
medium
Determine of biological
attributes and
performence
treating with the lysozyme for
prokaryote and snail digestive enzyme
for eukaryote results in the formation
of protoplast（ G+） or spheroplast（ G-).
Remove of the cell wall in the
protoplast fusion
Gene recombination of eukaryotes
Sexual hybridization
Parasexual hybridization
二、真核微生物 的基因重组
有性杂交不同遗传型微生物的性细胞发生接合和重组的现象
准性杂交不同遗传型微生物体细胞的核融合而导致的一种低频率基因重组现象
The process of parasexual
hybridization
Anastomosis（菌丝联结）
The formation of heterocaryon
（ plasmogamy）（形成异核体或质配）
Nuclear fusion（ caryogamy核融合或核配）
Somatic crossing-over（体细胞交换）
Section D
Genetic Engineering
基因工程 就是有意识地把一个生物体中有用的目的基因转入另一个生物体,使后者获得新的遗传性状或表达所需要的产物,
重组 DNA操作
1.获得目的基因（外源基因） gene of interest
2.形成重组 DNA分子
3.转化受体细胞
4.对转化子的筛选和鉴定
5.对获得外源基因的细胞或生物体通过发酵、
细胞培养、养殖或栽培等，最终获得所需要的遗传性状或表达出所需要的产物。
获得所需的目的基因从基因文库中钓取反转录合成 cDNA
聚合酶链式反应（ PCR）
细胞内总 DNA的提取、分离与基因文库的构建总 DNA的抽提、沉淀、纯度检测及浓度测定限制性内切酶酶切
DNA片段插入质粒或噬菌体 DNA
质粒或噬菌体导入细菌构建基因文库细胞内总 DNA提取的一般方法
1.细胞破碎或消化
2.蛋白质去除
3.氯仿进一步去除残留的蛋白质和微量苯酚
4.Rnase降解 RNA
5.乙醇使 DNA沉淀,
洗涤,干燥后 -20备用
6,DNA溶液的浓度和纯度的测定
DNA对 260nm紫外光的特征吸收在提取分离到的总 DNA中，目的基因片段含量很少，且难以检出和分离。因此，科学家们用限制性内切酶将总 DN A切开成为许多小的片段，把这些片段分别插入到环状 DNA载体即质粒（ plasmid）中，这些质粒载体可以转入细菌并随着细菌的繁殖而复制，这种过程又称为 基因克隆 。
基因克隆与基因文库将总 DNA包含的基因组各片段分别克隆在质粒和噬菌体载体上，便构成了该生物的 基因文库。
基因组文库由一批细菌组成，每个细菌带有人
DNA的一个小片段。为简化起见，
只显示了几个代表性片段（彩色的）的克隆。事实上，所有的灰色 DNA片段都要被克隆。
人基因组文库的构建科学家一般用与目的基因部分序列互补并标记了放射性同位素的一小段单链 DNA为探针（ probe）对克隆的基因文库进行杂交实验，从中选出目的基因。以后在做重组 DNA操作时，可以从基因文库中调用所需要的目的基因。
钓取目的基因构建基因文库并获取目的基因费时费事，
也会出现其他问题，如真核生物细胞的基因组都含有较多非编码蛋白的内含子序列，给构建基因文库并获取目的基因增添了难以克服的麻烦。为了避免这一问题，科学家发明了用反转录人工合成互补 DNA的方法 。
Synthesis of the cDNA
A cDNA (short for
complementary
DNA or copy DNA)
is a DNA copy of an
RNA,usually an
mRNA,It can be
synthesized from
an mRNA template
using reverse
transcriptase.
此合成反应的引物是由单链
DNA的 3端提供的，它能自身折叠，形成几个如图所示的随机互补碱基对。聚合酶为大肠杆菌 DNA-PolI的 Klenow
片段。
该过程称为反转录引物为 oligo（ dT）
模板为 mRNA
酶为逆转录酶
（ reverse transcriptase）
在细胞分化的不同阶段，根据代谢反应的特殊需要，细胞往往特异性地转录产生编码特殊蛋白的 mRNA。因此，用 cDNA方法获取的
DNA片段是具有特定功能的目的基因，这是反转录人工合成互补 DNA方法的优势。
advantages
inventor,invented by Kary Mullis (American )
and his colleagues in the year of 1988
features,rapid,efficient,specifitic
application,widely used in the field of
biology,iatrology,archeology,anthroplogy
and law.
polymerase chain reaction
(聚合酶链式反应 )
polymerase chain reaction
(聚合酶链式反应 )
Definition,it’s a technique using the enzyme
DNA polymerase to amplify a region of DNA
between two predetermined sites,Oligonucleotides
complementary to these sites serve as primers for
synthesis of copies of the DNA between the sites,
Each cycle of PCR doubles the number of copies of
the the amplified DNA until a large quantity has
been made,This process (exponential increase)
obviously leads to very hign numbers in only a short
time.
Mullis 的联想使他以后发明了
PCR技术
PCR的发明是操作技术的革命，因为这一重大创新成果,Mullis教授于 1993年获得了诺贝尔奖。
PCR system
DNA模板 +4种核苷酸 +引物 +DNA聚合酶
template 4dDNTPS primer DNA polymerase
Principles
denaturation（ 95℃ ）
3 stages annealing （ 55℃ ）
extension （ 72℃ ）
30 cycles
变性 延伸退火 denaturation
annealing
extention
每一轮 PCR可使目的基因片段增加一倍,30轮循环可获得 230（ 1.07*109）个基因片段。
PCR仪利用
PCR与分子杂交标记快速准确地检测出病原性物质构建重组质粒和基因克隆基因重组和克隆操作最重要的工具限制性内切酶载体宿主菌限 制 性 内 切 酶
Arber,Smith和 Nathans因为在发现限制酶方面开创性的工作而共同获得了 1978年的诺贝尔奖。迄今为止，人们已发现和鉴定出了 200多种限制性核酸内切酶。
发现者和 DNA 技术的大部分工具相似，限制酶最先是由研究者们在研究他们感兴趣的特殊生物学问题时发现的。当将一些细菌 DNA导入某些细菌后，这些
DNA总会被降解掉。寻找这种降解原因时揭示了在宿主细菌内存在一类新型的核酸酶。这些核苷酸酶的最重要的特征是它们只在某些核苷酸序列处切割 DNA。
细菌自身的 DNA在这些序列上做了化学修饰因而免受切割。 因为这些酶限制了 DNA在某些细菌菌株之间的
DNA转移，所以它们被命名为限制酶。 不同的细菌菌株含有不同的限制酶，每一种酶的识别序列和切割位点均不同。
限制性内切酶发 现 及 命名限制性内切酶这些核苷酸酶的最重要的特征是它们只在某些核苷酸序列处切割 DNA。被比喻为 DNA操作的分子手术刀。这使得它们对现代 DNA技术的所有方面都有决定性的作用，因为一个特定的限制酶总是在同样的位点上切割一个特定的 DNA分子，这样，对来源于生物体的 DNA样品，
一个特定的限制酶总是产生一批相同的
DNA片段。
作 用 特 点
DNA技术中应用的限制酶主要来自细菌，而且因为它们的靶序列较短（通常为
4-8个核苷酸对）它们将完全随机地出现在任何一个足够长的 DNA分子中，这样它们可以被用来分析任何来源的 DNA。
限 制 性 内 切 酶来 源平 末 端 与 粘 性 末 端限制性内切酶和 T4连接酶对 DNA的作用此酶产生的粘性末端使得两个片段的末端能相互正确配对，极大地促进了它们的连接，此连接反应也重建原先的酶切位点。
DNA连接酶能把两个 DNA片段连到一起而不管 DNA的来源。
体外重组 DNA分子的形成为什么需要载体被限制酶切开的 DNA并不能直接进入到细菌等宿主细胞中，切出的目的基因连入到一个合适的载体中，才可能被转入宿主细胞中并且随之繁殖而复制。
载体是运送目的基因片段进入宿主细胞的工具。
细菌质粒目前最常用的载体 噬菌体
cosmid质粒 等载体质粒是细菌细胞中自然存在与染色体外可以自主复制的一段环状 DNA分子为了适于基因工程操作，需要将质粒加以修饰和改造 。
质粒载体
PUC18特点,
1,该质粒比较小，可以插入一段较长的 DNA片段。
2,进入宿主细菌细胞后,pUCl8在每个细胞中可复制形成大约 500个拷贝,于是也大大增加了插入在该质粒中的外源目的基因的拷贝数 。 质粒载体上的外源基因片段在宿主细胞中大量复制过程称为克隆 (clone)。
PUC18特点
PUC18特点
3．在 pUCl8中有一小段人为设计和插入的具有多种限制性酶切位点的序列，即多克隆位点 (图 11— 12)。这一段序列为根据目的基因片段的需要，选用不同的限制性内切酶分别对质粒和外源 DNA进行切割，产生相互匹配的末端，为进一步的连接提供了方便。
PUC18特点之三含有多克隆位点
PUC18中的多克隆位点
4,含有一个 LacZ基因，多克隆位点区就位于 lacZ基因之中。 lacZ基因编码 半乳糖苷酶,它可以使细菌在含有 X-gal(5— 溴 -4-氯 -3-吲哚 — p-D-半乳糖 )底物的培养基上形成蓝色的菌落，即 X-gal被 lacZ基因编码产生的 — 半乳糖苷酶水解成 蓝色 。当外源 DNA片段插入到多克隆位点时就隔断了 lacZ基因，使其失去活性和表达功能。因此，凡是插入了外源 DNA片段 pUCl8重组质粒进入到宿主细菌后，这些细菌由于不能利用
X— gal，结果在含有 X-gal底物的培养基上形成了白色的菌落，而不是蓝色的菌落。如此，利用这一特性很容易将携带重组质粒的细菌克隆从没有携带重组质粒的细菌中筛选出来 。
PUC18特点之四含有筛选标记
5,pUCl8还携带了氨卞青霉素抗性基因
(ampR),它使细菌能在含氨卞青霉素的培养基中生长,而没有转入 pUCl8的细菌则全部会被杀死 。 因此,用含氨卞青霉素的培养基可很方便地对携带 pUCl8的细菌做筛选 。
PUC18特点之五含有抗药性标记
ampR
利用 lacZ基因的插入失活筛选重组质粒为什么需要宿主菌 DNA技术并非仅仅在细胞体外将 DNA分子与载体切开,再连接成重组分子,而是要做外源目的基因的克隆,然后使其在合适的宿主生物中表达 。 基因克隆过程是通过将重组 DNA
分子转入到宿主细胞中来完成的 。
宿主菌有一些原核生物和真核生物或噬菌体都可以是基因克隆的宿主 。 在 实验室中,大肠杆菌是一种常用的宿主菌 。
为什么需要宿主菌类型将目的基因克隆到大杆肠菌中的操作步骤感受态细胞
1,用特定的限制性内切酶酶解 (切割 )已获取的目的基因片段和质粒载体；
2,用 T4连接酶将已形成特定末端序列的目的基因与质粒相互连接,形成重组 DNA分子,即重组质粒；
3,用物理方法处理大肠杆菌细胞,使其易于接纳外源重组
DNA分子 (制备感受态细胞 ),在大肠杆菌细胞中加入了少量重组 DNA分子后,使其进入到宿主细胞中,这一过程又称为转化；
以大肠杆菌为宿主菌进行基因的克隆
DNA重组转化质粒载体目的基因重组质粒大肠杆菌感受态细胞
4,培养大肠杆菌,让重组 DNA分子及其外源目的基因形成大量的拷贝；
5,用抗生素和 X-gal筛选出含重组质粒的大肠杆菌细胞,将细菌中的质粒分离出来,进行 DNA分子的检查,或直接对已克隆了目的基因的大肠杆菌细胞进行鉴定 。
以大肠杆菌为宿主菌进行基因的克隆基因克隆筛选和鉴定一般克隆基因的检查和鉴定方法实 验 室 内 一 般 常 用 酶 切 和 电 泳
(electrophoresis)方法来检查克隆的基因除了从细菌细胞中提取质粒做酶切电泳分析外,
还可以利用 DNA杂交的技术直接鉴定带有重组质粒的细菌克隆 。
基 因 酶切和电泳细菌克隆 DNA杂交用类似于提取分离细胞中总 DNA的方法将克隆了目的基因的重组质粒从大肠杆菌中分离出来酶 切 和 电 泳根据载体部分和目的基因片段上已知的酶切位点，
选择合适的限制性内切酶对重组质粒进行酶解利用琼脂糖凝胶电泳的方法对大小不等的酶解片段进行分离和鉴定质粒提取酶切电泳琼脂糖凝胶电泳原理,DNA片段上的磷酸基团都带有负电荷，
把大小不同的 DNA片段装入琼脂糖凝胶 (包含电解质的多孔支持介质 )一端并将其置于静电场中，
DNA分子便向阳极移动。 DNA长度增加，来自电场的驱动力和来自凝胶的阻力之间的比率就会降低，不同长度的 DNA片段就会表现出不同的迁移率，因而就可依据 DNA分子的大小来使其分离。
分离、纯化和鉴定 DNA片段最常规的实验技术琼脂糖凝胶电泳该过程可以通过把示踪染料和分子质量标准参照物与样品一起进行电泳而得到检测 。
标准分子质量参照物可以提供一个用于确定 DNA片段大小的依据 。 最后,根据电泳条带分析的结果,可知重组质粒经酶切后产生了大小为多少的几段 DNA分子,是否与所设计构建的重组质粒结构相吻合 。
正在进行的 DNA电泳凝胶电泳
DNA杂交技术鉴定带有重组质粒的细菌克隆根据重组质粒上目的基的部分 DNA序列,
人工合成 (或用 PCR技术合成 )与之互补的一小段单链 DNA(或 RNA),利用带有 32p_磷酸的
dATP使其标记上放射性同位素,这一小段与重组质粒上目的基因的部分序列互补的核酸分子称为 DNA探针 。
目的基因的部分序列
DNA杂交
DNA探针利用核酸杂交技术鉴定克隆基因的具体操作
(1) 将琼脂培养基上的菌落原位印迹 (转移 )到一张滤膜上； （ 原位印迹 ）
（ 2） 对滤膜及细胞做原位物理和化学处理,使其 DNA暴露并变性成单链； （ 物化处理 ）
（ 3） 将同位素探针液加入到滤膜上保温一定时间使之与克隆的目的基因杂 交,冲洗滤膜,除去多余的探针分子 ； （ 与探针杂交 ）
(4)将滤膜铺放在光学胶片上,有放射性同位素的杂交分子造成光学胶片的原位局部暴光 （ 放射自显影 ）
(5)根据光学胶片上放射自显影的具体斑点位置,确定和挑选出克 隆了目的基因的菌落 。
（ 确定阳性菌落 ）
利用核酸杂交技术鉴定克隆基因通过 DNA体外重组技术构建的重组质粒还可以直接用以转化蓝藻 (又称蓝细菌 )等原核生物或其他一些原生生物如单细胞绿藻等利用核酸杂交技术鉴定克隆基因的具体操作原位印迹物化处理与探针杂交放射自显影确定阳性菌落转化受体细胞和转化子的筛选基因克隆以后便有了大量目的基因,下一步就是使其在合适的宿主细胞中表达,产生需要的基因表达产物或使宿主生物具备所需要的性状,同时,该外源目的基因还能在宿主细胞中稳定地遗传,这一过程就是 遗传转化 。
所谓合适的 宿主细胞 就是接纳外源 DNA
的细胞,它们可以是细菌等原核生物,也可以是植物和动物细胞 。
如果需要让克隆的基因表达和产生大量的编码蛋白,
科学家们往往将该基因插入到一种 表达载体 中,用带有目的基因的表达载体再转化 大肠杆菌,对大肠杆菌进行大量培养使该目的基因在大肠杆菌细胞中大量表达和积累,通过对表达产物的分离纯化,便可以获得想要的产品 。
DNA体外重组技术构建的重组质粒还可以直接用以转化 蓝细菌 等原核生物或其他一些原生生物如 单细胞绿。
原 核 生 物 的 遗 传 转 化大肠杆菌目的基因插入表达载体 转化宿主细胞 蓝细菌单细胞绿藻诱导表达植物和动物的遗传转化常用的方法载体法转化基因直接转移基因的直 接转移方法
(1)高压电脉冲法：利用高压电脉冲的电激穿孔作用把外源 DNA引 入动植物细胞或组织中；
(2)基因枪法：用粒子枪把表面吸附有外源
DNA的金属微粒高速地射人动植物细胞或组织中 。
(3)微注 射法：利用显微注射仪等将外源
DNA直接注入细胞核或细胞质中 。
基因直接转移的基因枪法基因枪利用高压气体冲击包裹 DNA的金或钨颗粒，当压力超过破裂盘的承受能力，气体喷出，
经过加速的颗粒射向靶细胞，将 DNA
转入目的细胞中植物细胞,常用除去了细胞壁的原生质体；
为受体动物细胞,常 用的受体包括受精卵,胚胎干细胞等 。
与目的基因在大肠杆菌或蓝藻等原核生物中的转化和表达相比较,对植物或动物的遗传转化往往要困难 和复杂得多 。
宿 主 细 胞转化子的分析 —— Southern杂交通过 PCR,酶切,连接,克隆,转化等一系列 DNA的操作,获得了转基因的生物 (又称转化子 )
后,常用核酸杂交的方法对转基因生物中外源目的基因的情况进行检测和分析 。 为了纪念其发明者 Edward Southern,这种核酸杂交的技术称为
Southern杂交 (Southern blot)
Southern杂交 具 体 操 作 过 程
(1)从转化子中提取出总 DNA；
(2)根据外源目的基因片段上和前后的酶切位点情况选择合适的限制性内切酶，对总 DNA进行酶解；
(3)对酶解产物做琼脂糖凝胶电泳，与重组质粒酶切后只形成很少的 DNA片段不同，总 DNA上有许多相同的酶切位点，酶解后产生出许多 DNA片段。因此，在琼脂糖凝胶上形成许多连续无法辨认的条带，其中可能有外源目的基因的片段和相应的酶切位点；
(4)在电泳后的凝胶上覆盖一片杂交滤膜，上下铺盖许多纸巾并压一重物，通过纸巾的毛细管吸水作用，琼脂糖凝胶上的全部 DNA条带便转移印迹到滤膜上；
(5)用碱性溶液对滤膜及 DNA做变性处理，使双链
DNA解开成为单链分子；
(6)用与外源目的基因的部分序列互补,并带有放射性同位素的核酸分子作为 DNA探针,将同位素探针液加入到滤膜上保温一定时间使之与外源目的基因的 DNA变性单链杂交 形成双链分子；
(7)用缓冲液冲洗滤膜,使其他不能互补的多余放射性 探针分子被除去；
(8)把杂交后的滤膜铺放在光学胶片上,具有放射性 同位素的杂交分子造成光学胶片的原位局部暴光,即放射自显影
(9)根据光学胶片上放射自显影的具体条带位置,
以野生型细胞总 DNA的 Southern杂交实验结果为对照,分析确定在转化子总 DNA中是否存在外源目的基因及相应的酶切位点 。
生物技术 —— 现代生命科学的革命生物科学成为当今世界自然科学的热点和重点 主要由于两方面的原因,(1)20世纪后叶，
分子生物学 领域一系列突破性成就，使生命科学在自然科学中的地位发生了革命性的变化；
(2)建立在实验室研究基础上的 生物技术
(biotechnology)的发展为人类带来了巨大的利益和财富。
生物技术的成果和成功应用首先需要实验室大量复杂的基础研究工作,生物技术还是微生物学,分子生物学,化学工程,材料科学等多学科交叉的综合性学科 。 高技术 (精细和密集的复杂技术 ),高投入 (尤其 是前期科研投入高 ),高利润是生物技术产业的显著特点 。
科学界广泛地认识到：作为 21世纪发展最快的学科,
生物技术将是未来经济发展的新动力,它们将在以下领域形成巨大的产业,
A 农业
B 动物饲养业,
C 能源,
D 生物法处理环境污染,
E 纤维和包装材料,
F 药物和医学等 。
从以开发利用石油和金属为主的传统工业经济过渡到开发利用基因的经济将从根本上改变我们的文明 。
生物技术将是未来经济发展的新动力
Product line using
biotechnology
几百年前，以蒸汽机为标志的工业革命被称为人类的第一次技术革命，它解放了人的双手，使人类从繁重的体力劳动中解脱出来；
几十年前，以计算机和网络为标志的电子和信息技术革命被看作是人类的第二次技术革命，它扩张了人的大脑，极大地提高了人们获取和交流信息的速度和广度；
科学家们预言，以重组 DNA和基因克隆为标志的生物技术是人类历史上第三次技术革命，生命的复制和改造将极大地提高人类生活的质量，这一次革命更重大的意义在于，人类不但可以改造客观世界，还可以改造自身。
生物技术是人类历史上的第三次技术革命一,生物技术定义生物技术是应用自然科学及工程学的原理,依靠微生物,动物,植物体作为反应器将 物料进行加工以提供产品为社会服务的技术 。
1982年，国际合作与发展组织应用生物或来自生物体的物质制造或改进一种商品的技术,其中还包括改良有重要 经济价值的植物与动物和利用微生物改良环境的技术 。
美国政府技术顾问委员会 (OAT)
一,生物技术定义强调了生物技术的商品属性生物技术商业化特点
1997年 2月,英国科学家 Wilmut等人完成了首例哺乳动物 —— 绵羊,多莉,的克隆这个神奇的故事立刻上了各传播媒介的首页和头条,一夜之间,全球大多数生物技术公司的股票价值成倍地上升 。
自从世界上第一个生物技术公司 Genentech公司于
1976年成立以来,现在世界上已涌现出了几千个生物技术公司,估计年产值高达 6000亿美元 。 目前世界 著 名 的 生 物 技 术 公 司 包 括 Dupont 公司,
Medicago公司,Monsanto公司,Genentech公司,
Novo Nordisk公司等等 。
生物技术可获得高利润
Section E
degeneration,rejuvenation and
preservation of the stock culture
degeneration
Lose typical morphology
Slow growth and decreased spore number
Minus mutation with decreased metabolites
Weakened ability of infection to the host
Lowered resistant ability to the adverse
environment
features
Prevention of degeneracy
Control the numbers of transplantation
Create better culture condition
Transplant using spore rather than hypha
Preserved by effective methods
rejuvenation
Pure culture isolation
Rejuvenated by culture in host
Eliminated the degenerated individules
Preservation
(conservation,maintenance)
Lyophilization,freeze-drying 冷冻干燥保藏
Liquid nitrogen cryo-preservation 液氮超低温冷冻保藏
Routine slant transplantation method 常规斜面定期移植法
Stab semi-solid agar preservation 半固体穿刺保藏
Covered by liquid paraffin 石蜡油封藏法
Glycerol suspension 甘油悬液保藏法
Sand and oil preservation 砂土保藏法
heredity,variation and breeding of
microbes
Heredity(inheritance) and variation is the most
intrinsical atrributes of all living organisms.
Let’s get married our
babies will be as beautiful
as me and as intelligent as
you……,
If they are as ugly as
me and as foolish as
you……,
The genotype(遗传型又称基因型 ) of an organism is the
genetic information stored in its genetic material,the
exact sequence of the bases or nucleotides in its DNA,
This sequence dictates what proteins or RNA molecules
can be produced by the organism,how the genes may be
regulated and the functioning of the protein once it is
synthesized,Not all proteins are made at the same time
and the full genotype of an organism cannot be
discovered unless the genome is sequenced,
genotype
The phenotype(表型 ) of an organism is the expression or
manifestation of that genotype as it can be observed or
measured; the manifestation of the genotype in the form of
characteristics which is observable and measurable,for
example,whether or not it produces a capsule or is resistant
to an antibiotic,The most common form of an organism is
often referred to as the wild-type,
Alterations to the genotype can therefore be seen as changes
in the phenotype,(遗传性的改变可通过表型改变来观察 )
phenotype
Any change to the sequence of bases (genotype) in
the DNA of an organism is a mutation or variation(突变或变异 ),The change may be a single-base change
called a point mutation or involve a much larger
sequence of DNA in which case it is called a multi-site
mutation,DNA may be deleted,inserted,inverted,
duplicated or substituted,(that means the change of
structure or amount),
Variation or
mutation
Modification(饰变 ) is the changing of phenotype
occurs only on the transcriptional and translational
levels but not involve the change of the genotype,
that is,the genetic material or the nucleotides,thus
it’s only a ornamental alteration.
Modification
Different nomenclatures/abbreviations are used
for describing genes and mutations in different
organisms,but in bacteria the following
convention is used:
Phenotype is described using a three-letter
abbreviation of roman letters with the first letter
capitalized,A superscript + or - is used to designate
the presence or absence of this phenotype;
For example,Phe- indicates the inability to
synthesize phenylalanine or Lac+ indicates
the ability to utilize lactose as a carbon
source,The superscripts 's' and 'r' may also
be used to designate sensitive or resistant.
Genotype is indicated by three lower case letters
(usually reflecting the phenotype) with a fourth
upper case letter to indicate specific genes
involved,all of which are italicized,for example,
pheA or lacZ.
Although,naturally,there may be a large number of
individuals of a species with slightly different phenotypes,
microbiologists and in particular microbial geneticists try
to work with organisms that are genetically identical,
Consequently,in the laboratory,we work with pure
cultures where microbes are descendent from the same
original parent to minimize the genetic variability,That
parent may often be referred to as the wild-type.
Section A
substantial basis for genetic variation
Part A
Three classical experiments
1,Transformation
2,Bacteriophage infection
3,Reconstruction of tobacco mosaic viruses
Our knoelege that DNA is the genetic material
has come from the experiments based on the fact
that some species of bacteria are capable of
naturally taking up DNA from their surrouding
medium,and incorprating it into their genomes,by
a process called transformation.
Fred Griffith in the late 1920s first showed that a
rough,non-capsulated,avirulent strain of
Streptococcus pneumo-niae could be converted into
a smooth,capsulated,virulent strain by the addition
of heat-killed smooth cells,
Oswald Avery and his group in a series of
experiments in the 1930s showed that the
material that carried this transforming
ability was DNA and not the proteins of the
cell.
transformation
Bacteriophage infection
转化试验
Transformation experiments
噬菌体感染试验
T4 bacteriophage are infectng E.coli
Part B
Genetic material form and site in the cell
1,Seven levels
① cell
② nucleolus
③ chromosome
④ nucleic acid
⑤ gene
⑥ genetic codon
⑦ nucleotide
2,Plasimid
① definition and features
② utilization in genetic
engineering
③ isolation and identification
④ classification
⑤ typical plasma
① Cell
Most DNA of prokaryotes and eukaryotes located in the
nucleolus （ eukayoote） or nucleoid（ prokayote）,The
number of the nucleolus or nucleoid varies for different
microbes and different cells of the same species.
some fungus are single-nucleolused such as Saccharomyces
cerevisiae(酿酒酵母 ),Aspergillus niger(黑曲霉 ) A.nidulans(构巢曲霉 ),Penicillium chrysogenum(产黄青霉 ),
While others are multi-nucleolused such as Neurospora
crasssa(粗糙脉孢霉 ) and A,oryzae (米曲霉 )
Algae and actinomyces,the hypha has more than one
nucleolus,while their spore has only one.
For bacterium,most bacillus has two nucleoids,and
coccus is single.
Genome
Mitochondrum
Cytoplasm gene
Choloroplast
coexisting organism
2μm plasmid(circle)
F plasmid
R factor
Col plasmid
prokaryote Ti plasmid
Ri plasmid
Mega plasmid
Degradative plasmid
eukaryote
Non-
nucleolus
chromosome
Types of genetic
material
② nuleolus
③ chromosome
A,The number of chromosome varies
greatly for different species.
B,Chromosomal set—— Heoloid and diploid
heploid has only one set of chromosome in
one cell and diploid has two same set of
chromosomes in one cell.
④ nucleic acid
A,Categories
B,Structure
C,length
⑤ gene
promoter
operon operator genes
structural genes
regulatory genes
Gene regulatory system
prokaryotes
exon
Gene
intron
eukaryotes
6,Genetic code(遗传密码 )
Specific nucleotide sequence in the DNA that determining
every amino acid,The information unit for genetic code is
condon (遗传密码的信息单位是密码子 )
A codon is composed by a triplet (三联体 )nucleotides.
A codon is described using a three uninterrupted nucleotide
sequence.
non-sense codon (UAA,UAG,UGA)
7,nucleotide
Functional unit for genetic—— gene
Information unit for genetic—— codon
The minimum unit for mutation and exchanging—
— nucleotide or base
AMP
TMP
GMP
CMP
2,Plasmid
2.1 definition：
prokaryote
free from the genome
independent replication（ independent
replicon独立性的复制子 ）
ccc DNA （ circular covalently closed DNA）
dsDNA
2.2 features：
Shape,Linear plasmid later be found in actinomyces such
as streptomyces coelicoler（天蓝色链霉菌） and the
prokaryote such as Borrelia hermsii（赫氏婢疏螺旋体）
Structure,twisted supercoil
Size,1.5~300kb,
Relative molecular weight,106~108,≈1% genome
Funtion,carrring non essential genes
Replicon
stringent replication control （ 1~2 plasmids）
relaxed replication control（ 10~15 plasmids even more）
Plasmid curing or elimination：
一些染料,丝裂霉素 C,UV，利福平，重金属离子,or
high tempreture
Integration （ episome附加体） F facfor
Utilization in genetic engineering
Plasmid has the following advantages in the manipulation of
genetic engineering,
1,Small volume facilitated the isolation of DNA and operation；
2,Circular—— keeping stability during the isolation
3,Replication origin independent of the genome
4,Multi-copy
5,Carring selective marks
Many cloning vectors are the modification of plasmid.
Isolation and identification of plasmid
Cell lysis
Removing of
RNA and
proteins
Seperation of
plasmid DNA and
chromosome DNA
identification
EM,
Agarose electrophoresis
PAGE
Density gradient centrifugation
Isolation
MW
measurement
Restrictive
endonulease
fragments
analysis
Kinds of plasmid
1.Conjugative plasmid
F plasmid (E.Coli)
pfdm and k plasmid (pseudomonas)
P plasmid (vibrio cholerae)
SCP plasmid (streptomyces)
2,antibiotic and
heavy metal ion
resistant plasmid
R plasmid (enterobacteria and
staphylococcus)
3,bacteriocin or antibiotic producing plasmid
enterobacteria
clostridium
streptomyces
4,Plasmid of physiological funtion
Utilize lactose,sugar,urea and nitrogen fixation
Decompose octane,camphor,naphthalene,水杨酸
Producing pigment
Symbiotic nitrogen fixing
5,toxin-producing plasmid
① exotoxin,endotoxin,K（ capsule） antigen—— E.coli
② burl-causing (致瘤 )—— Agrobacterium tumefaciens(根瘤土壤杆菌 )
③ tooth decay—— Streptococcus mutans(变异链球菌 )
④ 凝固酶,hemolysin,fibrinlysase溶纤维蛋白酶 和 enterotoxin—
— Staphylococcus aureus
① F plasmid (sex factor,fertility factor,F factor)
② R plasmid (resistance plasmid,R factor)
③ Col plasmid (colicin plasmid,colicinogenic factor,col factor)
④ Ti plasmid (tumor inducing plasmid诱癌质粒 )
⑤ Ri plasmid (root inducing plasmid)
⑥ megaplasmid (巨大质粒 )
⑦ degradative plasmid
typical
plasmids
Section B
gene mutation and breeding by induced
mutation
Part A gene mutation
a kind of variation
heritable changing
molecular structure and
amount of the genetic material
occur spontaneously or be induced
at a rather low frequency（ 10-6~10-9）
Definition
Wild type strain (野生型菌株,简称野生型 )is
isolated from the nature,wild type for short,
Mutant(突变株,突变体或突变型 ) derived from the
wild type via mutation and carries new traits.
A1 types of mutation
auxotroph
selectable mutant resistant mutant
conditional lethal mutant
morphological mutant
non-selectable mutant antigenic mutant
metabolite quantitative mutant
plus-mutant
minus-mutant
There are many genes in the cell whose gene product is
essential for cell growth such as proteins necessary for
DNA replication,It is therefore impossible to isolate
mutations in these genes,which totally inactivate the gene
function,as the cell would die,Conditional mutants can
be used in these circumstances,These are mutations which
are expressed only under certain circumstances,the most
common being high growth temperature,
Conditional
mutants
These temperature-sensitive mutants have a wild-
type phenotype at low temperatures but when
switched to a higher temperature the mutation is
manifested which allows the effect of the mutation to
be studied.
A2 mutation rate
definition
description
detection
A3 features of gene mutation
1,non-correspondent不对应性
2,rare 稀有性
3,independent独立性
4,inducible可诱变性
5,stable稳定性
6,reversible可逆性
7,Spontaneous 自发性
A4 mechanisms of gene mutation
transition
transversion
deletion
insertion
deletion
inserftion
translocation
inversion
substitution
frame-shift
mutation
point mutation
aberration
induced
mutation
Spontaneous
mutation
Gene
mutation
Replacement of a purine with another purine (or a
pyrimidine with a pyrimidine) is called a transition,
The change of a purine to a pyrimidine or vice versa
is a transversion,
Trransition and
transversion
The consequence of a point mutation on the
phenotype of the cell depends on how it alters the
protein sequence,The possibilities are,
same-sense,
mis-sense,
nonsense
frameshift mutations.
Consequence of a
point mutation
Point mutation - single base change
Deletion - loss of DNA
Insertion - addition of DNA
Inversion - DNA rearrangement
Duplication - repetition of a DNA sequence
Fig,1,Possible changes to DNA sequence that may result in mutation.
A mutation is any change in the nucleotide sequence
of the DNA in a cell,This change may be seen as an
alteration in the phenotype of the cell but it may be
silent if the change occurs in a non-essential part of a
protein sequence or in a non-coding part of the genome,
Mutations in essential genes such as those necessary for
DNA replication may be lethal and therefore never
isolated,
mutation
The changes in the DNA sequence range from single
base changes,normally called point mutations,to large
rearrangements of the genome,sometimes called multi-
site mutations,which involve short or long stretches of
DNA and may affect a number of genes,These multi-site
mutations may be deletions,insertions,inversions,
substitutions and duplications of sequence and occur as a
result of recombination(重组 ) or transposition(转座 ) in
the genome.
point mutation and
multi-site mutation
Point mutations may be the change of a purine for
another purine (A G) or a pyrimidine for another
pyrimidine (C T),called transitions,whereas the
changes of a purine to a pyrimidine and vice versa are
called transversions,The effect of a base change in the
genotype on the phenotype depends on the nature of the
mutation and where it occurs on the genome,There are
four types of point mutation illustrated in the following
figure and described below.
point mutation
1,Same-sense mutations are due to the redundancy( 冗余性 ) in the genetic code; the same amino acid is inserted
into the protein so no change in the phenotype is seen.
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Same sense mutation - same amino acid inserted
5 -AUG CCU UCA AGA UGU GGA CAA-3'
Met Pro Ser Arg Cys Gly Gin
Same-sense mutation
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Missense mutation - different amino acid inserted
5 -AUG CCU UCA GGA UGU GGG CAA-3'
Met Pro Ser G]y Cys Gly Gln
missense mutation
2,Missense mutations occur when a different amino acid is
inserted into the chain,The consequence of this change is
dependent on whether the alter ation is in an essential or non-
essential part of the protein,In the first case,protein function
may be altered or lost but in the latter no change to,the
phenotype may be observed.
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Nonsense,the creation of a termination codon - protein synthesis
stops
5-AUG CCU UCA AGA UGA GGG CAA-3
Met Pro Ser Arg STOP
nonsense mutation
3,Nonsense mutations result when the change in the base
sequence alters an amino acid-encoding codon to a STOP codon
(UAA,UAG,UGA),Protein synthesis terminates prematurely,
leading to the production of a truncated protein.
4,Frameshift mutations occur when one or two
nucleotides are deleted or inserted into the DNA
sequence,therefore altering the translational
reading frame and producing a completely
altered amino acid sequence from the point of
change.
frameshift mutation
Original sequence
5 -AUG CCU UCA AGA UGU GGG CAA-3
Met Pro Ser Arg Cys Gly Gln
Frame shift mutation - from the point that a base is inserted or
deleted the amino acids are altered
Deletion or insertion of one or two bases
5'-AUG CCU UCA AGU GUG GGC AA-3'
Met Pro Ser Ser Val Gly etc.
Fig,The nature of point mutations as seen in the mRNA
sequence and their consequence on protein sequence.
frameshift mutation
Transposable element
It includes the following forms:
insertion sequence (插入序列,IS) prokaryotes
transposon(转座子,Tn) prokaryotes
transposable phage(转座噬菌体 ) Mu phage of E.coli
Transposable element(转座因子,TE) is also
known as moveable gene(可移动基因 ) and mobile
genetic element(可移动遗传因子 ) or jumping
gene(跳跃基因 )
The effect of a mutation can be reversed by a number of
means,The simplest is by reversion where a back
mutation (回复突变 )occurs to give back the original base
sequence (the wild-type),Selection for reversions is a good
test to indicate if the original mutation was a point
mutation as deletions do not revert easily.
reversion of
mutations
Another way of achieving a wild-type phenotype is by a
mechanism called suppression in which a second mutation
occurs at a different point in the genome,which
compensates for the effect of the first mutation,The second
mutation may be at a different site in the same gene in
which case it is called an intragenic suppressor or in a
completely different gene when it is termed an intergenic
suppressor.
第二节 微生物的基因突变
突变的机理
碱基的置换
移码突变
诱发突变的机理
辐射类诱变剂
碱基类似物诱变剂
引起 DNA碱基发生化学变化的诱变剂
自发突变的机理
Part B breeding by mutation
B1 breeding by spontaneous mutation
B2 breeding by induced mutation
Breeding by spontaneous mutation
1,Breeding in the production
Plus mutant (正突变株 ) is metabolite quantitative
mutant (产量突变株 )which has better productive
attributes than the original strain(原株 ).
2.Directional cultivation of better strains (定向培育优良菌株 )
Breeding by spontaneous mutation
2.Directional cultivation of better strains (定向培育优良菌株 )
based on the spontaneous mutation
under specific selective condition
constantly transplant
Time consuming and is a passive way of breeding,the
result is inpredictable,so is replaced by modern
breeding techniques.
BCG vaccine (卡介苗 )
B2 Breeding by induced mutation
1,treatment,mutagen (physical or chemical)
2,Microbe,evenly distributed or dispersed
3,Mutation rate,remarkably increased
4,Screening,by simple but efficient and rapid ways
5,For,mutant is utilized in later scienticific research and
production
6,Significance,nearly all strains used in the fermentation
industry are mutant famed by penicillium
chrysogenum(青霉素生产菌株,产黄青霉 )
1,Basic steps of breeding by induced breeding
die
unchange
survive minus
mutated a little
plus unsuitable
a lot
suitable for
productionoriginal strain
mu
tage
n
2.Principles of breeding by induced mutation
① select simple,easy and efficient mutagen
② use better original strain
③ treat the single-cell or single-spore suspension
④ use optimum mutating agent
⑤ take advantage of synergism of complex treatment
⑥ utilize and create the correlation of morphology,
physiology and yield
⑦ design high-efficient screening scheme
3,Means of screening
Although mutations occur spontaneously in
bacteria this is at a very low frequency so it is usually
desirable to use a mutagen such as UV light to
increase the chance of isolating the mutation of
interest,Identification of the presence of that
mutation depends on the nature of the gene of interest,
but generally these fall into three categories:
isolation of mutants
1,Mutants that are resistant to some toxic
compound such as antibiotics or to the
infection by a bacteriophage,These
mutations can be selected by growing in the
presence of the toxic agent,Only the
resistant mutant strains will grow.
2,Auxotrophic mutants that are unable to
synthesize a compound essential for growth such
as an amino acid or vitamin,These mutants
cannot be isolated directly but can be screened
for by replica plating(超连 ) as described below.
3,Mutants that are unable to use particular
substrates such as lactose or maltose for growth,
These may also be identified using replica plating.
Mutants resistant to toxic compounds
can be selected for by growth in the
presence of that agent,Mutations
leading to altered growth requirements
may be screened for by replica plating.
Colonies of bacteria are transferred,using
a sterile velvet pad,to two sets of media; one
on which the mutant will not grow and the
other on which it will,The mutant Colony is
identified by its lack of growth on the former
medium but purified from the latter.
1.Screening for metabolite quantitative mutant
2.Screening for resistant mutant
3.Screening for auxotroph mutant
琼脂块培养法
Gradient plate （梯度平板法）
4 steps for auxotroph mutant screening
① treating with mutagen
② eliminating the wild type
③ detecting the auxotroph
④ identification of the auxotroph
4 methods for the detection of the auxotroph
① layer plating method
② 限量补充培养法
③ 逐个检出法
④ replica plating影印培养法
营养缺陷型的鉴定 （ 生长谱法）
A 1 2 3 4 5 6 7
B 2 8 9 10 11 12 13
C 3 9 14 15 16 17 18
D 4 10 15 19 20 21 22
E 5 11 16 20 23 24 25
F 6 12 17 21 24 25 26
G 7 13 18 22 25 26 27
A
B
C
D
E
F
G
基础培养基
Layer plating method（夹层培养法及其结果）
This is a method used to screen a large number of colonies
for the presence of a particular mutation,Bacteria are spread
on to plates,containing medium on which both the parent and
the mutants will grow,at a dilution such that individual
colonies can be seen on the plate,After incubation,the
colonies are transferred to plates containing media that will
allow the detection of the presence of the mutation,using a
sterile velvet pad (丝绒布包裹的园柱章的园垫 ),
replica plating
The nature of that media depends on the mutation
required,In the case of auxotrophic mutations the
colonies may be replica plated on to plates with and
without a particular growth factor,Mutant strains that
are unable to synthesize that factor will not grow on the
minimal medium but will grow on the minimal medium
that does contain the growth factor,Having identified the
mutant colony by its lack of growth,it may then be picked
off and purified from the plate on which it has grown.
Putative
mutants
unable to
grow on
selective
medium
original plate
replica plate
nonselective
medium
selective
medium
replica plating for
the isolation of
mutant colonies
replica plating
Section C
Gene recombination and breeding by
hybridization
Part A gene recombination of prokaryote
① fragmental
② one direction from the donator to the recipient
③ unique and diverse mechanism ——
conjugation，
transformation
transduction
features
1,transformation
Definition
Microbes
Competence
Transforming principle
Process
transfection
Transformation is the result of the uptake of
free DNA from the surrounding medium and
the incorporation of that DNA into the genome,
In eukaryotes the ability of cells to take up
DNA is called transfection.
definition
转化,受体菌直接接受来自供体菌的 DNA片段，将其组合到基因组中，从而获得供体菌部分遗传性状的现象转化的必要条件
外部吸附
协助进入
不被降解
可以重组
Our knowlege that DNA is the genetic material
has come from the experiments based on the fact
that some species of bacteria are capable of
naturally taking up DNA from their surrounding
medium,and incorporating it into their genomes,
by a process called transformation.
Fred Griffith in the late 1920s first showed that a
rough,non-capsulated,avirulent strain of
Streptococcus pneumo-niae could be converted into
a smooth,capsulated,virulent strain by the addition
of heat-killed smooth cells,
Oswald Avery and his group in a series of
experiments in the 1930s showed that the
material that carried this transforming
ability was DNA and not the proteins of the
cell.
They were fortunate in their choice of
experimental microorganism as only a few species
are naturally able to take up DNA,including
Streptococcus,Bacillus,Neisseria,Haemophilus
and some species of archaebacteria,Uptake of
DNA by eukaryotic cells is usually referred to as
transfection.
microbes
The ability of a cell to take up DNA is dependent
on the cell being in a particular state called
competence,Competence is related to the presence
of receptors for DNA being present on the surface
of the cell,Other bacteria,such as ￡,cod,can be
induced to competence by chemical treatment
under cold conditions.
Depending on the species,DNA binding and
transfer into the cell may be of specific or non-
specific sequences,The DNA bound is normally
ds but in most cases it is made ss as it enters the
cell,Haemophilus influenzae takes up ds DNA,
The incoming DNA is integrated into the
chromosome by recombination.
Not all species within a genera will necessarily be
capable of transformation nor all cells within a
population,The ability of a bacterial cell to take up
DNA is associated with the development of a
competent state in which DNA receptors on the cell
surface and other transformation-specific proteins are
present,For example,in Neisseria gonorrhoeae,only
piliated cells are competent for transformation.
competence
Competence is generally dependent on growth
conditions,Competence may occur in only a few
cells in a culture (B,subtilis) or in nearly all cells
(S,pneumoniae) depending on the genera,
Competence may last for a few minutes (S,
pneumoniae) or for several hours (B,subtilis).
Competence can be induced in species that are
not naturally transformable,for example,￡,
coli,using chemical treatments such as ice-cold
Cad; treatment followed by a brief heat shock,
This competent state is not the same as natural
competence.
The nature of the DNA taken up is dependent on the
genera,Most bacteria will take up any DNA molecule,
but Haemophilus requires the presence of a 10 bp
specific sequence on the DNA,Haemophilus,a Gram-
negative bacterium,binds and takes up ds DNA in
contrast to the Gram-positive bacteria,Bacillus and
Streptococcus,which bind ds DNA but degrade one
strand as the other strand is imported into the cell,
Transforming
principle
The incoming linear DNA is protected from
degradation by competence-specific proteins and
is integrated into the genome by recombination
involving RecA protein,During artificially
induced competence in E,coli,circular plasmid
DNA are preferentially taken up and in this case
remain independent of the chromosome.
fate of DNA
Incomingsimple-
strandedDNA
Specific proteins
prevent DNA
degradation
homologousDNA
recipient chromosome
Transformation is probably an important
mechanism by which genes are transferred
naturally in the environment,Transformation
has also been used in genetic mapping as genes
that are close together are more likely to be taken
up on the same fragment of DNA and
incorporated together into the chromo some of a
recipient cell.
A mechanism by which DNA can be moved from one
cell to another is through the agency of a phage particle;
this is called transduction,Phage can pick up pieces of
chromosomal or plasmid DNA from one bacterium,
called the donor,and transfer it to a recipient bacteria
where it may be integrated into the recipient genome by
recombination,
transduction
Transduction has been found in a wide range of bacteria and
phage and is thought to play a significant role in the transfer of
genetic information in nature,Transduction is also an
important tool for geneticists as it allows the transfer of genes
between bacteria and the mapping of bacterial genomes; genes
that are close together will be transferred to a recipient cell
together,Many of the original uses of transducing phage have
been replaced by gene cloning,but they are still useful tools
today for the creation of bacterial strains and the analysis of
genomes.
roles of transduction
Generalized transduction is the transfer of any piece
of host DNA to a recipient by a phage particle,This is
due to the fact that some phage,at the point of phage
assembly,will accidentally package a phage-size
piece of host DNA into the phage head instead of the
phage genome,These transducing phage,on release
from the host,are capable of attaching to another cell
and injecting the DNA into this recipient,
普遍转导一、原核微生物 的基因重组
转导,以噬菌体为载体，将供体菌的部分
DNA传递给受体菌的过程普遍性转导局限性转导
The DNA,however,is not phage DNA and is
not capable of replicating,It will therefore only
survive if it is integrated into the host
chromosome by recombination.
Fig,1,Generalized transduction showing the transfer of
bio genes to a recipient.
Normal phage
replication and lysis No bio
+ transduction bio+ transduction
Frequently,especially if the donor and
recipient are closely related,the new DNA
will not make a significant difference to the
recipient,but if the donor and recipient
have different genotypes,a gene or genes
of the recipient cell may be converted to
that of the donor,
Fig 2 integration of transducing DNA into the host
chromosome by recombination
× ×
×
Transduced DNA
Chromosome
Recombination
cross-overs
Integration of incoming
DNA into chromosome
bio+
bio
bio+
Transduction is easy to carry out in the laboratory,A phage
preparation is added to a culture of the donor strain which is
allowed to incubate until complete lysis has been achieved,
The phage lysate containing a few transducing phage is then
used to infect a recipient culture of bacterium and this is
plated out on agar plates,Bacteria that receive the wild-type
phage will be lysed,but bacteria that receive transducing
phage will be able to grow as colonies,
The transfer of genes of interest may be
selected by using appropriate media,The
frequency at which a particular gene may be
transferred is about one cell in every 106 to 108,
Phage P22 in Salmonella typhimurium and
phage PI in E,coli are two systems frequently
used in the laboratory.
Specialized transduction is a rare occurrence
associated with a number of lysogenic phage that
integrate into the host chromosome,Normally when a
prophage is induced it excises from the chromosome in
a very precise fashion to form a complete phage
genome,Very rarely an excision event occurs which is
inaccurate; a piece of chromosome adjacent to the
phage is removed and a piece of the phage is left in the
chromosome,
specialized transduction
The excised phage is replicated normally,
packaged into phage heads and released to infect
another cell,The nature of the genes lost from the
phage particle is dependent on which end of the
genome is lost,In the example shown in Fig,3
where λ is integrated at its normal att site,on
excision,it may pick up gal or bio genes to form
λgal or λpbio,
Fig,3,Production of specialized transducing phage.
Theλgal phage have lost essential genes for
making mature phage so are incapable of
replicating and forming plaques in a new host
unless these functions are provided by a helper
λphage,The λbio phage are plaque-forming,
hence the 'p' designation,without any additional
functions being provided,as the genes that are
lost are not essential for phage replication.
The fate of the DNA in the recipient is various as shown
in Fig,4,The transducing phage may enter the lytic cycle
and produce many copies of the phage and the genes it
carries or it may integrate into the host chromosome,
Integration may be by site-specific recombination at the
att site to form a lysogen (Fig,4a) in which case there are
now two copies of the transduced gene(s) (partial diploid),
or recombination may occur between genes on the phage
and in the recipient chromosome (Fig,4b).
In contrast to generalized transduction,the particles
produced by specialized transduction are capable of
moving genes at a high frequency of transduction in a
specific manner,as all phage in a prepa ration carry the
same piece of DNA,Using a variety of genetic techniques
specialized transducing phage have been produced for
most areas of the ￡,coli chromosome which were used in
the past for the production of large amounts of DNA for a
particular gene,the transfer of genes between strains and
for the creation of partial diploid strains in order to study
gene function.
Fig,4,(a) formation of A lysogen;
Some of the fates of a specialized transducing phage on infections
of a recipient:
(b) replacement of recipient chromosomal DNA with
DNA carried on A by generalized recombination.
Transduction is the movement of host DNA
from one bacterial cell (donor) to another cell
(recipient) by a bacteriophage.
defective phage (缺陷噬菌体 )
summarization
Generalized transduction is the result of a rare
error during phage assembly,leading to the
packaging of a piece of host genome into the phage
head instead of the phage DNA,Such transducing
phage are capable of infecting new hosts and
injecting in the DNA but,as this DNA is not phage
DNA,it will be lost unless the DNA is integrated
into the host genome by recombination.
Generalized
transduction（普遍转导）summerization
This is a feature of some lysogenic phage which spend
some time integrated into sites in the host chromosome,At
induction,instead of the phage genome accurately excising
from the chromosome,it excises a piece of adjacent
chromosome as well,Phage particles containing these
genes will infect recipient cells where the whole phage may
integrate into the chromosome to form a lysogen,or the
chromosomal DNA may be integrated by recombination.
局限转导summarization
Special transduction has two categories:
① Low frequency transduction
② High frequency transduction
Donor,λdgal（转导噬菌体）
Recipient,E.coli K12gal-
Frequency,10-1~10-6
Transductant,gal+
Low frequency transduction
High frequency transduction
Based on F plasmid,E.coli can be divided into
four types:
① F+ strain
② F- strain
③ Hfr strain (high frequency recombination)
④ F- strain
研究细菌接合方法的基本原理
Integration and excision of F plasmids from the
chromosomes showing the production of F’
factors by aberrant excision.
Conjugation is the mechanism by which genetic
material is transferred between two bacterial cells
by plasmids,A cell containing a conjugative plasmid
(F+ fertility+) forms a mating pair with a cell that
does not contain a conjugative-plasmid (F-) by means
of an F-pilus on the surface of the cell,The pilus
contracts,pulling the two cells into contact,and the
DNA is transferred from the plasmid-containing cell,
the donor,to the recipient,The tra genes,carried
on the F plasmid,contain all the information for the
conjugative process.
Key notes——conjugation
The DNA that is transferred is single
stranded and is synthesized by rolling-circle
replication,The plasmid is nicked in one
strand at a site called oriT,origin of transfer,
The intact strand acts as a template for DNA
synthesis while the nicked strand is displaced
and transferred into the recipient cell,Once
inside the recipient cell,a complementary
strand of DNA is synthesized to form a new F
factor.
Key notes —— DNA transfer
F factors can integrate into specific sites in the E,
coli chromosome by homologous recombination between
insertion sequences found on the chromosome and the
plasmid,Even though integrated,the plasmid is still
capable of initiating the rolling-circle mechanism of
replication at oriT,followed by the transfer of single-
stranded DNA into a recipient,(continue)
Key notes integration and H fr strains
The whole chromosome therefore acts like a
large F plasmid,allowing the transfer of
chromosomal genes to another bacterium,Genes
closest to the plasmid are transferred first,In
the recipient,the donor chromosomal DNA is
integrated into the recipient chromosome by
recombination,Strains which have an F plasmid
integrated into the chromosome are called Hfr
(high-frequency recombination) strains.
Key notes
F plasmids integrated in the chromosome
normally excise themselves as precisely as they
entered to give an intact F plasmid,However,in
some cases,the excision is not precise and a
piece of the chromosome is inte grated into the
F plasmid,These are called F' factors.
Key notes F’ factors
一、原核微生物 的基因重组
接合,供体菌与受体菌的完整细胞之间直接接触而传递和重组 DNA的过程
F+和 F-的接合一、原核微生物 的基因重组
接合,Hfr菌株与 F-接合一、原核微生物 的基因重组
接合,F’转导
An important mechanism by which DNA can be
transferred between cells is conjugation,
mediated by some plasmids,which requires
direct contact between cells,Conjugative
plasmids,typified by the F plasmid of E,coli,
have the ability to transfer themselves
between cells and,in some cases,also to
transfer pieces of chromosomal DNA,
conjugation
F plasmids and their relatives carry a group of
genes called the tra genes which encode all the
proteins required to form a mating pair with another
bacterium that does not contain an F plasmid,DNA is
then transferred from the plasmid-containing cell,
called the donor or F+ cell,to the recipient,F- cell,A
map of the F plasmid of E.coli is shown in Fig,The F
plasmid also contains a number of insertion
sequences distributed throughout the molecule,
A simplified map of an F plasmid
The plasmid is large,95
kb in size,and normally
exists as one or two
copies in the cell,
Vegetative replication of
the plasmid at cell
division is by normal
semi-conservative,
bidirectional DNA
synthesis from oriV
followed by segregation
of the plasmid into the
daughter cells.
Cells containing F plasmids (F+) produce specialist F-pili on
their surface which are encoded by the tra genes,These are
proteinaceous,filamentous structures that attach to the
surface of F- cells to form a mating pair (Fig,a),They will not
form mating pairs with F+ due to a phenomenon called surface
exclusion which is mediated by a pair of tra-gene encoded
proteins in the outer membrane of F+ cells,The F-pilus
retracts on contact,pulling the two cells into close contact.
电镜照片显示两个细菌细胞的接合。
为了更易看见，性丝已经用与它特异结合的病毒标记。
A single-stranded copy of the F plasmid is
transferred into the recipient cell,It is produced by
rolling-circle replication,A single-strand nick is made
in the DNA at a site called oriT (origin of transfer)
which is located beside the tra genesThe 3' end of the
nicked DNA is used as a primer to start synthesis of a
new strand of DNA,using the intact strand as a
template by rolling-circle replication,The nicked
strand is displaced and transferred through a pore into
the recipient cell (Fig.b).
DNA transfer
Transfer of chromosomal genes to a recipient
by Hfr strains
Synthesis of a complementary strand of DNA
occurs in the recipient cell,The order of
transfer of the F plasmid is such that the last
genes to be transferred are tra genes,The
plasmid circularizes in the recipient to give an F+
cell and the mating-pair separates (Fig c).
Stages in conjugation,(a) a mating pair; (b)
DNA transfer;(c) separation
Integration and Hfr strains
As mentioned previously,the F plasmid has a
number of insertion sequences scatttered
about the molecule as does the E.cvoli
chromosome,Occasionally,recombination
between these homologous sequences leads th
the integration of the F plasmid into the
genome at specific sites,
Strains that have an F plasmid integrated into the
chromosome are called Hfr strains (standing for
high-frequency recombination for the reasons
described below),The F plasmid,once integrated,is
still capable of forming mating pairs and initiating
transfer
replication at oriT,However,in this case,the
chromosome plus plasmid acts like a large F
factor so F-plasmid DNA followed by the E,coli
chromosome is transferred into the recipient
(Fig,4),Chromosomal genes closest to the oriT
are transferred first,The order of transfer can
be clockwise or anticlockwise,depending on the
orientation in which the F plasmid is inserted.
The number of genes transferred depends on how
long the mating pair is held together but,in theory,
it takes 100 min to transfer the whole E,coli
chromosome,The last genes to be transferred are
the tra genes,In practice,the mating pairs break
at random and,normally,only part of the DNA is
transferred,The incoming single-stranded,
chromosomal DNA cannot recircularize to form a
plasmid or a second chromosome,However,it can
integrate into the recipient chromosome by
homologous recombination,thus allowing the
transfer of genes from donor to recipient.
This is why the strains are named high-frequency
recombination strains as they allow the transfer of
chromosomal genes from one strain to another at
high frequency,Hfr strains are used in genetic
mapping for determining the position of genes on the
chromosome of ￡,coli as the order of genes can be
determined by the time at which they enter a
recipient during mating.
F factors excise themselves from the chromosome by a
process which is the reverse of integration,Normally
this is an accurate process but occasionally an error is
made and the plasmid picks up a piece of the adjacent
chromosome to form an F' factor,These F' factors
can be used to transfer chromosomal genes into a
recipient cell and are used by bacterial geneticists
particularly for creating partial diploids of genes in a
cell for investigating dominance/reces sive
relationships between alleles of the same gene.
F’ factors
Protoplast fussion(原生质体融合 )
Definition
Microbes
Stages
通过人为的方法，使遗传性状不同的两个细胞的原生质体进行融合，借以获得兼有双亲遗传性状的稳重组子的过程，称为原生质体融合。由此法获得的重组子称为融合子（ fusant）。
Bacteria Actinomycetes
Yeast,mold,mushroom,
Plant cell
Animal cell（ without the cell wall）
Parent strain selecting （ placed in isotonic solution）
Remove of
the cell wall centrifugation
Addition of PEG to
facilitate the fusion
Diluting with
isotonic
solution
Plating on the
minimum medium
Replica
plating
Plating on
the selective
medium
Determine of biological
attributes and
performence
treating with the lysozyme for
prokaryote and snail digestive enzyme
for eukaryote results in the formation
of protoplast（ G+） or spheroplast（ G-).
Remove of the cell wall in the
protoplast fusion
Gene recombination of eukaryotes
Sexual hybridization
Parasexual hybridization
二、真核微生物 的基因重组
有性杂交不同遗传型微生物的性细胞发生接合和重组的现象
准性杂交不同遗传型微生物体细胞的核融合而导致的一种低频率基因重组现象
The process of parasexual
hybridization
Anastomosis（菌丝联结）
The formation of heterocaryon
（ plasmogamy）（形成异核体或质配）
Nuclear fusion（ caryogamy核融合或核配）
Somatic crossing-over（体细胞交换）
Section D
Genetic Engineering
基因工程 就是有意识地把一个生物体中有用的目的基因转入另一个生物体,使后者获得新的遗传性状或表达所需要的产物,
重组 DNA操作
1.获得目的基因（外源基因） gene of interest
2.形成重组 DNA分子
3.转化受体细胞
4.对转化子的筛选和鉴定
5.对获得外源基因的细胞或生物体通过发酵、
细胞培养、养殖或栽培等，最终获得所需要的遗传性状或表达出所需要的产物。
获得所需的目的基因从基因文库中钓取反转录合成 cDNA
聚合酶链式反应（ PCR）
细胞内总 DNA的提取、分离与基因文库的构建总 DNA的抽提、沉淀、纯度检测及浓度测定限制性内切酶酶切
DNA片段插入质粒或噬菌体 DNA
质粒或噬菌体导入细菌构建基因文库细胞内总 DNA提取的一般方法
1.细胞破碎或消化
2.蛋白质去除
3.氯仿进一步去除残留的蛋白质和微量苯酚
4.Rnase降解 RNA
5.乙醇使 DNA沉淀,
洗涤,干燥后 -20备用
6,DNA溶液的浓度和纯度的测定
DNA对 260nm紫外光的特征吸收在提取分离到的总 DNA中，目的基因片段含量很少，且难以检出和分离。因此，科学家们用限制性内切酶将总 DN A切开成为许多小的片段，把这些片段分别插入到环状 DNA载体即质粒（ plasmid）中，这些质粒载体可以转入细菌并随着细菌的繁殖而复制，这种过程又称为 基因克隆 。
基因克隆与基因文库将总 DNA包含的基因组各片段分别克隆在质粒和噬菌体载体上，便构成了该生物的 基因文库。
基因组文库由一批细菌组成，每个细菌带有人
DNA的一个小片段。为简化起见，
只显示了几个代表性片段（彩色的）的克隆。事实上，所有的灰色 DNA片段都要被克隆。
人基因组文库的构建科学家一般用与目的基因部分序列互补并标记了放射性同位素的一小段单链 DNA为探针（ probe）对克隆的基因文库进行杂交实验，从中选出目的基因。以后在做重组 DNA操作时，可以从基因文库中调用所需要的目的基因。
钓取目的基因构建基因文库并获取目的基因费时费事，
也会出现其他问题，如真核生物细胞的基因组都含有较多非编码蛋白的内含子序列，给构建基因文库并获取目的基因增添了难以克服的麻烦。为了避免这一问题，科学家发明了用反转录人工合成互补 DNA的方法 。
Synthesis of the cDNA
A cDNA (short for
complementary
DNA or copy DNA)
is a DNA copy of an
RNA,usually an
mRNA,It can be
synthesized from
an mRNA template
using reverse
transcriptase.
此合成反应的引物是由单链
DNA的 3端提供的，它能自身折叠，形成几个如图所示的随机互补碱基对。聚合酶为大肠杆菌 DNA-PolI的 Klenow
片段。
该过程称为反转录引物为 oligo（ dT）
模板为 mRNA
酶为逆转录酶
（ reverse transcriptase）
在细胞分化的不同阶段，根据代谢反应的特殊需要，细胞往往特异性地转录产生编码特殊蛋白的 mRNA。因此，用 cDNA方法获取的
DNA片段是具有特定功能的目的基因，这是反转录人工合成互补 DNA方法的优势。
advantages
inventor,invented by Kary Mullis (American )
and his colleagues in the year of 1988
features,rapid,efficient,specifitic
application,widely used in the field of
biology,iatrology,archeology,anthroplogy
and law.
polymerase chain reaction
(聚合酶链式反应 )
polymerase chain reaction
(聚合酶链式反应 )
Definition,it’s a technique using the enzyme
DNA polymerase to amplify a region of DNA
between two predetermined sites,Oligonucleotides
complementary to these sites serve as primers for
synthesis of copies of the DNA between the sites,
Each cycle of PCR doubles the number of copies of
the the amplified DNA until a large quantity has
been made,This process (exponential increase)
obviously leads to very hign numbers in only a short
time.
Mullis 的联想使他以后发明了
PCR技术
PCR的发明是操作技术的革命，因为这一重大创新成果,Mullis教授于 1993年获得了诺贝尔奖。
PCR system
DNA模板 +4种核苷酸 +引物 +DNA聚合酶
template 4dDNTPS primer DNA polymerase
Principles
denaturation（ 95℃ ）
3 stages annealing （ 55℃ ）
extension （ 72℃ ）
30 cycles
变性 延伸退火 denaturation
annealing
extention
每一轮 PCR可使目的基因片段增加一倍,30轮循环可获得 230（ 1.07*109）个基因片段。
PCR仪利用
PCR与分子杂交标记快速准确地检测出病原性物质构建重组质粒和基因克隆基因重组和克隆操作最重要的工具限制性内切酶载体宿主菌限 制 性 内 切 酶
Arber,Smith和 Nathans因为在发现限制酶方面开创性的工作而共同获得了 1978年的诺贝尔奖。迄今为止，人们已发现和鉴定出了 200多种限制性核酸内切酶。
发现者和 DNA 技术的大部分工具相似，限制酶最先是由研究者们在研究他们感兴趣的特殊生物学问题时发现的。当将一些细菌 DNA导入某些细菌后，这些
DNA总会被降解掉。寻找这种降解原因时揭示了在宿主细菌内存在一类新型的核酸酶。这些核苷酸酶的最重要的特征是它们只在某些核苷酸序列处切割 DNA。
细菌自身的 DNA在这些序列上做了化学修饰因而免受切割。 因为这些酶限制了 DNA在某些细菌菌株之间的
DNA转移，所以它们被命名为限制酶。 不同的细菌菌株含有不同的限制酶，每一种酶的识别序列和切割位点均不同。
限制性内切酶发 现 及 命名限制性内切酶这些核苷酸酶的最重要的特征是它们只在某些核苷酸序列处切割 DNA。被比喻为 DNA操作的分子手术刀。这使得它们对现代 DNA技术的所有方面都有决定性的作用，因为一个特定的限制酶总是在同样的位点上切割一个特定的 DNA分子，这样，对来源于生物体的 DNA样品，
一个特定的限制酶总是产生一批相同的
DNA片段。
作 用 特 点
DNA技术中应用的限制酶主要来自细菌，而且因为它们的靶序列较短（通常为
4-8个核苷酸对）它们将完全随机地出现在任何一个足够长的 DNA分子中，这样它们可以被用来分析任何来源的 DNA。
限 制 性 内 切 酶来 源平 末 端 与 粘 性 末 端限制性内切酶和 T4连接酶对 DNA的作用此酶产生的粘性末端使得两个片段的末端能相互正确配对，极大地促进了它们的连接，此连接反应也重建原先的酶切位点。
DNA连接酶能把两个 DNA片段连到一起而不管 DNA的来源。
体外重组 DNA分子的形成为什么需要载体被限制酶切开的 DNA并不能直接进入到细菌等宿主细胞中，切出的目的基因连入到一个合适的载体中，才可能被转入宿主细胞中并且随之繁殖而复制。
载体是运送目的基因片段进入宿主细胞的工具。
细菌质粒目前最常用的载体 噬菌体
cosmid质粒 等载体质粒是细菌细胞中自然存在与染色体外可以自主复制的一段环状 DNA分子为了适于基因工程操作，需要将质粒加以修饰和改造 。
质粒载体
PUC18特点,
1,该质粒比较小，可以插入一段较长的 DNA片段。
2,进入宿主细菌细胞后,pUCl8在每个细胞中可复制形成大约 500个拷贝,于是也大大增加了插入在该质粒中的外源目的基因的拷贝数 。 质粒载体上的外源基因片段在宿主细胞中大量复制过程称为克隆 (clone)。
PUC18特点
PUC18特点
3．在 pUCl8中有一小段人为设计和插入的具有多种限制性酶切位点的序列，即多克隆位点 (图 11— 12)。这一段序列为根据目的基因片段的需要，选用不同的限制性内切酶分别对质粒和外源 DNA进行切割，产生相互匹配的末端，为进一步的连接提供了方便。
PUC18特点之三含有多克隆位点
PUC18中的多克隆位点
4,含有一个 LacZ基因，多克隆位点区就位于 lacZ基因之中。 lacZ基因编码 半乳糖苷酶,它可以使细菌在含有 X-gal(5— 溴 -4-氯 -3-吲哚 — p-D-半乳糖 )底物的培养基上形成蓝色的菌落，即 X-gal被 lacZ基因编码产生的 — 半乳糖苷酶水解成 蓝色 。当外源 DNA片段插入到多克隆位点时就隔断了 lacZ基因，使其失去活性和表达功能。因此，凡是插入了外源 DNA片段 pUCl8重组质粒进入到宿主细菌后，这些细菌由于不能利用
X— gal，结果在含有 X-gal底物的培养基上形成了白色的菌落，而不是蓝色的菌落。如此，利用这一特性很容易将携带重组质粒的细菌克隆从没有携带重组质粒的细菌中筛选出来 。
PUC18特点之四含有筛选标记
5,pUCl8还携带了氨卞青霉素抗性基因
(ampR),它使细菌能在含氨卞青霉素的培养基中生长,而没有转入 pUCl8的细菌则全部会被杀死 。 因此,用含氨卞青霉素的培养基可很方便地对携带 pUCl8的细菌做筛选 。
PUC18特点之五含有抗药性标记
ampR
利用 lacZ基因的插入失活筛选重组质粒为什么需要宿主菌 DNA技术并非仅仅在细胞体外将 DNA分子与载体切开,再连接成重组分子,而是要做外源目的基因的克隆,然后使其在合适的宿主生物中表达 。 基因克隆过程是通过将重组 DNA
分子转入到宿主细胞中来完成的 。
宿主菌有一些原核生物和真核生物或噬菌体都可以是基因克隆的宿主 。 在 实验室中,大肠杆菌是一种常用的宿主菌 。
为什么需要宿主菌类型将目的基因克隆到大杆肠菌中的操作步骤感受态细胞
1,用特定的限制性内切酶酶解 (切割 )已获取的目的基因片段和质粒载体；
2,用 T4连接酶将已形成特定末端序列的目的基因与质粒相互连接,形成重组 DNA分子,即重组质粒；
3,用物理方法处理大肠杆菌细胞,使其易于接纳外源重组
DNA分子 (制备感受态细胞 ),在大肠杆菌细胞中加入了少量重组 DNA分子后,使其进入到宿主细胞中,这一过程又称为转化；
以大肠杆菌为宿主菌进行基因的克隆
DNA重组转化质粒载体目的基因重组质粒大肠杆菌感受态细胞
4,培养大肠杆菌,让重组 DNA分子及其外源目的基因形成大量的拷贝；
5,用抗生素和 X-gal筛选出含重组质粒的大肠杆菌细胞,将细菌中的质粒分离出来,进行 DNA分子的检查,或直接对已克隆了目的基因的大肠杆菌细胞进行鉴定 。
以大肠杆菌为宿主菌进行基因的克隆基因克隆筛选和鉴定一般克隆基因的检查和鉴定方法实 验 室 内 一 般 常 用 酶 切 和 电 泳
(electrophoresis)方法来检查克隆的基因除了从细菌细胞中提取质粒做酶切电泳分析外,
还可以利用 DNA杂交的技术直接鉴定带有重组质粒的细菌克隆 。
基 因 酶切和电泳细菌克隆 DNA杂交用类似于提取分离细胞中总 DNA的方法将克隆了目的基因的重组质粒从大肠杆菌中分离出来酶 切 和 电 泳根据载体部分和目的基因片段上已知的酶切位点，
选择合适的限制性内切酶对重组质粒进行酶解利用琼脂糖凝胶电泳的方法对大小不等的酶解片段进行分离和鉴定质粒提取酶切电泳琼脂糖凝胶电泳原理,DNA片段上的磷酸基团都带有负电荷，
把大小不同的 DNA片段装入琼脂糖凝胶 (包含电解质的多孔支持介质 )一端并将其置于静电场中，
DNA分子便向阳极移动。 DNA长度增加，来自电场的驱动力和来自凝胶的阻力之间的比率就会降低，不同长度的 DNA片段就会表现出不同的迁移率，因而就可依据 DNA分子的大小来使其分离。
分离、纯化和鉴定 DNA片段最常规的实验技术琼脂糖凝胶电泳该过程可以通过把示踪染料和分子质量标准参照物与样品一起进行电泳而得到检测 。
标准分子质量参照物可以提供一个用于确定 DNA片段大小的依据 。 最后,根据电泳条带分析的结果,可知重组质粒经酶切后产生了大小为多少的几段 DNA分子,是否与所设计构建的重组质粒结构相吻合 。
正在进行的 DNA电泳凝胶电泳
DNA杂交技术鉴定带有重组质粒的细菌克隆根据重组质粒上目的基的部分 DNA序列,
人工合成 (或用 PCR技术合成 )与之互补的一小段单链 DNA(或 RNA),利用带有 32p_磷酸的
dATP使其标记上放射性同位素,这一小段与重组质粒上目的基因的部分序列互补的核酸分子称为 DNA探针 。
目的基因的部分序列
DNA杂交
DNA探针利用核酸杂交技术鉴定克隆基因的具体操作
(1) 将琼脂培养基上的菌落原位印迹 (转移 )到一张滤膜上； （ 原位印迹 ）
（ 2） 对滤膜及细胞做原位物理和化学处理,使其 DNA暴露并变性成单链； （ 物化处理 ）
（ 3） 将同位素探针液加入到滤膜上保温一定时间使之与克隆的目的基因杂 交,冲洗滤膜,除去多余的探针分子 ； （ 与探针杂交 ）
(4)将滤膜铺放在光学胶片上,有放射性同位素的杂交分子造成光学胶片的原位局部暴光 （ 放射自显影 ）
(5)根据光学胶片上放射自显影的具体斑点位置,确定和挑选出克 隆了目的基因的菌落 。
（ 确定阳性菌落 ）
利用核酸杂交技术鉴定克隆基因通过 DNA体外重组技术构建的重组质粒还可以直接用以转化蓝藻 (又称蓝细菌 )等原核生物或其他一些原生生物如单细胞绿藻等利用核酸杂交技术鉴定克隆基因的具体操作原位印迹物化处理与探针杂交放射自显影确定阳性菌落转化受体细胞和转化子的筛选基因克隆以后便有了大量目的基因,下一步就是使其在合适的宿主细胞中表达,产生需要的基因表达产物或使宿主生物具备所需要的性状,同时,该外源目的基因还能在宿主细胞中稳定地遗传,这一过程就是 遗传转化 。
所谓合适的 宿主细胞 就是接纳外源 DNA
的细胞,它们可以是细菌等原核生物,也可以是植物和动物细胞 。
如果需要让克隆的基因表达和产生大量的编码蛋白,
科学家们往往将该基因插入到一种 表达载体 中,用带有目的基因的表达载体再转化 大肠杆菌,对大肠杆菌进行大量培养使该目的基因在大肠杆菌细胞中大量表达和积累,通过对表达产物的分离纯化,便可以获得想要的产品 。
DNA体外重组技术构建的重组质粒还可以直接用以转化 蓝细菌 等原核生物或其他一些原生生物如 单细胞绿。
原 核 生 物 的 遗 传 转 化大肠杆菌目的基因插入表达载体 转化宿主细胞 蓝细菌单细胞绿藻诱导表达植物和动物的遗传转化常用的方法载体法转化基因直接转移基因的直 接转移方法
(1)高压电脉冲法：利用高压电脉冲的电激穿孔作用把外源 DNA引 入动植物细胞或组织中；
(2)基因枪法：用粒子枪把表面吸附有外源
DNA的金属微粒高速地射人动植物细胞或组织中 。
(3)微注 射法：利用显微注射仪等将外源
DNA直接注入细胞核或细胞质中 。
基因直接转移的基因枪法基因枪利用高压气体冲击包裹 DNA的金或钨颗粒，当压力超过破裂盘的承受能力，气体喷出，
经过加速的颗粒射向靶细胞，将 DNA
转入目的细胞中植物细胞,常用除去了细胞壁的原生质体；
为受体动物细胞,常 用的受体包括受精卵,胚胎干细胞等 。
与目的基因在大肠杆菌或蓝藻等原核生物中的转化和表达相比较,对植物或动物的遗传转化往往要困难 和复杂得多 。
宿 主 细 胞转化子的分析 —— Southern杂交通过 PCR,酶切,连接,克隆,转化等一系列 DNA的操作,获得了转基因的生物 (又称转化子 )
后,常用核酸杂交的方法对转基因生物中外源目的基因的情况进行检测和分析 。 为了纪念其发明者 Edward Southern,这种核酸杂交的技术称为
Southern杂交 (Southern blot)
Southern杂交 具 体 操 作 过 程
(1)从转化子中提取出总 DNA；
(2)根据外源目的基因片段上和前后的酶切位点情况选择合适的限制性内切酶，对总 DNA进行酶解；
(3)对酶解产物做琼脂糖凝胶电泳，与重组质粒酶切后只形成很少的 DNA片段不同，总 DNA上有许多相同的酶切位点，酶解后产生出许多 DNA片段。因此，在琼脂糖凝胶上形成许多连续无法辨认的条带，其中可能有外源目的基因的片段和相应的酶切位点；
(4)在电泳后的凝胶上覆盖一片杂交滤膜，上下铺盖许多纸巾并压一重物，通过纸巾的毛细管吸水作用，琼脂糖凝胶上的全部 DNA条带便转移印迹到滤膜上；
(5)用碱性溶液对滤膜及 DNA做变性处理，使双链
DNA解开成为单链分子；
(6)用与外源目的基因的部分序列互补,并带有放射性同位素的核酸分子作为 DNA探针,将同位素探针液加入到滤膜上保温一定时间使之与外源目的基因的 DNA变性单链杂交 形成双链分子；
(7)用缓冲液冲洗滤膜,使其他不能互补的多余放射性 探针分子被除去；
(8)把杂交后的滤膜铺放在光学胶片上,具有放射性 同位素的杂交分子造成光学胶片的原位局部暴光,即放射自显影
(9)根据光学胶片上放射自显影的具体条带位置,
以野生型细胞总 DNA的 Southern杂交实验结果为对照,分析确定在转化子总 DNA中是否存在外源目的基因及相应的酶切位点 。
生物技术 —— 现代生命科学的革命生物科学成为当今世界自然科学的热点和重点 主要由于两方面的原因,(1)20世纪后叶，
分子生物学 领域一系列突破性成就，使生命科学在自然科学中的地位发生了革命性的变化；
(2)建立在实验室研究基础上的 生物技术
(biotechnology)的发展为人类带来了巨大的利益和财富。
生物技术的成果和成功应用首先需要实验室大量复杂的基础研究工作,生物技术还是微生物学,分子生物学,化学工程,材料科学等多学科交叉的综合性学科 。 高技术 (精细和密集的复杂技术 ),高投入 (尤其 是前期科研投入高 ),高利润是生物技术产业的显著特点 。
科学界广泛地认识到：作为 21世纪发展最快的学科,
生物技术将是未来经济发展的新动力,它们将在以下领域形成巨大的产业,
A 农业
B 动物饲养业,
C 能源,
D 生物法处理环境污染,
E 纤维和包装材料,
F 药物和医学等 。
从以开发利用石油和金属为主的传统工业经济过渡到开发利用基因的经济将从根本上改变我们的文明 。
生物技术将是未来经济发展的新动力
Product line using
biotechnology
几百年前，以蒸汽机为标志的工业革命被称为人类的第一次技术革命，它解放了人的双手，使人类从繁重的体力劳动中解脱出来；
几十年前，以计算机和网络为标志的电子和信息技术革命被看作是人类的第二次技术革命，它扩张了人的大脑，极大地提高了人们获取和交流信息的速度和广度；
科学家们预言，以重组 DNA和基因克隆为标志的生物技术是人类历史上第三次技术革命，生命的复制和改造将极大地提高人类生活的质量，这一次革命更重大的意义在于，人类不但可以改造客观世界，还可以改造自身。
生物技术是人类历史上的第三次技术革命一,生物技术定义生物技术是应用自然科学及工程学的原理,依靠微生物,动物,植物体作为反应器将 物料进行加工以提供产品为社会服务的技术 。
1982年，国际合作与发展组织应用生物或来自生物体的物质制造或改进一种商品的技术,其中还包括改良有重要 经济价值的植物与动物和利用微生物改良环境的技术 。
美国政府技术顾问委员会 (OAT)
一,生物技术定义强调了生物技术的商品属性生物技术商业化特点
1997年 2月,英国科学家 Wilmut等人完成了首例哺乳动物 —— 绵羊,多莉,的克隆这个神奇的故事立刻上了各传播媒介的首页和头条,一夜之间,全球大多数生物技术公司的股票价值成倍地上升 。
自从世界上第一个生物技术公司 Genentech公司于
1976年成立以来,现在世界上已涌现出了几千个生物技术公司,估计年产值高达 6000亿美元 。 目前世界 著 名 的 生 物 技 术 公 司 包 括 Dupont 公司,
Medicago公司,Monsanto公司,Genentech公司,
Novo Nordisk公司等等 。
生物技术可获得高利润
Section E
degeneration,rejuvenation and
preservation of the stock culture
degeneration
Lose typical morphology
Slow growth and decreased spore number
Minus mutation with decreased metabolites
Weakened ability of infection to the host
Lowered resistant ability to the adverse
environment
features
Prevention of degeneracy
Control the numbers of transplantation
Create better culture condition
Transplant using spore rather than hypha
Preserved by effective methods
rejuvenation
Pure culture isolation
Rejuvenated by culture in host
Eliminated the degenerated individules
Preservation
(conservation,maintenance)
Lyophilization,freeze-drying 冷冻干燥保藏
Liquid nitrogen cryo-preservation 液氮超低温冷冻保藏
Routine slant transplantation method 常规斜面定期移植法
Stab semi-solid agar preservation 半固体穿刺保藏
Covered by liquid paraffin 石蜡油封藏法
Glycerol suspension 甘油悬液保藏法
Sand and oil preservation 砂土保藏法
