第1页：生物技术在生物农药上的应用
在生物农药上的应用生物技术
第2页：生物技术的诞生
The birth of biotechnologyStanford medical professor Stanley Cohenand biochemist Herbert Boyerfrom the University of California, San Francisco, were in Honolulu to attend a meeting on plasmids, the ringlets of DNA contained in bacteria.Stanley Cohen
第3页：生物技术产业的诞生
Birth of an industry 1953DNA structure proposed by James Watson and Francis Crick 1960Arthur Kornberg synthesizes DNA in vitro1970Hamilton Smith and Kent Wilcox isolate the first restriction enzyme1971The first biological engineering company, Cetus, founded 1972Paul Berg uses a restriction enzyme to form a hybrid circular molecule 1973Stanley Cohen and Herbert Boyer develop DNA cloning and recombinant DNA
第4页：生物农药的定义
生物农药的定义
- 生物农药定义
- 化学农药专家：应包括微生物活体、昆虫天敌、部分植物源农药；不包括农用抗生素、植物生长调节剂、转基因农药。
- 国外：农用抗生素列为化学农药的范畴。争论点？
第5页：新型生物农药品种
新型生物农药品种
- 寡糖
- 丙烷脒
- 激活蛋白
- 多粘类芽孢杆菌
- 地衣类芽孢杆菌
- 海洋地衣芽孢杆菌
- 嗜线虫致病杆菌
- 绿色木霉
- 抑霉菌素
- 放线菌新菌株
- 植物活性物质新型生物农药品种
第6页：生物农药的功用
防病防止病害生物农药的功用杀虫防止虫害除草防止草害提高自然产物的生物农药活性生物技术
第7页：DNA克隆概述
1 An overview of DNA cloningSome conception need to knowSome useful technology important to gene manipulationEnzymes for DNA cloningVectors
第8页：DNA克隆的基本概念
ConceptionDNA cloningis to place a relatively short fragment of a genome, which might contain the gene or other sequence of interest, in an autonomously replicating piece of DNA, known as a vector, forming recombinant DNA, which can be replicated independently of the original genome, and normally in other host species altogether. Propagation of the host organismcontaining the recombinant DNA forms a set of genetically identical organism, ora clone. This process is called DNA cloning.
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第10页：克隆宿主细胞
克隆宿主细胞
- Host organism/cell: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning.
- Hosts for DNA cloning vectorProkaryotic host : E. coli( most cases)Eukaryotic host : Yeast (Saccharomyces cerevisiae),large fragments of human genome
第11页：载体的基本特征
General features of a Vector1.autonomously replicating DNAindependent of host’s genome.2.Easily to be isolatedfrom the host cell3.Most are circular, some are linear4.Contains at least oneselective marker, which allows host cells containing the vector to be selected amongst those which do not. 5.Contains amultiple cloning site(MCS)
第12页：载体类型
载体类型
- Types of vectors Cloning vectors: allowing the exogenous DNAto be inserted, stored, and manipulated at DNA level. E. colicloning vector: plasmids, bacteriophages (λand M13), plasmid-bacteriophage l hybrids (cosmids).Yeast cloning vector: yeast artificial chromosomes (YACs) Expression vectors: allowing the exogenous DNA to be inserted and expressed.Promoterand terminatorfor RNA transcription are required.
- bacterial expression vectors
- yeast expression vectors
- mammalian expression vectors
第13页：整合型载体
整合型载体
- Integration vectors: allowing the exogenous DNA to be inserted andintegratedinto a chromosomal DNA after a transformation. The integration is conducted by homologous recombinationbetween the homologous sequence shared by the plasmid and the genome of the recipient cells.
- bacterial integration vectors(Agrobacterium tumefaciensTi plasmid is used to integrate DNA into plant genome)
- yeast integration vectors
- Mammalian integration vector: virus based
第14页：亚克隆
SubcloningTransfer of a fragment of cloned DNA from one vector to another.1.Enables us to investigate a short region of a large cloned fragment in more detail.2.To transfer a gene from one plasmid to a vector designed to express it in a particular species.
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第16页：DNA文库
DNA文库
- DNA librariesare sets of DNA clones, each of which has been derived from the insertion of a different fragment into a vector followed by propagation in the host.
- A cloneis a genetically distinct individual or set of identical individualsGenomic libraries cDNA libraries
第17页：基因组文库
Genomic librariesprepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNAcDNA librariesDNA copies (cDNA) synthesized from the mRNA by reverse transcription are inserted into a vector to form a cDNA library. Much more efficient in identifying a gene, but do not contain DNA coding functional RNA or noncoding sequence.
第18页：DNA克隆常用技术流程
DNA克隆常用技术流程
- Technology
- Plasmid preparation
- Restriction digests
- Agarose gel electrophoresis
- DNA ligation
- Transformation
- Selection
第19页：质粒DNA的制备
Preparation of plasmid DNAPlasmids: small, extrachromosomal circular molecules, from 2 to ~200 kb in size, which exist in multiple copies within the host cells.Characters: contain an originof replication and replicate independentlyUsually carry a few genes, one of which may confer resistance to antibacterial substance.Example: amprgene encoding the enzyme b-lactamase which degrades penicillin antibiotics such as ampicillin.
第20页：大肠杆菌质粒小量制备
大肠杆菌质粒小量制备
- Plasmid minipreparation from E. coli Plasmids
- ~2-20 kb in length that much smaller than E. coli chromosomal DNA (4600 kb), and independently supercoiled
- Resistant to shearing forceand chemical denaturation, thus can be isolated from the chromosomal DNA easily such as alkaline lysis. Minipreparation1.Growthof the cells containing plasmids2.Collectthe cells by centrifugation3.Alkaline lysisresuspension alkaline lysis neutralization4.Phenol extractionto get rid of the protein contaminants5.Ethanol precipitationto concentrate the nucleic acids remained. (Please noted that RNase A is very bad for the lab working with RNA)
第21页：碱裂解法提取质粒
碱裂解法提取质粒
- Alkaline lysis
- Resuspendthe cells in a buffer solution
- Lysozyme to digest the cell wall (optional)
- Cell lysisin lysis buffer containing SDS (disrupts cell membrane and denatures proteins) and NaOH (denatures DNA)
- Neutralizationbuffer containing KAc (pH 5): renaturation of plasmid DNA (supercoiled) and precipitation of denatured proteins and chromosomal DNA which can not be renatured because of its size and physical property of easily being sheared.
第22页：氯化铯密度梯度离心
氯化铯密度梯度离心
- Cesium chloride gradient centrifugation1.CsCl gradient purification is the last step of large scale plasmid DNA purification2.Laborious3.Best for the production of very pure supercoiled plasmid DNA4.The presence of ethidium bromide (EB) is important.
- Binding of EB to DNA will unwind the DNA and reduce the DNA density
- Supercoilded DNA bind less EB than linear DNA or nicked DNA, thus has a higher densitySupercoiled DNA may be purified from protein,RNAchromosomal DNA and nicked plasmid DNA in one step!!
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第24页：限制性内切酶
Restriction endonuclease1.Bacterial enzymeswhich cut DNA into defined and reproducible fragments2.Identified in the 1960s and early 1970s3.Key discoverywhich allowed the DNA cloning to become a reality
第25页：限制与修饰系统
限制与修饰系统
- Restriction-modification systemsOccur in many bacterial species, a natural defense mechanism of bacteria to against the introduction of foreign DNAinto the cell
- Restriction endonuclease: recognize a short, symmetrical DNA sequence, and cut DNA backbone in each strand at a specific site within that sequence (kill foreign DNA)Recognition sequences (1)Recognize 4-8 bp. Most recognition sequences are 6 bp which occurs at a rate of 46=4096 bp. (2) Highly specific
- Mythylase: methylates C or A of the cellular DNA
第26页：黏性末端与平末端
5’ protruding ends3’ protruding endsCohensive ends5’-CCCGGG-3’3’-GGGCCC-5’5’-CCC-OH3’-GGG-pp -GGG-3’OH-CCC-5’SmaI+blunting ends
第27页：琼脂糖凝胶电泳
Agarose gel electrophoresisAgrose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (0.5%-3%)
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第29页：转化与筛选
Transformation and selectionCompetent cells: E. colicells treated with Ca2+solution are susceptible to take up exogenous DNA. Enzymes involved in host cell defending, such as restriction-modification system are suppressed. Transformation: a process of uptake of exogenous DNA by competent cells. Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103/μgto more than 108/μg. 105/μgis adequate for a simple cloning. Heat-shock: After the DNA is uptaken, the cells shall be put at 42oC for 1 min in order to induce the suppressed enzymes for cell defending
第30页：抗生素抗性筛选
Selection with antibiotic resistance (ampr)
第31页：DNA克隆常用酶
DNA克隆常用酶
- Enzyme for DNA cloning
- Alkaline phophatse
- removes the phosphate groups from the 5’-endsof the vector DNA linearized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followed ligation
- Single restriction enzyme directed cloning
第32页：碱性磷酸酶防止载体自连
The use of alkaline phosphate to prevent religation of vector molecules
第33页：DNA连接酶与连接反应
•LigaseDNA ligationCovalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5’-ends have phosphate groups.
第34页：克隆载体
Vector
第35页：克隆载体的特征
Characters of cloning vectors
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第37页：蓝白斑筛选
Blue-white screening
第38页：表达载体的特征
Characters of expression vectors
第39页：融合蛋白
Fusion protein
第40页：重组克隆的鉴定
2 Analysis and uses of cloned DNACharacterization of clonesDetermining various properties of a recombinant DNA molecule,such as size, restriction map,nucleotide sequence, whether containing a gene (transcribed sequence), the position and polarity of any gene.Preparation of pure DNA is the first step of any characterization
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第42页：限制性酶切图谱
限制性酶切图谱
- Restriction MappingCleavage pattern of the insert DNA by restriction enzymes. Useful in determining order of multiple fragments (genes).
- 1. Combinational enzyme digestion
- 2. Partial digestion
第43页：部分酶切与组合酶切
PartialdigestionCombinationalenzymedigestion
第44页：目的基因的文库筛选
目的基因的文库筛选
- Searching the genes of interest in a DNA libraryHybridization to identify the interested DNA or its RNA product1.Radiolabeled probes which is complementary to a region of the interested geneProbes:
- An oligonucleotidederived from the sequence of aprotein product of the gene
- A DNA fragment/oligo from a related gene of another species2.Blotting the DNA or RNA on a membrane 3.Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane
第45页：Southern与Northern印迹
Southern and Northern blotting
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第48页：目的基因蛋白产物鉴定
Identify the protein product of an interested gene1.Protein activity2.Western blotting using a specific antibody
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第50页：核酸测序方法
Nucleic acid sequencingDNA sequencingTwo main methods:Maxam and Gilbert chemical methodthe end-labeled DNA is subjected to basespecific cleavage reactions prior to gel separation.Sanger’s enzymic method the latter uses dideoxynucleotides as chain terminators to produce a ladder of molecules generated by polymerase extension of primer.
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第54页：RNA测序
RNA sequencingIt is sometimes necessary to sequence RNA directly, especially to determine the position of modified nucleotides present in, eg, tRNA and rRNA.This is achieved by base-specific cleavage of 5’-end-labeled RNA using RNases (ribonuclease) that cleave 3’to a particular nucleotide. Partial digestion is required to generate a ladder of cleavage products which are analyzed by PAGE.
第55页：序列数据库
Sequence database1 sequence database online2 sequence database software (DNA club、GCG、DNAman)
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第57页：PCR聚合酶链式反应
PCR聚合酶链式反应
- PCR (polymerase chain reaction)The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers eachcomplementary to one end of the the DNA target sequence.The PCR cycle
- Denaturation: The target DNA (template) is separated into two stands by heating to 95℃
- Primer annealing: The temperature is reduced to around 55℃ to allow the primers to anneal.
- Polymerization(elongation, extension): The temperature is increased to 72℃ for optimal polymerization step which uses up dNTPsand required Mg++.
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第60页：PCR模板与引物
PCR模板与引物
- Template
- Any source of DNA that provides one or more target molecules can in principle be used as a templatefor PCR
- Whatever the source of template DNA, PCR can only be applied if some sequence information is known so that primers can be designed.
- Primers
- PCR primers need to be about 18 to 30 nt long and have similar G+C contents so that they anneal to theircomplementary sequences at similar temperatures.They are designed to anneal on opposite strands of the target sequence.
- Tm=2(a+t)+4(g+c): determine annealing temperature. If the primer is 18-30 nt, annealing temperature can be Tm±5oC
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