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1、在生物农药上的应用 生物技术 The birth of biotechnologyThe birth of biotechnology Stanford medical professor Stanley Cohen and biochemist Herbert Boyer from the University of California, San Francisco, were in Honolulu to attend a meeting on plasmids, the ringlets of DNA contained in bacteria. Stanley Cohen Birth
2、 of an industry Birth of an industry 1953 DNA structure proposed by James Watson and Francis Crick 1960 Arthur Kornberg synthesizes DNA in vitro 1970 Hamilton Smith and Kent Wilcox isolate the first restriction enzyme 1971 The first biological engineering company, Cetus, founded 1972 Paul Berg uses
3、a restriction enzyme to form a hybrid circular molecule 1973 Stanley Cohen and Herbert Boyer develop DNA cloning and recombinant DNA 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 ？ 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
4、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 1 An overview of DNA cloning vSome conception need to know vSome useful technology important to gene manipulation vEnzymes for DNA cloning vVectors Conception DNA cloning is to place a relatively short fragment of a genome, which might contain the
5、 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 organism containing the recombinant DNA f
6、orms a set of genetically identical organism, or a clone. This process is called DNA cloning. Host organism/cell: where the plasmids get multiplied and propagated faithfully, which is crucial for DNA cloning. Hosts for DNA cloning vector Prokaryotic host : E. coli ( most cases) Eukaryotic host : Yea
7、st (Saccharomyces cerevisiae), large fragments of human genome General features of a Vector autonomously replicating DNA independent of hosts genome. Easily to be isolated from the host cell Most are circular, some are linear Contains at least one selective marker, which allows host cells containing
8、 the vector to be selected amongst those which do not. Contains a multiple cloning site (MCS) Types of vectors Cloning vectors: allowing the exogenous DNA to be inserted, stored, and manipulated at DNA level. E. coli cloning vector: plasmids, bacteriophages ( and M13), plasmid-bacteriophage l hybrid
9、s (cosmids). Yeast cloning vector: yeast artificial chromosomes (YACs) Expression vectors: allowing the exogenous DNA to be inserted and expressed. Promoter and terminator for RNA transcription are required. bacterial expression vectors yeast expression vectors mammalian expression vectors Integrati
10、on vectors: allowing the exogenous DNA to be inserted and integrated into a chromosomal DNA after a transformation. The integration is conducted by homologous recombination between the homologous sequence shared by the plasmid and the genome of the recipient cells. bacterial integration vectors (Agr
11、obacterium tumefaciens Ti plasmid is used to integrate DNA into plant genome) yeast integration vectors Mammalian integration vector: virus based Subcloning Transfer of a fragment of cloned DNA from one vector to another. Enables us to investigate a short region of a large cloned fragment in more de
12、tail. To transfer a gene from one plasmid to a vector designed to express it in a particular species. DNA libraries are 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 clone is a genetically distinct i
13、ndividual or set of identical individuals Genomic libraries cDNA libraries Genomic libraries prepared form random fragments of genomic DNA, which may be inefficient to find a gene because of the huge abundance of the non-coding DNA cDNA libraries DNA copies (cDNA) synthesized from the mRNA by revers
14、e 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. Technology vPlasmid preparation vRestriction digests vAgarose gel electrophoresis vDNA ligation vTransformation vSelection
15、 Preparation of plasmid DNA Plasmids: small, extrachromosomal circular molecules, from 2 to 200 kb in size, which exist in multiple copies within the host cells. Characters: contain an origin of replication and replicate independently Usually carry a few genes, one of which may confer resistance to
16、antibacterial substance. Example: ampr gene encoding the enzyme b-lactamase which degrades penicillin antibiotics such as ampicillin. 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 s
17、hearing force and chemical denaturation, thus can be isolated from the chromosomal DNA easily such as alkaline lysis. Minipreparation Growth of the cells containing plasmids Collect the cells by centrifugation Alkaline lysis resuspension alkaline lysis neutralization Phenol extraction to get rid of
18、the protein contaminants Ethanol precipitation to concentrate the nucleic acids remained. (Please noted that RNase A is very bad for the lab working with RNA) Alkaline lysis Resuspend the cells in a buffer solution Lysozyme to digest the cell wall (optional) Cell lysis in lysis buffer containing SDS
19、 (disrupts cell membrane and denatures proteins) and NaOH (denatures DNA) Neutralization buffer 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
20、being sheared. Cesium chloride gradient centrifugation CsCl gradient purification is the last step of large scale plasmid DNA purification Laborious Best for the production of very pure supercoiled plasmid DNA The presence of ethidium bromide (EB) is important. Binding of EB to DNA will unwind the D
21、NA and reduce the DNA density Supercoilded DNA bind less EB than linear DNA or nicked DNA, thus has a higher density Supercoiled DNA may be purified from protein,RNA chromosomal DNA and nicked plasmid DNA in one step! Restriction endonuclease Bacterial enzymes which cut DNA into defined and reproduc
22、ible fragments Identified in the 1960s and early 1970s Key discovery which allowed the DNA cloning to become a reality Restriction-modification systems Occur in many bacterial species, a natural defense mechanism of bacteria to against the introduction of foreign DNA into the cell Restriction endonu
23、clease: 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: met
24、hylates C or A of the cellular DNA 5 protruding ends 3 protruding ends Cohensive ends 5-CCCGGG-3 3-GGGCCC-5 5-CCC-OH 3-GGG- p p -GGG-3 OH-CCC-5 SmaI + blunting ends Agarose gel electrophoresis Agrose: a polysaccharide derived from seaweed, which forms a solid gel when dissolved in aqueous solution (
25、0.5%-3%) Transformation and selection Competent cells: E. coli cells 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 competen
26、t cells. Transformantion efficiency: number of colonies formed per microgram (mg) of input DNA. Ranges from 103/g to more than 108/g. 105/g is 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 c
27、ell defending Selection with antibiotic resistance (ampr) Enzyme for DNA cloning Alkaline phophatse removes the phosphate groups from the 5- ends of the vector DNA linearized by a single restriction enzyme to prevent the self-ligation of the vector DNA upon the followed ligation Single restriction e
28、nzyme directed cloning The use of alkaline phosphate to prevent religation of vector molecules Ligase DNA ligation Covalently join the DNA molecules with the base-pairing cohesive ends, or blunt ends, if the 5-ends have phosphate groups. Vector Characters of cloning vectors Twin antibiotic resistanc
29、e Blue-white screening Characters of expression vectors Fusion protein 2 Analysis and uses of cloned DNA Characterization of clones Determining various properties of a recombinant DNA molecule,such as size, restriction map,nucleotide sequence, whether containing a gene (transcribed sequence), the po
30、sition and polarity of any gene. Preparation of pure DNA is the first step of any characterization Restriction Mapping Cleavage pattern of the insert DNA by restriction enzymes. Useful in determining order of multiple fragments (genes). 1. Combinational enzyme digestion 2. Partial digestion Partial
31、digestion Combinational enzyme digestion Searching the genes of interest in a DNA library Hybridization to identify the interested DNA or its RNA product Radiolabeled probes which is complementary to a region of the interested gene Probes: An oligonucleotide derived from the sequence of a protein pr
32、oduct of the gene A DNA fragment/oligo from a related gene of another species Blotting the DNA or RNA on a membrane Hybridize the labeled probe with DNA membrane (Southern) or RNA (Northern) membrane Southern and Northern blotting Identify the protein product of an interested gene Protein activity W
33、estern blotting using a specific antibody Nucleic acid sequencing DNA sequencing Two main methods: Maxam and Gilbert chemical method the end-labeled DNA is subjected to basespecific cleavage reactions prior to gel separation. Sangers enzymic method the latter uses dideoxynucleotides as chain termina
34、tors to produce a ladder of molecules generated by polymerase extension of primer. RNA sequencing It 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-labele
35、d 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. Sequence database 1 sequence database online 2 sequence database software (DNA club、GCG、 DNAman) PCR (polymerase chain react
36、ion) The polymerase chain reaction(PCR) is to used to amplify a sequence of DNA using a pair of primers each complementary 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
37、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 dNTPs and required Mg+. Template Any source of DNA that provides one or more target molecules can in principle be used as a
38、 template for 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 their complementary 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 Tm5oC