基因工程chapter07

1、 Cloning vectors for Eukaryotes1.2.3.Vectors for yeast and other fungiCloning vectors for higher plants Cloning vectors for animal cells7.1Vectors for yeast and other fungi 2 m plasmid of yeast Saccharomyces cerevisiae Vectors based on the 2 m plasmid Other types of yeast cloning vector Yeast artifi

2、cial chromosomeThe yeast 2 m plasmidREP1 and REP2 function as plasmid replication;FLP function asplasmid form change (A B);The function of D gene is unknown.70200copies/cell No selectable markerGenerally one that codes for an enzyme involved in amino acid biosynthesis(auxotrophic mutant)异丙基苹果酸脱氢酶Trp

3、1,His3,Ura3亮氨酸酸Using the LEU2 gene as a selectable markerVectors based on the 2um plasmid- yeast episomal plasmidsYeast episomal plasmids ( YEps ): Some YEps contain the entire 2 um plasmid Othersjust contain the 2um origin of replicationsuch as YEp13Figure 7.3A yeast episomal plasmid, YEp1320-50 co

4、piesYEp13A Shuttle vectorcan replicate and be selected in more than one host, such as yeast and E.coliFigure 7.4Cloning withE. coli-yeast shuttle vector such as YEp13. Transformation of yeast cells1.2.3.Yeastwas inoculated into 10 ml YPD medium，30 overnight。RT，3000 rpm 5 min。3.5xvolume plate solutio

5、n（1 x volume 10xTE, pH7.5；1x volume 1M LiAC；8x volume water) resuspant。4.100lresuspantcellsinto1.5mleppendorftube ， add10lrecombinant DNA（about 2g ）and 600 l TE-LiAC-PEG solution, mixed。30 200 rpm 30 min，42 15min，on-ice 10min。5.6.3000rpm5，rejectsupernant，add0.5mlTE（pH7.5），mixed200 l on selective pla

6、te, 30 2-4 d 2.67% Minimal SD Base,0.69% His-Do Suplemetationwell7.YEp13 可整合进入酵母染色体Recombination between plasmid and chromosomal LEU2 genes can integrate YEp13 into yeast chromosomal DNA. 2um circle Vectors based on the 2um circle Other types of yeast cloning vector Yeast integrative plasmids ( YIps

7、 ) Yeast replicative plasmids ( YRps ) Yeast artificial chromosome7.1.Vectors for yeast and other fungi乳清苷酸脱羧酶forfor色氨酸生物合成嘧啶核苷酸生物合成ori.1 copy5-100 copiesYIp5YRp7Three factors come into play when deciding which typeof yeast vector is most suitablecloning experiment.for a particularTransformation fre

8、quencyYEps have the highest transformation ; YRps 1000 and 10,000 transformants per g, YIps usualy only 110 transformants per g.Copy numberYEps and YRps have the highest copy numbers: 2050 and 5100, respectively. In contrast, a YIp is usually present at just one copy per cell.StabilityYIps produce v

9、erystable recombinants, In contrast, YRp and YEps recombinants are extremely unstable. 2m circle Vectors based on the 2um circle Other types of yeast cloning vector Yeast artificial chromosome7.1.Vectors for yeast and other fungiEukaryotic Chromosome StructureFigure 7.7 Chromosome structure.yeast ar

10、tificial chromosome (YAC)bacterial artificial chromosomes (BACs) P1-derived artificial chromosomes (PACs)The structureand application of a YAC vectorFigure 7.8A YAC vector and the way it is used to clone large piceces of DNA.原生质转化法Insertional inactivation of SUP4 leads towhite colonies (recombinants

11、) and activation of SUP4 red colonies (nonrecombinants)Application of YAC vectorStudy the structure and behaviour ofchromosome.Act as vector that can be inserted with large fragment , 1400kb 。YACs can reduce the gemonic library to 6500colonies。1987年 Burke等构建了能克隆200kb大片段人体DNA分子的YAC( Yeast Artificial

12、Chromosome), 比载体容量大10倍,克隆600kb的片段，特殊的类型可以携带1400kb片段，将人类基因文库的克隆数降至6500个。1992年Shizuya等构建了能克隆300kb大片段的细菌人工染 色体BAC (Bacteria Artificial Chromosome).可高效转化。人类的基因文库30000个克隆。1994年Ioannou等发展了基于原噬菌体P1的人工染色体PAC , 能容纳100300kb， 容易转化。人类基因文库克隆数目257000个（粘粒）降低到90000个。1997年Harrington 等报道合成了人类人工染色体HAC (Human Artificia

13、l Chromosome). 长度610Mb 相当于正常染色体的1015分之一。The development of artificial chromosome1.2.3.Vectors for yeast and other fungiCloning vectors for higher plantsforinsectandCloningvectorsanimal cellsCloning vectors for organisms other than E.coliGenetically modified (GM) cropsCloning vectors for higher plant

14、s Ti plasmid of Agrobacterium Direct gene transfer Plant virus vectorsAlthough no naturally occurring plasmids are known in higher plants, one bacterial plasmid, the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens, is of great importance.ThestructureofTiplasmid( Tumour inducing )8 genesPart

15、 of the Ti (tumor inducing) plasmid is integrated into the plant chromosomal DNA after its infection, called the T-DNA(transfer DNA).15-30kbThe Ti plasmid integration into the plant chromosomal DNA after Agrobacterium tumefaciens infectionHow to use Ti plasmid to introduce new genesinto plant cell?T

16、wo strategies:The binary vector strategy1.双元载体The binary vector strategy is based on the observation that the T-DNA does not need to be physically attached to the rest of the Ti plasmid.2. The cointegration strategy共整合机制The co-integration strategy uses an entirely new plasmid, based on an E. coli ve

17、ctor, but carrying a small portion ofthe T-DNA 农杆菌介导的植物基因转化 外植体转化（叶圆盘转化法）Figure7.13Transformation of plant cells by recombinant Agrobacterium tumefaciens.regeneration 悬浮细胞转化In fact, the only parts of the T-DNA that are involved in infection are two 25 bp repeat sequences found at the left and right

18、borders of the region integrated into the plant DNA.shuttle vectorDisarmed vectorsRi plasmid of Agrobacterium rhizogenesResults in hairy root diseaseThe two plasmids are difficult in transformation inmonocots1.2.Vectors for yeast and other fungi Cloning vectors for higher plants Ti plasmid of Agroba

19、cteriumRi plasmid Direct gene transfer Plant virus vectorsforinsectand3.cellsCloningvectorsanimalCloning vectors for organisms other than E.coli1984年 Figure 7.15Direct gene transfer.The integration occur randomly at any position in any of the plant chromosomes随机整合 PEG 沉淀法，利用胞吞作用（Endocytosis)For few

20、days Recombinant DNAFigure7.16Direct gene transfer by (a) precipitation of DNA on tothe surfaces of protoplasts, and (b) fusion with DNA-containing liposomes. Liposome method脂体法 Eletroporation(电击法） Microinjection（微注射法）1. Vectors for yeast and other fungi2. Cloning vectors for higher plants Ti plasmid of Agrobacterium Direct gene transfer Plant virus vectors3.Cloningvectorsforinsectandanimal cellsCloning vectors for organisms other than E.coli