Study On Isolation Of Cowpea Trypsin Inhibitor Gene And Its Transformation Into Mustard(Brassicajuncea Coss)

By means of plant genetic engineering, foreign insects resistance gene can be transferred into plant cell.we cloned the CpTI gene and transferred it into mustard by Agrobacterium-mtdi&ted transformation method.and obtained the transgenic mustard plants.The main results are as follows: 1.Isolation of CpTI geneTotal RNA was isolated from cowpea seedss cotyledons and leaves.The CpTI gene fragment was amplified by RT-PCR using sequences of its two sides as primers. 2.Cloning of the PCR productsThe PCR products were purified by agarose gel electrophoresis and was ligated with pUCm-T vector.By the method of PCR and enzyme digest analysis.the result shows that the plasmid containing CpTI gene was transferred into E.coli DHso. 3.The sequencing and analysis of CpTI geneThe sequencing of the single recombinant bacterium containg the inserted CpTI gene fragment was carried out by Daliang bio-engineering Co.Ltd.The result showed the fragment has 326bp,encoding 108 amino acid, the nucleotide acid homology was 99%,the ammo acid homology was 79%,comparing to CpTI gene isloated by other researcher before. 4.The construction of middle-clone vector and expression vectorThe pUC-Cp and pGEM-7Z plasmid were digested by Kpnl and BamHI, and collected the digested CpTI fragment and the pGEM-7Z, then ligated by T4 DNA ligase and formed the pGEM-CP. By the same method, the expression vector pBI121-Cp was constructed from pGEM-Cp and pBI121 with Xbal and SacI digestion.After that ,the pBI121-Cp was transferred into Agrobacterium LBA4404 strain by freeze-thaw method. The PCR amplification indicated the LBA4404 strain containning CpTI gene.The LBA4404 strain was used in genie transformation of mustard. S.Regeneration system of mustardHypocotyls and cotyledons of 4 days and leaves of 25 days were used as transformation materials , and MS as basic medium supplying different BA^ NAA and KT concentration , we got the optimum explant adventitious bud inducing medium(MS+6-BA3.0mg/L+NAA0.2 mg/L).Among the three kinds of mustard explants.the cotyledons showed the highest adventitious bud regeneration capacity.and the regeneration capacity of leaf explantswas weaker.and the weakest of hypocotyls.6.Transformation system of mustardA serials of kanamycin concentration was added to optimum medium to test the explants resistance capacity of two kinds of mustard.The transformation procedures described were derived from numerous regeneration and trasformation designed to test factors that might affect shoot regeneration,which including length of co-cultivation.Those producing the best result parameters were described as below:After the mustard explants were precultured on regeneration medium for 2 days.they were inoculated with Agrobacterium for 20 minutes.Inoculated explants were co-cultivated for 4 days and in shadow at first 2 days.then transferred to the same medium plus 30 mg/L kanamycin and 500mg/L Garb. All of them were transferred to fresh medium every 2 weeks.The kan-resistant plants were regenerated. T.Identification of charactrization of transgenic mustard plantsThe putative transformant regeneration plants were assayed by PCR and PCR-Southern blot analysis.Both analysis the target bands were observed.So the integration of the CpTI gene into mustard genome DNA was confirmed.The result of insect-resistance showed that the transgenic plants are more resistant than non-transgenic plants.