The Studies On Constructing Expression Vector Of Nematode Resistance Gene And Genetic Transformation

This study is for genetic transformation of nematode resistance gene, including four parts: I. Constructing expression vectors; II.Experiment on the genetic transformation of sugarcane and soybean with Agribacterium tumefaciens; III. Studies on the genetic gransformation of sugarcane and soybean embryogenic calli via microprojectile bombardment; IV.Molecular detection of kanamycin resistance seedlings. Cloning vector P 1832 was isolated from E.coii DH5a and cut with restriction Enzyme NcoI, filled in 3?end of double-stranded DNA with DNA polymerase I Large Fragment (Klenow LF) and cut off again with restriction Enzyme Sad. The reaction mixture was electrophoresed with low melting gel and reclaimed the short fragment and stored in ?0 C. Expression vector pBI 121 was extracted from E.coli DHSa and cut off with two restriction Enzymes SinaI and Sad. The large fragment was reclaimed by low melting gel electrophoresis. The short fragment of P1832 and tile Large fragment of pBIl2l were mixed in proportion 3 to 1 and ligated with T4 DNA ligase which was incubated at 11 C for 16-18 hours. When the reaction finished the mixture was added into E.coli DH5a which were offered receptity by CaCI, treatment. The treated E. coil was smeared on the surface of LB solid medium containing 100 mg.L?Kanamycin. The plasmid was isolated from the single colonies and identified with restriction Enzymes marked in the recombined plasmid. The recombined plasmid we wanted was singled out (Which was named pBl 1812 ) and then transformed into Agrobacterium LBA4404. The single Agrobacterium colonies growing on YEB solid medium (K~ lOOmg/L) were detected with restriction enzymes and Polymerase Chain Reaction. The colony of 53 LBA4404 containing pBI1812( the promotor is CaMV35s) was used for transformation of dicotyledon. The recombined plasmid using Ubi promoter was constructed with cloning vector P1832 and expression vector pBIL-2. The procedure is similar to the procedure above excepting from restriction enzyme KpnI and T4 DNA polymerase for blunting the ends of DNA fragment. The recombined expression vector with Ubi promoter was named pBIl8-2 which was used for transformation of monocotyledon. Sugarcane genotypes RQCI6 and Funong 93- 3608 were selected as testees for gene transformation. The leaf meristems from top of stalks were cut into pieces and induced in MS+2,4-D 3mg/L and the tissues were cocultivated with Agrobacterium turncfaciens strain~ LBA 4404(pAL4404:pBIl8-2) for two days. Three treatments namely sugarcane tissues cocultivated with LBA4404~ gene gun bombardment and cocultivation with LBA4404 gene gun bombardment were designed for gene transformation. The treated tissues were selected with MS? 2,4-D 2mg/L+ K~300mg/L(Cefotaxime was added into medium to eliminate LBA 4404). The resistant calli to kanarnycin were differentiated into seedlings in MS+BA2mg/L+ NAAO.lmg/L+ K~300mg/L. Sixteen albino sugarcane seedlings were obtained from the calli and three albino seedlings were proved to be transgenic seedlings. Soybean genotype Kefeng 9 was bombarded with gene gun. Due to the differentiation of soybean is very difficult no regenerative seedlings were differentiated. Soybean nodes were cut off and cocultivated with Agrobacterium turnefaciens strain LBA4404(pAL4404:pB[ 1812). The explants were differentiated and kanamycin resistant regenerated seedlings were obtained.