Construction Of The Insect-resistant Gene Vector And Study On The Transgene Of Chinese Cabbage

Spider insecticidal protein (SIP) gene can encode a small polypeptide containing 37 amino acids. The polypeptide has strong toxicity to many insects. Plasmid pBBBast-PinII has the Potato proteinase inhibitor II (Pin II ) gene which encodes potato proteinase inhibitor. The SIP gene was digested by restriction enzyme Hindlll and inserted into the vector pBBBast-Pin II . So a plant expression vector with two insect-resistant genes -pBBBast-Pin II -SIP was constructed. A plant expression vector - pBBBast-SIP with one insect-resistant gene was also constructed by inserting the SIP gene into the plasmid pBBBast. Restriction enzyme digestion analysis and PCR analysis showed that these two plant expression vectors constructed were correct.49Caixin, one cultivar of the Brassica campestris L. ssp. chinensis , was transformed by vacuum infiltration method. 7 herbicide resistant plants were obtained. PCR, PCR-Southern blotting analysis showed that the target genes were integrated into the plant genome. The segregation ratio in T1 of two examed transformants was 3:1. It revealed that these two transformants were heterozygote, and the foreign gene inserted just to on locus in the plant genome. As the observed ratio of progeny of two transformants which on selfing was 3:1, it was proved to be heterozygote for two transformants.Several factors influencing the vacuum infiltration transformation efficiency in Brassica campestris L. ssp. chinensis were also studied. The developmental status of seedlings, vacuum infiltration time, type of Agrobacterium tumefaciens used, all influence the transformation frequency. It was concluded that using the nopaline type of Agrobacterium (e.g. C58), 5min vacuum infiltration, and using the plants with just several blooming flowers would give the best results.