形質轉換에 의한 殺蟲性遺傳子 cryⅡA의 배추로의 導入

Transformation of Bacillus thuringiensis serovar. kurstaki cryⅡA insecticidal gene to Chinese cabbage (Brassica campestris ssp. pekinensis)

This study was carried out to find out the method to control the diamond backmoth (Plutella xylostella L.) in Chinese cabbage by introduction of biotechnological techniques. Firstly, plant expression vector containing Bt toxin gene was constructed and tissue culture system of Chinese cabbage (Brassica campestris ssp. pekinensis) was established. Bt toxin gene was transformed through infection of Agrobacterium tumefaciens. And the gene expression of transformed plants were confirmed by PCR analysis, southern blot analysis and bioassay. The results obtained are summarised as follows : Expression vectors pSRL2 (14.4 kb) containing insecticidal toxin gene cry Ⅱ A were introduced via infection of Agrobacterium strain LBA 4404. E. coli containing cry Ⅱ A gene was investigated on toxicity against four Lepidopteran insects. The results showed 100% insecticidal activity against Plutella xylostella, Heliothis assulta and Spodoptera litura, whereas only 12.0% insecticidal activity was expressed against Spodoptera exigua. In selection of transformed Chinese cabbage, optimum concentration of kanamycin was 10 mg/L, optimum density of Agrobacterium tumefaciens was 2×10⁷ cells/mL and optimum pH of selection medium was pH 5.6. MS medium supplemented with 1 mg/L NAA and 4 mg/L BAP was the most effective on cocultivation and selection medium. Transformation efficiency between two explants such as cotyledons and hypocotyls showed big difference and especially in hypocotyls continuous biweekly subculture was essential after infection of Agrobacterium. PCR analysis of regenerated Chinese cabbage on selection medium showed 1. 1 kb bands suggesting that it has cry Ⅱ A gene. When genomic southern blot analysis was conducted on three transformants, the plants containing GUS gene showed 3.0 kb bands and the plants containing cry Ⅱ A gene showed 2.2 kb bands, respectively. Bioassay was conducted on Chinese cabbage transformed with cry Ⅱ A. It was confirmed that insecticidal activities of transformants are higher so far than that of control plants. And in progeny test of transformants, twenty nine percent of T₁ seeds were resistant on selection medium containing 30 mg/L kanamycin.