Studies on Co-transfer of Bt-toxin, D-Amino Acid Oxidase, Transketolase, and Heat Shock Protein into Cabbage via Agrobacterium

Abstract

本試驗以農桿菌共同轉移法，同時轉移以CaMV35S或rbcS為啟動子之蘇力菌殺蟲體蛋白(Bt)、D型胺基酸氧化酵素(DAO)、轉酮醇酵素(TKL)及熱休克蛋白(HSP)等四種基因，依照啟動子的不同組合二種(Bt及DAO)、三種(Bt、DAO及TKL)或四種(Bt、DAO、TKL及HSP)基因至'高峰'甘藍。試驗結果顯示，經農桿菌共同感染之'高峰'甘藍下胚軸之轉殖再生率判於1.6~2.8%之間。共同轉移二種、三種、四種基因之轉殖植物，以南方墨點slot分析，進行初步篩選基因轉殖植物，以PCR電泳分析證實轉移基因已插入到轉殖植物染色體上。北方墨點電泳分析轉殖植株顯示，共同轉移二基因之轉殖率為14%，同時轉移三種基因之共同轉移率約為14%，四種基因的共同轉移率為10~15%。以西方墨點電泳分析含有DAO與TKL的共同轉移植株，均可偵測到轉殖基因的蛋白質表現。本研究証實利用農桿菌混合感染培殖體可達到多基因同時轉移到同一棵甘藍，且能正常表現RNA及蛋白質。Attempts had been made to co-transfer the Bt-toxin (Bt), D-amino acid oxidase (DAO), transketolase (TKL), and heat shock protein (HSP) genes into the cabbage. The objectives of this study are to establish the gene co-transfer technology and to study the possibility for improvement of cabbage with insect, disease, and stresses resistance by gene transformation. Regenerated plants were obtained after co-transformation with four kinds of genes (Bt + DAO + TKL + HSP) into the cabbage and screened by Southern slot hybridization. The transgenic plants were further examined by PCR and Northern hybridization. Several transgenic plants contained four, three, and two kinds of genes in one plant were inserted into the chromosome of transgenic plants and expressed Bt transcript with 1.35 kb RNA, DAO transcript with 1.1 kb RNA, TKL transcript with 2.2 kb RNA, HSP transcript with 2.1 kb RNA. The study demonstrated that two, three and four foreign genes could be co-transferred into cabbage plant simultaneously via Agrobacterium mediated transformation