根癌农杆菌介导的转双价抗虫基因(CryIA＋CpTI)苎麻

Transgenic of Ramie with Synthetic CryIA+CpTI Gene by Agrobacterium tume-faciens-Mediated

利用苎麻下胚轴高频再生体系,将携带人工合成的CryIA杀虫基因和CpTI基因的高效双价杀虫基因的植物表达载体pGBI4ABC转化到苎麻主栽品种中苎1号中。经根癌农杆菌侵染和共培养后,用50mgL-1卡那霉素+300mgL-1头孢霉素筛选共获得32株抗性植株;PCR检测显示26株为阳性,占80%。Southern杂交结果证实,外源基因已经整合到苎麻的基因组中。这项研究为最终创造兼抗鳞翅目及鞘翅目等害虫的苎麻种质材料奠定了基础。

Ramie [Boehmeria nivea L. （Guad.）] is a perennial herbaceous plant of the urticaceae family. Ramie affected by many insect pests above and below the ground during the growing season. The pests damage has become an important factor affecting ramie yield and quality in China. Application of chemical insecticides not only is less effective and costly, but also causes environmental pollution. Development of a genetic transformation system to introduce desired genes （e.g. toxin genes） into ramie could improve its agromonic traits such as resistance to insects. The study was conducted to transfer CryIA gene and CpTI gene into the ramie variety Zhongzhu 1. The CryIA gene encodes Bt toxin protein from Bacillus thuringiensis toxinof Vitis pseudoreticulata; while, the CpTI gene encodes a cowpea trypsin inhibitor produced by Vigna unguiculata. Synthesized CryIA geneand CpTI gene were inserted into plant expression vectorpGBI4ABC whichcarried high efficient bivalent insect resistant genes. With high frequency of adventitious shoot induction and plant regeneration, ramie hypocotyls of the variety Zhongzhu 1 were transformed with Agrobacterium tumefaciens strain LB4404 harboring the plant expression vector pGBI4ABC, and then shoot buds formed from calli induced from hypocotyls were selected with the mixed solution of 50 mg L^–1 Kanamycin and 300 mg L^–1 Cefotaxime. The result showed that 32 Kanamycin-resistant plants were obtained, and therein 26 plants were positive in PCR test, indicating that 80% of the Kanamycin-resistant plants contain CryIA gene and CpTI gene. Southern blot and GUS analysis also verified that the foreign genes of CryIA and CpTI were successfully integrated into the genome of the transgenic plants and expressed.