关键词: 载体构建, Cry 1 Ac基因, Cry 3 Aa基因, 双Bt 741杨, 抗虫性

Abstract:

In this study, plasmid pBCC3 with Cry3Aa gene was used. A Cry3Aa gene was cloned and inserted into the middle of CaMV35S promoter and NOS terminator of pCAMBIA1305 by using PCR and DNA recombination technology, and inserted into the pCAMBIA1305- Cry3Aa plant expression vector. This vector was introduced into Agrobacterium tumefaciens EHA105. Through Agrobacterium-mediated transformation, the target Cry3Aa gene was transferred into the genome of transgenic poplar pB29 (hybrid 741 line expressing CryIAc+API gene). Nine regenerated lines with hygromycin resistance were obtained and named pCCA1-pCCA9. The presence and expression of Cry1Ac gene and Cry3Aa gene have been verified using PCR and ELISA analysis. Toxicity evaluation on Plagiodera versicolora (Coleoptera) and Hyphantria cunea (Lepidoptera) by feeding fresh detached leaves showed double resistance. The deleterious ability was classified into three groups with high, medium and low level according to mortality. Toxicity of pCCA2, pCCA5, pCCA6 and pCCA9 showed high level to both P. versicolora and H. cunea; pCCA3, pCCA4 and pCCA7 showed medium or low toxicity to P. versicolora,but high to H. cunea; while pCCA1 showed extremely low toxicity to H. cunea, but high to P. versicolora.

Key words: vector construction, Cry 1 Ac gene, Cry 3 Aa gene, double Bt 741 poplar, insect-resistance