双价抗病基因植物表达载体构建及在烟草中表达的初步研究

Vector Construction with Two Disease-resistance Genes and Their Expression in Transgenic Tobacco

多基因转化是基因工程研究热点之一。本研究应用DNA重组技术,将两个抗病机制不同,抗菌谱较广的抗病基因(天麻抗真菌蛋白GAFP和兔防御素NP1基因)构建在一个植物表达载体pBin35SGAFP-NP1上,两者具有各自的CaMV35S启动子和Nos终止子。通过根癌农杆菌介导,采用叶盘法转化烟草,PCR和PCR-Southern分析证明已将NP1和GAFP基因整合到烟草基因组中。离体抑菌实验表明转基因植株对真菌和细菌表现出一定的抗性。以上结果表明通过该表达载体进行遗传转化可获得含双价抗病基因植物,并能有效表达,提高转基因植物抗病能力。

Genetic transformation with multiple genes is one of the popular researches in gene engineering. Difensin which is resistant to many kinds of virus, bacterium and fungal, and Gastrodia Antifungal Protein （GAFP） which has the resistant ability to more than twenty kinds of fungal were used for the vector construction. Also, the mechanisms of anti-disease by two genes are different from each other. By use of DNA recombination method a vector （ pBin35SGAFP- NP1 ） that contains the two anti-disease genes： NP1 and GAFP was constructed. Mediated by Agrobacteriurn tumefaciens, the two disease-resistance genes were transferred into tobacco using leaf-disk method. Results of PCR and PCR-Southern determination indicated that NP1 and GAFP genes were integrated into the tobacco genome. In vitro testing of transgenic plants to virus, bacterium and fungal showed that they had the resistance to the growth of Agrobacteriurn tumefaciens, Trichoderma reesei, Sphaeropsis sapinea, and it was appeared that NP1 and GAFP genes were expressed efficiently in transgenic tobacco and the transgenic plants had disease-resistant ability to pathogens. Therefore, by using this vector transgenic plants with the two anti-disease genes were obtained which were proved to be expressed efficiently and the ability of anti-disease could be increased.