高效双价(Bt+cpti)表达载体的构建及其表达分析

以已有的中间载体和表达载体为基础,对Bt、cpti基因进行了修饰。在cpti基因的5′端增加了大豆胰蛋白酶抑制剂SKTI信号肽序列,在3′端增加了内质网滞留信号肽KDEL序列,然后在Bt基因的5′端增加了叶绿体靶向肽序列,构建了带有信号肽的双价表达载体PGBIC(K).B.4A和不含信号肽的对照载体PGBIC.B.4A。通过农杆菌介导的喷花法转化陆地棉Y18,Southern杂交结果显示,外源基因已经整合到了受体植物基因组中;ELISA结果显示,所获得的5株阳性株蛋白表达量分别提高了1~8倍不等。在杀虫实验中,修饰的双价载体的转基因植株1/4-1由于蛋白表达量的积累而显示了较高的棉铃虫抗性。为获得更高抗性的双价转基因抗虫棉提供了一种新的方法,并为基因工程中提高外源蛋白积累量的研究。

Construction of High-performance Bivalent (Bt+cpti) Expression Vector and its Expression Analysis

In this study, the Bt and cpti gene were modified on the base of existing vector and expression vector. Soybean trypsin inhibitor SKTI signal peptide sequence was added at 5′ end of cpti, KDEL signal peptide sequence was added at its 3′ end, and at Bt 5′ end chloroplast targeting peptides was added, and then a bivalent expression vector PGBIC(K).B.4A with signal peptides and a control vector PGBIC.B.4A without signal peptides were constructed. Through pollen tube pathway mediated by agro-bacterium Gossypium hirsutum Y18 was transformed. The result of southern blot showed that the exogenous gene had been integrated into the receptor with genome different copies. By ELISA, it was found that the protein expression was increase by 1~8 times in 5 positive strains. In the insecticidal tests, the modified binary vector transgenic plants1/4-1showed higher resistance to the bollworm because of the accumulation of protein. This study provided a new method to obtain the higher resistance of bivalent transgenic cotton, and basis to improve the accumulation of other foreign protein in genetic engineering.