Epsps基因为筛选标记的多基因抗虫表达载体构建

Construction of Plant Expression Vector Containing Multiple Insect-resistant Genes with Epsps as Selection Marker

目的:为了获得水稻的广谱抗虫性,同时也避免由潮霉素抗性基因引起的安全性问题。构建以抗除草剂基因(Epsps基因)为筛选标记的多基因抗虫植物表达载体pCTSK。方法:以双元载体pCAMBIA1300为基础,将潮霉素抗性基因hpt替换成携带烟草叶绿体转运肽序列TSP的草甘膦抗性突变基因aroAM12(编码细菌5-烯醇式丙酮酸莽草酸-3-磷酸合成酶Epsps)作为筛选标记基因,获得中间表达载体pCTSM1300。然后再连接上3个抗虫基因(马铃薯蛋白酶抑制剂基因Pin-Ⅱ、苏云金杆菌毒蛋白基因Bt cryI(A)及雪花莲外源凝集素基因GNA)的完整表达片段(包含各自的启动子和终止子)。结果:通过PCR方法和酶切方法鉴定已成功地构建了该表达载体;通过不同实验方案的数据分析,得出在载体构建的大分子连接中优化的一次连接法连接效率高于二次连接法的结论。

Objective：For the security of hygromycin phosphotransferase gene hpt was oppugned, the herbicide-resistant gene （ Epsps gene） was used as a selection marker in comtruction of the plant expression vector pCTSK which contained multiple insect - resistant genes to endow rice with broad-spectrum insect resistance. Methods： Firstly, the hpt gene in binary vector pCAMBIA1300 was replaced by glyphosate resistant gene aroAM12 （a mutant of 5 - enolpyruvylshikimate- 3 - phosphate synthase gene） leading by chloroplast transit peptide of tobacco （ TSP ） for obtaining the medium vector pCISM1300. Results： Then, the complete expression frame of potato proteinase inhibitor gene Pin Ⅱ , Bacillus thuriniensis endotoxin gene cryⅠ（A ） and Calanthus riwalis agglutinin gene GNA were inserted into pCTSM1300 which resulted in the achievement of vector pCTSK. The ligation method of large fragment DNA was improved in construction of the vector.