双价抗虫植物表达载体p3300-bt-pta转化苜蓿的初步研究  

A Primary Study on Transferring Plant Expression Vectors Containing Binary Insect Resistance Genes Bt CryIA(a)-pta or Bt CryIA(c)-pta into Alfalfa

作　　者：熊恒硕[1] 康俊梅[1] 杨青川[1] 孙彦[2] 唐克轩[3] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100094 [2]中国农业大学动物科技学院草地研究所,北京100094 [3]复旦大学生命科学院,上海200433

出　　处：《中国草地学报》2008年第1期21-26,共6页Chinese Journal of Grassland

基　　金：中国农业科学院北京畜牧兽医研究所基本科研业务费专项基金创新团队项目(ywf-td-3)

摘　　要：以中苜1号无菌苗子叶为外植体,采用农杆菌介导的叶盘法,将含有CryIA(a)/CryIA(c)基因、半夏凝集素(pta)基因和bar基因的双价抗虫植物表达载体p3300-Bt-pta导入苜蓿子叶中,在含除草剂的筛选培养基中连续筛选,获得抗性转化植株。研究了农杆菌菌液浓度、浸染时间、共培养时间等因素对苜蓿转化效率的影响,结果表明:各因素对苜蓿转化率均有不同程度的影响,当转化的菌液浓度OD600为0.6、浸染时间为10min、共培养时间为3d时转化效率较好。载体的抗性基因为除草剂草丁膦抗性的Bar基因,对子叶外植体的最佳筛选浓度为8mg/L;抑制农杆菌所用Carb的有效工作浓度为400mg/L,以后继代逐次减少用量到100mg/L;按此方法以根癌农杆菌菌株EHA105介导,将双基因Bt和pta导入紫花苜蓿品种"中苜1号",最终获得122棵除草剂草丁膦(ppt)抗性的植株,经过初步的PCR及RT-PCR分子生物学检测,有30棵检测到特异性条带,表明外源基因已成功整合到苜蓿基因组中,并在转录水平得到表达。Agrobacteriurn-mediated gene transformation is the most widely used technique for gene transferring into plants. The expression vectors with binary insect resistance genes Bt CryIA（a）-pta or Bt CryIA（c）-pta were transformed into the explant of alfalfa cotyledon. In this study the effects of influencing factors on genetic transformation frequency of alfalfa were investigated, such as Agrobacterium concentration, infection time, co-culture time. The results showed that the transformation efficiency can be clearly improved if using the following transformation conditions. Agrobacteriurn concentration OD600≈0.6, infection time 10min, co-culture time 3 days, phosphinothricin concentration 8mg/L, and Carbenicillin concentration 400mg/L（decreased gradually to 100mg/L）. Leaf discs of alfalfa were infected by Agrobacteriurn turnefaciens strains EHA105 containing these plant expression vectors and Bar gene cassette encoding phosphinothricin acetyltransferase. 122 phosphinothricin resistant plants from individual transformation events were obtained and PCR and RT-PCR checking confirmed the integration of foreign genes.

关 键 词：双价抗虫 转基因植株 CryIA(a/c)基因 pta基因 紫花苜蓿 

分 类 号：Q943.2[生物学—植物学] S541[农业科学—作物学]