农杆菌介导抗除草剂基因bar和抗虫基因cry1ab/ac转化甘蓝型油菜的研究  

Transgenic of Brassica napus with Herbicide-resistant bar Gene and Insect-resistant cry1ab/ac Mediated by Agrobacterium tumefaciens

作　　者：陈张彬 张振乾[1,2] 陈浩 刘忠松[1,2] 熊兴华[1] 邬贤梦 官梅[1,2] 陈社员[1] 官春云 肖钢[1,2] CHEN Zhangbin;ZHANG Zhenqian;CHEN Hao;LIU Zhongsong;XIONG Xinghua;WU Xianmeng;GUAN Mei;CHEN Sheyuan;GUAN Chunyun;XIAO Gang(College of Agriculture,Hunan Agricultural University,Changsha 410128,China;Hunan Provincial Key Laboratory of Rice and Rapeseed Breeding for Disease Resistance,Changsha 410128,China)

机构地区：[1]湖南农业大学农学院,湖南长沙410128 [2]水稻油菜抗病育种湖南省重点实验室,湖南长沙410128

出　　处：《华北农学报》2018年第5期76-81,共6页Acta Agriculturae Boreali-Sinica

基　　金：2018年转基因重大专项(SQ2018ZD080068)

摘　　要：为了获得同时具备抗虫和抗除草剂特性的甘蓝型油菜新种质材料,以湘油15下胚轴为外植体,将人工合成的抗虫融合基因cry1ab/ac与带有草铵膦抗性基因bar的植物表达载体pFGC5941连接,构建了植物表达载体pFGC-Bt。利用农杆菌介导油菜下胚轴遗传转化体系,将载体pFGC-Bt转入到油菜品种湘油15愈伤组织中。经愈伤组织分化和植株再生,获得了7株T0转基因植株。分株收获这7株转基因油菜的种子,种于花盆中,用除草剂basta喷洒,对存活下来的植株用检测引物进行PCR检测。挑选11株2个基因检测结果都呈现阳性的植株,进行Cry1Ab/Ac蛋白双抗体夹心免疫层析技术检测,在这11株油菜中都检测到了Cry1Ab/Ac蛋白。进行转基因油菜抗菜粉蝶幼虫(菜青虫)的离体鉴定,以非转基因湘油15为对照,结果显示,啃食了转基因油菜叶片的菜青虫停止运动和生长,大概7 d后陆续死亡,而非转基因油菜叶片上的菜青虫表现正常,3 d后叶片基本被其啃食干净。饲喂鉴定结果表明,转抗虫融合基因cry1ab/ac油菜对菜青虫具有明显抗性。得到了同时具有菜青虫和草铵膦双抗性的转基因植株,且抗性显著,为油菜的抗性育种提供了新的材料。In order to obtain a new germplasm material of Brassica napus with both insect-resistance and herbicide-resistance properties,the hypocotyls of Xiangyou 15 were used as explants.The synthetic insect-resistant cry1ab/ac fusion gene was inserted into the expression vector pFGC5941 to form pFGC-Bt.pFGC-Bt was transformed into Brassica napus cultivar Xiangyou 15 by Agrobacterium tumefaciens infection of hypocotyls.In order to obtain novel materials of Brassica napus with insect-resistance and herbicide-resistance,the synthetic insect-resistant cry1ab/ac fusion gene was inserted into the expression vector pFGC5941 to form pFGC-Bt.pFGC-Bt was transformed into Brassica napus cultivar Xiangyou 15 by Agrobacterium tumefaciens infection of hypocotyls.After the callus differentiation and plant regeneration,seven T 0 transgenic plants were obtained.The plants were harvested individually,and the seeds were then planted in pots,when seedlings were 4-leaf-stage,herbicide basta was applied.The surviving plants were then detected by PCR with bar gene primers.Eleven positive plants were selected for further test of Cry1Ab/Ac protein double antibody sandwich immunochromatographic assay.Cry1Ab/Ac protein was detected in all the eleven transgenic plants.The entomocidal activity was evaluated by insect feeding bioassays performed with larvae of Pieris rapae.The transgenic plants and non-transgenic control leaves were used in the insect bioassay.The Pieris rapae larvae that fed on transgenic plants were observed to stop growing and died after 7 days of feeding,whereas larvae fed on control plants grew normally.After 3 days,the leaves were basically eaten clean.The results of feeding identification indicated that the transgenic insect fusion gene cry1ab/ac was highly resistant to Pieris rapae.The results identified the transgenic plants had double resistance to herbicide basta and insect.

关 键 词：甘蓝型油菜 cry1ab/ac基因 BAR基因 遗传转化 

分 类 号：Q78[生物学—分子生物学] S635.03[农业科学—蔬菜学]