华北农学报 ›› 2018, Vol. 33 ›› Issue (5): 76-81. doi: 10.7668/hbnxb.2018.05.010

所属专题: 油料作物 生物技术

• 论文 • 上一篇    下一篇

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

陈张彬1, 张振乾1,2, 陈浩1, 刘忠松1,2, 熊兴华1, 邬贤梦1,2, 官梅1,2, 陈社员1, 官春云1, 肖钢1,2   

  1. 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
  • 收稿日期:2018-05-14 出版日期:2018-10-28
  • 通讯作者: 肖钢(1978-),男,湖南长沙人,副教授,博士,主要从事油菜遗传育种研究。
  • 作者简介:陈张彬(1994-),男,湖南常德人,在读硕士,主要从事油菜分子育种研究。
  • 基金资助:
    2018年转基因重大专项(SQ2018ZD080068)

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

CHEN Zhangbin1, ZHANG Zhenqian1,2, CHEN Hao1, LIU Zhongsong1,2, XIONG Xinghua1, WU Xianmeng1,2, GUAN Mei1,2, CHEN Sheyuan1, GUAN Chunyun1, XIAO Gang1,2   

  1. 1. College of Agriculture, Hunan Agricultural University, Changsha 410128, China;
    2. Hunan Provincial Key Laboratory of Rice and Rapeseed Breeding for Disease Resistance, Changsha 410128, China
  • Received:2018-05-14 Published:2018-10-28

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。

关键词: 甘蓝型油菜, cry1ab/ac基因, bar基因, 遗传转化

Abstract: 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 T0 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.

Key words: Brassica napus, cry1ab/ac gene, bar gene, Genetic transformation

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陈张彬, 张振乾, 陈浩, 刘忠松, 熊兴华, 邬贤梦, 官梅, 陈社员, 官春云, 肖钢. 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[J]. 华北农学报, 2018, 33(5): 76-81. doi: 10.7668/hbnxb.2018.05.010.

CHEN Zhangbin, ZHANG Zhenqian, CHEN Hao, LIU Zhongsong, XIONG Xinghua, WU Xianmeng, GUAN Mei, CHEN Sheyuan, GUAN Chunyun, XIAO Gang. Transgenic of Brassica napus with Herbicide-resistant bar Gene and Insect-resistant cry1ab/ac Mediated by Agrobacterium tumefaciens[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2018, 33(5): 76-81. doi: 10.7668/hbnxb.2018.05.010.

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