基于GR79 EPSPS筛选标记的抗虫抗除草剂表达载体构建及抗性鉴定

doi:10.13304/j.nykjdb.2017.0819

中国农业科技导报 ›› 2018, Vol. 20 ›› Issue (11): 22-28.DOI: 10.13304/j.nykjdb.2017.0819

基于GR79 EPSPS筛选标记的抗虫抗除草剂表达载体构建及抗性鉴定

杨羊1,2,梁成真2,孟志刚2,陈全家1,罗淑萍1,张锐2,郭三堆2*

1.新疆农业大学农学院, 乌鲁木齐 830052; 2.中国农业科学院生物技术研究所, 北京 100081

收稿日期:2017-11-27 出版日期:2018-11-15 发布日期:2018-06-14
通讯作者: *通信作者：郭三堆，研究员，博士生导师，研究方向为植物分子生物学与基因工程。E-mail：guosandui@caas.cn
作者简介:杨羊，硕士研究生，研究方向为植物基因工程。E-mail：115173026@qq.com。
基金资助:
国家转基因生物新品种培育重大专项（2016ZX08005-004）资助。

Construction of Insect and Herbicide Resistant Expression Vector Based on GR79 EPSPS Screening Marker and Identification of Resistance

YANG Yang1,2, LIANG Chengzhen2, MENG Zhigang2, CHEN Quanjia1, LUO Shuping1, ZHANG Rui2, GUO Sandui2*

1.College of Agronomy, Xinjiang Agricultural University, Urumuqi 830052;2.Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

Received:2017-11-27 Online:2018-11-15 Published:2018-06-14

摘要/Abstract

摘要： 虫害和草害是农业生产中两大主要危害。目前，尽管转基因抗虫和抗除草剂作物相继报道和应用，然而我国抗虫抗除草剂复合性状作物培育仍然明显滞后，而且多依靠传统杂交选育的手段，费事费力、周期长。根据苏云金芽孢杆菌（Bacillus thuringiensis, Bt）Cry1Ac蛋白结合结构域和毒性结构域特征，人工合成杀虫基因cry1Ac-2.5。同时，利用抗除草剂基因GR79 EPSPS替换卡那霉素筛选标记，构建基于草甘膦除草剂筛选标记的复合抗虫抗除草剂植物表达载体，并转化烟草。qRT-PCR结果表明转基因烟草cry1Ac-2.5和GR79 EPSPS基因转录水平均成功表达，经Bt和GR79试纸条检测表明上述基因在蛋白水平正确翻译。抗性实验表明，转基因烟草愈伤经草甘膦筛选，阳性率达到80%，且转基因烟草耐受100 mg/L草甘膦处理。抗虫实验表明，饲喂cry1Ac-2.5转基因烟草4 d后，棉铃虫幼虫死亡率为90%左右，表明人工基因cry1Ac-2.5具有显著的的抗虫效果。以上结果表明，构建的抗虫抗除草剂植物表达载体（Cry1Ac-2.5+GR79 EPSPS）将抗虫基因和抗除草剂基因有效合并，对于快速培育抗虫抗除草剂作物具有指导意义。

关键词: Bt, cry1Ac, GR79 EPSPS, 烟草, 抗虫, 抗草甘膦

Abstract: Pests and weeds are 2 main hazards in agricultural production. At present, although transgenic insect and herbicide resistant crops have been reported and applied, cultivation of crops with resistance both to insect and herbicide in China is still lagging behind. The traditional hybrid breeding means are mainly adopted, which are energy/time consuming with long cycle. According to the binding and toxicity domain of Bacillus thuringiensis (Bt) Cry1Ac protein, this study synthesized the insecticidal gene cry1Ac-2.5; used GR79-EPSPS gene to replace the kanamycin screening marker; and build the insect and herbicide resistant plant expression vector based on glyphosate herbicide screening marker, then transformed to tobacco. The results of real-time fluorescence quantitative PCR (qRT-PCR) showed that transgenic tobacco with cry1Ac-2.5 and GR79-EPSPS gene were expressed successfully at transcription level. The further Cry1Ac and GR79-EPSPS strips test showed that the above genes were translated correctly at the protein level. The resistance experiment showed that the transgenic tobacco callus could be screened by glyphosate. The positive rate was 80%, and transgenic tobacco was tolerant to treatment with 100 mg/L herbicide. The insect-resistant experiments showed that the mortality of Helicoverpa armigera larvae was about 90% 4 d after feeding Cry1Ac-2.5 transgenic tobacco for 4 d, indicating that the artificial gene cry1Ac-2.5 was significantly insect-resistant. All above results showed that the insecticidal herbicide plant expression vector (Cry1Ac-2.5+GR79 EPSPS) constructed in this study had combined the plant screening markers and herbicide resistance effectively. This study was of guiding significance for rapid cultivation of insect and herbicide resistant crops.

Key words: Bacillus thuringiensis, Cry1Ac, GR79 EPSPS, tobacco, insect resistance, herbicide resistance

杨羊1,2,梁成真2,孟志刚2,陈全家1,罗淑萍1,张锐2,郭三堆2*. 基于GR79 EPSPS筛选标记的抗虫抗除草剂表达载体构建及抗性鉴定 [J]. 中国农业科技导报, 2018, 20(11): 22-28.

YANG Yang1,2, LIANG Chengzhen2, MENG Zhigang2, CHEN Quanjia1, LUO Shuping1, ZHANG Rui2, GUO Sandui2*. Construction of Insect and Herbicide Resistant Expression Vector Based on GR79 EPSPS Screening Marker and Identification of Resistance[J]. Journal of Agricultural Science and Technology, 2018, 20(11): 22-28.

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基于GR79 EPSPS筛选标记的抗虫抗除草剂表达载体构建及抗性鉴定

Construction of Insect and Herbicide Resistant Expression Vector Based on GR79 EPSPS Screening Marker and Identification of Resistance

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