| 二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合 |
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| 引用本文： | 周浩,李博,牛林,邱林,王永.二化螟钙黏蛋白CAD1的原核表达及与Cry1Ac、Cry2Aa蛋白的结合[J].生物安全学报,2018,27(4):249-254. |
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| 作者姓名： | 周浩 李博 牛林 邱林 王永 |
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| 作者单位： | 湖北工程学院生命科学技术学院/特色果蔬质量安全控制湖北省重点实验, 湖北 孝感 432000;华中农业 大学植物科学技术学院/昆虫资源利用与害虫可持续治理湖北省重点实验室, 湖北 武汉 430070,广州 海关隶属南沙海关, 广东 广州 511400,中国农业科学院棉花研究所/棉花生物学国家重点实验室, 河南 安阳 455000,湖南农业大学植物保护学院, 湖南 长沙 410128,湖北工程学院生命科学技术学院/特色果蔬质量安全控制湖北省重点实验, 湖北 孝感 432000;华中农业 大学植物科学技术学院/昆虫资源利用与害虫可持续治理湖北省重点实验室, 湖北 武汉 430070 |
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| 基金项目： | 湖北省教育厅科研项目（Q20162707） |
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| 摘 要： | 【目的】二化螟是水稻的重要害虫之一,钙黏蛋白(cadherin,CAD)是一类重要的Bt杀虫蛋白受体,在获得二化螟钙黏蛋白基因(Cs CAD1)的基础上,明确Cs CAD1蛋白与Cry1Ac和Cry2Aa蛋白的结合能力。【方法】利用PCR技术克隆Cs CAD1基因片段,将构建的p ET-28a-(+)-Cs CAD1重组质粒转入原核表达菌株BL21(DE3)中,IPTG诱导表达。目的蛋白经Ni柱亲和纯化后SDS-PAGE电泳检测,利用western blot和ligand blot技术分析其与Cry1Ac和Cry2Aa蛋白的结合能力。【结果】重组载体可在表达菌株BL21中表达一个约44 ku的蛋白,原核表达载体构建成功。SDS-PAGE显示该蛋白条带单一,且纯度较好。Ni柱亲和层析纯化该目的蛋白后进行Ligand blot分析,结果显示Cs CAD1重组蛋白可以与Cry1Ac和Cry2Aa蛋白结合。【结论】Cs CAD1蛋白可以与Cry1Ac和Cry2Aa蛋白结合,是潜在的Cry蛋白受体,所得结果有助于阐明Cry1Ac和Cry2Aa蛋白对二化螟的作用机制。
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| 关 键 词： | 二化螟 钙黏蛋白 原核表达 蛋白结合 |
| 收稿时间： | 2018/9/15 0:00:00 |
| 修稿时间： | 2018/10/27 0:00:00 |
Prokaryotic expression of the cadherin gene in Chilo suppressalis and binding analysis with Cry1Ac and Cry2Aa proteins |
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| ZHOU Hao,LI Bo,NIU Lin,QIU Lin and WANG Yong.Prokaryotic expression of the cadherin gene in Chilo suppressalis and binding analysis with Cry1Ac and Cry2Aa proteins[J].Journal of Biosafety,2018,27(4):249-254. |
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| Authors: | ZHOU Hao LI Bo NIU Lin QIU Lin and WANG Yong |
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| Institution: | Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables, College of Life Science and Technology, Hubei Engineering University, Xiaogan, Hubei 432000, China;Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China,Guangzhou Customs Attached to Nansha Customs, Guangzhou, Guangdong 511400, China,Institute of Cotton Research, Chinese Academy of Agricultural Sciences/State Key Laboratory of Cotton Biology, Anyang, Henan 455000, China,College of Plant Protection, Hunan Agricultural University, Changsha, Hunan 410128, China and Hubei Key Laboratory of Quality Control of Characteristic Fruits and Vegetables, College of Life Science and Technology, Hubei Engineering University, Xiaogan, Hubei 432000, China;Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan, Hubei 430070, China |
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| Abstract: | Aim] Chilo suppressalis is one of the most important insect pests in rice, and is sensitive to Bt insecticidal proteins through its cadherin (CAD) receptor. To clarify the role of the receptor in toxicity, the binding ability of CsCAD1 protein to Cry1Ac and Cry2Aa was clarified.Method] The CsCAD1 gene fragment was cloned by PCR. The constructed pET-28a-(+) CsCAD1 recombinant plasmid was transferred into the prokaryotic expression strain BL21 (DE3) and the protein was induced by IPTG. The binding ability of the recombinant protein to Cry1Ac and Cry2Aa was analyzed by western blot and ligand blot.Result] The recombinant vector could express a 44 ku protein in the BL21 strain, which indicated that the prokaryotic expression vector was successfully constructed. SDS-PAGE showed a single protein band, indicating purity. Ligand blot results showed that CsCAD1 recombinant protein could bind to Cry1Ac and Cry2Aa.Conclusion] The CsCAD1 protein can bind to Cry1Ac and Cry2Aa proteins, and proved to be a potential Cry protein receptor. These results may help to elucidate the mechanism of Cry1Ac and Cry2Aa proteins on C. suppressalis. |
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| Keywords: | Chilo suppressalis cadherin prokaryotic expression protein binding |
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