甜菜夜蛾SeABCB3基因的克隆、表达及与Bt Cry1Ab35的结合特性分析

doi:10.16409/j.cnki.2095-039x.2023.01.052

摘要/Abstract

摘要： 为了深入解析Cry1Ab蛋白对主要农业害虫甜菜夜蛾的作用机制，本研究开展了甜菜夜蛾ABC转运蛋白（ ATP-binding cassette transporter）与Bt Cry1Ab35蛋白的结合特性研究。首先，本研究克隆获得了甜菜夜蛾ABC转运蛋白SeABCB3全长基因序列（ GenBank登录号为OP948097），开放阅读框为3915 bp，编码1304个氨基酸，预测蛋白分子量为142.66 kDa。进一步克隆了编码膜外区域SeABCB3-OM，并构建了原核表达载体pET30a-SeABCB3-OM，成功表达38 kDa的重组蛋白；并且Ligand blot及ELISA分析结果显示重组蛋白SeABCB3-OM能与Bt Cry1Ab35活化蛋白特异性结合，推测其为Bt Cry1Ab35蛋白的受体蛋白。实时荧光定量PCR（ quantitative Real-time PCR，qRT-PCR）结果表明，SeABCB3在甜菜夜蛾的各龄期和不同组织中均有表达，其中在卵期和马氏管的表达量最高；本研究克隆获得甜菜夜蛾SeABCB3基因，分析了各龄期及组织结构的表达情况，明确了SeABCB3蛋白与Bt Cry1Ab35蛋白的结合特性，为进一步深入研究Bt Cry蛋白对甜菜夜蛾的杀虫作用机制提供了基础。

关键词: 甜菜夜蛾, ABC转运蛋白, 克隆与表达, 表达分析

Abstract: The mechanism of Cry1Ab protein against Spodoptera exigua was studied in this study through analysis of the binding characteristics of ATP-binding cassette transporter with Bt Cry1Ab35 protein.A full-length cDNA sequence of SeABCB3(GenBank accession number OP948097) was cloned with a open reading frame of 3915 bp,which encodes 1304 amino acids.The SeABCB3 protein is predicted to have a molecular weight of 142.66 kDa.A SeABCB3-OM cDNA sequence coding for the extracellular region was further cloned and the prokaryotic expression vector pET30a-SeABCB3-OM was successfully constructed.Both Ligand blot and ELISA analysis indicated that SeABCB3-OM recombinant protein exhibited a specific binding to Bt Cry1Ab35 activated protein,indicating that it may be the receptor of Bt Cry1Ab35 protein.Quantitative Real-time PCR was used to analyze the expression level of SeABCB3 in different instar larvae,tissues,and structures.The results showed that SeABCB3 was expressed in all instar larvae and different tissues and structures of S.exigua,with the highest expression in the egg stage and the Malpighian tubule.In this study,a full-length cDNA of SeABCB3 in S.exigua was cloned,the SeABCB3-OM expression pattern was clarified in different instar larvae,tissues,and structures,and the binding characteristic of SeABCB3-OM protein to Bt Cry1Ab35 protein was determined,which paves a scientific basis for further exploration of the Bt Cry protein action mechanism against S.exigua.

Key words: Spodoptera exigua, ABC transporter, cloning and expression, expression analysis

中图分类号:

Q965

郭晓昌, 赵丹, 王倩, 王冠迪, 温晶鑫, 吴涵, 郭巍. 甜菜夜蛾SeABCB3基因的克隆、表达及与Bt Cry1Ab35的结合特性分析[J]. 中国生物防治学报, 2024, 40(2): 327-336.

GUO Xiaochang, ZHAO Dan, WANG Qian, WANG Guandi, WEN Jingxin, WU Han, GUO Wei. Cloning and Expression of ABCB3 Gene in Beet Armyworm,Spodoptera exigua and Analysis of Its Binding Affinity with Bt Cry1Ab35[J]. Chinese Journal of Biological Control, 2024, 40(2): 327-336.

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