棉蚜AgosOBP5基因克隆、表达纯化及结合特性研究  

Cloning,expression,purification and binding characteristics of the AgosOBP5 gene in Aphis gossypii

作　　者：王铁矿 李峰奇 刘思雨 李增凤 李秋荣 WANG Tie-Kuang;LI Feng-Qi;LIU Si-Yu;LI Zeng-Feng;LI Qiu-Rong(Agriculture and Forestry Science Academy of Qinghai University,Provincial Key Laboratory of Agricultural Integrated Pest Management,Scientific Observing and Experimental Station of Crop Pest in Xining,Ministry of Agriculture and Rural Affairs,Xining 810016,China;National Key Laboratory of Green Pesticide,Key Laboratory of Green Pesticide and Agricultural Bioengineering,Ministry of Education,Center for R&D of Fine Chemicals of Guizhou University,Guizhou 550025,China;Dulan Linsheng Sand Prevention and Control Co.,Ltd,Dulan 816100,China)

机构地区：[1]青海大学农林科学院,青海省农业有害生物综合治理重点实验室,农业农村部西宁作物有害生物科学观测实验站,西宁810016 [2]贵州大学绿色农药全国重点实验室,精细化工研究开发中心,贵阳550025 [3]都兰林晟防沙治沙有限责任公司,都兰816100

出　　处：《应用昆虫学报》2025年第1期123-133,共11页Chinese Journal of Applied Entomology

基　　金：海西州重点研发与转化计划(2023-YZ-Q02);青海省昆仑英才·乡村振兴人才项目。

摘　　要：【目的】棉蚜Aphis gossypii是枸杞Lycium chinense、棉花Gossypium herbaceum等作物上的一种成灾性害虫。明确气味结合蛋白与气味分子的结合特征及其在棉蚜嗅觉识别过程中发挥的作用,可为今后利用化学挥发物制作诱芯防治棉蚜提供理论依据。【方法】基于转录组数据对棉蚜气味结合蛋白(OBPs)AgosOBP5基因进行全长克隆并开展生物信息学分析;构建原核表达载体并用十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)检测;利用荧光竞争结合实验研究AgosOBP5重组蛋白与水杨酸甲酯、苯乙醇、苄醇、壬醛、1-辛烯-3-醇、棕榈酸这6种植物源气味分子的结合特征。【结果】棉蚜AgosOBP5基因全长为675 bp,编码224个氨基酸,含有12个保守半胱氨酸,预测理论相对分子量为54.50 kD。序列比对和系统进化树分析表明,棉蚜AgosOBP5氨基酸序列与大豆蚜Aphis glycines AglyOBP5同源性最高。经异丙基-β-D-硫代半乳糖吡喃糖苷(IPTG)诱导和SDS-PAGE检测,证明了AgosOBP5重组蛋白是可溶性蛋白,能够在上清液中表达。6种气味分子均可将AgosOBP5与N-苯基-1-萘胺(1-NPN)荧光探针体系的荧光强度值降到50%以下,不同的是水杨酸甲酯、2-苯乙醇、苄醇、壬醛、1-辛烯-3-醇这5种化合物从初始滴加,荧光值即开始下降,随后一直保持不断下降的趋势,而棕榈酸在浓度低于96μmol·L-1时荧光值呈升高趋势,随自身浓度不断升高后转为下降趋势。【结论】本研究明确了棉蚜AgosOBP5基因的核苷酸及其编码氨基酸序列,根据AgosOBP5与植物源挥发物水杨酸甲酯、2-苯乙醇、苄醇、壬醛、1-辛烯-3-醇的良好结合能力,推断AgosOBP5蛋白可能在棉蚜的嗅觉信息识别中起着重要的作用。[Aim] Aphis gossypii is a serious pest of the Chinese wolfberry,cotton and other crops.The aim of this study was to identify the binding characteristics of odorant-binding proteins with odor molecules and establish their role in the olfactory recognition process in A.gossypii.The findings will establish a theoretical basis for using chemical volatiles in developing lures aimed at the future prevention and control of A.gossypii.[Methods] Using transcriptome data,a full-length sequence of the odorant-binding proteins(OBPs) AgosOBP5 gene was obtained and bioinformatics analysis was performed.The prokaryotic expression of AgosOBP5 was constructed and detected by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis(SDS-PAGE).Fluorescence competitive binding experiment was used to study the binding characteristics of the AgosOBP5 recombinant protein with six different odor molecules derived from plants.[Results] The total length of the A.gossypii gene AgosOBP5 was 675 bp,encoding 224 amino acids,including 12 conserved cysteines.The predicted theoretical relative molecular weight was 54.50 kD.Sequence alignment and phylogenetic tree analysis show that the amino acid sequence of AgosOBP5 in A.gossypii has the highest homology with the AglyOBP5 in soybean aphid A.glycines.By Isopropyl β-D-thiogalactoside(IPTG) induction and SDS-PAGE detection,it demonstrates that the recombinant protein AgosOBP5 is soluble and can be expressed in the supernatant.The test results show that these six odor molecules can reduce the fluorescence intensity value of the AgosOBP5 and N-phenyl-1-naphthylamine(1-NPN) fluorescent probe system to less than 50%.Starting from the initial dropwise addition of the five compounds including methyl salicylate,2-phenylethanol,benzyl alcohol,nonanal,and 1-octen-3-ol,the fluorescence value began to decrease and continued to maintain a downward trend.However,palmitic acid initially showed an increasing trend at the concentrations below 96 μmol·L-1,this trend reversed later and showed a continuous decrea

关 键 词：棉蚜 气味结合蛋白 基因克隆 表达纯化 荧光竞争结合 

分 类 号：S433.39[农业科学—农业昆虫与害虫防治]