小地老虎化学感受蛋白AipsCSP2配体结合特性分析

Ligand Binding Characteristics of the Chemosensory Protein AipsCSP2 from Agrotis ipsilon(Hufnagel)(Lepidoptera: Noctuidae)

为了明确小地老虎化学感受蛋白基因AipsCSP2在雌、雄成虫各组织中的表达情况,解析Aips CSP2蛋白的配体结合特性并探讨其功能。本文基于小地老虎性腺转录组数据,利用PCR技术克隆AipsCSP2基因,并进行生物信息学和系统进化分析;采用q PCR技术测定该基因在小地老虎雌、雄成虫不同组织中的表达水平;利用原核表达技术体外表达并纯化Aips CSP2蛋白;最后采用荧光竞争性结合试验测定该蛋白与小地老虎和其他鳞翅目昆虫性信息素组分以及主要寄主植物挥发物的结合能力。结果显示AipsCSP2基因开放阅读框(open reading frame,ORF)长度为360 bp,编码119个氨基酸,氨基酸序列中具有4个保守半胱氨酸位点,是典型的化学感受蛋白;组织表达谱结果显示AipsCSP2基因在雌成虫性腺和雄成虫附腺中特异高表达,在其他组织中表达量较低;荧光竞争性结合试验结果表明,Aips CSP2蛋白和小地老虎性信息素组分顺-11-十六碳乙酸酯的合成前体顺-11-十六碳醛有着极其强的结合能力,Ki值为1.48μmol/L,此外AipsCSP2蛋白和其他鳞翅目昆虫的醇类和醛类性信息素如十四烷醇、顺-9-十六碳醛、顺-7-十六碳醛也有着极其强的结合能力,Ki值分别为1.10μmol/L、0.63μmol/L和1.51μmol/L,和小地老虎性信息素组分顺-7-十二碳乙酸酯、顺-8-十二碳乙酸酯的结合能力为中等,与小地老虎主要寄主植物挥发物结合能力较弱或不结合。推测Aips CSP2蛋白可能主要参与了小地老虎性信息素的合成与识别过程,并在生殖活动中起着重要的作用。

To clarify the expression of AipsCSP2 gene in male and female adult tissues of Agrotis ipsilon(Hufnagel),we investigate the ligand binding characteristics of the AipsCSP2 and explore its functions.According to the pheromone gland transcriptome data,the AipsCSP2 gene was cloned and analyzed by bioinformatics and phylogenetic analysis.The expression level of the gene in different tissues of male and female adult was determined by qPCR.The recombinant protein was expressed and purified with prokaryotic expression system.Finally,the binding ability of this protein to the sex pheromone components of A.ipsilon and other Lepidoptera insects,as well as the volatiles of the main host plants were determined by fluorescence competitive binding assay.Results showed that the open reading frame(ORF)of AipsCSP2 gene was 360 bp and it encoded 119 amino acids.It has four conserved cysteines in the amino acid sequence and is a typical chemosensory protein.The tissue expression patterns showed that it was specifically high expressed in the female adult pheromone gland and male adult accessory gland,low expressed in other tissues.Fluorescent competitive binding assay results showed that AipsCSP2 has a very strong binding ability to cis-11-hexadecylaldehyde,the precursor of A.ipsilon sex pheromone cis-11-hexadecylacetate,with Ki of 1.48μmol/L.It also has a strong binding ability to alcoholic and aldehydic sex pheromone of other Lepidoptera insects,such as tetradecanol,cis-9-hexadecaldehyde and cis-7-hexadecaldehyde,with Ki of 1.10μmol/L,0.63μmol/L and 1.51μmol/L.It has a moderate ability to the sex pheromone components of A.ipsilon cis-7-dodecanoacetate and cis-8-dodecanoacetate.The binding ability to the volatiles of the main host plants of A.ipsilon was weak or not.It is suggested that AipsCSP2 may be involved in the synthesis and recognition of sex pheromone in A.ipsilon,and play an important role in reproductive activities.