斜纹夜蛾化学感受蛋白SlitCSP2的表达模式定位及配体结合特性分析

Expression Pattern and Ligand Binding of Chemosensory Protein SlitCSP2 of Spodoptera litura(Lepidoptera:Noctuidae)

研究斜纹夜蛾Spodoptera litura化学感受蛋白Slit CSP2的表达模式、组织定位及与配体结合特性,为揭示SlitCSP2在化学通讯中的功能提供理论依据。利用RT-qPCR检测了SlitCSP2在蛹及雌、雄成虫各组织的基因表达水平;Westernblot方法检测了SlitCSP2在幼虫期和雄成虫头部、触角和足部的蛋白表达水平;利用免疫组织化学技术对Slit CSP2在幼虫各龄期的分布情况进行定位分析;利用荧光竞争结合试验检测Slit CSP2与13种非挥发性化合物的结合特性。RT-qPCR结果表明,SlitCSP2在3龄幼虫表达量最高,3~6龄表达量逐渐降低;成虫阶段雄成虫足部及胸部和雌成虫头部及翅的表达量最高,且雄成虫头部、足部和胸部的表达量显著高于雌成虫。Western blot结果显示,SlitCSP2在3龄幼虫头部(去触角)的蛋白表达水平较高。免疫组化结果表明,Slit CSP2在幼虫阶段主要分布于头部、口器和胸足;在雄成虫中,Slit CSP2主要分布于足部前跗节和足部外骨骼内侧。荧光竞争结合试验表明,Slit CSP2与单宁(Ki=1.77)、花青素(Ki=2.34)和叶酸(Ki=4.56)有很强的结合能力。本研究明确了Slit CSP2在幼虫各龄期和成虫各组织的表达特征。根据免疫定位及荧光竞争结合试验结果,我们推测Slit CSP2可能在斜纹夜蛾寄主选择和解毒过程中发挥重要作用。

The expression pattern,tissue localization and binding characteristics of nonvolatile compounds of SlitCSP2 were investigated for revealing the function of SlitCSP2 in chemical communication.The expression levels of SlitCSP2 in larval,pupal,male and female adult tissues were detected by RT-qPCR.The expression levels of SlitCSP2 protein in larval stages,and heads,antennae and legs of male adults were detected by Western blot.The distribution of SlitCSP2 in legs of male adults and larval stages were analyzed by immunohistochemistry.The binding characteristics of SlitCSP2 with 13 nonvolatile compounds were analyzed by fluorescence competition binding assay.RT-qPCR results showed that SlitCSP2 was expressed the highest in the third instar larvae,and gradually decreased from the third to the sixth instar larval stages.In adult stage,SlitCSP2 was expressed the highest in legs and thoraxes of male adults as well as in heads and wings of female adults.Moreover,the expression levels in legs,heads and thoraxes of male adults were significantly higher than those of female adults.Western blot results showed that SlitCSP2 protein was expressed higher in the third instar larvae and heads(without antennae).Immunohistochemistry results showed that SlitCSP2 was mainly distributed in heads,mouthparts and thoracic legs in larval stages.In male adults,SlitCSP2 was mainly distributed in anterior tarsus and the inside of the exoskeleton of legs.Fluorescence competitive binding experiments showed that SlitCSP2 had strong binding ability with tannin(Ki=1.77),anthocyanin(Ki=2.34) and folic acid(Ki=4.56).In this study,the expression characteristics of SlitCSP2 in larval stages and adult tissues were determined.Based on the results of immunolocalization and fluorescence competition,we speculate that SlitCSP2 plays an important role in host selection and detoxification in S.litura.