桃蛀螟性信息素结合蛋白CpunPBP3的表达模式及性信息素结合特性  

Expression patterns and pheromone-binding properties of the pheromone binding protein CpunPBP3 in Conogethes punctiferalis(Lepidoptera:Crambidae)

作　　者：陈秋应 杨喜 游东蕊 杨沐 徐志峰[1,2] 肖伟[1,2] CHEN Qiu-Ying;YANG Xi;YOU Dong-Rui;YANG Mu;XU Zhi-Feng;XIAO Wei(Key Laboratory of Entomology and Pest Control Engineering,College of Plant Protection,Southwest University,Chongqing 400715,China;Academy of Agricultural Sciences,Southwest University,Chongqing 400715,China)

机构地区：[1]西南大学植物保护学院,昆虫学及害虫控制工程重点实验室,重庆400715 [2]西南大学农业科学研究院,重庆400715

出　　处：《昆虫学报》2021年第3期318-326,共9页Acta Entomologica Sinica

基　　金：国家重点研发计划(2017YFD0202002)。

摘　　要：【目的】本研究旨在对桃蛀螟Conogethes punctiferalis性信息素结合蛋白CpunPBP3进行鉴定和定性分析,完善对桃蛀螟性信息素感受机制的理解。【方法】扩增、分析桃蛀螟CpunPBP3的cDNA序列,并与其他草螟科昆虫的同源蛋白氨基酸序列进行比较。利用qRT-PCR测定桃蛀螟不同日龄雄成虫触角中CpunPBP3的表达量变化,以及该基因表达的昼夜波动;以性信息素反-10-十六碳烯醛(E 10-16∶Ald)(150 ng)和顺-10-十六碳烯醛(Z 10-16∶Ald)(6 ng)对雄成虫进行诱导,并检测诱导后24 h内不同时间节点时触角中CpunPBP3的表达量。构建重组表达载体pET-30a(+)/CpunPBP 3,通过大肠杆菌Escherichia coli表达获得重组蛋白,并通过荧光竞争实验检测纯化重组蛋白CpunPBP3与上述2种性信息素成分的结合能力。【结果】系统发育分析结果显示,CpunPBP 3与已经鉴定的桃蛀螟PBP基因CpunPBP 2和CpunPBP 5分布在不同分支中,但与其他昆虫物种的PBP基因亲缘关系较近;qRT-PCR结果表明,雄成虫触角中CpunPBP3的表达量在羽化后0-8 d呈先升高后下降的趋势,24 h光周期内17∶00时的表达量显著高于1∶00时的,其余时间节点间的表达量无显著差异;150 ng的E 10-16∶Ald诱导3和6 h后的表达量显著下降,6 ng的Z 10-16∶Ald诱导6和24 h后的表达量显著增加。荧光竞争结合实验结果表明,CpunPBP3重组蛋白与性信息素E 10-16∶Ald和Z 10-16∶Ald均有强结合能力,K i值分别为9.267和8.656μmol/L。【结论】本研究明确了CpunPBP3的核苷酸和氨基酸序列及其表达模式,能够响应性信息素的诱导而表达,而且CpunPBP3重组蛋白能够很好地结合性信息素,表明CpunPBP3是桃蛀螟的一条性信息素结合蛋白。【Aim】This study aims to better understand the sex pheromone perception mechanisms by identifying and characterizing a sex pheromone binding protein(PBP)in the yellow peach moth,Conogethes punctiferalis(CpunPBP3).【Methods】The cDNA sequence of CpunPBP3 of C.punctiferalis was amplified and analyzed,and the amino acid sequence was compared to those of the homologous proteins in other Crambidae species.The day-age-dependent changes and circadian fluctuations in the expression levels of CpunPBP3 in the male adult antenna of C.punctiferalis,and the changes in the expression level of CpunPBP3 in the antenna over 24 h-period following exposure of adult males to the sex pheromones E 10-16∶Ald(150 ng)and Z 10-16∶Ald(6 ng)were examined by qRT-PCR.The recombinant expression vector pET-30a(+)/CpunPBP 3 was constructed,and the recombinant CpunPBP3 was expressed in Escherichia coli.The binding capacity of the purified recombinant protein CpunPBP3 with the above two sex pheromones was evaluated by fluorescence competitive binding assay.【Results】The phylogenetic analysis result revealed that CpunPBP3 and the previously identified C.punctiferalis PBP genes CpunPBP2 and CpunPBP5 clustered in different branches,but CpunPBP3 is similar to PBP genes in other insect species.The qRT-PCR results showed that the expression level of CpunPBP3 in the male adult antenna increased first and then decreased from day 0 to 8 after adult eclosion,with significantly higher expression level at 17∶00 than at 1∶00,but with no significant difference at other time points within 24-h photoperiod.However,the expression level of CpunPBP3 in the male adult antenna significantly decreased after induction by 150 ng E 10-16∶Ald for 3 and 6 h,and significantly increased after induction by 6 ng Z 10-16∶Ald for 6 and 24 h.Fluorescence competitive binding assay result showed that the recombinant CpunPBP3 had strong binding capacity with E 10-16∶Ald and Z 10-16∶Ald,with the K i values of 9.267 and 8.656μmol/L,respectively.【Conclusion】

关 键 词：桃蛀螟 性信息素结合蛋白 表达模式 昼夜节律 原核表达 荧光竞争结合实验 

分 类 号：Q966[生物学—昆虫学]