荔枝蒂蛀虫卵5个化学感受蛋白的基因克隆及除虫脲对其表达的影响

Molecular cloning of five chemosensory proteins from Conopomorpha sinensis eggs and gene expression after diflubenzuron treatment

【目的】获得荔枝蒂蛀虫Conopomorpha sinensis 5个化学感受蛋白(Chemosensory proteins,CSPs)基因CsinCSPs的全长序列,并解析除虫脲对其表达的影响。【方法】提取荔枝蒂蛀虫卵总RNA,利用转录组测序结果和cDNA末端快速扩增技术(Rapid amplification of cDNA ends,RACE)获得5个CsinCSPs基因的全长序列;对序列进行生物信息学分析,用Phyre2在线软件进行同源建模,用AutoDock软件对蛋白与农药分子进行分子对接预测;用荧光定量PCR方法分析在除虫脲处理荔枝蒂蛀虫卵后这5个基因的表达情况。【结果】从荔枝蒂蛀虫卵中克隆了5个CsinCSPs基因的全长序列,序列分析结果表明,这5个基因的编码蛋白具有昆虫化学结合蛋白的典型特征,其保守半胱氨酸位点排布模式均为C1-X6-C2-X18-C3-X2-C4,并分为了包含5个α-螺旋和6个α-螺旋的2个类群。软件模板分析表明,5个CsinCSPs蛋白的三维结构均由α-螺旋、β-折叠和卷曲环组成,且与甘蓝夜蛾Mamestra brassicae CSP2的三维结构相似度最高。分子对接数据表明,5个CsinCSPs与2个菊酯类药剂分子结合亲和力最强,与除虫脲的结合力最弱。亚致死剂量除虫脲处理荔枝蒂蛀虫卵后,CsinCSP3和CsinCSP5分别在处理后6、24 h表达上调超过40余倍,48 h则表达下降。【结论】化学感受蛋白CsinCSPs可能参与农药胁迫下荔枝蒂蛀虫卵的解毒过程。

【Objective】To obtain the full-length sequences of CsinCSPs genes of five chemosensory proteins(CSP)from Conopomorpha sinensis,and analyze the effect of diflubenzuron on CsinCSP gene expression.【Method】Total RNA was extracted from C.sinensis eggs.The full-length cDNA of five CsinCSP genes were cloned by transcriptome sequencing results and rapid amplification of cDNA ends(RACE).The putative amino acid sequences of CsinCSPs were analyzed by bioinformatics software.The three-dimensional(3D)structure of CsinCSPs were predicted using the Phyre2 online software.The binding capacities of CsinCSPs to pesticides were studied by AutoDock software.The changes of CsinCSP gene expressions after treating the C.sinensis eggs with diflubenzuron were analyzed by qRT-PCR.【Result】Full-length sequences of five CsinCSP genes were cloned from C.sinensis eggs.Sequence analysis results indicated these five genes encode proteins with typical characteristics of insect chemosensory proteins.The putative cysteines pattern of five CsinCSPs was C1-X6-C2-X18-C3-X2-C4.The five CsinCSPs could be classified to clades with fiveα-helix and sixα-helix.Based on analysis of model template,the 3D structures of five CsinCSPs were found to consist ofα-helix,β-strand and loop,and were most similar to the 3D structure of Mamestra brassicae CSP2.The molecular docking results revealed that the five CsinCSPs had the highest binding affinities with two pyrethroids,while the lowest binding affinity with diflubenzuron.The expressions of CsinCSP3 and CsinCSP5 were up-regulated by more than 40 times at 6 and 24 h after diflubenzuron treatment at sublethal dose to C.sinensis eggs,and decreased at 48 h after treatment.【Conclusion】The chemosensory protein CsinCSPs may be involved in the detoxification process after pesticide treatment in C.sinensis eggs.