沙葱萤叶甲化学感受蛋白基因的鉴定及表达谱分析

摘要

[目的]沙葱萤叶甲Galeruca daurica是一种在内蒙古草原上爆发成灾的新害虫.昆虫的化学感受蛋白(CSPs)是一类水溶性小分子蛋白,其主要功能是识别和传导环境中的化学刺激至受体,参与众多昆虫行为.本研究旨在鉴定沙葱萤叶甲化学感受蛋白基因,并对其表达谱进行分析.[方法]通过筛选本实验室组装的沙葱萤叶甲转录组鉴定沙葱萤叶甲化学感受蛋白基因;采用实时荧光定量PCR方法分析其在沙葱萤叶甲不同发育阶段(卵、1-3龄幼虫、蛹和成虫)和成虫组织[触角、头(去触角)、胸、腹、足和翅]中的表达水平.[结果]鉴定出10个化学感受蛋白基因,将其命名为GdauCSP1-10(GenBank登录号:KY885471-KY885480).10条编码蛋白的氨基酸序列一致性范围为17.27％ ～ 62.79％,彼此间分化程度较高.通过NCBI的Blast比对结果显示,GdauCSPs与榆黄毛萤叶甲Pyrrhalta maculicollis的PmacCSP氨基酸序列一致性最高,为90％.系统发育分析表明,4种GdauCSPs首先与榆黄毛萤叶甲的PmacCSPs聚为一支.实时荧光定量PCR结果显示,GdCSPs在不同发育阶段中的表达量有显著差异,GdauCSP4-5,GdauCSP7-8和GdauCSP10 5个GdCSPs在成虫中的表达量显著高于其他发育阶段,而GdCSP2在卵中的表达量显著高于其他发育阶段;GdCSPs不仅表达于成虫触角中,在头(去触角)、胸、腹、足、翅等其他部位也有表达,且10个GdCSPs在沙葱萤叶甲成虫各组织中有着不同的表达谱,其中GdauCSP2,GdauCSP4,GdauCSP5,GdauCSP8和GdauCSP9 5个GdCSPs在雌成虫触角中的表达量显著高于其他组织.[结论]结果提示化学感受蛋白在沙葱萤叶甲的生长发育和化学感受过程中可能起着不同的作用.本研究为进一步研究沙葱萤叶甲化学感受蛋白的生理功能及化学通讯的分子机理奠定了必要的基础.%[Aim] Galeruca daurica is a new pest with outbreak status in grasslands of Inner Mongolia,northern China.The chemosensory proteins (CSPs) of insects are a class of small and water soluble proteins whose functions are to recognize and transport environmental chemical stimuli to receptors,participating in many aspects of insect behavior.The objective of this study is to identify the chemosensory protein genes and to analyze their expression profiles in G.daurica.[Methods] The chemosensory protein genes in G.daurica were identified by screening the transcriptome data of G.daurica assembled in our laboratory,and qPCR was conducted to analyze their expression levels in different developmental stages (egg,1 st-3rd instar larva,pupa and adult) and adult tissues (antenna,head with antennae removed,thorax,abdomen,leg and wing).[Results] Ten chemosensory protein genes were identified from G.daurica,and named as GdauCSP1-10 (GenBank accession numbers:KY885471-KY885480),respectively.The amino acid sequence identity between the 10 GdauCSPs ranges from 17.27％ to 62.79％ with a high divergence between each other.The results of Blast in NCBI showed that GdauCSPs have the highest amino acid sequence identity (90％) with PmacCSP from Pyrrhalta maculicollis.Phylogenetic analysis showed that four of the 10 GdauCSPs were firstly clustered into a clade with PmacCSPs from P.maculicollis.The qPCR results proved that the expression levels of GdCSPs were significantly different in different developmental stages,and five GdCSPs including GdauCSP 4-5,GdauCSP7-8,and GdauCSP10 had significantly higher expression levels in the adult stage than in other developmental stages while GdCSP2 had significantly higher expression level in the egg stage than in other developmental stages.GdauCSPs were also expressed in the head with antennae removed,thorax,abdomen,leg and wing besides in the antenna,and 10 GdauCSPs had different tissue expression profiles but five out of 10 GdauCSPs,including GdauCSP2,GdauCSP4,GdauCSP5,GdauCSP8 and GdauCSP9,had significantly higher expression levels in female antennae than in other tissues.[Conclusion] These results suggest that GdauCSPs may play different roles in the development and chemosensory process of G.daurica.This study lays the necessary foundation for further research on the physiological function of CSPs and the molecular mechanism of chemical communication in G.daurica.