绿盲蝽TRPA1基因的鉴定及功能分析

doi:10.3864/j.issn.0578-1752.2015.12.009

摘要/Abstract

摘要： 【目的】克隆绿盲蝽（Apolygus lucorum）TRPA1基因，明确其在成虫不同组织中的表达分布，研究绿盲蝽TRPA1基因的功能，阐明绿盲蝽感受温度的分子基础。【方法】在绿盲蝽转录组测序的基础上，通过生物信息学方法分析转录组数据,得到编码绿盲蝽TRPA1基因的cDNA序列片段。根据已知的cDNA序列，设计引物。采用RT-PCR及RACE（rapid-amplification of cDNA ends）技术克隆TRPA1基因的全长序列，并通过DNAMAN软件及BLAST分析测序结果。测序正确后，使用在线工具SMART及TMpred预测TRPA1蛋白的锚蛋白重复序列（Ankyrin repeats）及跨膜结构域序列。通过序列比对，构建昆虫TRP（transient receptor potential）通道的家族进化树，分析绿盲蝽TRPA1与其他物种同源基因的相似性及进化地位。以GADPH为内参基因，利用RT-PCR检测绿盲蝽TRPA1在成虫触角、头、喙、足、腹等不同组织中的相对表达量。通过爪蟾卵母细胞体外表达结合双电极电压钳记录技术研究绿盲蝽TRPA1对温度刺激的反应。【结果】通过绿盲蝽转录组数据预测得到TRPA1基因的4个cDNA序列片段，经过RT-PCR及RACE技术克隆得到了绿盲蝽TRPA1的全长序列，并命名为AlucTRPA1。AlucTRPA1开放阅读框全长3 672 bp，编码1 223个氨基酸。通过在线工具SMART及TMpred预测分析结果表明，AlucTRPA1含有16个锚蛋白重复序列结构及6个保守的跨膜结构域。序列比对及进化树分析结果显示，昆虫TRPA家族包含TRPA1、Painless、Pyrexia及Water witch，AlucTRPA1属于昆虫TRPA1亚家族。AlucTRPA1与其他昆虫的同源基因具有很高的相似性，特别是与同属半翅目的豌豆蚜（Acyrthosiphon pisum）的同源性高达65.1%。成虫组织表达谱分析结果表明，AlucTRPA1在成虫触角中的表达量最高，头、喙、足、腹中也都有表达。爪蟾卵母细胞体外表达结合双电极电压钳记录结果表明20℃至40℃逐渐上升的温度可以直接激活AlucTRPA1通道，且两次连续重复的温度刺激未引起AlucTRPA1通道的钝化。【结论】AlucTRPA1在绿盲蝽成虫的触角、头等组织中均有表达，且在爪蟾卵母细胞表达的AlucTRPA1能被20℃至40℃逐渐上升的温度刺激直接激活，推测AlucTRPA1在绿盲蝽体内是直接的温度感受器，参与调控绿盲蝽对生活环境温度的感知行为。

关键词: 绿盲蝽, TRPA1, 温度, 爪蟾卵母细胞, 双电极电压钳

Abstract: 【Objective】 The objective of this study is to obtain the TRPA1 gene in Apolygus lucorum,characterize its expression profiles in different tissues of adult, illustrate its functions through heterologous expression system of Xenopus oocytes, and further to explain the molecular mechanism of thermosensation in A. lucorum.【Method】The transcriptome of A. lucorum was sequenced and analyzed, and several cDNA fragments encoding A. lucorum TRPA1 were identified. And then specific primers for RT-PCR and RACE were designed. The full-length cDNA sequence encoding AlucTRPA1 was cloned via RACE and RT-PCR techniques. The cDNA sequence encoding TRPA1 gene was analyzed by DNAMAN software and BLAST. Then ankyrin repeats and transmembrane domains were predicted with SMART and TMpred. The phylogenetic tree of TRPs in insects was constructed to analyze the evolutionary relationship between A. lucorum and other insects. RT-PCR was performed to detect the relative-expression of TRPA1 in different tissues of the adults. Two-electrode voltage clamp electrophysiological recording was introduced to demonstrate the function of TRPA1 in vitro. 【Result】 Four fragment sequences of the TRPA1gene were found according to the results of transcriptome analysis. The full-length sequence of TRPA1 gene named AlucTRPA1 was further obtained using RACE and RT-PCR techniques, which is 3 672 bp and encodes 1 223 amino acids. AlucTRPA1 contained 6 conserved transmembrane domains and 16 ankyrin repeats, which predicted by SMART and TMpred. The results of multiple alignment and phylogenetic tree analysis showed that TRPA of insects contains TRPA1, Painless, Pyrexia and Water witch, and AlucTRPA1 clustered into the branch of TRPA1. AlucTRPA1 is highly similar to the homologs from other insects, especially the pea aphids (Acyrthosiphon pisum), which also belongs to the Hemiptera, with the identity of 65.1%. Expression profiles showed that AlucTRPA1 was expressed in antennae at the highest level among different tissues. In the Xenopus-based functional study, AlucTRPA1 could be activated directly by an increasing temperature from 20℃ to 40℃ and didn’t show adaption after repeated temperature stimuli. 【Conclusion】 AlucTRPA1ors. expressed in all tested tissues, but highest in antennae. AlucTRPA1 could be activated directly by an increasing temperature from 20℃ to 40℃, indicating that AlucTRPA1 is a direct thermal sensor in A. lucorum and mediates their temperature sensing behavi

Key words: Apolygus lucorum, TRPA1, temperature, Xenopus oocytes, the two-electrode voltage-clamp

付婷，杨婷，刘杨，王桂荣. 绿盲蝽TRPA1基因的鉴定及功能分析[J]. 中国农业科学, 2015, 48(12): 2364-2373.

FU Ting, YANG Ting, LIU Yang, WANG Gui-rong. Identification and Functional Characterization of TRPA1 in Apolygus lucorum (Meyer-Dür)[J]. Scientia Agricultura Sinica, 2015, 48(12): 2364-2373.

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