小菜蛾三个普通气味受体基因的克隆及表达谱

doi:10.3864/j.issn.0578-1752.2014.09.008

摘要/Abstract

摘要： 【目的】克隆小菜蛾（Plutella xylostella）触角中3个气味受体基因，明确这3个气味受体在不同组织中的表达分布，进而推测这3个基因的功能，为进一步深入研究奠定基础。【方法】通过新一代高通量测序技术对小菜蛾成虫触角进行转录组测序，获得小菜蛾的转录组数据信息，通过对高质量序列的拼接组装、基因鉴定和功能注释，并通过Blastx基于数据库进行相似性比对分析，预测小菜蛾的气味受体候选基因；设计引物通过克隆得到3条普通气味受体基因的全长序列，并利用半定量RT-PCR研究其在雌、雄成虫9个不同组织中的表达。【结果】根据预测的基因序列，设计特异引物，克隆得到3条普通气味受体基因的全长序列，分别命名为PxylOR16、PxylOR17和PxylOR18（GenBank登录号分别为KF717601—KF717603）。PxylOR16开放阅读框全长为1 218 bp，编码406个氨基酸；PxylOR17开放阅读框全长为1 200 bp，编码400个氨基酸；PxylOR18开放阅读框全长为 1 191 bp，编码397个氨基酸。选择已报道的鳞翅目昆虫家蚕（Bombyx mori）、烟芽夜蛾（Heliothis virescens）和棉铃虫（Helicoverpa armigera）的气味受体，以及已报道的小菜蛾的6条性信息素受体与1条非典型气味受体与本实验克隆得到的3个气味受体进行序列比对和进化树分析，结果显示这3个气味受体基因与非典型气味受体和性信息素受体同源性低，而与其他的普通气味受体聚类在一起，且PxylOR16、PxylOR17和PxylOR18彼此同源性较低。半定量的结果显示，3个普通气味受体基因均在触角中表达量最大，在其他嗅觉感器，如喙、下唇须和头部中也有一定量的表达，雌、雄间无显著差异。另外，PxylOR17在雌蛾生殖器中，PxylOR18在雌蛾腹中也有一定表达。但3种气味受体基因在雌、雄蛾的胸、足和翅等组织中均不表达。【结论】鉴定和克隆了3个小菜蛾气味受体基因，明确这些气味受体基因在小菜蛾不同组织中的表达水平，通过进化树分析、序列比对和组织表达谱分析确定PxylOR16、PxylOR17和PxylOR18为3条普通气味受体，且在功能上高度分化。根据半定量RT-PCR的结果，推测这3个基因可能参与了普通气味分子的识别过程，此外PxylOR17和PxylOR18还可能参与了信息素的产生和释放过程。

关键词: 小菜蛾 , 触角 , 气味受体 , 基因克隆 , 半定量RT-PCR

Abstract: 【Objective】The objective of this study is to clone three odorant receptor genes and investigate their expression in different tissues of Plutella xylostella adults, and to provide basic knowledge for further functional studies of these three odorant receptor genes. 【Method】Antennal transcriptome was successfully used to identify candidate odorant receptors (ORs) by the next-generation sequencing. The gene was identified and the function was annotated by assembling the adult male and female antennal transcriptomes. Based on the database, the similarity was analyzed and comparison with Blastx and the candidate ORs of P. xylostella were identified. Based on the transcriptome sequencing results, the full length sequence of the odorant receptor genes were cloned. The expression profiles of these genes in different nine tissues were investigated by using semi-quantitative real-time PCR.【Result】Based on the predicted gene sequences, special primers were designed to clone the full length of odorant receptor genes. Three odorant receptor genes PxylOR16, PxylOR17 and PxylOR18 (GenBank accession number: KF717601-KF717603) were cloned. The open reading frame (ORF) of PxylOR16, PxylOR17 and PxylOR18 was 1 218, 1 200 and 1 191 bp, which encoded a polypeptide of 406, 400 and 397 amino acids, respectively. The sequences were aligned and the phylogenetic tree of the three odorant receptors was analyzed with the selected sequences of Lepidopteran insects, Bombyx mori, Heliothis virescens and Helicoverpa armigera odorant receptors which have been reported, and the reported P. xylostella six sex pheromone receptors and an olfactory receptor. The results showed that there were obvious differences between the three odorant receptor gene and atypical odorant receptors or sex pheromone receptor, and clustered together with other general odorant receptor. The three sequences had a low homology with each other and low sequence conservation. The results of semi-quantitative PCR showed that the three odorant receptor genes expressed in the antennae, heads, proboscises and labial palps and had no sexual specificity. The slight expression of PxylOR17 was detected in the female genitals. The expression of PxylOR18 could also be detected in female abdomens. No expression was observed between male and female. The three odorant receptor genes had no expressions in other tissues such as thoraxes, legs, abdomen and genitals.【Conclusion】Three odorant receptor genes were cloned in P. xylostella and the expression levels of them in different tissues were verified. It was identified that PxylOR16, PxylOR17 and PxylOR18 are three general odorant receptors by phylogenetic tree analyses and the sequences analyses. The three ORs are divergent in function. According to the results of RT-PCR, it was speculated that these three genes might be involved in the common odor molecules recognition process. In addition, it was also speculated that PxylOR17 and PxylOR18 might also participate in the process of identifying the male and female moths.

Key words: Plutella xylostella , antennae , odorant receptor , gene cloning , semi-quantitative reverse transcription PCR

孔畅仪1, 王桂荣2, 刘杨2, 严善春1. 小菜蛾三个普通气味受体基因的克隆及表达谱[J]. 中国农业科学, 2014, 47(9): 1735-1742.

KONG Chang-Yi-1, WANG Gui-Rong-2, LIU Yang-2, YAN Shan-Chun-1. Gene Cloning and Expression Analysis of Three Odorant Receptors in the Diamondback Moth (Plutella xylostella)[J]. Scientia Agricultura Sinica, 2014, 47(9): 1735-1742.

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