Scientia Agricultura Sinica ›› 2014, Vol. 47 ›› Issue (9): 1735-1742.doi: 10.3864/j.issn.0578-1752.2014.09.008

• PLANT PROTECTION • Previous Articles     Next Articles

Gene Cloning and Expression Analysis of Three Odorant Receptors in the Diamondback Moth (Plutella xylostella)

 KONG  Chang-Yi-1, WANG  Gui-Rong-2, LIU  Yang-2, YAN  Shan-Chun-1   

  1. 1、College of Forestry，Northeast Forestry University，Harbin 150040;
    2、State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
  • Received:2013-12-16 Online:2014-05-01 Published:2014-02-24

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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

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