Gene Cloning And Expression Analysis Of Odorant Receptors And Lonotropic Receptors In The Diamondback Moth (Plutella Xylostella)

:Insect population maintaining its large quantities can not live without a precise and sensitive, specific and complex olfactory system. Olfaction plays an indispensable role in mediating insect behavior such as food selection, predators and noxious agents avoidance, and appropriate mating partners choice. The Plutella xylostella is easy to evade predators, response keenly, breed rapidly and eat plenty of curciferous vegetables, which all driven by its sensitive precision of the olfactory system. Many crucifers are damaged much because of the moths’ invasion every year. In the process of recognizing smells, a number of proteins involved in olfactory signal transduction pathways. Odor receptors OR(odorant receptora), IR(ionotropic receptor) contain in the reaction specificity olfactory sensory neurons. In this research, we identified transcriptome of P. xylostella which is important pests of agricultural economy,54is verified OR selected genes, and16IR for selected genes, The main results were as follows:(1) In order to study the molecular mechanisms of olfactory recognition in P. xylostella, our laboratory transcriptome sequencing of the adult antennae of P. xylostella through second-generation DNA sequencing, procuring the P. xylostella transcriptome data information through the splice assembly and function of the high quality sequence annotation, to predict candidate olfactory genes of the P. xylostella. In this study we focus on the identification and sequence analysis of the receptor gene, including54and16IRs ORs gene gene. Received54of ORs in the selected genes,23cDNA gene is full length sequence, its open reading frame length (open reading frame,ORF)1156-2187bp base pairs in length, encoding163-473amino acids. Sequence analysis revealed that these ORs have transmembrane domains is5-8, in line with the typical structure of insect ORs characteristics in370-440between amino acids in length, which consists of seven transmembrane domains.At the same time, we get IRs12candidate genes, of which5IR for full-length sequences of genes. This is typical of IRs gene structure, of which9IRs there’s three transmembrane domains. When procuring candidate ORs and IRs gene of the P. xylostella, we verify the predicted receptor gene through semi-quantitative PCR technology, and preliminary analysis of their male, female insect antennal sensilla and expression of foot. We detected in the tentacles of all54Ors and16expression IRs,which proved the credibility of the results. Results show that54ORs gene expression in primary tentacles. Among them, PxylORl, PxylOR3, PxylOR4, PxylOR5, PxylOR6, and PxylOR7expression is higher in the male moth antennae, these6ORs are pheromone receptors (pheromone receptors,PRs), at the early stage work of the laboratory personnel have carried out detailed studies. PxylOR8specificly express in the female moth antennae. The remaining47ORs genes that expression of both male and female, the expression level of genes male and female are slightly difference. In contrast, almost no differences in male and female of the ORs,IRs gene in both the male and female antennae. PxylIR7d.3and PxylIR76b both have a lower levels of gene expression. Through this experiment proves the IRs is different from ORs,IRs in expression between male and female are similar. The same results also appeared in spodoptera litura (Spodoptera litura) IRs research. Therefore speculated that the IRs is relatively highly conserved sequences and expression, might imply that the IRs has conservative features.(2) Based on the predicted gene sequences, special primers were designed to clone the full length of odorant receptor genes. Three odorant receptor genes PxylOR16, PxylOR17and PxylOR18(GenBank accession number:KF717601-KF717603) were cloned. The open reading frame (ORF) of PxylOR16, PxylOR17and PxylOR18was1218,1200and1191bp, which encoded a polypeptide of406,400and397amino acids, respectively.Discovered through phylogenetic tree analysis of three gene do not accumulate in known pheromone receptor, because ordinary smell receptors are not as conservative as pheromone receptors, therefore filtered three genes are spread among various insects of the Lepidoptera of ORs. Through organized and expression profile analysis, PxylOR16, PxylOR17and PxylOR18are three common odor receptors, and highly differentiated in function. And the use of semi-quantitative PCR (RT-PCR) clearly indicate that these odor receptor genes in the P. xylostella show different levels of expression of nine adult male and female. Semiquantitative results showed that three ordinary odor receptor genes is expressed in the antenna the largest volume in other olfactory sensilla, such as the beak, and the head of the lower lip and there’s a certain amount of expression, no significant differences between male and female. In addition, PxylOR17on female genital mutilation, the PxylOR18also have a certain expression in the female abdominal. But three kinds of odor receptor genes in male and female breasts, feet, and wings of the moth and other organizations are not expressed. Speculated that three genes may be involved in the normal odor molecular recognition process, in addition to PxylOR17and PxylOR18also may have been involved in pheromone production and release process.