Molecular Cloning,Tissue Expression And Functional Study Of Gustory Receptor Genes From Plutella Xylostella(Lepidoptera:Plutellidae)

Plutella xylostella（Lepidoptera:Plutellidae）is an important agricultural pest in the world,which specifically damage cruciferous plants.Glucosinolates and its degradation products which are specific in cruciferous plants play an indicative role in host plant selection of P.xylostella.The mated female moths often touch the leaf surface with its legs before ovipositioning,which is considered to detect glucosinolates and other plant secondary substances.In addition,like most moths,P.xylostella females need to feed on nectar and other honey sources after emergence in order to achieve sexual maturity,and then calling and mating.Taste plays a key role in both nectar detection and oviposition medium selection.The identification of gustatory receptor（GR）genes is of great significance for elucidating the gustatory mechanism of P.xylostella feeding exclusively on cruciferous plants,and for developing pest control techniques based on taste interference.In order to identify the possible glucosinolates receptors on the foreleg of female P.xylostella moths,15 GR genes were obtained by analyzing the transcriptomes of female and male adult legs of P.xylostella and by verification with PCR and qPCR experiments.Four GR genes（PxylGR5,8,14,15）were highly expressed in female forelegs than male forlegs,and thus were considered as candidate glucosinolates receptors.Then the functional study by Xenopus oocytes-two electrode voltage clamp system showed that the four GR showed no obvious response to the four tested glucosinolates,and therefore,GRs for glucosinolates need to be further studied.For the identification of fructose receptor genes,cluster analysis showed that PxylGR8 and PxylGR43a（two splice variants of the same gene）were clustered in one branch with the known fructose receptors from other insect species.The RT-PCR analysis showed that the two splice variants were expressed in the antennae,head and foreleg of adults,but the expression in adult antennae was higher.Finally,12 sugars were measured by Xenopus oocyte-two electrode voltage clamp system.It was found that the two splice variants responded only to fructose and thus the two splice variants were confirmed to be fructose receptors.The results laid a foundation for further elucidating the gustatory mechanism of P.xylostella feeding exclusively on cruciferous plants.The main results are as follows:1.Cloning and sequence analysis of GR genes from the forelegs of P.xylostellaChemical sensilla are distributed on the tarsi of adult moths,which play an important role in the selection of oviposition media（host leaves,etc.）by females.In order to identify the possible glucosinolates receptor gene on the forelegs,the foreleg transcriptomes of female and male adults of P.xylostella were sequenced and analyzed.A total of 15 cDNA sequences of GR gene were identified,of which 13 were in full-length and 4 were highly expressed in female forelegs（FPKM:female/male>2.00）,and thus these 4 GR genes were selected as candidate glucosinolates receptors for further functional study.Eight of the 13 GR full-length cDNAs were verified by PCR,and full-length cDNA of PxylGR8 was successfully amplified by RACE-PCR.Cluster analysis showed that PxylGR8 and other functionally characterized insect fructose receptor genes were clustered in a distinct branch,indicating that PxylGR8 was a candidate fructose receptor for subsequent functional studies.In addition,NCBI BlastX alignment and genomic sequence analysis showed that PxylGR8 and reported PxylGR43a were two splice variants of a same gene.The N-terminus of PxylGR8 is 23 amino acids less than that of PxylGR43a,which may lead to the difference in the function.2.Tissue expression profile analysis of GR genes in P.xylostellaIn order to determine the temporal and spatial expression dynamics and the expression difference between male and female moths,the expression levels of 12 GR genes were measured by qPCR using P.xylostella RPS3（gene accession number:NW₀11952055.1）as the reference gene.The results showed that the expressions of 12 genes were very low in larval stage.Among different ages（1-4 instar）,PxylGR5,14,15 were the highest in first instar larvae.The analysis of the expression of different adult tissues（heads,antennae,forelegs,middle and hind legs）showed that 10 of the 12 GR genes were the highest in the antennae,and 2 GR genes（PxylGR14,15）were the highest in the adult heads（removal of antennae）.In addition,expression levels of two candidate fructose receptors PxylGR8 and PxylGR43a were measured by RT-PCR using P.xylostella Pxylβ-Actin（gene accession number:AB282645）as the reference gene.The results showed that the two splice variants were highly expressed in the antennae,which was consistent with the fact that P.xylostella adults feed flower nectar for sexual maturity.3.Functional analysis of GR genes in P.xylostellaBefore the functional study of four candidate glucosinolates receptors,the stimulating effect of GS crude extract of radish seeds on oviposition of female P.xylostella moth was determined.The results showed that GS crude extract could significantly increase the oviposition of P.xylostella,indicating that the female moths could sense GS to increase the oviposition.The proboscis extension reflex（PER）of female moths was measured by stimulating antennae with fructose solutions,showing that female moths could detect fructose through antennae and cause feeding behavior.After verifying the behavioral responses,the function of the candidate genes was analyzed by Xenopus oocyte-two electrode voltage clamp system.The four candidate glucosinolates receptor genes（Pxy1GR5,8,14,15）did not respond to the all four tested glucoside components,and therefore,the glucosinolate receptor genes need to be further studied,by measuring more GS genes with more glucosinolates.Two candidate fructose receptors all showed specific electrophysiological responses to fructose among 12 tested sugars at 0.1 M concentration.In comparison,the response of PxylGR8 to fructose was significantly higher than that of PxylGR43a,with the current value of the former was 3.85 times higher than that of the latter at 0.1 M sugar concentration.Dose-response curve analysis showed that the EC50 values of Pxy1GR8 and PxylGR43a to fructose were 0.089 M and 0.068 M,respectively.