Analysis And Functional Study Of Epdsx Gene Of Euzophera Pyriella

Euzophera pyriella belongs to the Pyralididae in the family Lepidoptera,which is a serious pest of pear trees,apple trees and other fruit trees.Since the rapid development of characteristic industry of forestry fruit in Xinjiang,Euzophera pyriella has seriously affected the growth of fruit trees and fruit yield.Sex determining genes are closely related to pest population outbreak and fecundity,doublesex gene is located at the bottom in sex determination mechanism,and produces female-specific protein DSX^Fand male-specific protein DSX^Mprotein through alternative splicing.The female-and-male specific protein regulates the sex differentiation of insects,the formation of insect reproductive organs,the courtship behavior of insects,and the development of insect neural networks.In order to explore the DSX protein,gene expression and gene function of the Epdsx gene of Euzophera pyriella,the research contents are as follows:1.Bioinformatic analysis of Epdsx gene of Euzophera pyriellaThe ORF Finder software of NCBI was used to analyze the open reading frame of the Epdsx gene sequence of Euzophera pyriella in transcriptome group.The physiological and biochemical characteristics of DSX protein of Euzophera pyriella were predicted by using bioinformatics analysis softwares.DNAMAN and MEGA X softwares were used for multiple alignment of amino acid sequences and phylogenetic tree construction.The results showed that Epdsx protein was an unstable hydrophilic protein composed of 273 amino acids.There were no signal peptides and transmembrane domains,and there were29 phosphorylation sites.The prediction results of Epdsx protein’s second and third structures were consistent,the random curl andα-helix were dominant.The similarity between Epdsx protein and a variety of Lepidoptera insects was 91.55%,indicating that Epdsx protein was highly conservative.In terms of homology,Epdsx protain forms a clade with Amyelois transitella and Ephestia Kuehniella,suggesting a relatively close evolutionary relationship.2.Analysis of Epdsx gene splicing pattern of Euzophera pyriellaThree types of transcripts were obtained by PCR amplification and gene cloning,encoding non-specific protein,female specific protein(Dsx ^F1)and male specific protein(Dsx ^M1),respectively.The amino acid sequences of Dsx ^M1and Dsx ^F1proteins were compared with those of 14 species of Lepidoptera insects.The amino acid sequences of Dsx ^M1and Dsx ^F1proteins were similar to those of other Lepidopteran insects,and they both possessed two highly conserved functional regions,DM domain and DSX-dimer.The phylogenetic tree constructed by MEGA X software showed that Dsx ^M1and Dsx ^F1proteins were in the same branch with Amyelois transitella,and were closely related to each other.3.Analysis of Epdsx gene expression profile of Euzophera pyriellaThe Epdsx gene expression levels in different developmental stages and tissues of male and female adults were detected by real-time fluorescence quantitative assay,using Tub as an internal reference gene.The results showed that Epdsx gene was expressed in different stages of male and female larvae,with the highest expression level in female pupal stage and male adult stage.In different tissue parts of adult,the expression level of female adult was the highest in chest and wing,and the expression level of male adult was the highest in chest.The lowest expression level was found in the head of female adult and in the wing of male adult.4.RNAi of Epdsx gene of Euzophera pyriellaThe gene function of Epdsx gene of Euzophera pyriella was preliminarily explored by ds RNA microinjection.The results showed that ds DSX injection affected the expression level of doublesex gene in both male and female larvae.Compared with the control,the expression level of Epdsx gene in female larvae reached the lowest value of 0.31 at 12h after injection,and the lowest value of Epdsx gene in male larvae reached the lowest value of 0.51 at 24h after injection,and then increased,but did not reach the normal expression level of Epdsx gene.