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Identification,Tissue Expression Profile And Protein Expression Research Of Glyphodes Pyloalis Olfactory Genes

Posted on:2020-06-07Degree:MasterType:Thesis
Country:ChinaCandidate:W M SongFull Text:PDF
GTID:2393330590950992Subject:Biology
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The mulberry pyralid,?Lepidoptera:Pyralidae?is one of a major pest of mulberry trees.Due to its short reproductive cycle,large amount of reproduction,and control difficulties,it can burst into huge destruction in a short time,thus it can cause serious harm for the development of the sericultural industry.Because chemical control can easily lead to pest resistance and silkworm pesticide damage,it is urgent to develop new methods for controlling this pest.In recent years,based on the principle of insect chemical ecology,new pest control methods represented by attractant technology have been paid more attention and achieved good control effects.In this paper,on the basis of constructing the antennal transcriptome database of Glyphodes pyloalis,we identified the chemoreceptor genes and odour-degrading enzyme genes in the antennae of Glyphodes pyloalis,analysed the sequence characteristics and phylogenetic relationship of the genes,verified the tissue expression patterns of representative olfactory related genes,constructed the prokaryotic expression system,and clarified the catalytic activity of odour-degrading enzyme genes in vitro.1.A total of 51,776,602 clean reads were obtained,and 33,461 unigenes were spliced.The N50 value was 1968.By blastx analysis,the similarity between unigenes and umbilical Amyelois transitella was the highest.A total of 25 GpOBPs,4 GpPBPs,4 GpGOBPs,20 GpCSPs,53 GpORs,19 GpIRs,and 2 GpSNMPs were identified from the antennal transcriptome of Glyphodes pyloalis.2.Real time quantitative PCR results showed that 20 GpOBPs,including GpPBPs and GpGOBPs,were highly expressed in the antennae,suggesting that these genes may play an important role in the chemical signal sensing process of Glyphodes pyloalis.Eleven GpOBPs were highly expressed in the abdomen of adult Glyphodes pyloalis,and two GpOBPs highly expressed in the thorax of adult Glyphodes pyloalis might be related to some developmental functions of Glyphodes pyloalis.It was speculated that these genes might be highly expressed in the abdomen of adult Glyphodes pyloalis.GpOBP1,GpOBP10,GpOBP13,GpOBP16 and GpOBP21 were simultaneously expressed in the antennae and abdomen of adult Glyphodes pyloalis,and the expression showed gender difference,which may play a role in the mutual recognition between the same sex and the opposite sex.According to the high expression levels of GpOBP12and GpPBP1 in the antennae of the male Glyphodes pyloalis,these two genes were further expressed and purified in vitro.These two genes were constructed into pEASY and pET32a expression vectors respectively,and then expressed in E.coli through IPTG induction.SDS-PAGE analysis showed that GpPBP1/pET32a and GpOBP12/pEASY had an obvious bold band at 33kDa and 18kDa,respectively.The recombinant protein was purified by HisTrapTMM FF crude affinity chromatography.The recombinant GpPBP1 protein has the ability to bind to?-ionone(IC50=28.7?M;Ki=2.0317?M).3.Eighteen odour-degrading enzyme genes,including 15 GpGSTs genes and 3GpAOXs genes,were identified from the antennal transcriptome of Glyphodes pyloalis.Among them,GSTs can be divided into six categories:Delta,Epsilon,Omega,Sigma,Theta and Zeta.Conservative structural domain analysis showed that 8 GpGSTs had GSH binding domain and 13 GpGSTs had substrate binding domain.Fluorescence quantitative PCR results showed that GpGST-D1 and 3 GpAOXs were highly specific expressions in the antennae of male and female adults,suggesting that they may be involved in the degradation process of odor and pheromone in the antennae of Glyphodes pyloalis.The expression levels of GpGST-O1 and GpGST-O2 in the antennae of female adults were higher than those of other tissues,and the expression levels of GpGST-D3 in the abdomen of female adults were the highest,but there was no significant difference with that in the antennae of male adults.In addition to GpGST-D3,the expression levels of 10 GpGSTs?GpGST-D2,GpGSTD4,GpGST-E1,GpGST-E2,GpGST-E3,GpGST-T1,GpGST-Z1,GpGST-S1,GpGST-S2,GpGST-S3?were also higher in the abdomen of adult Glyphodes pyloalis than other tissues,but there was gender difference.GpGST-D1 was further expressed and purified in vitro.The gene was constructed into pEASY expression vector and then expressed in E.coli through IPTG induction.SDS-PAGE analysis showed that GpGST-D1 had a thick band at 25kDa.The recombinant protein was purified by HisTrapTM FF crude affinity chromatography.For GpGST-D1 enzymology properties of recombinant protein was studied,the results show that,under the condition of in vitro when temperature is 50?,reached the highest enzyme activity was 44.95±0.67?mol/min/mg;when at pH 7.0,reached the highest enzyme activity was 36.68±0.38?mol/min/mg.The research on olfactory mechanism in Glyphodes pyloalis will not only help to reveal the functions and functions of different olfactory related genes in the olfactory system of Glyphodes pyloalis,but also provide reference for the research on chemical ecology of Glyphodes pyloalis,thus providing support for the research and development of new control methods of mulberry borer based on olfactory targets.
Keywords/Search Tags:Glyphodes pyloalis, Chemosensory genes, Odor degrading enzymes, Antennal transcriptome, Protein purification, Enzymatic properties
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