Cloning,Expression And Function Analysis Of Bursicon Gene In Myzus Persicae(Sulzer)

Bursicon is a functional protein that regulates epidermal sclerosis and spreading wings of insects,it plays a key role in epidermis hardening of insects after molting.In this study,we obtained the full-length of Bursicon genes?burs-?and burs-??,predicting their structures and functions and analyzing their evolutionary status.The study in the expression of Myzus persicae?Sulzer?in different developmental stages by real-time quantitative PCR technology.Finally,using RNAi technology to study the physiological function of bursicon gene in M.persicae?Sulzer?.The results are as belows:1.Transcriptome analysis of different winged of Myzus persicaeAim to understand the transcriptome features of M.persicae of different wing types?winged and apterous?,to enrich its transcriptome data,and to reveal the molecular mechanisms of wing dimorphism of this insect.The differences in transcriptome expression between nymphs,apterous adults,and winged adults of M.persicae were detected by high-throughput sequencing technology Illumina HiSeq^TM2000.The expression patterns of wing type related genes were analyzed by real-time quantitative PCR.Results:The percentage of nucleotides with the quality score over 20 was no less than 94%?GenBank accession numbers:SRR6112438,SRR6112436 and SRR6112437?.All reads were assembled into 128 065 unigenes with an average length of 607.08 bp.The resulting sequences were annotated to databases NR,NT,KOG,GO,KEGG,etc.,and a total of 128 065 unigenes were annotated.Gene expression analysis showed that the genes of flight proteins,peptide hormones,receptor proteins,etc.,qPCR analysis showed that there were significant differences in the expression levels of wing type differentiation related genes including Bursicon?bur-?and bur-??,Flightin?Fln?,Wntless?Wnt?,Distal-less?Dll?,Decapentaplegic?Dpp?,juvenile hormone epoxide hydrolase gene?JHEH?and ecdysone receptor gene?EcR?between winged adults and apterous adults.2.Cloning the full-length cDNA sequences of Mpburs-?and Mpburs-?We obtained the full-length sequence of cDNA burs-?and burs-?by reversing transcription PCR?RT-PCR?and rapid-amplification of cDNA ends?RACE?cloning technology;We named the obtained full-length sequence of 848bp Mpburs-??GenBank accession number:MF083566?and 841bp Mpburs-??GenBank accession number:MF083567?Mpburs-?and Mpburs-?respectively.Bioinformatics analysis shows that Mpburs-?contains a 483bp open reading frame?ORF?and encodes 160amino acid residues,which includes a short 5'-UTR of 63 bp,a much longer 3'-UTR of 302 bp with a canonical polyadenylation signal sequence AATAAA,a poly?A?tail,while Mpburs-?contains a 417bp open reading frame?ORF?and encodes 138 amino acids residues,which includes a short 5'-UTR of 124 bp,a much longer 3'-UTR of300 bp with a canonical polyadenylation signal sequence AATAAA,a poly?A?tail.3.Bioinformatics analysis of Bursicon geneThe bioinformatics analysis of the physicochemical properties and the higher structure of amino acids and proteins encoded by Mpburs-?and Mpburs-?genes was carried out.The results are as follows:The relative molecular weights of Mpburs-?and Mpburs-?are about 17.59 kDa and 15.45 kDa,respectively,Consists of 2424 and2148 atoms,respectively.The estimated theoretical isoelectric point PI is 7.96 and4.84,respectively.The chemical formula is C₇₅₉H₁₂₀₄N₂₁₀O₂₃₂S₁₉9 and C₆₇₂H₁₀₇₂N₁₈₀O₂₁₂S₁₂.The amino acid sequences of Mpburs-?and Mpburs-?are most consistent with the amino acid sequences of tanning hormone of pea aphids and wheat aphids.There is no transmembrane region in Mpburs-?and Mpburs-?genes,but both have signal peptides and phosphorylation sites.Phylogenetic tree analysis showed that the aphids were closely related to A.Pisum and Diuraphis noxia.4.Analysis of Bursicon gene expression at different agesThe expression patterns of Mpburs-?and Mpburs-?genes of Myzus persicae at different time points?nymphs from first instar to newly emerged adults?were analyzed by fluorescence quantitative PCR.The results show,Mpburs-?and Mpburs-?genes were expressed in different developmental stages of peach aphid,but the expression levels were different.The highest expression was found in the 1st instar and the lowest in the 4th instar.Compared with wingless adult aphids,we can see that the expression of Mpburs-?and Mpburs-?genes in wingless adult aphids is significantly higher than that in wingless adult aphids.5.Functional analysis of Bursicon geneThe second instar nymphs were fed with dsburs-?dsburs-?and PBs.the phenotypic changes and mortality rate were observed and recorded 72 hours later,the changes of target gene expression in some insect bodies were collected.The results showed that the expression of burs-?gene and burs-?gene decreased by 67.54%and50.46%,respectively,compared with the control fed with Pbs,indicating that burs-?gene and The expression of burs-?gene was significantly inhibited.The mortality statistics and phenotypic observation showed that when the final concentration of dsMpburs was 15 ng/?L,the mortality of the experimental group was not significantly different from that of the control group,and the color of the epidermis is abnormal,from reddish brown to pale green or transparent,and the degree of epidermis blackening is obviously reduced.It indicated that the epidermis tanning and blackening of Myzus persicae after dsMpburs gene were inhibited.In this paper,the whole long sequence of Burs-?and Burs-?genes of Myzus persicae was successfully obtained by sequencing of M.persicae transcriptional group,specific primers,RT-PCR and RACE techniques.In order to further study the function of Bursicon gene of M.persicae,it will provide a reference for the study of the new control technology of tannic hormone as the target.We study of their expression in the different developmental stages of M.persicae and the growth of M.persicae were studied by using fluorescence quantitative PCR and RNAi technology.