Cloning,Expression And Functional Analysis Of VgR Gene In Mythimna Separata (Walker)

Mythimna separata(Walker),an important worldwide agricultural pest,belongs to Noctuide of Lepidoptera.The frequent outbreak of M.separata in different regions is inextricably linked with strong fertility and special reproductive adaptability.Revolving around the question that reproductive potential and adaptability is determined by reproductive regulation,it is necessary to carry out research on reproductive-related genes.The vitellogenin receptor(VgR),an important role in the vitellogenesis and the development and maturation of the oocyte,is a key receptor to mediate the vitellogenin(Vg)transport between the fat body and the ovary.Therefore,there are important theoretical significance and practical application value to study VgR gene.The full-length cDNA of M.separata VgR gene is cloned,the expression pattern and function identificationis are established in this paper,which aims to provide the theoretical basis for insect reproductive mechanism of M.separata.In this paper,the full-length cDNA sequence of the M.separata VgR gene was cloned through everse transcription-PCR(RT-PCR)and rapid amplification of cDNA ends(RACE)technology,which is analyzed by bioinformatics method.Then using?-Tubulin as an internal reference gene,expression pattern of VgR gene in different developmental stages and different tissues of M.separata was established through Real-time fluorescent quantitative PCR technique(RT-qPCR).The VgR gene was silenced by RNA interference(RNAi)technology to further explore the fuction.1.The full-length cDNA of M.separata VgR gene,5380 bp,which contains an open reading frame of 5328 bp encodeing 1775 amino acids.The start codon ATG is at positions 16-18 and the stop codon is at positions 5341-5343.The molecular weight is 198.995 kDa and the isoelectric point is 5.11.154 phosphorylation sites and 16 glycosylation sites are contained in M.separata VgR gene.The analysis of conserved domains shows that M.separata VgR protein is a low-density lipoprotein receptor and lacks an O-glycosylation domain.M.separata VgR protein contains two ligand binding domains and two EGF-precursor homology domains(EGFP).The two LBD include 4 and 7 type-A repeat region respectively.One EGFD includs 4 type-B motif repeats and seven YXTD motif clusters,another one is consisted of three type-B repeat motifs and two YXTD motifs.Existing a transmembrane region(TMD),the protein is a transmembrane protein.The signal peptide of the VgR protein is located at the N-terminal and consists of 20 amino acids.The amino acid sequence is MKYECLILVVLVTWCAEFA.In the phylogenetic tree,the M.separata VgR is clustered in Lepidoptera,which has closely-related envolution reationship with Helicoverpa armigera and Spodoptera litura.2.In this experiment,the expression patterns of VgR in different tissues of M.separata larvae and female adults are established respectively.The Study show that VgR was expressed in all tissues of female adults,and the expression level of the ovary was significantly higher than that of other tissues,and the expression level was the lowest in the epidermis.In the M.separata larvae,the VgR gene of fat body is significantly expressed comparing to the head,epidermis,and intestine,but the expression level is low.3.The expression pattern of VgR in different development stages of female M.separata is established in this experiment.The result shows that the M.separata VgR gene mainly expresse in the adult stage,and the expression level increases first and then decreases as time goes on.The expression increases on the second day of emergence and decreases afther the peak of the fifth day,then declines on the sixth day.4.In this experiment,M.separata VgR gene is silenced by RNAi technology.The result shows that the expression level of VgR gene in treatment group(VgR-dsRNA)was significantly lower than that in negative control group(GFP-dsRNA)after the 48-60 h of injection,indicating that the interference iseffectively suppressed the expression of VgR gene.At the same time,the females in the two groups cultured until the third day of emergence is dissected to gain ovaries.Comparing with negative control group,the deposition of yolk protein in the eggs of the treatment group is significantly inhibited,which is consistent with the detection result of VgR gene expression.