Functional Study Of Vitellogenin And Its Receptor In The Reproductive Process Of Spodoptera Litura

Spodoptera litura Fabricius(Lepidoptera: Noctuidae),also known as Prodenia litura,Mythimna seperata etc.,is an important agricultural pest in the world,mainly endangering vegetables,cotton,corn,rice,tobacco and other crops.Reproduction is the way of pest population continuation.It has positive scientific significance and broad practical value to explore the reproduction law of pests,explore the gene regulation mechanism of pest reproduction,and realize the control of pest population based on reproduction gene regulation.Vitellogenin(Vg)and vitellogen receptor(VgR)play an important role in the process of insect ovary maturation,directly affect the reproductive capacity of insects,whether they are related to reproductive behavior,which is very important for further understanding of its mechanism of action on insect reproduction.In this study,Spodoptera litura was used as the research material.Through the cloning of Vg and VgR mRNA sequence,the temporal and spatial expression of Vg and VgR in Spodoptera litura,and the comparative analysis of the expression after RNAi interference,the functional analysis of Vg and VgR on Spodoptera litura reproduction was further discussed.The main results are as follows.(1)The protein encoded by Vg(SlVg)mRNA of Spodoptera litura consists of 1748 amino acids.The N-terminal contains Vitellogenin-N,DUF1934 and VWD(von Willebrand factor type D domain).Sl Vg isoelectric point is 8.75,including 100 phosphorylation sites,including 58 serine(Ser),25 threonine(Thr),17 tyrosine(Tyr).Sl Vg has high homology with other Lepidoptera VG protein sequences.The most similar insects are Helicoverpa armigera(67.78%),Antheraea pernyi(52.74%),Samia ricini(52.69%),Bombyx mori(49.46%),Bombyx mandarina(49.14%)and Conopomorpha sinensis(41.25%).Phylogenetic tree analysis showed that Sl Vg and Vg of cotton bollworm were on the same branch,and the self guidance value reached 100%.(2)The protein encoded by VgR(SlVgR)mRNA of Spodoptera litura consists of 1815 amino acids.The conserved domain of the protein was analyzed by online software SMART(http://smart.embl-heidelberg.de/),which showed that the protein belongs to the low density lipoprotein receptor(LDLR)gene family,including four classical domains: ligand binding domain(LBD),epidermal growth factor precursor homology domain(EGFD),transmembrane domain(TMDY)and cytoplasmic tail domain(CPD).Sl VgR contains two LBDs,29-217 aa and 942-1284 aa,respectively.The first LBD contains four A-type repeats and the second LBD contains seven A-type repeats.There is an EGFD homologous domain at the back end of two LBD regions,located at 263-938 aa and 1285-1663 aa.The former includes 5 YWTD motif clusters,and the latter includes 3 YWTD motif clusters.Sl VgR isoelectric point is 5.05,including 93 phosphorylation sites,including 40 serine(Ser),27 threonine(Thr),26 tyrosine(Tyr).According to the amino acid sequence alignment,the homology of Sl VgR with other insects is not high.The highest similarity is B.mori(48.78%),Colaphellus bowringi(25.30%),Blattella germanica(23.76%),Drosophila melanogaster(23.66%),Nilaparvata lugens(22.49%),Solenopsis invicta(22.31%).Phylogenetic tree analysis showed that the VgR gene of Spodoptera litura and the VgR gene of Bombyx mori were in the same branch,and the self guidance value reached 100%.(3)The real-time fluorescence quantitative analysis showed that the specific expression of SlVg mRNA in the fat body of female Spodoptera litura began from the pupa stage,and the expression level increased with the development stage of the body,reached the highest level in one-day-old adults,and then decreased gradually.SlVgR mRNA was specifically expressed in the ovaries of females,which was similar to SlVg mRNA expression pattern.It first appeared in pupa and reached the highest level in one day old adults.(4)The RNAi system of SlVg and SlVgR was constructed.Through the comparison of injection,feeding,soaking,transgene and other methods,the feeding methods with small damage,high efficiency and low cost were selected.The experimental group was fed with bacteria expressing SlVgdsRNA and SlVg R-dsRNA,and the control group was fed with DEPC water and EGFP-dsRNA.Fluorescence quantitative detection shows that the interference efficiency is high(50%-80%).(5)Under the interference of SlVg and SlVgR genes,the survival rate,pupation rate and emergence rate of Spodoptera litura larvae in the experimental group were detected.It was found that the egg laying amount of the female in the experimental group was only 15%-20% of that in the control group.The hatching rate was about 80% of the control group,but there was no significant effect on the survival rate,pupation rate and emergence rate of larvae.The results showed that SlVg and SlVgR had significant effects on egg formation and female fecundity.In many insects,including Lepidoptera,whether females accept male courtship is closely related to the maturation of their eggs.Therefore,Vg and VgR may indirectly affect the mating activity of females in the process of regulating egg maturation.Through behavioral analysis,it was found that RNAi of SlVg and SlVgR significantly inhibited courtship and mating behavior of females,and indirectly affected courtship and mating behavior of males.Vg and VgR families have sequence diversity and functional diversity.In this study,RNAi was used to investigate the role of these two genes in egg formation and fecundity,and it was found that these two genes may indirectly regulate female reproductive behavior,which is a new exploration for different functions of known genes.At the same time,this study uses bacterial mediated RNA interference technology,which has the advantages of low cost,high efficiency,and can be used in continuous culture after the successful construction of vector.It has application value in gene function research,and has broad prospects in pest control and promotion.