| As a member of the low-density lipoprotein receptor(LDLR)superfamily,vitellogenin receptor(VgR)is responsible for the uptake of vitellogenin(Vg)into developing oocytes to ensure the nutrient requirement for embryo development.Besides the function of regulating development and metamorphosis,juvenile hormone(JH)and its receptor(Methoprene-tolerant,Met)play important roles in insect reproduction,however,the research on the underlying mechanism is limited to model insects.In this paper,the full length cDNAs of CsVgR,CsMetl and CsMet2 have been cloned from Chilo suppressalis,and molecular structure characteristics,phylogenetic relationships and temporal-spatial expression patterns of these genes were analyzed.Furthermore,the role of CsVgR in the reproduction of C.suppressalis and the transcriptional regulation of CsVg and CsVgR by CsMets were studied by using RNA interference(RNAi).Meanwhile,sublethal effect of chlorantraniliprole on mRNA expression levels of CsVgR and CsMets was investigated.The research results not only provide insights into the molecular mechanism of JH signaling pathway in regulating insect reproduction,but also lay foundation for the development of RNAi-based control strategy for insect pestsThe full-length cDNA of CsVgR cloned by RT-PCR and RACE was 6,117 bp with a 5,484 bp open reading frame(ORF),encoding 1,827 amino acids(aa).The predicted molecular weight(Mw)and isoelectric point(pI)were 203.4 kDa and 5.02,respectively.Structural analysis revealed that CsVgR contained two ligand-binding domains(LBDs)with four Class A(LDLRa)repeats in the first LBD(LBDI)and seven LDLRA repeats in the second(LBDII),which was structurally different from most non-Lepidopteran insect VgRs with five and eight repeats in the LBDI and LBDⅡ,respectively.Multiple sequence alignment revealed that CsVgR shared 52.15%,27.13%,26.97%and 24.79%identities with SeVgR in Spodoptera exigua,AmVgR in Apis mellifera,Lm VgR in Leucophaea maderae and NlVgR in Nilaparvata lugens,respectively.Moreover,the developmental expression analysis showed that CsVgR expression levels was almost undetectable in the prepupal(PP)and 1-day-old pupae(1P),first detectable in 3-day-old pupae,sharply increased in newly emerged female adults,and reached a peak at 48 h after eclosion.Additionally,CsVgR was exclusively expressed in the ovary,which was consistent with most insect VgRs.These results indicated that CsVgR might be correlated with reproduction of C.suppressalis.To determine the role of CsVgR in the reproduction of C.suppressalis,dsCsVgR was injected into the 6-day-old female pupae.Compared with the control(dsEGFP-treated)group,the expression level of CsVgR was down-regulated by 45.73%(48 h)and 65.92%(72 h)in dsCsVgR-treated group.The ovaries dissected from 72 h-old female adults in control group were obviously mature,enriched with a large number of follicles,and the fecundity was 72.2±23.40 eggs/per female,while injection of dsCsVgR into late pupae resulted fewer follicles in the ovarioles and significantly reduced fecundity with 27.6± 12.28 eggs/per female,suggesting a critical role of VgR in the ovary development and reproduction of C.suppressalisThe full-length cDNA sequences of 2,673 bp CsMet1 and 2,824 CsMet2 bp were cloned from C.suppressalis by RT-PCR and RACE.The CsMet1 had a 1,587 bp ORF encoding a protein of 528 aa with a predicted Mw and p1 of 60.34 kDa and 8.35,respectively,while the CsMet2 had a ORF of 2,598 bp encoding a protein of 865 aa with a predicted Mw and pI of 97.37 kDa and 6.68,respectively.Structural analysis revealed that both CsMetl and CsMet2 exhibited typical bHLH(basic Helix-Loop-Helix),PAS(Per-Arnt-Sim,PAS-A,PAS-B)and PAC(PAS C terminal)domains,Alignment of amino acid sequences demonstrated that CsMet1 shared identity with other insect Met orthologues including Bombyx mori BmMet1(60.98%),BmMet2(21.88%),Drosophila melanogaster DmMet(27.49%)and Tribolium castaneum TcMet(28.30%).CsMet2 shared 20.55%,44.19%,17.19%and 22.90%identities with BmMet1,BmMet2,DmMet and TcMet,respectively.Furthermore,the amino acid identity between CsMetl and CsMet2 was 21.71%.The expression level of CsMet1 was relatively stable in the larval stage(3L-6L),sharply decreased in the prepupal stage(PP),maintained at a low level during the pupal stage(1P-5P),and reached a peak in 12 h-old female adults.The transcript level of CsMet2 was lower in the larval stage(4L-5L),and increased from 6th instar(6L)to 5-day-old pupae(5P),and reached a peak at 48 h after eclosion.Tissual expression pattern showed that CsMet1 was highly expressed in head,hemolymph and midgut of larva.The highest expression level of CsMet2 was observed in hemolymph,followed by head and malpighian tube.Further study showed that CsMet1 was significantly up-regulated at 1 h(2.74 times),6 h(2.41 times)and 12 h(1.63 times)after treatment with JH analogues methoprene Similarly,transcription level of CsMet2 was increased by 1.95,1.45 and 3.68 times at 1 h,6 h and 12 h post-treatment,respectively.These results indicated that CsMetl and CsMet2 were positively regulated by JH.To investigate the function of CsMetl and CsMet2 in the transcriptional regulation of CsVg and CsVgR,6-day-old female pupae and newly emerged female adults were selected for double-injection of dsCsMet1 or dsCsMet2.The results indicated that knockdown of CsMetl or CsMet2 suppressed the transcript levels of both CsVg and CsVgR,resulted in much less number of mature follicles.These findings preliminarily revealed the potential mechanism of JH signaling pathway in regulating reproduction of C.suppressalisThe diet incorporation method was used to determine the sublethal effect of chlorantraniliprole on transcript level of CsVgR,CsMetl and CsMet2 mRNA.The results showed that CsVgR in LC10 or LC30 chlorantraniliprole-treated group was down-regulated by 41.65%(LC10),35.82%(LC30)at 24 h after eclosion,respectively.The transcript level of CsMet1 in LC10 chlorantraniliprole-treated group was unexpectedly 1.52 times higher than control at 24 h after eclosion.CsMet2 was down-regulated by 54.62%at 24 h after eclosion in LC30 chlorantraniliprole-treated group.These results indicated that sublethal concentration of chlorantraniliprole might affect the development and reproduction of C suppressalis. |
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