Identification Of The COP9 Complex Subunit Gene And Function Analysis Of Them During Reproductive Development In Female Bactrocera Dorsalis

Bactrocera dorsalis(Hendel)is a kind of devastating pest of fruit and vegetable in China which feeds on a variety of host plant,and it is strong in fertility and difficult to control.B.dorsalis adults reach reproductive maturity after emergence by feeding on host plants;then mating and spawning are performed.Therefore,studying the molecular mechanism of the reproductive development of female B.dorsalis can not only provide new clues for enriching and perfecting insect reproductive regulatory mechanism,but also offer theoretical basis for efficient,simple and sustainable green prevention and control of B.dorsalis.In this study,we identified six COP9 signal complex subunit genes csn2,csn3,csn4,csn5,csn6,csn7,which were differentially expressed during the stages of new emergence and sexual maturation from the transcriptome library of B.dorsalis that was previously established in the laboratory.Real-time fluorescence PCR was used to investigate genes temporal and spatial expression patterns.Genes csn3 and csn5 which were highly expressed in sexual maturation stage and ovaries were chosen to study their function and molecular regulation mechanisms in ovary development,reproduction and other reproductive development process in female B.dorsalis using RNA interference,Hiseq sequencing and other molecular biological techniques.1 Identification and spatial and temporal expression pattern of the COP9 complex subunits genes in B.dorsalisGenes csn2,csn3,csn4,csn5,csn6,csn7 which were differentially expressed during emergence and sexual maturation were chosen from the transcriptome library of B.dorsalis.Then they were compared by NCBI and DNAman multisequence alignment.We found that six genes maintained more than 84 % homologous similarity with Tephritidae,such as Bactrocera latifrons,Bactrocera oleae,Bactrocera cucurbitae and Ceratitis capitata,and shared more than 67 % homology with Drosophila melanogaster.Then the amino acid sequences of six COP9 complex subunits genes were arranged for phylogenetic analysis along with homologous sequence from other insect species(D.melanogaster,C.capitate,A.aegypti,A.mellifera,P.xylostella,S.litura,T.castaneum,A.pisum,N.lugens and H.armigera).The csn2-csn7 subunit genes of different species clustered respectively on a large branch,indicating that the genetic distance of COP9 subunit genes is relatively close between different species.We detected the spatio-temporal expression patterns of six subunit genes in B.dorsalis through real-time fluorescence quantitative PCR.Expression patterns of different developmental stages results showed that all of the six subunit genes were highly expressed in egg stage,csn3 and csn5 were also highly expressed during sexual maturity;csn2,csn6,csn7 were also respectively highly expressed in prepupae,new emergence and sexual maturity stage.The results of the expression patterns of females and males in different periods showed that csn3,csn4 and csn5 were highly expressed in sexually mature and mated females,and there was no significant difference between male stages;the expression of csn2,csn6 and csn7 were not different in new emergence,sexual maturation and mating,and there was no significant difference between male and female.Different tissues expression patterns showed that each subunit gene was expressed in different tissues.Among them,csn3 and csn5 have significantly higher expression in the ovary than in other tissues,and csn7 has significantly higher expression in the testis.Adult ovary and fat body expression patterns of different reproductive stages showed that the expression of six COP9 subunit genes showed an increasing trend with the development and maturation of B.dorsalis,and all of them were highly expressed in the ovary 11-15 days after emergence.And six COP9 subunit genes were highly expressed in fat body of B.dorsalis adults of different days.csn2,csn3 and csn5 have higher expression at 11days;csn2,csn4 and csn6 have higher expression at 1 d after emergence.Based on the results of the above expression models,we selected two genes,csn3 and csn5,that were highly expressed in the ovaries of sexually mature females of B.dorsalis to study function.2 Functional analysis of csn3 geneTo further study the role of csn3 in the reproductive development of female B.dorsalis,we synthesized csn3 ds RNA in vitro using microinjection technique for RNA interference.Results showed that silencing csn3 gene significantly inhibited the amount of eggs laid compared to control group,but had no effect on ovary morphological development,mating rate and hatching ability.We further detected the expression of vitellogenin gene(Vg)vg1,vg2 and vitellogenin receptor gene yolkless by q RT-PCR and found that vg1,vg2 and yolkless were significantly down-regulated after silencing csn3.Above results indicate that by regulating the yolk deposition and oviposition,csn3 can in turn affect reproduction of female B.dorsalis.3 Functional analysis of csn5 geneTo further clarify the function of csn5 in B.dorsalis female reproductive development,we used RNAi technology to silence csn5 and the results showed that silencing the gene inhibited the ovarian morphology and the amount of eggs laid,but had no effect on the mating rate and hatching ability.To clarify the molecular mechanism of csn5 regulating the reproductive development of female B.dorsalis,we used RNAi technology to silence csn5 of female B.dorsalis and extracted total RNA from the ovary of the interference and control groups for high-throughput sequencing,to screen differential genes or differential signaling pathways.By analyzing the transcriptome data,a total of 398 differentially expressed genes which were involved in multiple signaling pathways and life activities were identified.According to GO,COG and other functional annotations,in these differentially expressed genes we found cytochrome P450 family genes,ecdysone kinase,vitellogenin genes and lipases that are involved in ecdysone synthesis.Among them,cytochrome P450 CYP301a1 and CYP313a4 were significantly down-regulated,and ecdysone kinase 1 Ec K1,ecdysone kinase 2 Ec K2 and ecdysone kinase 3 Ec K3 were significantly up-regulated in the interference group.We verified the expression of these genes in the interference and control groups by q RT-PCR,and the results were consistent with the transcriptome data.We further examined the expression of key upstream transcription factors Ec R,USP,E75,and vg1,vg2 in ecdysone signaling pathway.The results showed that the above genes were significantly down-regulated after silencing csn5.In summary,csn5 plays an important role in the reproductive process of female B.dorsalis.Silencing csn5 affected ecdysone synthesis and signal transduction,which in turn affected vitellogenin synthesis and yolk deposition,eventually leading to delayed ovarian development and decreased egg laying.