MiRNA Analysis Of Transitions Between Different Developmental Stages And The Molecular Mechanism Of MiR-309a Regulating The Ovarian Development In Bactrocera Dorsalis

microRNA（miRNA）is a kind of endogenous small noncoding RNA,which widely exists in all kinds of organisms,with a length of about 22 nucleotides,and it regulates gene expression at the post transcription level.In recent years,miRNA has gradually become a research hotspot in the field of insect developmental biology because of its role in regulating many physiological processes,such as insect growth,reproduction,immunity and resistance.The oriental fruit fly,Bactrocera dorsalis（Hendel）,is a destructive agricultural pest and causes severe economic loss to many kinds of fruits and vegetables,and it has been listed as a quarantine pest in many countries and regions.At present,to explore a new strategy for prevention and control is particularly urgent,because this pest has become a great threat to the healthy development of fruit and vegetable industry in China.Therefore,based on the high-throughput sequencing of transitions between different developmental stages in B.dorsalis,miRNAs were identified and then the differentially expressed miRNAs were analyzed;the expression profile of miRNAs related to the egg and pupae development were analyzed by qRT-PCR,and miRNA analogues/inhibitors were used to explore the function of miRNAs in regulating pupation or eclosion;finally,targeting miR-309a,a series of techniques and methods were utilized,including qRT-PCR,target gene prediction,dual luciferase reporter system,RNA binding protein immunoprecipitation and exogenous hormone treatment,to reveal the molecular mechanism of miR-309a involved in the regulation of ovary development of B.dorsalis.The main results in this study are listed as follows:1 miRNAs identification of transitions between different developmental stages in B.dorsalisUsing high-throughput sequencing technology,the samples between hatching,molting,pupation and eclosion of B.dorsalis were analyzed,including late egg（EL）,early 1^st instar larvae（L1E）,late 1^st instar larvae（L1L）,early 2^nd instar larvae（L2E）,late 2^nd instar larvae（L2L）,early 3^rdd instar larvae（L3E）,late 3^rd instar larvae（wandering stage,WS）,early pupa（PE,prepupa）,late pupa（PL）and early adult（AE）,to conduct the miRNAs identification,and 250 miRNAs,including 167 novel miRNAs,and 47isomiRs were found.The obtained miRNAs were systematically verified and analyzed.It enriches the information of miRNAs in B.dorsalis,and it is the first time to identify the isomiR in this species,which provides a useful resource for the future research.2 Differentially expressed miRNAs analysis of transitions between different developmental stages in B.dorsalisBased on transcriptome sequencing results,differentially expressed miRNAs were analyzed,and 29 differentially expressed miRNAs（DEMs）were identified in hatching,44 DEMs in molting,6 DEMs in pupation and 3 DEMs in eclosion.The target genes of miRNAs were predicted and annotated by software and are mainly enriched in energy metabolism and signal transduction pathways,in corresponding to the characteristics of development stages transition.The expression patterns of 12 randomly selected miRNAs,7 DEMs with miRNA-mRNA negative regulation patterns and their predicted target genes were analyzed by qRT-PCR,and the results were consistent with the sequencing expression profile.In addition,in the resequencing analysis of the samples at three time points during pupation,miR-981,bdo-novel-mir-55,let-7a-3p,bdo-novel-mir-24 participated in the regulation of B.dorsalis pupation.3 Investigation on the specifically expressed miRNA function in different developmental stages of B.dorsalisThe expression profiles of miRNAs with high specific expression in EL（10）and PE/PL（14）were analyzed in egg and pupa stages by qRT-PCR,respectively,and the miRNAs with potential regulatory functions were selected.The pupation and eclosion rates were significantly reduced by injecting mimic-100 into last instar larvae,and the phenotypes of the larvae died with failing to jump out and unable to pupate after jumping out were observed;after injecting antago-317,the pupation rate was significantly reduced and the pupation time was shortened,and the phenotypes of the larvae unable to pupate after jumping out and the deformed pupa were observed.These results showed that miR-100 and miR-317 are involved in the regulation of B.dorsalis pupation.In addition,by injecting miR-100 and miR-285 mimic/antagomir into pupae,respectively,the eclosion rate significantly reduced as well.It suggests that miR-100and miR-285 are involved in the regulation of B.dorsalis eclosion.4 miR-309a in regulation of ovary developmentBased on the expression profile of miR-309a in egg stage,the injection of mimic-309a into female adults resulted in the obstruction of ovary development and the reduction of egg production,which indicated that miR-309a was involved in the regulation of ovary development of B.dorsalis.The target genes of miR-309a were predicted by four miRNA-targets prediction softwares,and 11 target genes predicted by all softwares were obtained.After mimic-309a injection,the expression of pnr and tret were significantly downregulated.Meanwhile,the expression profiles of the two predicted target genes were negatively correlated with that of miR-309a,which was in line with the expected miRNA-mRNA negative regulation pattern;through the dual luciferase reporter system in vitro,miR-309a combines with pnr but not with tret;finally,the combination of miR-309a and pnr was verified in vivo by RIP assay.It was found that pnr was significantly enriched after mimic-309a treatment,which proved that miR-309a and pnr interacted in vivo.These results showed that miR-309a regulates ovary development through the target gene pnr.By using RT-PCR,the expression levels of Vg1,Vg2,Vg3 and VgR decreased significantly after the mimic-309a injection,suggesting that Vg related genes are involved in the regulation of miR-309a on the ovary development.In addition,methoprene treatment inhibited the expression of miR-309a,upregulated the expression of pnr and Vg related genes and rescued the phenotype of ovary development obstruction and oviposition amount decline after miR-309a mimic injection,which revealed that exogenous JH was involved in the regulatory pathway of miR-309a and affected the downstream genes expression to control the ovary development of B.dorsalis.In conclusion,in this dissertation,based on the high-throughput sequencing of transitions between different developmental stages in B.dorsalis,miRNAs were identified and then the differentially expressed miRNAs were analyzed;four miRNAs were chosen to explore their function in pupation and eclosion,and B.dorsalis isomiR were identified for the first time;the study clarified the molecular mechanism of miR-309a targeting pnr to regulate ovary development in B.dorsalis.The results not only enrich the miRNA research system in B.dorsalis,promote the understanding of miRNA and pnr transcription factors in regulating the ovarian development of insects,but also lay a foundation for developing the new insecticides with miRNA as the target.