Synthesis Of MiRNAs And Regulation Mechanism On Genes Involved In Chitin Metabolism In Locusta Migratoria

MicroRNAs(miRNAs)are small(approximately 22 nt at length),noncoding RNA molecules that play important roles in a wide range of biological process by post-transcriptional regulation.The migratory locust,Locusta migratoria,is a worldwide insect pest.The periodical synthesis and degradation of the cuticle is critical to maintaining normal molting.Chitin metabolism is a complex biological process,and chitin synthesis and degradation is precisely regulated by two crucial enzymes,chitin synthase(CHS)and chitinase(CHT).Our previous studies have confirmed that two chitin metabolic genes,namely chitin synthase1(CHS1)and chitinase10(CHT10),are responsible for chitin synthesis and degradation and play key roles in molting development in the migratory locust.Furthermore,we reported that deppression of LmDicer1,the coding enzyme catalyzes the final step of miRNA biosynthesis,inhibited the synthesis of mature mi RNAs and induced a molting defect.So we propose the hypothesis: miRNA could regulate the key genes involved in cuticle metabolism during molting process and affect normal molting in insects.In this thesis,the migratory locust was used as the research system to determine the influences of the key factors LmDicer1 and LmAGO1 on miRNA synthesis,to identify and update the locust miRNAs library and to reveal the molecular mechanism of miRNA regulation on key genes involved in cuticle metabolism.The main contents are as follows:1.Construction of small RNA library of locust and identification of miRNAsThrough high-throughput sequencing and experimental approaches,we performed the small RNA transcriptomes in developmental stages.We identified a total of 833 miRNAs and found a large portion of them are specific for locust,suggesting that the genome size expansion in locusts results in an increase of mi RNA number.Furthermore,we silenced the expression of Drosha,a key gene in miRNA synthesis pathway and performed miRNA expression analysis in locust.Compared with those in dsGFP injected controls,the significant depression of miRNA expression by Drosha RNAi further proved the authenticity of identified miRNAs in locust.These results serve as a theoretical and resource fundaments for the future studies on miRNA-mediated regulation of biological functions.2.The effects of LmDicer1 on miRNA synthesis and locust molting developmentIn order to elucidate the role of Dicer1,a key factor in miRNA biosynthesis pathway,in miRNA synthesis and its effect on the life activities of locust.RNA interference(RNAi)and real time quantitative PCR(RT-qPCR)were used to analyze the expression of LmDicer1 and its effect on miRNA synthesis in locust.We found that silencing of LmDicer1 results in reduced levels of mature miRNAs and severely molting defects in the migratory locust.3.The effects of Argonaute 1 on mi RNA synthesis and locust developmentIn order to elucidate the role of Argonaute 1(AGO1),a key factor in miRNA biosynthesis pathway,in mi RNA synthesis.RNA interference(RNAi)and real time quantitative PCR(RT-qPCR)were applied to analyze the expression of LmAGO1 and its effect on miRNA synthesis in locust.we found that besides the role in RSIC formation,LmAGO1 can enhance the processing of miRNA maturation and regulate the normal development in the migratory locust.4.Regulation mechanism of miRNAs on chitin synthase(CHS)and chitinase(CHT)genes in locustFurther,we studied the function of mi RNAs during the molting development of locust.The bioinformatic prediction showed miR-71 and miR-263 have binding sites in CHS1 and CHT10 in locusts.The RT-qPCR analyses indicated that the expressions of miR-71 and miR-263 were negatively correlated with the expression of CHS1 and CHT10.The luciferase assay and immunoprecipitation(RIP)analysis showed that miR-71 and miR-263 are directly interacted with LmCHS1 and LmCHT10,respectively.The locusts injected with agomir-71 or agomir-263 displayed a distinct molting defect phenotype.These results suggested that miR-71 and miR-263 control chitin synthesis and degradation by targeting LmCHS1 and LmCHT10,resulting in a deformed molting and final death in locust.5.Regulation mechanism of miRNAs on genes responsible for cuticle metabolismTo investigate whether miRNAs are also responsible for the expression regulation of key genes involved in cuticle metabolism.We selected four key genes including fatty acid synthase(FAS),UDP-N-acetylglucosamine pyrophorylase(UAP),asparagine-linked glycosylation protein 5(ALG5)and Sinuous.Potential miRNAs that target key genes involved in cuticle metabolism were identified using bioinformatic approaches.The RT-qPCR,the RIP experiment and the luciferase reporter experiment were performed to validate the potential binding relationship betwen miRNAs and their target genes in vivo or in vitro.The results showed that the four cuticle metabolism related genes,namely,UAP,ALG5,FAS and Sinuous,contained potential binding sites for miRNA-2796,miRNA-275,miRNA-276 b and mi RNA-184,respectively.These miRNAs interact with the four cuticle metabolic genes,which may regulate the expression of these target genes.The results systematically illustrated the synthesis of locust mi RNAs and revealed the molecular regulatory mechanisms of miRNAs in molting development.These innovative discoveries will provide important scientific values,and be used to screen for potential small molecule targets in locust control and to design environmentally friendly insecticides in future.