The Mechanism Of MiR-927-5p Targeting Stalky To Regulate Spermatogenesis In Zeugodacus Cucurbitae

The melon fly,Zeugodacus cucurbitae(Coquillett),a worldwide agricultural pest,is affiliated to the Diptera Tephritidae and can harm diverse fruits and vegetables,such as bitter melon,pumpkin and towel gourd,represented by Cucurbitaceae.As the main site of spermatogenesis,testis is very important to growth,development and reproduction.Identifying the critical genes or proteins regulating reproduction in testis is one of the current research hotspots in insect physiology.Micro RNA(miRNA),a short RNA in organisms that does not directly encode proteins,can regulate gene expression at the posttranscriptional level.In this thesis,a highly expressed miRNA,miR-927-5p in testis was identified by analyzing miRNA libraries of different tissues from melon fly;using RTq PCR,overexpression and inhibition,the sperm number was decreased by 53.10% after overexpression of miR-927-5p.Stalky was systematically validated as a target gene of miR-927-5p using a combination of bioinformatics prediction,transcriptome sequencing,RT-q PCR,RNA-pull down,and dual luciferase reporter assays.The effect of Stalky on spermatogenesis in melon fly was explored using RNAi.Ultimately,we confirmed that miR-927-5p regulates spermatogenesis in melon fly by targeting Stalky.The main findings of this thesis are as follows:1 Analysis of miRNA expression of Z.cucurbitae testisBased on the established miRNA libraries of different tissues of melon fly,highly expressed miRNAs in testis were screened out by clustering analysis,and 41 miRNAs were identified to be highly expressed in testis.Based on the expression among tissues,between females and males(testis/ovary),a candidate miRNA miR-927-5p was selected as the potential miRNA to regulate male fertility in melon fly.RT-q PCR was used to validate the high expression in testis.Homology analysis of miR-927(-5p)sequences in several insect species using sequence alignment revealed that the mature sequence of miR-927-5p is highly conserved within species such as Drosophila melanogaster and Bombyx mori,suggesting a similarity in their biological functions.2 Effect of miR-927-5p on spermatogenesis in Z.cucurbitaeThe expression profile of miR-927-5p at different day-olds in male adults of melon fly was dissected using RT-q PCR.It was found that miR-927-5p was expressed in male adults during the sexual maturation period.The localization of miR-927-5p in testis was analyzed using fluorescence in situ hybridization(FISH).A positive signal was found in the transformation zone,meaning it might be involved in sperm maturation and transformation.After inhibition of miR-927-5p,there was no significant change in sperm number of melon fly,but after overexpression,sperm number decreased by 53.10%.3 Analysis of the regulation of miR-927-5p targeting Stalky in Z.cucurbitaeThe target genes of miR-927-5p were predicted using three bioinformatics prediction softwares(miRanda,PITA and RNAhybrid);12 candidate genes were predicted when combined with the transcriptomic sequencing after miR-927-5p overexpression.Comprehensively using RT-q PCR,RNA-pull down,and dual luciferase reporter system to validate candidate target genes,one target gene Stalky was finally confirmed to be a target gene for the following study.Dual luciferase reporter system results showed that the binding site between Stalky and miR-927-5p was located in the variable splicing region(Stalky＿1 and Stalky＿2).After that,the RNA-pull down revealed that miR-927-5p could only directly target Stalky＿1.According to the overexpression and inhibition results,there was a negative regulatory relationship between miR-927-5p and Stalky＿2,implying that miR-927-5p could indirectly regulate Stalky＿2.4 Functional analysis of Stalky in spermatogenesis in Z.cucurbitaeThe characteristic of Stalky at the bioinformatic level revealed the presence of one transmembrane domain and one conserved domain with an unknown function in the amino acid sequence.The temporal expression profile of Stalky＿1 by RT-q PCR revealed a stable expression during sexual maturation in male adults of melon fly,which was similar to the expression of miR-927-5p.The relative expression of Stalky is low in testis,but higher in fat body and ovary.A positive signal of Stalky＿1was found in the transformation zone,as same as miR-927-5p,meaning it might be involved in sperm maturation and transformation using FISH.Using siRNA to interfere with the expression of Stalky,the results showed that sperm number was significantly reduced by 70.64%.In summary,miR-927-5p of melon fly affected male fertility during spermatogenesis by targeting Stalky.The results of this thesis enrich the research on the reproductive physiology of melon fly.They provide a new theoretical basis for integrated pest control and sterile insect technique and a new insight into the environmental control of melon fly.