MicroRNAs在橘小实蝇精子发育中的作用研究

发布时间：2018-03-10 08:48

本文选题：MicroRNAs　切入点：橘小　出处：《华中农业大学》2015年博士论文　论文类型：学位论文

【摘要】：橘小实蝇是广泛分布于东亚和太平洋地区的最重要的园艺害虫之一。在其防治策略中,雄性不育技术(SIT)具有高特异、环境友好的巨大优势,然而传统的SIT依赖于辐照和化学不育剂,容易损伤不育雄虫的交配竞争力,从而限制了其防治效果。近年来,基于遗传改造的SIT在橄榄实蝇的防治中表现优异,显示了在实蝇防治应用中的巨大潜力。另一方面,microRNAs(miRNAs)作为一种负向调控目标基因表达的关键性的转录后调控因子,参与调控了各种生命过程,其中就包括对精子发生的调控。因此,基于精子发生关键mi RNA的遗传改造技术对于SIT具有重要的开发价值。然而到目前为止,橘小实蝇中精子发生调控相关miRNA的研究还很缺乏。本研究首先对性成熟期(FM)、初羽化(IM)、生殖前期(MA)橘小实蝇的精巢组织small RNA进行了深度测序,并整合构建了总small RNA转录组数据库,预测了172个已知和78个未知的miRNA,并分析了它们在精子发生过程中的表达动态。然后,通过对三个发育时期精巢组织small RNA转录谱进行两两比较,分别发现了在FM与IM、FM与MA、IM与MA发育时期之间差异表达的24、15、14个精巢mi RNA。通过生物信息学分析,进一步预测了13个转录差异最大的miRNA的124个靶标基因,并从中随机选择了6个miRNA和其预测调控的基因进行qRT-PCR验证分析,发现有4个miRNA与其靶标基因的转录模式相反。进一步的,对这4个miRNA中的mi R-8-3p调控精子发生的功能进行了深入研究。首先,我们预测出mi R-8-3p的靶标基因,B.dorsalis mitoferrin(bmfrn),推测miR-8-3p通过负调控bmfrn来调节精子发生。接下来,我们通过HeLa细胞的dual-luciferase报告系统验证了mi R-8-3p通过结合bmfrn的3′UTR区抑制后者的表达。而且,miR-8-3p的类似物(antagomi R)或bmfrn的dsRNA注射处理都能显著抑制mitoferrin的转录,减少精子数量,降低精子活性,最终抑制了橘小实蝇雄虫的生育能力。总之,本研究首次开展了不同生殖发育时期的雄性橘小实蝇精巢的miRNA转录组比较分析,描述了与精子发生相关的miRNA转录谱。同时,本研究筛选出调控精子发生的mirna,即mir-8-3p,并证实其通过结合bmfrn的3'utr区来抑制后者的表达,而最终影响精子发育。本研究对基于遗传改造的sit技术具有重要的理论和实际意义。
[Abstract]:Fruit fly is one of the most important horticultural pests widely distributed in East Asia and the Pacific. However, the traditional SIT relies on irradiation and chemical sterilizers, which can easily damage the mating competitiveness of male sterile insects, thus limiting its control effect. In recent years, SIT based on genetic transformation has performed well in the control of olive-fly. On the other hand, microRNAsmiRNAsS, as a key post-transcriptional regulator of negative regulation of target gene expression, is involved in regulating various life processes. This includes regulation of spermatogenesis. Therefore, genetic modification based on the key mi RNA in spermatogenesis has important development value for SIT. The studies of spermatogenesis and regulation of spermatogenesis related miRNA in fruit fly were still lacking. In this study, the small RNA of spermatozoa tissue of the fruit fly was deeply sequenced, and the total small RNA transcriptional database was constructed. 172 known and 78 unknown miRNAs were predicted, and their expression dynamics during spermatogenesis were analyzed. Then, the small RNA transcriptional profiles of spermatozoa during three developmental stages were compared. A total of 24, 15 and 14 spermary miRNA differentially expressed between FM and IMFM and MAHIM and MA were found respectively. By bioinformatics analysis, 124 target genes of 13 miRNA with the largest transcriptional difference were further predicted. Six miRNA and its predictive regulatory genes were randomly selected for qRT-PCR validation analysis. It was found that 4 miRNA were reverse to the transcription patterns of their target genes. The regulation of spermatogenesis by miR-8-3p in these four miRNA was studied. Firstly, we predicted the target gene of miR-8-3p, B.dorsalis mitof errinine bmfrnn, and speculated that miR-8-3p regulates spermatogenesis through negative regulation of bmfrn. We confirmed by dual-luciferase reporting system of HeLa cells that miR-8-3p inhibits the expression of the latter by binding to the 3UTR region of bmfrn. Moreover, either the injection of bmfrn or dsRNA can significantly inhibit the transcription of mitoferrin, decrease the number of sperm, and decrease the activity of sperm. In conclusion, the comparative analysis of miRNA transcriptome of male spermatozoa at different reproductive stages was carried out for the first time, and the miRNA transcriptional profiles associated with spermatogenesis were described. In this study, mirna, or mir-8-3p, was selected to regulate spermatogenesis, and it was confirmed that it inhibited the expression of bmfrn by binding to the 3tutr region of sit, and ultimately affected sperm development. This study has important theoretical and practical significance for sit based on genetic modification.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：S433

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