microRNA-430对黄鳝性腺发育及性逆转的调控作用

MicroRNA (miRNA) are endogenous small RNAs that can regulate target mRNAs by binding to their sequences in the 3’ untranslated region (UTR). The expression of miRNAs and the biogenetic pathway are involved in sex differentiation and the regulation of the development of primordial germ cells and gonadal somatic cells. miR-430 target a large number of maternally expressed mRNAs and promote their deadenylation and degradation. miR-430 is a well characterized miRNA, which is expressed at maternal-zygotic transition stage. Interestingly, miR-430 is only observed in the fish species base on the miRbase datebase. The rice field eel (Monopterus albus) undergoes a natural sexual transformation from female to male via an intersex stage during its life cycle. Our pervious study found that the miR-430 family may be involved in the sexual transformation of the rice field eel. However, the mechanism of this physiological phenomenon is not clear, especially through the miRNA. We will investigate the gene and protein differential expression at transcription and protein level through the miR-430 family members cloning, knocking out the precursor sequence of miR-430, and the over expression of miR-430. We will study the effect of miR-430 on the aromatase gene expression in gonad tissue, the activity of aromatase, and sex hormones synthesis. And the binding sites between potential target genes and miR-430 will be validated, clarifying the molecular mechanism of miR-430 on gonadal development during the course of sexual reversal in rice field eel.

microRNA（miRNA）的表达及其生物途径涉及动物性腺分化和生殖细胞的发育。而miRNA-430被认为是在母型-合子型过渡阶段表达的miRNA，调控着大量母系表达的mRNA。黄鳝是具有天然性逆转现象的鱼类，雌性在性成熟产卵后，会经过间性最终转化为雄性性别。前期研究发现，黄鳝miRNA-430家族及其靶基因可能涉及黄鳝性逆转这一生物过程，但是其介导的性逆转期间的性腺发育机制并不清楚。本项目对黄鳝miRNA-430家族成员进行克隆分析，并验证miRNA-430的性别调控靶基因的结合位点， 通过抑制miRNA-430表达和过表达miRNA-430的生物学研究，检测对黄鳝性腺发育相关基因的转录和蛋白水平的表达以及性激素合成途径的影响，明确miRNA-430对性别相关基因表达和性别分化的调控作用，阐明miRNA-430在黄鳝性逆转过程中对性腺发育的调控机制。

microRNA是生物体内合成的一类不编码蛋白质的短链RNA，结合靶mRNA 3’端非编码区（3’ UTR），进而干扰靶mRNA。miR-430参与了配子形成、原始生殖细胞（PGC）的迁移等重要的生殖发育过程。本研究利用黄鳝胚胎分别显微注射miR-430过表达模拟物（agomir430a、agomir430b和agomir430c）或miR-430敲降抑制剂（antagomir430a、antagomir430b和antagomir430c）后，RNA-seq结果显示，差异基因显著富集到GnRH、ErbB、Wnt、PPAR和PI3K-Akt等与类固醇生成相关的信号通路；类固醇生成相关基因17β-hsdb3、17β-hsdb7、17β-hsdb12和cyp19a1b在miR-430敲降组显著下调，表明miR-430可以改变类固醇生成和性腺发育相关基因，在转录水平上介导黄鳝的类固醇生成；miR-430可能通过靶向调控cyp19a1b直接或通过靶向调控foxl2间接调控cyp19a1b促进类固醇激素的生成和生物合成。并且，本研究利用RNA定位表达，首次在活体黄鳝中观察到PGC的迁移，并证明含有斑马鱼nanos3 3'UTR和黄鳝dnd 3'UTR的融合mRNA比含有黄鳝vasa 3' UTR的mRNA更适用于黄鳝PGC的可视化；利用Morpholino基因敲降证明，dnd表达降低可导致PGC数量减少，说明dnd在黄鳝PGC的发育过程中起重要作用。这些成果将为揭示鱼类的性别决定和性别分化机制提供了理论基础和技术支持。