三倍体虹鳟不育相关miRNA的鉴定与表达调控研究

Argonaute(Ago)and miRNA could bind to mRNA (RNA-induced silencing complex, RISC)which plays a role in regulating the post-transcriptional level of protein expression. Triploid Rainbow trout showed several improved characteristics such as sterility,fast growth rate, good flesh quality and strong disease resistance.Previous studies indicate that the dysfunction of hypothalamic pituitary gonadal(HPG) axis is related to the sterile of triploid rainbow trout. Thus, the study on AGO-miRNA pathway in reproductive cycles and its regulation in triploid rainbow trout will unveil the mechanism of the sterile appearance. In order to find the correlation among Ago gene, miRNA and fertility in different ploidy, we assayed Ago and miRNA expression in triploids and diploids. By prediction and confirmation of target genes, the miRNA roles in gonads will be assayed. In addition, by detecting HPG axis regulation on Ago genes, miRNA and target genes, the effects of HPG axis on Ago-miRNA pathway will be demonstrated. This research would contribute to uncover the mechanism of post-transcriptional level in sterile triploid and provide evidence of molecular mechanism for gonad development in teleosts.

miRNA通过与AGO结合，形成RNA诱导基因沉默复合物（RISC），行使转录后水平调节的功能。人工诱导的三倍体虹鳟具有不育、生长快、肉质好、抗病性好等优良品性，显示了多倍体育种在鱼类养殖上的应用前景。下丘脑-垂体-性腺（HPG）轴的紊乱与三倍体虹鳟不育相关。因此，对AGO-miRNA信号通路在三倍体虹鳟鱼生殖周期中的表达及调控机制进行研究有望取得三倍体鱼不育机制的突破。本项目拟对三倍体虹鳟与二倍体虹鳟的AGO和miRNA的表达特征进行研究，以期找到AGO基因和miRNA和三倍体虹鳟的不育性的关联。通过靶基因预测和验证，证实miRNA在性腺中扮演的重要调节作用。另外，通过HPG轴对AGO基因、miRNA和相关靶基因的研究，明确生殖调控轴对AGO-miRNA信号通路的影响。本项目的开展将为进一步揭示三倍体虹鳟不育在转录后调控水平的发生机制，同时也为探讨鱼类miRNA在生殖中的作用提供参考。

虹鳟是世界性的重要养殖鱼类之一。人工诱导的三倍体虹鳟具有不育、生长快、肉质好、抗病性好等优良品性，显示了多倍体育种在鱼类养殖上的应用前景。而miRNA通过与AGO结合，形成RNA诱导基因沉默复合物（RISC），行使转录后水平调节的功能。本研究对AGO-miRNA信号通路在三倍体虹鳟鱼生殖周期中的表达及调控机制进行研究。通过HPG轴的关键激素对AGO基因进行诱导，发现HPG轴对虹鳟的AGO有明显的调控作用。此外，通过靶位点结合实验，证明了纺锤体蛋白-1的表达为miR-1388-5p所结合。miR-1388-5p在三倍体虹鳟卵巢中的miR-1388-5p表明明显升高。通过HPG轴下游的雌二醇E2激素注射发现，E2注射可提高Spin-1的表达，同时抑制miR-1388-5p的表达，而二倍体血清中的雌二醇浓度相对高于三倍体血清中的雌二醇浓度。因此，从miRNA的视角本研究证明了miR-1388-5p参与三倍体虹鳟不育的关键作用，为进一步揭示三倍体虹鳟不育在转录后调控水平的发生机制，同时也为探讨鱼类miRNA在生殖中的作用提供参考。