调控荷斯坦公牛microRNAs生物学合成和精子活力的SNPs筛选与功能验证

Sperm motility is a determining factor in normal male fertility and a vital parameter for the evaluation of sperm quality, which is useful for the diagnosis and treatment of low fertility and infertility. However, the molecular mechanisms controlling sperm motility are still limited. Single nucleotide polymorphisms (SNPs) around the processing sites, and within the mature miRNA, especially the seed sequence in the human and animal genomes, can impair or enhance miRNA processing and alter the processing sites as well as affect biogenesis and function of miRNA. In our previous study, by using our self-developed miRNAQTLsnp software, we have found 105 SNPs which are located, just right, in the pre-miRNA, mature miRNA and seed sequences as well as within the QTLs that are invovled in sperm motility in the bovine genome. As a consequence, we suggest that the candidate SNPs would affect biogenesis and function of miRNAs, and subsequently modulate their target genes and signal pathways that are related to sperm moltility, thus alter sperm moltility and semen quality. In order to comfirm above idea, a few experiments need to be performed in the proposal. First of all, we will investigate potenital new SNPs within the pre-miRNAs and sperm-molitity-related QTLs and confirm some of them in the Holstein bulls using the direct sequencing and miRNAQTLsnps sofware. Second, we are about to determinate the expression and location of candidate miRNAs in the low-motile and high-motile sperms, testis and Leydig cell lines using the qRT-PCR, Fluorescence in situ hybridization, and the relationship between miRNA expression and genotype of SNP. Third, to bulid mutants of pre-miRNAs and transfect them into Leydig cells and to validate the target genes and signal pathways involved in sperm motility at cell level by making use of the Luciferase report system, Western-blotting and RNA sequencing experiments, etc. Finally, we aim to clarify the regulatory function of the SNPs on sperm motility and their underlying molecular mechanisms, and to excavate the functional markers that are associated with semen quality, which can be used for molecular breeding and fecundity forecast in Holstein bulls.

精子活力是精液质量评价的关键参数，但分子调控机制尚不明了。单核甘酸多态性（SNPs）可影响microRNAs（miRNAs）的加工合成。我们前期通过自主开发的miRNAQTLsnp软件，在牛基因组上筛选出108个SNPs恰好位于miRNAs基因序列内以及与精子活力有关的QTLs内。我们推测上述SNPs可能通过影响miRNAs的生物学合成，调控下游靶基因和信号通路，最终影响精子的活力。本项目拟①应用测序和miRNAQTLsnp软件鉴定潜在影响miRNAs合成的SNPs；②应用qRT-PCR、荧光原位杂交等分析候选miRNAs在高低活力精子、睾丸间质细胞中的表达和定位；③构建突变体，应用荧光素酶报告系统、蛋白印迹等在细胞水平上验证miRNAs与精子活力有关靶基因和信号通路；④阐明SNPs的功能及其对精子活力的调控作用与分子机制，挖掘功能性标记，为高精液品质公牛的分子育种和繁殖力预测提供依据。

精子活力是衡量精液品质的关键指标，而调控精子活力的分子遗传机制尚不清楚。MicroRNA（miRNA）的基因序列中的单核苷酸多态性（SNP）可影响miRNA的生物学合成及其表达调控模式，进而影响种公牛精液品质和繁殖力。本项目利用目标区域测序的方法对8个候选miRNA的遗传变异进行筛选，鉴定到bta-miR-6531是与精液品质相关的关键miRNA，并在其前体序列上发现了功能性SNPs；利用Targetscan7、miRWalk等生物信息学软件筛选了bta-miR-6531的靶基因，同时分析了这些靶基因主要靶向钙离子信号通路和cAMP信号通路；利用双荧光素酶报告系统、睾丸间质细胞中验证发现miR-6531主要通过靶向ATP2A2来调控细胞钙离子的流入；利用奶牛群体分析了miR-6531前体序列上的SNP位点与精液品质相关。此外，项目基于高活力和低活力荷斯坦奶牛精子转录组、miRNAs和piRNA的差异表达分析结果，筛选出了与精子活力相关基因EFNA1、SESN3、miRNAs和piRNA簇，鉴定了在精子活力QTL位点上或附近的6个差异表达piRNA簇。本项目阐明了miRNA及其前体区SNPs与奶牛精子活力的关系，挖掘出影响精子活力的miRNA及其功能性分子标记。

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