中国荷斯坦牛HIBADH和TNP1基因的遗传变异及其对精液品质影响的研究

发布时间：2018-07-28 10:23

【摘要】：本课题研究对象是公牛的HIBADH和TNP1基因，重点鉴定它们功能区的遗传变异，筛选与公牛精液品质密切相关的功能性分子标记，并尝试探究这些遗传变异可能的分子调控机理，为未来培育高繁殖力的公牛提供参考及依据。 1.中国荷斯坦公牛HIBADH基因核心启动子、功能性SNPs及甲基化的探究本研究首先通过western blot、免疫组化，发现HIBADH在公牛睾丸和附睾中均有表达。免疫荧光进一步验证HIBADH集中表达于精子中部和颈部，少量表达于头部。考虑到大量线粒体聚集在精子中部，提供能量，使鞭毛运动。我们初步猜测HIBADH可能与精子活力有关。随后扫描HIBADH基因5'侧翼区SNPs，发现g.-165TC突变。关联分析发现g.-165TC-CC基因型的个体鲜精活力显著低于TT基因型个体(P0.05)。生物信息学预测此突变位于HIBADH基因的核心启动子区，推测可能影响启动子区的活性。启动子缺失片段实验表明HIBADH核心启动子区域处于-703bp~+175bp处。将含有TT、CC基因型的启动子片段分别连接pGL3-Basic，转染表明TT基因型有最高的启动子活性。g.-165TC突变后增加了转录因子E47结合位点。核心启动子区甲基化试验表明高和低精子活力两组甲基化水平差异不显著（P0.05），均呈现的是低甲基化态势。但是高活力组CpG岛第7位点基本都呈现甲基化的状态，极显著的高于低活力组（P0.01）。这种特殊模式作用机制尚不清楚，需进一步探究。综上所述，我们推测启动子区功能性SNP（g.-165TC）可以调控HIBADH基因表达，进而影响公牛精子活力，核心启动子区域甲基化模式可能并非起主要的作用。 2. Bta-miR-204和bta-miR-532与中国荷斯坦公牛TNP1基因3’UTR的相关探究公牛TNP1基因的3’UTR区探测到2个多态性位点，分别是g.442AG和g.528GA。分析表明，g.528GA-GG基因型的个体精子畸形率显著比AA和GA基因型个体低(P0.05)；而g.442AG对公牛精液品质影响不显著。单倍型组合分析表明，H1H3和H1H1型公牛的精子畸形率显著比其他单倍型组合个体低(P0.05)。由于g.442AG和g.528GA均位于3’UTR区，我们推测它们可能通过相应的miRNAs发挥相应的调控作用。 miRNAs预测软件发现bta-miR-204和bta-miR-532可以与公牛TNP1基因3’UTR结合，且g.442AG和g.528GA分别存在于结合靶序列内。构建bta-miR-204质粒，，β-gal质粒，以及包含g.442AG野生型和突变型的3’UTR表达质粒共转MLTC-1细胞；同时构建bta-miR-532质粒，β-gal质粒，以及包含g.528GA野生型和突变型的3’UTR表达质粒共转MLTC-1细胞。结果与cmir0001-MR04对照相比，荧光活性降低。表明bta-miR-204和bta-miR-532可以和公牛TNP1基因3’UTR结合，降低TNP1的表达，并且g.442AG和g.528GA突变后增加了两个miRNAs与靶序列结合能力，使TNP1表达量更低。我们又构建分别含H1，H2，H3和H4四种单倍型的3’UTR表达质粒，将它们分别与bta-miR-204质粒，β-gal质粒，bta-miR-532质粒，共转MLTC-1细胞。结果单倍型H4有最高的miRNAs结合能力，显著高于单倍型H1（P0.05）。Q-PCR方法检测不同单倍型组合个体TNP1mRNA的含量，揭示H1H1个体mRNA水平显著高于H4H4个体（P0.05）。Q-PCR结果与单倍型试验结果一致。此外，bta-miR-204和bta-miR-532在性成熟牛的睾丸组织中的表达量比性未成熟牛的睾丸组织分别下调1.6和5倍。总上所述，TNP1基因3’UTR的两个SNPs均可影响bta-miR-204和bta-miR-532与TNP1基因3’UTR区的结合能力，调节TNP1的表达，继而影响种公牛精液品质，是功能性的位点。 3. HIBADH基因多态性与中国荷斯坦公牛精液品质的相关性我们选取404头公牛样本，利用PCR-RFLP、直接测序等技术扫描HIBADH基因全序列，进行多态性分析。在公牛HIBADH基因中发现了2个SNPs位点，分别是g.26736TC和g.90209CT。g.26736TC位于内含子4上；g.90209CT位于外显子5上，突变前后，第165位氨基酸未发生改变，故为同义突变。分析表明，g.26736TC位点的TC基因型个体鲜精活力显著高于TT和CC基因型个体(P0.05)；g.90209CT与冻后活力关系紧密，CC基因型个体冻后活力显著比TT个体高(P0.05)。单倍型构建分析，发现4种单倍型和9种单倍型组合。H1H3的鲜精密度、鲜精和冻后活力显著比其他单倍型组合高(P0.05)。因此，H1H3是优质单倍型组合，是有效分子标记，将来可参与辅助育种。
[Abstract]:The object of this study is the HIBADH and TNP1 genes of bulls, focusing on the identification of genetic variations in their functional areas, screening functional molecular markers closely related to the quality of bull semen, and trying to explore the possible molecular regulation mechanism of these genetic variations, providing a reference and basis for the future breeding of high reproductive bulls.
1. core promoter, SNPs and methylation of HIBADH gene in Chinese Holstein bulls
This study first showed that HIBADH was expressed in the testis and epididymis of the bulls by Western blot. The immunofluorescence further demonstrated that HIBADH was expressed in the middle and neck of the sperm and expressed in a small amount on the head. The sperm motility was related. Then the HIBADH gene 5'flanking SNPs was scanned and the g.-165TC mutation was found. The association analysis found that the fresh sperm motility of the g.-165TC-CC genotype was significantly lower than that of the TT genotype individual (P0.05). Bioinformatics predicted that the mutation was located at the core promoter region of the HIBADH gene. It is presumed that the activity of the promoter region may be affected. The fragment experiment showed that the core promoter region of HIBADH was at -703bp~+175bp. The promoter fragment containing TT and CC genotype was connected to pGL3-Basic respectively. The transfection showed that the TT genotype had the highest promoter active.G.-165TC mutation and increased the E47 binding site of the transcription factor. The core initiation subregion methylation test showed that the high and low sperm motility was found. The difference of methylation level in the two groups was not significant (P0.05), and all showed the trend of methylation. However, the seventh loci of the high activity group were basically methylation status, which was significantly higher than that of the low activity group (P0.01). This special mode of action mechanism is not clear and need to be further explored. In summary, we speculate that the functional SNP of the subregion is started. G.-165TC) can regulate the expression of HIBADH gene and further affect the motility of bull sperm. The core promoter region methylation pattern may not play a major role.
Correlation between 2. Bta-miR-204 and bta-miR-532 and 3 'UTR of TNP1 gene of Holstein bull in China
2 polymorphic loci were detected in the 3 'UTR region of the TNP1 gene of the bulls. The analysis of g.442AG and g.528GA. showed that the individual sperm abnormality rate of the g.528GA-GG genotype was significantly lower than that of the AA and GA genotype individuals (P0.05), and g.442AG had no significant influence on the semen quality of the bull. The haplotype combination analysis showed that the sperm malformation rate of the H1H3 and H1H1 type bulls. It was significantly lower than the other haplotype individuals (P0.05).
Since both g.442AG and g.528GA are located in the 3 'UTR region, we speculate that they may play a corresponding regulatory role through the corresponding miRNAs.
MiRNAs prediction software found that bta-miR-204 and bta-miR-532 can be combined with the bull TNP1 gene 3 'UTR, and g.442AG and g.528GA exist in the binding target sequence respectively. Construction of bta-miR-204 plasmids, beta -gal plasmids, and g.442AG wild type and mutant 3' UTR expression plasmids are converted to MLTC-1 cells. The plasmids, as well as the 3 'UTR expression plasmid containing the g.528GA wild type and the mutant type 3' UTR, were transferred to the MLTC-1 cells. The results were lower than that of cmir0001-MR04, indicating that bta-miR-204 and bta-miR-532 could be combined with the bull TNP1 gene 3 'UTR to reduce the expression of TNP1, and two miRNAs and target sequences were added after the g.442AG and g.528GA mutation. The expression of TNP1 was lower. We also constructed 3 'UTR expression plasmids containing four haplotypes of H1, H2, H3 and H4 respectively, and converted them to bta-miR-204 plasmids, beta -gal plasmids, bta-miR-532 plasmids and CO MLTC-1 cells. The content of the haplotype individual TNP1mRNA revealed that the mRNA level of the individual H1H1 was significantly higher than that of the H4H4 individual (P0.05).Q-PCR results and the haplotype test results. In addition, the expression of bta-miR-204 and bta-miR-532 in the testis tissues of sexually mature cattle was 1.6 and 5 times lower than that of the immature bovine testis. All above, the TNP1 gene 3 'UT was described. Two SNPs of R can affect the binding ability of bta-miR-204 and bta-miR-532 to the TNP1 gene 3 'UTR region, regulate the expression of TNP1, and then affect the semen quality of the bulls, which are functional sites.
Correlation between 3. HIBADH gene polymorphism and semen quality of Holstein bulls in China
We sampled 404 bull samples and scanned the complete sequence of HIBADH gene by PCR-RFLP and direct sequencing.
2 SNPs loci were found in the HIBADH gene of the bulls, which were g.26736TC and g.90209CT.g.26736TC in the intron 4, and g.90209CT was located on exon 5. Before and after the mutation, the 165th amino acids did not change, so it was synonymous mutation. The analysis showed that the fresh sperm vitality of the TC genotype individual at the g.26736TC site was significantly higher than that of TT and CC genotype individuals. (P0.05); g.90209CT was closely related to the vitality after the freeze. The viability of the CC genotype individual was significantly higher than that of the TT individual (P0.05). The haplotype construction analysis showed that the fresh sperm density of 4 haplotypes and 9 haplotype combinations was higher than that of the other haplotypes (P0.05). Therefore, H1H3 was a high quality haplotype combination and an effective fraction. Sub markers that may be involved in auxiliary breeding in the future.
【学位授予单位】：山东师范大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S823

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