中國(guó)荷斯坦牛HIBADH和TNP1基因的遺傳變異及其對(duì)精液品質(zhì)影響的研究
發(fā)布時(shí)間：2018-07-28 10:23
【摘要】：本課題研究對(duì)象是公牛的HIBADH和TNP1基因，重點(diǎn)鑒定它們功能區(qū)的遺傳變異，篩選與公牛精液品質(zhì)密切相關(guān)的功能性分子標(biāo)記，并嘗試探究這些遺傳變異可能的分子調(diào)控機(jī)理，為未來(lái)培育高繁殖力的公牛提供參考及依據(jù)。 1.中國(guó)荷斯坦公牛HIBADH基因核心啟動(dòng)子、功能性SNPs及甲基化的探究 本研究首先通過(guò)western blot、免疫組化，發(fā)現(xiàn)HIBADH在公牛睪丸和附睪中均有表達(dá)。免疫熒光進(jìn)一步驗(yàn)證HIBADH集中表達(dá)于精子中部和頸部，少量表達(dá)于頭部。考慮到大量線粒體聚集在精子中部，提供能量，使鞭毛運(yùn)動(dòng)。我們初步猜測(cè)HIBADH可能與精子活力有關(guān)。隨后掃描HIBADH基因5'側(cè)翼區(qū)SNPs，發(fā)現(xiàn)g.-165TC突變。關(guān)聯(lián)分析發(fā)現(xiàn)g.-165TC-CC基因型的個(gè)體鮮精活力顯著低于TT基因型個(gè)體(P0.05)。生物信息學(xué)預(yù)測(cè)此突變位于HIBADH基因的核心啟動(dòng)子區(qū)，推測(cè)可能影響啟動(dòng)子區(qū)的活性。啟動(dòng)子缺失片段實(shí)驗(yàn)表明HIBADH核心啟動(dòng)子區(qū)域處于-703bp~+175bp處。將含有TT、CC基因型的啟動(dòng)子片段分別連接pGL3-Basic，轉(zhuǎn)染表明TT基因型有最高的啟動(dòng)子活性。g.-165TC突變后增加了轉(zhuǎn)錄因子E47結(jié)合位點(diǎn)。核心啟動(dòng)子區(qū)甲基化試驗(yàn)表明高和低精子活力兩組甲基化水平差異不顯著（P0.05），均呈現(xiàn)的是低甲基化態(tài)勢(shì)。但是高活力組CpG島第7位點(diǎn)基本都呈現(xiàn)甲基化的狀態(tài)，極顯著的高于低活力組（P0.01）。這種特殊模式作用機(jī)制尚不清楚，需進(jìn)一步探究。綜上所述，我們推測(cè)啟動(dòng)子區(qū)功能性SNP（g.-165TC）可以調(diào)控HIBADH基因表達(dá)，進(jìn)而影響公牛精子活力，核心啟動(dòng)子區(qū)域甲基化模式可能并非起主要的作用。 2. Bta-miR-204和bta-miR-532與中國(guó)荷斯坦公牛TNP1基因3’UTR的相關(guān)探究 公牛TNP1基因的3’UTR區(qū)探測(cè)到2個(gè)多態(tài)性位點(diǎn)，分別是g.442AG和g.528GA。分析表明，g.528GA-GG基因型的個(gè)體精子畸形率顯著比AA和GA基因型個(gè)體低(P0.05)；而g.442AG對(duì)公牛精液品質(zhì)影響不顯著。單倍型組合分析表明，H1H3和H1H1型公牛的精子畸形率顯著比其他單倍型組合個(gè)體低(P0.05)。 由于g.442AG和g.528GA均位于3’UTR區(qū)，我們推測(cè)它們可能通過(guò)相應(yīng)的miRNAs發(fā)揮相應(yīng)的調(diào)控作用。 miRNAs預(yù)測(cè)軟件發(fā)現(xiàn)bta-miR-204和bta-miR-532可以與公牛TNP1基因3’UTR結(jié)合，且g.442AG和g.528GA分別存在于結(jié)合靶序列內(nèi)。構(gòu)建bta-miR-204質(zhì)粒，，β-gal質(zhì)粒，以及包含g.442AG野生型和突變型的3’UTR表達(dá)質(zhì)粒共轉(zhuǎn)MLTC-1細(xì)胞；同時(shí)構(gòu)建bta-miR-532質(zhì)粒，β-gal質(zhì)粒，以及包含g.528GA野生型和突變型的3’UTR表達(dá)質(zhì)粒共轉(zhuǎn)MLTC-1細(xì)胞。結(jié)果與cmir0001-MR04對(duì)照相比，熒光活性降低。表明bta-miR-204和bta-miR-532可以和公牛TNP1基因3’UTR結(jié)合，降低TNP1的表達(dá)，并且g.442AG和g.528GA突變后增加了兩個(gè)miRNAs與靶序列結(jié)合能力，使TNP1表達(dá)量更低。我們又構(gòu)建分別含H1，H2，H3和H4四種單倍型的3’UTR表達(dá)質(zhì)粒，將它們分別與bta-miR-204質(zhì)粒，β-gal質(zhì)粒，bta-miR-532質(zhì)粒，共轉(zhuǎn)MLTC-1細(xì)胞。結(jié)果單倍型H4有最高的miRNAs結(jié)合能力，顯著高于單倍型H1（P0.05）。Q-PCR方法檢測(cè)不同單倍型組合個(gè)體TNP1mRNA的含量，揭示H1H1個(gè)體mRNA水平顯著高于H4H4個(gè)體（P0.05）。Q-PCR結(jié)果與單倍型試驗(yàn)結(jié)果一致。此外，bta-miR-204和bta-miR-532在性成熟牛的睪丸組織中的表達(dá)量比性未成熟牛的睪丸組織分別下調(diào)1.6和5倍。總上所述，TNP1基因3’UTR的兩個(gè)SNPs均可影響bta-miR-204和bta-miR-532與TNP1基因3’UTR區(qū)的結(jié)合能力，調(diào)節(jié)TNP1的表達(dá)，繼而影響種公牛精液品質(zhì)，是功能性的位點(diǎn)。 3. HIBADH基因多態(tài)性與中國(guó)荷斯坦公牛精液品質(zhì)的相關(guān)性 我們選取404頭公牛樣本，利用PCR-RFLP、直接測(cè)序等技術(shù)掃描HIBADH基因全序列，進(jìn)行多態(tài)性分析。 在公牛HIBADH基因中發(fā)現(xiàn)了2個(gè)SNPs位點(diǎn)，分別是g.26736TC和g.90209CT。g.26736TC位于內(nèi)含子4上；g.90209CT位于外顯子5上，突變前后，第165位氨基酸未發(fā)生改變，故為同義突變。分析表明，g.26736TC位點(diǎn)的TC基因型個(gè)體鮮精活力顯著高于TT和CC基因型個(gè)體(P0.05)；g.90209CT與凍后活力關(guān)系緊密，CC基因型個(gè)體凍后活力顯著比TT個(gè)體高(P0.05)。單倍型構(gòu)建分析，發(fā)現(xiàn)4種單倍型和9種單倍型組合。H1H3的鮮精密度、鮮精和凍后活力顯著比其他單倍型組合高(P0.05)。因此，H1H3是優(yōu)質(zhì)單倍型組合，是有效分子標(biāo)記，將來(lái)可參與輔助育種。
[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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823
本文編號(hào)：2149786
[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.
【學(xué)位授予單位】：山東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：S823
【參考文獻(xiàn)】
相關(guān)期刊論文 前10條
1 郭睿;李喜霞;王惠珍;;小鼠睪丸特異基因在生精細(xì)胞中階段性表達(dá)的定量分析[J];動(dòng)物學(xué)雜志;2009年01期
2 王啟貴;李輝;李寧;王宇祥;唐志權(quán);;雞Myogenin基因單核苷酸多態(tài)檢測(cè)及群體遺傳分析[J];東北農(nóng)業(yè)大學(xué)學(xué)報(bào);2008年06期
3 淮亞紅;王淑輝;許尚忠;高雪;任紅艷;陳金寶;;精液品質(zhì)相關(guān)基因的研究進(jìn)展[J];中國(guó)畜牧獸醫(yī);2008年03期
4 范覺(jué)鑫;張彬;李麗立;袁曉雪;王水蓮;謝婷;耿梅梅;;大豆異黃酮對(duì)香豬睪丸形態(tài)及精子發(fā)生標(biāo)志基因表達(dá)的影響[J];動(dòng)物營(yíng)養(yǎng)學(xué)報(bào);2012年10期
5 趙銳峰;任久生;徐靖博;柳思源;于汪洋;鄧瓊;馬騰壑;姜昊;高妍;趙志輝;張嘉保;;GSTs-Mu基因的單核苷酸多態(tài)性與種公牛精液品質(zhì)的相關(guān)性分析[J];黑龍江動(dòng)物繁殖;2010年06期
6 張雁峰;張銳;宿兵;;MicroRNA基因簇的多樣性與進(jìn)化[J];中國(guó)科學(xué)(C輯:生命科學(xué));2009年01期
7 周國(guó)權(quán);徐敬哲;趙高俊;高磊;馬欣;秦懇;錢漢超;;影響種公牛精液品質(zhì)的因素[J];現(xiàn)代畜牧獸醫(yī);2012年04期
8 余慶鋒;楊學(xué)習(xí);李粉霞;葉祿偉;吳英松;毛向明;;PRM1基因SNP位點(diǎn)-190C-＞A多態(tài)性對(duì)精子畸形率的影響[J];中華男科學(xué)雜志;2012年04期
9 秦立紅;任穎;劉暢;張金玉;張嘉保;趙志輝;;牛AR基因5′UTR SNP分析及與精液品質(zhì)關(guān)系[J];吉林農(nóng)業(yè)大學(xué)學(xué)報(bào);2010年03期
10 周凡;莊詩(shī)美;;microRNA與腫瘤[J];生命科學(xué);2008年02期
相關(guān)博士學(xué)位論文 前2條
1 韓鳳桐;牛Sry基因核心啟動(dòng)子定位及其基因功能研究[D];東北農(nóng)業(yè)大學(xué);2008年
2 劉英;鼻咽癌易感基因miRNA結(jié)合位點(diǎn)多態(tài)性研究[D];南方醫(yī)科大學(xué);2009年
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