【作者】 马勇；

【导师】 刘树真； 王长法；

【作者基本信息】 山东师范大学 ， 动物学， 2015， 硕士

【摘要】 在现代奶牛群体改良中，种公牛是影响奶牛群体遗传品质的主要因素，其对奶牛群体遗传进展的贡献率高达70%以上。精液品质性状是种公牛选育工作中最基本的一项衡量指标，受到多基因控制，而常规育种手段选育良种公牛效率低，因此结合分子生物学技术，筛选出影响种公牛精液品质的候选基因作为分子标记，通过分子标记辅助选择育种，为未来定向选育高品质精液的种公牛提供参考。粘着斑激酶（Focal adhesion kinase, FAK）是一类胞质非受体蛋白酪氨酸激酶（PTKs），它在细胞信号转导中发挥重要作用，它是胞内外信号出入的中枢，介导多条信号通路。FAK还参与精子的释放和精子的获能等过程，在机体睾丸中具有较高的表达量，据此推测FAK基因可能是影响种公牛精液品质的一个候选基因。因此本课题以218头中国荷斯坦种公牛精液为样本，通过PCR和DNA测序技术筛选FAK基因的5’侧翼区和3’UTR区中的突变位点，运用SAS软件统计分析这些突变位点与公牛精液品质的关系，通过实验最终鉴定出功能性的突变位点。1、中国荷斯坦公牛FAK基因3’UTR SNP的鉴定及其与精液品质的关联性分析本实验通过PCR和DNA测序技术对FAK基因的3’UTR区进行了SNPs位点的扫描，发现了1个SNP，即g.129462₁29463insGAA。对该g.129462₁29463insgaa SNP位点与种公牛精液品质关联分析发现，野生基因型BB种公牛个体的射精量和鲜精密度显著高于AA基因型种公牛。2、3’UTR SNP g.129462₁29463insGAA对bta-miR-21调控FAK基因表达的影响利用miRNA在线软件预测与FAK基因3’UTR区域结合的miRNAs。结果表明FAK基因3’UTR上的突变位点g.129462₁29463insGAA位于bta-miR-21的种子序列区，而且发现此SNP突变前后影响FAK基因和bta-miR-21的结合能力。为了更好的验证分子标记g.129462₁29463insGAA对bta-miR-21与FAK基因3’UTR片段结合能力的影响，利用细胞瞬时转染实验对上述预测结果进行了验证。分别将含有突变型和野生型两种基因型的FAK基因3’UTR片段连入pMIR-ReportTM Luciferase报告载体中，与bta-miR-21真核表达载体共转染MLTC-1细胞，通过双荧光素报告系统检测发现，与pMIR-REPORTLuciferase-FAK野生型重组质粒相比， bta-miR-21质粒与pMIR-REPORTLuciferase-FAK突变后的重组质粒共转染可使细胞的荧光素酶活性下降。通过Q-PCR检测g.129462₁29463insGAA位点不同基因型个体中FAK mRNA的含量，结果发现，BB基因型个体中FAK mRNA的含量极显著（P<0.01）高于AB基因型。上述的关联分析也表明，BB基因型个体的射精量和鲜精密度显著高于AA型种公牛，因此我们推测此SNP可能通过影响bta-miR-21与FAK基因3’UTR的结合能力，影响FAK基因的表达量，从而影响精子的释放。3、中国荷斯坦公牛FAK基因的启动子区SNP与精液品质的关联性分析启动子作为基因必不可少的元件，可以影响基因转录的启动和表达的强度。本研究利用生物信息学方法预测FAK基因的启动子区，发现启动子区一些序列可与各种转录因子结合，包括NFATC1，STAT1，STAT3，FOXP1，SOX18，SP1等，这些位点的存在可能涉及FAK基因的转录调控机制。设计引物扩增该预测的启动子区，测序发现在该预测启动子区的靠近起始密码子处发现一个g.-58C>T突变。通过测序分型及关联分析发现，该SNPs位点中，对种公牛个体的鲜精活力来说，CC基因型个体值显著低于TT基因型个体值（P＜0.05）。更多还原

【Abstract】 In the modern improvement of dairy cattle, the bulls are the main factors to affectthe genetic quality of dairy cows and the rate of its contribution to cow populationgenetic is up to more than70%. The semen quality traits, which are controlled bypolygenic, are ecnomically important traits with a complex environmental and geneticbackground. Genetic markers may be useful in selection of breeding bulls with highquality semen traits due to the low efficiency of conventional methods to breed bulls..Focal adhesion kinase（FAK） is a kind of cytoplasmic non receptor proteintyrosine kinases （PTKs）. FAK has a high expression in testis and is involved in therelease of the sperm and its capacitation. FAK plays an important role in signaltransduction, crucial for embryonic development, regulation protein tyrosinephosphorylation in stallion sperm, and etc. It suggests that FAK gene may be acandidate gene which affect the semen quality of bull. In this research, we firstscanned single nucleotide polymorphisms（SNPs） of FAK gene5’ flanking region and3’UTR region by PCR and DNA sequencing technology from semen samples of218Chinese Holstein bulls,then we studied the relationship between SNPs loci and bullsemen quality, and selected the functional SNPs associated with bull semenquality,which can provide reference for the subsequent bulls breeding.1.Identification of FAK gene3’UTR SNP of Chinese Holstein bulls and itscorrelation analysis with sperm quality traits.In this experiment,we scanned the SNPs loci of3’ UTR region of FAK gene byPCR and DNA sequencing technology, a new SNP （g.129462₁29463insGAA） wasfound. Association analysis showed that sperm quality traits in Chinese Holstein bulls wassignificantly affected by the this SNP, bulls with BB genotypes had higher density of freshsemen than that bulls with AAgenotype at g.129462₁29463insgaa SNP loci. 2.The SNP g.129462-129463insGAA is located within bta-miR-21binding site inFAK3’UTR and affects FAK mRNAexpressionSNPs in MicroRNA （miRNA） binding region of gene3′-UTR may contribute tophenotypic differences. The phenotypic differences are caused by altering the expression ofmiRNAs and their targets. Bioinformatic analysis with the miRNA online predictionsoftware predicted that the g.129462₁29463insGAA site in the FAK3’UTR is locatedin the bta-miR-21binding region, and also found that the SNP mutations altersed thebinding to the seed sequence of bta-miR-21.combination of FAK gene and bta-miR-21. Inorder to verify the expression of the FAK gene by regulation with g.129462₁29463insGAA and bta-miR-21, we used transient transfection cell experiments tovalidate the prediction results.We amplified the FAK gene3’UTR fragments with thewild or mutant alleles, and cloned into the pMIR-ReportTM Luciferase REPORTvector respectively and then cotransfected into MLTC-1cell with bta-miR-21eukaryotic expression vector. Compared with the combination of bta-miR-21plasmid,dual luciferase reporter system of experimental group is lower thancontrol group. The quantitative real-time polymerase chain reaction results showed that therelative expression of FAK mRNA in bulls with the genotypes AB （increasing three basesof the hybrid type） is significantly lower than bulls with the genotype BB （wild type）（P <0.05） in the g.129462₁29463insGAA site,,which was consistency with the transfectionexperiment results. The high expression of FAK is conductive to spermatogenesis andrelease, and is consistent with the association analysis results. The results showed that theSNP g.129462₁29463insGAA in the FAK3′-UTR affected the mRNA expression of FAKby altering the binding of FAK, and bta-miR-21and further affects the release of thesperm.3. SNP g.-58C>T of Chinese Holstein bulls FAK gene promoter region and itscorrelation analysis with semen quality traits.To explore genetic variations in the5’-flanking region of FAK gene as well astheir frequent genotypes in the Chinese Holstein Bulls and determine whether suchvariations have an impact on bovine sperm quality traits and transcriptional activity,DNA samples were collected from Chinese Holstein Bulls and sequenced for identifying genetic variants in the5’-flanking region of FAK. One genetic variantsg.-58C>T which are both located in the core promoter region and in the transcriptionfactor binding sites through the bioinformatic analysis, was identified and genotypedin normal Chinese Holstein Bulls. The association results showed that the geneticvariation has significant impact on the sperm quality traits, the bulls with TT genotypeis significantly higher motility of fresh semen than the bulls with CC genotypes.更多还原