Genetic Variations Of Chinese Holstein Bulls In Fak Gene Regulation Zone Identification And Its Associated With Semen Quality Analysis

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.