Variation Of CD9and TNP2and Its Associations With Sperm Quality Traits In Chinese Holstein Bulls

This study was carried out to investigate the relationship between the variants ofthe bull TNP2and CD9gene and sperm quality traits in Chinese Holstein bulls.Single nucleotide polymorphisms (SNPs) of the TNP2and CD9genes wereresearched in392Chinese bulls by PCR-RFLP and DNA sequencing methods, for thepurpose of selecting molecular maker information to improve the breeding efficiencyof high sperm quality.1. Associations analysis between the polymorphisms of TNP2gene and spermquality traits in Chianese Holstein bullsDuring spermiogenesis, major restructuring of chromatin takes place. It isbelieved that the transition proteins (TNP1and TNP2) participate in the removal ofthe nucleohistones and in the initial condensation of the spermatid nucleus. Later inthe spermatogenesis, transition proteins are in turn replaced by the protamines, Prm1and Prm2. The TNP2gene in bulls is1560bp, which only contains one intron, and themRNA is transcribed in round spermatids and stored for up to5days in an inert statebefore translation is activated in elongated spermatids.In the present study we detected three single-nucleotide polymorphisms (SNPs)of TNP2gene in392Chinese Holstein bull, and they were g.269G>A, g.480C>Tand g.1536C>T. Linkage disequilibrium and haplotype construction were analyzedusing SHEsis software. Eight haplotypes were constructed, and eighteen haplotypecombinations were found. The volume of semen, vitality and density of fresh semen,vitality of frozen semen and rate of teratosperm were selected for the sperm qualityindex. Association analysis showed that sperm quality traits in Chinese Holstein bullwas significantly affected by the three polymorphism sites. The SNP g.269G>A wasidentified as a non-synonymous mutation (codon62, Arg> His) at the Arginine-richdomain. In this position, the vitality of fresh semen of bull with GA genotype waslower than that of GG or AA (p<0.05), the rate of teratosperm of bull with GA genotype was higher than that of GG or AA genotype (p<0.05). Bulls with TTgenotype had lower teratosperm rate (p<0.05) at the480position. In the g.1536C>Tsites, bulls with CC genotype had lower volume of semen and vitality of frozen semen(p<0.05). Bull s with H7H8and H8H4haplotype combination had higher vitality offresh semen than H6H4, H6H6and H6H8haplotype combination (p<0.05). H1H4haplotype combination had higher density of fresh semen than H6H4and H5H2haplotype combination (p<0.05).2.The interaction between bta-miR-154and TNP2-3’UTRmiRNAs are a class of small non-coding endogenous RNA molecμLes (18~24ntin length) that mainly bind to partially complementary sequences in the3’-untranslated region (UTR) to inhibit the target mRNA translation into protein oraccelerate its degradation. Many studies have proven that miRNAs take part in regμLating the quality of semen.Bioinformatic analysis predicted that the1536position was located in themicroRNA binding (bta-miR-154) region. Single-nucleotide polymorphisms (SNPs)associated with polygenetic disease can create, destroy, or modify miRNA bindingsites. By detection the luciferase activity of different vectors demonstrated thatg.1536C＞T mutation caused the target binding of microRNA, resμLting in thetranscriptional repression, which, in turn, resμLted in induced expression. Our findingrevealed that the TNP2gene possibly contributed to conducting association analysisand can be used as molecμLar marker in sperm quality traits and other performancefor animal breeding.We compared the relative expression of TNP2mRNA in the testis of bulls withthe three genotypes using Q-PCR. The relative expression of TNP2mRNA inHolstein bulls with the genotype CT and CC is significantly higher (P<0.05) than thatin bulls with the genotype TT. The high expression of TNP2is conducive tospermatogenesis, this consequence is consistant with association analysis above.3. Cloning and Identification of Core Area of CD9Gene PromoterCluster-of-differentiation antigen9(CD9) gene which expressed in the male germline stem cells is crucial for sperm–egg fusion, and was therefore selected as an candidate gene for bull sperm quality. There is no TATA box in the5’ flanking regionof CD9in bull, but containing the GC-rich region, the highest GC content between-302to-183is88%, which some sequence can be combined with varioustranscription factors, including the sPl, AP2, GATA, NF-kB binding sites, such as thepresence of these loci may be involved in gene transcriptional regulation mechanism.In this research, we cloned the5’flanking region of CD9gene including the5’UTR and partial coding sequence, and screening the SNPs of the region. g.-298C＞G and g.-943C＞A were found, the mutation rate of g.-298C＞G was too high to bestatistically significant. The sequence was analyzed in MLTC-1and kidney cell (293T)respectively. There is promoter activity in both MLTC-1and293T cell, but thepromoter activity in MLTC-1is stronger than in293T. The studies showed that thesequence from-605—+50bp had the basal promoter activity in MLTC-1. We inferredthat there were negative regulatory domains between-1640to-1125bp.The promoteractivity decreased when g.-943C＞A mutation, the reason maybe due to the impact ofthe combination of SP1.