Genetic Variations Of PRM1and PRM2Genes And Their Associations With Semen Quality Traits In Chinese Holstein Bulls

A bull in the genetic improvement of modern dairy cattle herds play a key role, semenquality is not only decided to economic benefit of bull stations but also the importantfactors that affect the cow conception rate. Bull semen quality traits were influenced byheredity, environment, nutrition and and feeding management so on. Improving bull semenquality traits by nutrition, breeding management and conventional breeding methods is verydifficult, long cycle and low efficiency. Bull semen quality traits are quantitative traits andregulated by multigene, and the spermatogenesis process is complex. So screening for maineffect genes or loci for enhancing the bull semen quality traits is extremely important.Protamine(PRM) as a major nuclear proteins in sperm cells, closely related to malereproductive that is crucial to development of sperm and the formation of normal sperm. Inthis study, PRM1and PRM2genes had been chosen as candidate genes to analyze theimpact of genetic variation in bull semen quality traits by at the levels of promoters,alternative splicing, and SNPs, and try to analyze the molecular regulation mechanism forthe bull semen quality traits. The main research results follow as:1. PRM2gene polymorphism in Chinese holstein bullsScreening SNPs of PRM2genes in Chinese holstein bulls by DNA sequencingtechnology，discovered a SNP (g.+478A>G) in exon2, is a missense mutation, mutationcaused the arginine (Arg) into glycine (Gly); Genotyping by PCR-RFLP technique for theSNP，carry out correlation analysis with phenotypic trait data, The results showed that thevolume of semen, vitality of fresh semen and vitality of frozen semen of GG genotypeindividuals is significantly higher than AA genotype and AG genotype (P <0.05), thedeformity rate significantly lower than AA and AG (P <0.05). Sperm quality traits of PRM2GG genotype individuals is higher than other, can be used as a molecular marker to assistbreeding bulls.2. Quantitation and identification of PRM2gene splicing variants In order to explore expression pattern, differently expressed in adult bulls testitis andnewborn bulls testitis of the different splice variant(PRM2-CT and PRM2-TV1). RT-PCRwas used to explore the expression of the two splice variant in various tissues of bull. Theresult showed that PRM2-CT and PRM2-TV1were only expressed in testitis. qRT-PCR wasused to explore the relative expression of PRM2-CT and PRM2-TV1in adult bulls testitisand newborn bulls testitis. The result showed that PRM2-CT and PRM2-TV1were washighly expressed in adult bulls testitis; The expression of PRM2-TV1was significantlyhigher than PRM2-CT in adult bulls testitis and newborn bulls testitis(P<0.05).3. Activity analysis of PRM1gene promoters and identification of functional SNPs inChinese Holstein bullsBioinformatics predicted a promoter region was exists in PRM1gene. Two SNPs，g.-138.A> G and g.-111.G> A, were detected in the promoter of PRM1gene. They arecompletely linked in the tested populations, and they could be inherited as a unitSNP-1(g.-138.A> G) for following study. Bioinformatics analyses predicted that newtranscriptional factors (NF-Zc and H4TF-2) binding site disappeared in the promoter regiondue to the present of mutation, while previous studies have found the NF-Zc and H4TF-2has the function of promoting gene transcription. In the study, gene clone and Luciferasereporter gene system were used to construct different deleted segments covering predictedpromoter and a fragment with different point mutation A in g.-138.A> G and G in g.-111.G>A as the recombinant reporter plasmids, and transiently expressed in MLTC-1cells todetermine the promoter activity of each fragment, respectively, then luciferase activities oftransfected cells were analyzed, which revealed that g.-230~g.-89was confirmed as PRM1gene core sequence, and a lower fluorescence intensity of the G-allele in g.-138.A> G and A-allele in g.-111.G> A were revealed (P<0.05). The g.-138.A> G were genotypinged by RFLP,and then correlation analysis with the sperm quality traits showed that bulls with thegenetype. The vitality of fresh semen, vitality of frozen semen and density of fresh semen ofbulls with wild homozygous in g.-138.A> G and GG in g.-111.G> A were significantlyhigher than that of mutant homozygote and heterozygous (P <0.05), the deformity rate wassignificantly lower. Significant associations indicates that the A allele in g.-138.A> G and Gallele in g.-111.G> A might improve the vitality of fresh semen. The result indicated that the newly-discovered two SNPs could be regarded as a new molecular marker associated withsperm quality traits for Chinese Holstein Bulls breeding programs.4. Identification and expression analysis of alternative splice variants of the PRM1gene inChinese Holstein bullsThe study initially identified different alternative splice variants of PRM1gene andexplored their expression pattern in different tissues of adult bulls and calves by RT-PCR andgene sequencing, then detected the relative expression quantities of different splice variantsin testis tissue of adult bulls and newborn bulls by qRT-PCR. The sequencing result a novelsplice variant (PRM1-TV1) that lacked78bp of parts of5’UTR and exon1was identified.RT-PCR detected the expression of the bull PRM1exhibits tissue variability. The result byRT-PCR indicated: in adult bulls, PRM1-CT was expressed at higher level in all tissues.intriguingly, PRM1-TV1was little expresstion and present in testes and heart; in newbornbulls; PRM1-CT was expressed predominantly but less in all tissues, PRM1-TV wasdisplayed lower or undetectable level of activity except testes. The result by qRT-PCRindicated: the PRM1-TV1and PRM1-CT expression was significantly different between thetwo groups, a higher expression of PRM1-TV1and PRM1-CT was observed in the testis ofadult bull, in contrast to the testis of newborn bulls; PRM1-CT displayed higher expressionthan did PRM1-TV1in both samples. PRM1-TV1was hardly expressed in the testis ofnewborn bulls; These findings were matched with the RT-PCR results in the present study,suggesting that PRM1-CT was the primary transcript in the process of transcriptionregulation.