Genetic Variations Of KATNAL1 And GSK3?/? Genes And Their Associations With Semen Quality Traits In Chinese Holstein Bulls

In modern improvement system of dairy cow population, bullsare the main factor affecting the genetic quality of dairy herds and reproductive traits.The reproductive performance of bullisa key factor in determining the economic benefits of herd sire station. Bull fertility traits are quantitative traits of low heritability and regulated by multiple genes. In addition, their breeding takes a long selective cycle and its efficiency is too low. Therefore, it is urgent for us to exploit some novel breeding techniques and methods. Molecular breeding technology has become a developmental trend of the world's biological breeding science, and the directional breeding of high semen quality traits is an important developmental direction of dairy cattle breeding industry.In this study, KATNAL1 and GSK3?/? genes had been chosen as candidate genes in relation to bull semen quality traits on the basis of the results of domestic and overseas "-omics" studies and early analytic results of laboratory gene chips, combined with their physiological and biochemical function. At the levels of promoters, alternative splicing, miRNA and SNPs, the study explored the functional SNPs which could be used in the breedingsemen qualitytraits of Chinese Holstein bullsand tried toinvestigatethe molecular regulatory mechanisms of candidate gene expression because of these functional SNPs.1. Identification and expression analysis of alternative splice variants of the KATNAL1 gene inChinese Holstein bullsKATNAL1 gene plays an important role in the process of spermatogenesis. The study initially identified different alternative splice variants of KATNAL1 gene and explored their expression pattern in different tissues of adult bulls and calves by RT-PCR, then detected the relative expression quantities of different splice variants and gross mRNA in testis tissue of adult bulls and calves by RT-qPCR. The results showed thata novel splice variant, KATNAL1 transcript variant 2 (KATNAL1-TV2) of the retained 68 bp in intron 2, was identified by RT-PCR and compared with KATNAL1 transcript variant 1 (KATNAL1-TV1, NM 001192918.1) in various tissues. Bioinformatics analyses predicted that the translation initiation sites of KATNAL1-TV2was delayed and KATNAL1-TV2 encoded a shorter protein (364aa) lack of a MIT domain.RT-PCR results showed that the bovine KATNAL1 gene expression exhibits tissue variability. In adult bull tissues, KATNAL1-Gwas highly expressed in liver, kidney and testis tissues, and KATNAL1-G was more highly expressed in homologous tissues of adult bull than in calf. qRT-PCR results revealed that a higher expression of KATNAL1-G, KATNAL1-TV1, and KATNAL1-TV2 was observed in adult bull testis tissue compared with that in calf testis tissue (P<0.05), and the expression of the KATNAL1-G was higher than KATNAL1-TV1 and KATNAL1-TV1 in adult bovine, identical to that in calf (P<0.05). In addition, KATNAL1-TV1 manifested higher expression than KATNAL1-TV1 in adult bull testis. However, KATNAL1-TV1 was not significantly different when compared with KATNAL1-TV2 in calf testis (P>0.05). These findings suggested that KATNAL1 mRNA expression had obvious temporalspecificity.2. Activity analysis of KATNAL1 gene promoters and identification of functional SNPsin Chinese Holstein bullsTwo SNPs, c.163-210 T>C(rs 836312085) and c.323+6655 T>C(rs135559540), were detected in the promoter of KATNAL1-TV2 and in retained intron respectively. Bioinformatics analyses predicted that a new transcriptional factor (DeltaE) binding site appeared in the promoter region due to c.163-210 T>C and two splicing factors (SRSF1 and SRSF2) binding sites appeared in the proximal region of the retained intronowing toc.323+6655 T>C, which may promote the formation of the KATNAL1-TV2. In the study, gene clone, DNA sequencing and Luciferase reporter gene system were used to construct different deleted segments covering predicted promoter 1,2 and two fragments with different point mutation C and T in c.163-210as the recombinant reporter plasmids, and transiently expressed in MLTC-1 cells to determine the promoter activity of each fragment, respectively, then luciferase activities of transfected cells were analyzed, which revealed that c.-575?c.-180 and c.163-40-c.323+69 were confirmed as the core sequence of P1 and P2, respectively, and a higher fluorescence intensity of the C-allele was revealed (P<0.05). The two SNPs were genotypinged by Bi-PASA and RFLP respectively, and then correlation analysis with the sperm quality traits showed that bulls with the genetype CC of the two SNPs had a greater sperm deformity rate than bulls with the genotype TT (P<0.05). In addition, the association analysis between nine combined haplotypes and bull reproductive traits showed that the H1H1 haplotype with sperm motility was higher than that with the H1H4, H2H4, H3H3, H3H4 and H4H4 haplotype combination(P<0.05); but the H1H1 haplotype with deformity rate was lower than that with the H1H4, H2H4, H3H3, H3H4 and H4H4 haplotype combination (P<0.05). The result indicated that the newly-discovered two SNPs could be regarded as a new molecular marker associated with sperm quality traits for Chinese Holstein Bulls breeding programs.3. Effects of one SNP in the 3'UTR of the KATNAL1 gene on target gene expression regulated by bta-mir-22-5p in Chinese Holstein BullsMature miRNAs are binded to the 3'untranslated regions (3'UTR) of target gene mRNAs in two ways (incomplete complementarity and complete complementarity with target), which will lead to translational repression and mRNA degradation respectively. A novel SNP(*399 A>C) was identified in the 3'UTR of KATNAL1 gene by RT-PCR and sequencing technology.Bioinformatics analyses predicted thatbta-miR-22-5p had different binding capacities on the wild and mutant 3'UTR of KATNAL1 gene via miRNA targeted gene prediction software. Two genetypes (AA and CC) of 3'UTR were respectively inserted into the PMIR-ReportTM luciferase report vector, transfected MLTC-1 cells with bta-mir-22-5p expression vector. The result of dual luciferase report system analysis showed *399 A>C decreased the binding capacity on the 3'UTR of KATNAL1 gene(P<0.05). qRT-PCR results showed that the KATNAL1 expression of bulls with the genotype AA was lower than that of bulls with the genotype AC and CC (P<0.05). However, it was contrary to the results that SNP*399 A>C had no association with semen quality traits. Therefore, it is needed to explore the miRNA regulatory mechanism and select larger samples for association analysis, in order to determine whether the SNP could be a potentially functional molecular marker for semen quality traits breeding of Chinese Holstein Bulls or not.4. Identification and expression analysis of alternative splice variants of the GSK3? gene in Chinese Holstein bullsAlternative splicing can change the composition of gene coding region, generate different proteins and alter gene functions. In order to explore initially the expression pattern of GSK3? gene in bull testis, we cloned partial cDNA sequence including the comlete coding frame of bull GSK3? gene, and three novel alternative splicing variants were identified, named as GSK3?2 (E11b was retained), GSK3?3(E11 was missing),GSK3?4(E9 was missing) respectively; RT-qPCR results showed that the expression of GSK3? gene mRNA had obvious tissue variability and temporal specificity, and GSK3?4 was the main transcript combined with the results of monoclone. Bioinformatics analyses predicted that GSK3? gene had high homology and similar potential function among different species, and four transcripts were different in C-terminal. These results may lay a foundation for exploring the role of different transcripts in spermatogenesis further.5. A SNP G12A in pre-miRNA-320b is associated with sperm density by targeting GSK3? gene inChinese Holstein bullsSNPs in miRNA gene may affect miRNA expression levels, lead to changes in the ability of targeted regulation and finally result in changes of the organism traits. One SNP G12A (rs43416688) was identified in pre-miR-320b. Subsequently, genotyping and relevance analysis revealed that bulls with the genotype AA was higher than bulls with the genotype GA in sperm density (P<0.05).Bioinformatics analyses predicted that GSK3? gene could be a targeting gene of bta-miR-320b.After different combination of mimics, inhibitor, pre-miR-320b-G, pre-miR-320b-A and PMIR-GSK3?-3'UTR were cotransfected to MLTC-1 cell, dual luciferase activity assay showed that GSK3? gene was targeting gene of bta-miR-320b, and allele A was nonadvantageous for pre-bta-miR-320b processing. Stem-loop RT-qPCR of miRNA showed that mimics and inhibitor had been used in over-expression and inhibitory expression of bta-miR-320b in cell level, and the efficiency that mature bta-miR-320b originated in pre-miR-320b-G was higher than that originated in pre-miR-320b-A (P<0.05). Otherwise, in testis tissues, the expression level of bta-miR-320b in adult bulls and calves with the genotype AA was significantl lower than that in adult bulls and calves with the genotype GA (P<0.05), and the expression level of bta-miR-320b in calves was higher than that in adult bulls (P<0.05), which illustrated that the process of pre-miR-320b-GA was easier than that of pre-miR-320b-GA in vivo, and the expression of bta-miR-320b had temporal variability. RT-qPCR of GSK3? showed that, in testis tissues, the expression level of GSK3? mRNA in adult bulls and calves with the genotype AA was significantl higher than that in adult bulls and calves with the genotype GA (P<0.05), and the expression level of GSK3? mRNA in adult bulls was higher than that in calves (P<0.05), which illustrated that the expression of bta-miR-320b and GSK3? were influenced by SNP G12A, and their expression had temporal variability. Therefore, we speculated that SNP G12A in pre-miRNA-320b may be associated with sperm density inChinese Holstein bulls by targeting GSK3? gene.6. Activity analysis of the GSK3a gene promoter and identification of functional SNPs in Chinese Holstein bullsPromoter, as an indispensable element of one gene, can command the starting point of gene transcription and the degree of expression. SNPs in gene coding region and intron can influence the expression and function of gene in different ways. This study predicted gene promoter region by bioinformatics methods, and identified the core promoter and negative regulatory region by constructing different fragment length into pGL3-Basic luciferase report vector,being transfectedtransiently to MLTC-1 cells and determiningdual luciferase activity; Bioinformatics predicted that it had some negative regulatory elements including c-MYC, USF and YY1, and GSK3a gene was located in a CpG island. DMR methylation of GSK3a gene between high motility sperm and low motility sperm showed that GSK3a gene promoter was in low methylation condition, and methylation levelof low motility sperm was higher than that of high motility spermin the CpG site of GATA (one negative element) (p<0.05). Two SNPs (g.+4321 A>G and g.+7207 A>G) were detected in the fourth intron and eighth exonrespectively, and the latter was synonymous mutation. Genotyping and association analysis showed that three genetypes of g.+4321 A>G had no difference in semen quality traits (p>0.05), and bulls with g.+7207 A>G-GG were higher than bulls with g.+7207 A>G-AA in two traits of initial semen motility and frozen semen motility (P>0.05), and bulls with H2H2?H2H4 and H4H4 haplotype combinations were higher than bulls with other haplotype combinations (P<0.05). Therefore, we infered that promoter DMR methylation level and functional SNPs altered the expression and function of GSK3a gene, ultimately influenced sperm motility of bulls.