| The semen quality of Chinese Holstein bulls significantly affected the reproductive efficiency of cows. So in the past few decades, with the continuous development of molecular biology techniques, the candidate gene approach in genetics and breeding has been widely popularized. With related researches, this study selected INCENP gene as candidate genes to explore functional SNPs associated with bull semen quality and analysis molecular regulation mechanism of functional SNPs. 1. Promoter activity analysis of INCENP gene in Chinese Holstein bullsAccording to the biological information software, we found a promoter in 5'-flanking region of INCENP gene. We identified that g.-532 ~ g.-145 was the core promoter responsible for most of the promoter activity in 5'-flanking region by building the missing fragment pGL3 plasmid and transiently transfected into MLTC-1 cells. At the same time, we identified two SNPs: g.-556 G> T and g.-692 C> T in the promoter region by sequencing. Association analysis using SAS software was performed to analyze the correlations of SNPs with semen quality and the results showed that g.-556 G> T was significantly correlated with initial sperm motility. In addition, dual luciferase reporter gene approach was performed to verify function of two SNPs found in 5'-flanking region and we found that the TG haplotype demonstrated the highest luciferase activity, suggesting that g.-556 G>T and g.-692 C>T are functional mutations which could regulate INCENP gene expression by affecting promoter activity and then affect semen quality traits.2. Identification of INCENP gene splice variants and functional verification of related SNP in Chinese Holstein bulls.One novel splice variants was identified in the Chinese Holstein bulls through RT-PCR and clone sequencing, named INCENP-TV. QRT-PCR was performed to detect the expression levels of INCENP gene in heart, liver, spleen, lung, kidney and testis. The result showed that although there is no tissue specificity in INCENP gene, the expression level of INCENP gene in the spleen and testes were significantly higher than other organizations. The reference transcript of INCENP(INCENP-reference) and INCENP-TV newly discovered were expressed in adult bull testis as well as calf testis, but expression of INCENP transcripts in adult bull testis was significantly higher than calf testis. In addition, INCENP-TV was more highly expressed than INCENP-reference in adult bull while the difference between INCENPTV and INCENP-reference in calf was not significant, suggesting that INCENP gene was closely related with spermatogenesis.To explore the mechanism of alternative splicing, we found a SNP site(g.19970 A> G) located in intron 11 by sequencing. Using ESEfinder 3.0 software we predicted that the introduction of G allele of g.19970 site increases the binding sites of SRSF1, SRSF1(IgMBRCA1) and SRSF5. By transfection experiments, RT-PCR technology and cloned sequencing, we detected two transcripts INCENP-reference and INCENP-TV when SNP of g.19970 was A allele. But when A mutated to G, only INCENP-TV was detected, so we speculated this SNP may enhance the activity of splicing factor. In addition, the result of association analysis indicated that g.19970 A> G was significantly correlated with initial sperm motility. 3. Regulation analysis between bta-miR-378 and 3'UTR of INCNEP in Chinese Hostein bullsBy sequencing, we discovered a SNP(g.34078 T> G) in 3'UTR region of INCENP gene. The result of association analysis indicated that g.34078 T>G was significantly correlated with initial sperm motility. Mature miRNA has effect on the expression levels of target gene mRNA. We predicted by RNA22 and RNAhybrid software that bta-miR-378 can combined with 3'UTR of INCENP gene, and it has different affinity for g.34078 T>G-T and g.34078 T>G-G. Wild-type and mutant 3'UTR was respectively connected into PMIR-ReportTM luciferase report vector and co-transfected into MLTC-1 cells with bta-miR-378 vector. Through dual luciferase report system analysis, we found that bta-miR-378 can bind to 3'UTR of INCENP, and reporter gene expression of mutation-type was lower than wild-type, indicating that miR-378 has a stronger affinity with g.34078 T>G-GG than g.34078 T>G-TT. It is reasoned that, bta-miR-378 can be combined with the 3'UTR of INCENP, thereby reducing the expression level of INCENP gene. 4. Association analysis of between INCNEP gene haplotype combinations and semen qualityAccording to g.-556 G>T, g.-692 C>T, g.19970 A>G and g.34078 T>G identified in INCENP, haplotype combinations were constructed and we found 7 haplotypes and 13 haplotype combinations in the experiment group. The result of correlation analysis indicated that ejaculate volume of H1H12 and H2H2 was significantly greater than H2H10 and H11H11(P<0.05). Furthermore, the initial sperm motility of H11H11 and H2H10 was greater than H2H2(P<0.05), suggesting thatg.-692 C>T, g.-556 G>T, g.19970 A>G and g.34078 T>G are functional mutations which have an important role in regulating sperm quality traits. 5. The mRNA expression of different haplotype combinations of INCENP in Chinese Hostein bullsThe qRT-PCR was performed to illustrate the relative mRNA expression levels of INCENP in the sperm cells from different haplotype combinations of bulls. The result showed that INCENP with the mutant haplotype H11H11(TTTTGGTT) and H1H12(CTGTAGTG) showed a significantly(P < 0.05) higher mRNA expression compared with the haplotype combinations H2H10(CTGTAAGG), H2H2(CCGGAAGG) and H9H12(TTTTAGTG), suggesting that g.-692 C>T, g.-556 G>T, g.19970 A>G and g.34078 T>G are functional mutations which regulating expression levels of INCENP gene. |
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