The Relationship Between The Analysis Of Yak Boule Gene Alternative Splicingan And Its DNA Methylation And Male Sterility Of Cattle-yak

In this study, healthy adult male yak and cattle-yak was studied,we use RT-PCR to clone Boule gene, analyze the structure and fanction of the sequence and its encoded protein, use real-time fluorescence quantitative PCR to detect the expression level of alternative splicing variant of Boule gene in the yak and cattle-yak and use the methods of homologous amplification, and PCR to clone the sequence of differences methylated region （DMR） of Boule gene in the testicular tissue of yak,use sodium bisulfite sequencing to detect methylation status of Boule gene DMR,that provide a theoretical basis of study the relationship between male sterility of cattle-yak and DNA methylation.1The screen of Boule gene alternative splicing variantsIn this experiment,according to Boule gene sequence of cattle （NM₀01102115） we design two pairs of primers, use RT-PCR to amplify the open reading frame of Boule gene,and the results are that the first pairs of primers are802bp and766bp, the second pair of primers amplify the sequence is362bp, after assemble the two fragment sequence the length are:1037bp and1001bp, GenBank accession number are GU187434; GU187435.we have found the standard "GT","AG" site which strictly follow the "GT-AG" splicing rule after analyze these two sequences.Therefore, we can conclude that these two sequences of the Boule gene is two alternative splicing variants, named Boulel, Boule2.2The analysis of the biological information of Boulel、Boule2nucleotide sequence and proteinWith bioinformatics biology of network resources and associated software we filter out open reading frame of Boulel the length is849bp, compared with the normal sequence that absence5’end of exon3the length is36bp bases, that result in12amino acid residues deletion in open reading frame and we have found the splicing site in RRM domain, but that do not affect the RNP1, RNP2region, There are no splicing in Boule gene Reapt area; The length of Boule2open reading frame is813bp, that absent5’end of exon3compared with the normal sequence the length is72bp base, that result in24amino acid residues deletion in open reading frame and we have found the splicing site in Reapt domain, There are no splicing in Boule gene RRM domain.With the analysis of two protein we found Boule1lose a a-helix compaired with Boule2and their three-dimensional structure of RRM also confirmed this point, so we speculate Boule1have a close relationship with male sterility3The expression levels of Boulel and Boule2mRNA in the testis of yak and cattle-yak.Quantitative analysis of Boulel and Boule2mRNA expressed in testis of yaks and cattle-yak by Real-time PCR, showes that the expression of Boulel in Yak testis is higher than in cattle-yak, and the expression levels of cattle-yak is significantly differ from yak（P<0.01）. likewise, expression levels of spliceosome Boule2is higher in Yak testis than that in cattle-yak, while, the expression levels of cattle-yak is no significant differ from Yak（P>0.05）.4Cloning and sequence analysis of CpG island of yak Boule5’endIn this study, according to cattle Boule gene sequences （NW₀01494657.1） we design primers to amplify5’end sequence of Boule gene in yak, and analyze the methylation level of Boule gene in yak, cattle, and cattle-yak,The results is that:CpG islands exist in Boule gene5’end of Yak;CpG island length2000bp, containing the promoter region, first exon and first intron; cattle-yak Boule5’ end of DNA methylation level （17.5%） is very significantly higher than cattle （5.2%） and yak （7.0%）（P<0.01）. These results demonstrated that the hypermethylation of Boule gene was in accordance with the phenotype of Boule deficient expression and male infertility in cattle yaks, and indicated that the methylation status of Boule gene played an important role in Boule transcriptional regulation and maybe have a severe effect on meiotic process of spermatogenesis and male sterility in cattle yaks.