Expression, Cloning And Promoter Methylation Status Of The Genes Correlated With Meiotic Recombination In The Testis Of Cattle And Cattle-Yak

Cattle-yaks, the F1hybrid between cattle and yaks, exhibit significant hybrid vigor, but the F1cattle-yak is infertile. Researches showed that meiosis disturbance of spermatocyte and the synaptonemal complex can not form normally, probably had relationships with the infertility of cattle-yak. The homologous recombination have started before the homologous chromosomes synapsis in meiosis, however the study about meiotic homologous recombination of cattle-yak has not been reported. To study the relationship between meiotic recombination and infertility of cattle-yak, we showed the expression levels of the genes correlated with meiotic recombination (Spoll、Meil、Dmc1、 Rad51、 Msh4、Msh5、Mlh1and Exo1) in testes of cattle and cattle-yak hybrid with Real-time qPCR. The coding regions of the differentially expressed genes in cattle and yaks were cloned and the structures were analyzed by bioinformatics method. Laboratory preliminary study found that abnormal methylation of cattle-yak testicular tissue genomic and imprinted genes, abnormal expression of imprinted genes, and genomic imprinting abnormalities may be one of the main cause cattle-yak male sterility. In order to further reveal the relationship between methylation with cattle-yak male sterility, the methylation statute of promoter region of the differentially expressed genes of cattle and cattle-yak were detected by bisulfite sequence PCR. Then using the luciferase reporter gene system and in vitro methylation experiments we detect the core promoter regions of differentially methylated genes. Finally we analysis of promoter methylation on gene expression regulation, in order to provide a theoretical basis to clarify mechanism of cattle-yak male sterility. Results as below:1. mRNA expression of genes correlated with meiotic recombination in testes of cattle and cattle-yak hybrid We investigate the mRNA expression of genes correlated with meiotic recombination by Real-time PCR. The results showed that the mRNA expression levels of genes correlated with meiotic recombination (Spoll、Meil、 Dmcl、 Rad51、Msh4、Ms1i5、Mlhl and Exol) in cattle-yak with character of male sterilize were lower than cattle with normal meiosis. The levels of Spo11、 Meil、 Dmcl、Msh4and Mlhl in cattle-yak testicular tissue were significantly lower than cattle (P<0.01), and the levels of Msh5and Exol in cattle-yak were lower (P<0.05) than that in cattle, so that peculated that mRNA expression levels of Spol1, Meil, Dmcl, MSH4, MSH5, MLH1and Exol in testicular tissue may with cattle-yak male sterility have a certain relationship.2. Gene clone and bioinformatics analysis of the5genes correlated with meiotic recombination in cattle and cattle-yakBy cloning and sequencing technology on the five homologous recombination genes (Spoil, Meil、 Dmcl、 Msh4and MLH1) of cattle and cattle-yak testis that mRNA expression level of significant difference, we cloning and Analysis their coding region. The result showed that the translation regions of Spoll、Meil、 Dmcl、 Msh4and Mlhl were1188bp、3837bp、1023bp、2785bp and2294bp. The homologies of the coding region nucleotide sequences residues between the cattle and cattle-yak were100%respectively, and having a high homology with other species of mammals. The Spol1、Meil、Dmcl、. Msh4and Mlh1were located at chromosome13,5,5,3and22in bovine by chromosomal location, and the genes’ structure similar to other species mammals. Protein SPO11, MEI1, DMC1, MSH4and MLH1consists395,1278,340,934and758amino acid residues respectively, amino acid sequence also have high homology with other mammalian species, and contains typical structural domains same with other species of the mammals.3. Methylation level of the five genes correlated with meiotic recombination promoters analysis in yak and cattle-yak testicular tissuePromoter CpG island prediction found that the five genes (Spoil, Msh4, Meil, Dmc1, Mlh1and Exo1) correlated with meiotic recombination in bovine5’-regulatory region all contained CpG islands. We detected the promoter methylation of the five genes in the testes of cattle and cattle-yak hybrid by bisulfite sequencing techonology, the results showed that Spoll, Meil, Dmcl and Msh4in the testes of cattle-yak hybrid gene promoter of methylation were higher than that of cattle, which methylation level of Spoll, Meil and Msh4gene in cattle-yak testis were significantly higher than cattle (P<0.01). The methylation level of Dmc1gene in cattle-yak testis was higher than cattle (P<0.05), and the methylation levels of Mlhl gene were low both in cattle and cattle-yak testis, and the difference is not significant.4. The methylation of the Spoll and MSH4gene core promote in cattle influenced the activity of genes promoter regionAs the research object to Spoll and Msh4genes that were extremely significant difference to the degree of methylation in the cattle and cattle-yak testis, we analysis of methylation in promoter regulate the genes correlated with meiotic recombination in cattle. We detected the promoter methylation of Spoll and Msh4in the liver of cattle and cattle-yak hybrid by bisulfite sequencing techonology, the results showed that methylation level of Spoll and Msh4gene in cattle-yak livers (96.25%,67.39%)were significantly higher than cattle testis (P<0.01), further prove that Spoll and Msh4promoter region methylation is closely related to expression. We cloned1359bp of the5’flanking region sequence of Spoll and3480bp of the5’flanking region sequence of Msh4in cattle. The construction of four dual lucifease expression vector of Spoll were built, and the construction of three dual lucifease expression vector of Msh4were built, then transiently transfected COS-7and GC-1cells respectively. The results showed that the core promoter region of Spoll at-62nt～-293nt and the core promoter region of Msh4at-8nt～-160nt. To further study core promoter methylation on the expression level of the Spoll and Msh4, we modified core promoter region of dual luciferase expression vector in vitro methylation by M. SSSI methyl transferase, then we transiently transfected COS-7cells and GC-1cells. We found that the expression of the methylation vectors activity reduced significantly (p<0.01). And the results demonstrated the regulatory role of methylation in the gene expression.