| YY1is a ubiquitously expressed and highly conserved transcription factor that has been implicated in a variety of transcriptional regulatory roles, both as an activator and silencer of gene expression. To understand the expression pattern of YY1gene in buffalo preimplantation embryos, and its effect on DNA methylation regulation, in this study, the ORF sequences of buffalo YYl gene were cloned, the shRNA fragments were screened, and a eukaryotic expression vector of YY1was constrcucted. The effects of over-expression of YY1on buffalo embryo development and genomic DNA methylation were studied.1. The buffalo YY1CDS region was cloned, a fusion expression vector of buffalo YY1gene was constructed. The cDNA of BFF is used as template, the CDS full-length sequence of buffalo YY1gene was amplified by using RT-PCR and touchdown PCR methods, its fusion expression vector pYY1-EGFP-N1was constructed, then it was transfected into293T and BFF cells. The expression level of YYl gene in293T and BFF cells was detected by fluorescence observation and qRT-PCR method respectively. The results showed that the cloned buffalo YY1CDS was1248bp length, the homology of YYl gene among the compared species of Bos taurus, Homo sapiens, Mus musclus, Rattus norvegicus and Gallus gallus was above90%. The YY1recombinant plasmid could express in293T and BFF cells, the YY1gene can activate the expression of DNMT1gene on the level of buffalo somatic cells.2. Screening shRNAs of buffalo YY1gene. Two shRNAs targeting buffalo YY1gene were designed and constructed as lentiviral vector, named as pSicoR-GFP-YY1shRNA1/2. Recombinant plasmid of buffalo YY1(pYY1-EGFP-N1) and its shRNA lentiviral expression vectors (pSicoR-GFP-YYlshRNAl/2) were co-transfected into293T cells at the ratio of1:1and1:6respectively, by using Lipo-LTX reagent, pSicoR-GFP and pSicoR-GFP-1864plasmids were used as blank and negative control respectively. The results showed that both of the designed shRNAs could inhibit the expression of buffalo YY1gene when detected by qRT-PCR and Western-blot methods, shRNA2fragment had higher silence effect than that of shRNA1.3. Effect of YY1over-expression on buffalo embryo development and genomic DNA methylation. The linearized recombinant plasmid (pYY1-EGFP-N1) was injected into the buffalo embryos which were at the stage of8-10h after IVF. The effect of plasmid injection concentration, different DNA plasmids on buffalo preimplantation embryo development and exogenous gene expression was analyzed, effect of over-expression of YY1on buffalo embryo development and expression of DNMT1gene was explored. The results showed that there was no significant difference on blastocyst rate (P>0.05) when25μg/mL pYYl-EGFP-N1plasmid was injected into the cytoplasm of fertilized egg, which would obtain a significantly higher EGFP positive blastocyst rate compared with other injection concentration (5,15,50μg/mL)(P<0.05). There had no significant difference between the two DNA plasmids on the exogenous gene expression rates in blastocyst (P>0.05), and no difference between the YY1over-expression and no-injected groups on blastocyst rate(P>0.05). The expression patterns of YY1and DNMT1gene in buffalo preimplantation embryos showed an opposite trend. With the development of buffalo preimplatation embryos, the expression of YY1gene increased gradually, the highest expression was at the blastocyst stage, reversely, the expression of DNMT1gene was higher before the morula stage, the blastocyst had the lowest level. The over-expression of YY1gene could promote the expression of DNMT1gene in buffalo embryos, as well as the DNA methylation level.In conclusions:the CDS sequences of buffalo YY1gene is highly conserve during biological evolution. The YY1recombinant plamid can express in BFF cells, the buffalo YY1gene can activate the expression of DNMT1gene on the level of buffalo somatic cells. Two shRNAs targeting buffalo YY1are screened. The expression patterns of YYl and DNMT1gene in buffalo preimplantation embryos reveal an opposite trend. The over-expression of YY1gene can promote the expression level of DNMT1gene in buffalo embryo, as well as the DNA methylation level. |
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