The Expression Spectrum Analysis And Primary Identification Of Target Genes Of Mirna-451in Dairy Goat

It was reported that miRNA involved in the cell proliferation, differentiation, biology development and many metabolism of organism. Meanwhile, it also played an important role in lactation.In this research, based on Solexa high-throughput sequencing technology, the statistics of small RNA in peak lactation and dry period in dairy goat were observed. A series of methods including quantitative PCR, gene clone, adenovirus recombination, cell culture and bioinformatics analysis were used to predict and verify the expression spectrum and target genes of the miRNA-451.1. Analysis of expression spectrum of miRNA-451in different organizations and different lactation periods in dairy goatThe stem-loop real-time fluorescence quantitative PCR was used to detect the expression of miRNA-451in different tissues and different lactation periods. The results indicated that the expression levels of miRNA-451were very low in liver, spleen, lung, kidney, fat, ovarian tissues. The change of the expression level of miRNA-451in four lactation periods (early lactation, peak lactation period, late lactation period and dry period) was similar with lactation curve and had a tendency of gradually reduce during four lactation periods. In general, the expression level of miRNA-451in dairy goat mammary gland tissue was very high, and had a significant difference among different lactation periods, which had a similar trend with that obtained from the high-throughput sequencing.2. Prediction and verification of the miRNA-451target genesING3, CAST and FLT-1were confirmed to be the target genes of the miRNA-451by bioinformatics analysis based on database of bovine target genes. Target sites of ING3and CAST predicted were the same with those verified in cloning and sequencing in dairy goat. Moreover, the predicted target genes of the miRNA-451were verified using dual fluorescence report assay experiment. The results indicated that miRNA-451did not react with the target site of ING3gene but CAST gene. Overlap-PCR site-directed mutation technology was used to mutate the seed region of CAST gene target site. The later results of dual fluorescence report assay showed that miRNA-451could not reduce luciferase activity of mutation plasmid, indicating that miRNA-451could react with the target site of CAST gene. Therefore, CAST gene was the target gene of miRNA-451.3. Construction of recombinant adenovirus containing dairy goat miRNA-451In this study, the sequence of precursor miRNA-451which was404bp from dairy goat was cloned into recombinant adenovirus. Pre-miRNA-451and pAdTrack-CMV were respectively double enzyme digested then recombined to construct the pAdTrack-CMV-miRNA-451plasmid. Then the recombinant plasmid pAd-miRNA-451which containing the sequence of precursor miRNA-451of dairy goat was achieved by pAdTrack-CMV-miRNA-451and pAdEasy-1homologous recombination in E.coli BJ5183completent cells. Then pAd-miRNA-451was linearized by Pac I, and transfected into293A package cells by lipofectamine TM2000, in which the packing was completed. The poison with dairy goat miRNA-451inner was collected for four times. The expression of precursor miRNA-451was analyzed by real-time fluorescent quantitative PCR after adding different amount of adenovirus into293T cells. The results showed that the best Multiplicity of Infection (MOI) of pAd-miRNA-451was200while that of pAd-Control was100. The expressing of pAd-miRNA-451in293T was27times more than that of pAd-Control after72hours infection.