Homogenetic Amplification And Expression Study On Some Testicular Mirnas In Large White Pig

MicroRNAs(miRNAs) are 22nt,endogenous non-coding RNA(ncRNA) molecules that act by binding to the complementary sequence of target messenger RNAs(mRNAs) and regulate gene expression at post-transcriptional level in eukaryotes.Research revealed that these small molecules played very important regulatory roles in the processes of physical development,proliferation and differentiation of cells,cell apoptosis,carcinogenesis.As research of identifying and determining the function of miRNAs is improving rapidly,more and more evidences show that miRNAs are involved in the course of animal testis differentiates,spermatogenesis,which is one of the most complicated cell developments containing the processes of both cell proliferation and differentiation,and might play key roles in the course.However,the reports still remain limited in in the genetic mechanism of microRNAs by targeting key gene mRNAs for cleavage or translational repression in spermatogenesis,especially in pigs.Our research includes target gene prediction,expression pattern of different developmental stages of testis development and construction of miRNA expression vectors.The results are as follows:1.Identifying miRNA genes from porcine testis and bioinformatics analyzingWe chose miRNA genes according to the expression status of genes in human and mouse testis and the prediction information in miRBase.Sequences were cloned and sequenced using cDNA from testis tissue of 35-day,60-day,90-day,and 180-day Large White pig.And 10 miRNA genes were cloned and sequenced from porcine testes.To enquiry the targets genes of in the internet with the RNAhybrid and RNA22,six putative mRNA targets for the testicular miRNAs were predicted.The 3'UTR of SPAM-1 mRNA contains potential binding site for let-7c.The 3'UTR of AQN-1 mRNA contains potential binding site for miR-20.The 3'UTR of HAS3 mRNA contains three potential binding sites for miR-29b,miR-34b and miR-136.The 3'UTR of RNF4 mRNA contains potential binding site for miR-145.The 3'UTR of ELSPBP1 and SMCP mRNA contains potential binding sites for miR-136.2.Expressional analysis of some testicular miRNAs in different developmental stages of Large White pigspri-miRNAs expression were measured using RT-PCR,and miRNAs expression were measured using qRT-PCR relying on primer extension conversion of RNA to cDNA by reverse transcription in the testes of 35-day,60-day,90-day,and 180-day Large White pigs.The levels of pri-miRNAs and miRNAs showed different developmental patterns of expression,miR-20(p＜0.01) was down-regulated in the four stages and miR-23a(p＜0.01) was up-regulated obviously in the first three-stage.3.Construction of 4 porcine miRNA expression vectorspri-miRNA amplified products were connected to plasmid pcDNA3.1(+),the recombinant plasmids would be sent to some company to fulfill the sequences analysis. Four recombinant plasmids i.e.plet-7c,pmiR-20,pmiR-23a and pmiR-122 were transfected into swine testicular cell.qRT-PCR were used to detect the three miRNAs expression in the transfected swine testicular cell,and the data showed that three miRNA expression vectors could drive functional miRNA expression,and they could be used to screen functional miRNAs.