| microRNAs (miRNAs) are 18-22nt endogenous, high conservative, non-coding small RNA (ncsRNA) molecules that exist in eukaryotic genome widespread. miRNAs play an important role in animal testis development, differentiation of cells, and the process of spermatogenesis. However, the genetic mechanism of miRNAs targeting key mRNAs in spermatogenesis has not been fully understood, and the reports remain limited in pig miRNAs gene and miRNAs related to the production traits of pigs. Therefore, the isolation and function research on miRNAs in pigs testis is a new topic that is worth investigating. Our research includes isolating and verifying a batch of miRNAs in pigs testis that have different expression in different stages by miRNAs microarray and Real-time PCR; predicting target gene and analysing expression pattern of target genes in different stages of testis development; constructing of miRNAs expression vectors and Dual-Luciferase Reproter vectors. The main results are as follows:1. Identifying differential expression miRNAs from pig testis by miRNAs array and Real-time PCRWe chose 60-day and 180-day Large White pig testis as research materials, synthesize miRNAs microarray covering 1260 miRNAs in LC Sciences. One hundred and twenty-nine miRNAs represented by 164 reporter miRNAs were expressed differently (p<0.1). Fifty-one miRNAs were much up-regulated and 78 miRNAs were down-regulated in mature testes. The total RNA were 3’-extended with a poly (A) tail using poly (A) polymerase, and then reverse transcribed to cDNA using the RT-PCR System. Nine from those differentially expressed miRNAs such as mir-762 were validated using qPCR assay. Pearson correlation coefficient is above 0.5.2. Target genes prediction and expression analysis of target genes in different developmental stages of pig testisFour hundred and fouty-five pig cDNA sequences were randomly selected from GenBank and a GO term and KEGG pathway annotation was performed using the DAVID gene annotation tool (http://david.abcc.ncifcrf.gov/). GO and KEGG analysis showed that these genes were involved in the life process including multi-organism process, reproductive process and reproduction, and played important roles in 17 pathways. We used the whole cDNA sequences to match with the differentially expressed miRNA sequence by the RNA22 to predict the putative target genes and target sites, and totally 15919 putative miRNA-target sites were detected. About 76.95% of these binding sites located on the CDS of genes.Seven putative target genes associated with testis development and spermatogenesis were selected to confirm their expression patterns by qPCR in the testes of 35-day,60-day, 90-day, and 180-day Large White pigs. The levels of target genes showed different developmental patterns of expression. AQN-1 gene and HAS3 gene were up-regulated in the first three-stage, but down-regulated obviously in sexual mature testes. SPAG1 gene was exactly opposite to them. SMCP gene was up-regulated obviously in sexual mature testes. SPAM-1 gene was up-regulated with the age. There is no specific pattern in the expression of DAZL gene. RNF4 gene was high expressed in 60-day, and down-regulated with the age. By comparing with the expression profiling of miRNAs and target genes, about half of these putative target genes from bioimfomatics analysis were flase positive.3. Construction of miRNA expression vector and Dual-Luciferase Reproter vectorsAmplifiy the pri-miRNA of miR-762 and construct miRNA expression vector, named pmiR-762. Then transfect into swine testicular cell. qPCR were used to detect their miRNAs expression in the transfected swine testicular cell, and the data shows that miRNA expression vectors drive functional miRNA expression. The result shows the recombinant plasmid could be used to screen functional miRNAs. The RNF4 3’-UTR amplified products were linked to plasmid pRL-TK. The recombinant plasmids pRL-RNF4 and pmiR-762 were co-transfected swine testicular cell. Then Dual-Luciferase Detection System was used to detect the interaction between miR-762 and RNF4 gene. The result shows that miR-762 is a negative regulator of RNF4 gene, and miR-762 has different inhibit effection with different genotypic of RNF4 gene. |
