Sequencing, Expression Analysis Of Small RNAs In Porcine Testis And Mutation Detection Of PIWIL1 Gene

Piwi-interacting RNAs （piRNAs） are a class of small RNAs exclusively expressed in male germ cells. piRNAs are between 26 nt and 31 nt of size, which can bind to PIWI class of Argonaute proteins to silence gene and maintain germ cells. At present, the research in piRNAs focused on germinal tissues and the effect on breeding character in small animals such as drosophila and mice, the isolation and identification of porcine piRNAs is poorly inverstigated. Our research includes Solexa seep sequencing in porcine testis day 180, data analysis and chosing candidate piRNAs, verification in different expression of piRNAs/miRNAs in porcine testis of different stages and different tissues by RT-PCR, piRNA binding protein-PIWIL1 gene sequencing and mutations analysis. The main results are as follow:1. Solexa deep sequencing of porcine testis and bioinformatics data analysisTotal RNA in porcine testis from 3 Large White boars at the age of 180 day were isolated and mixed equally, and then sent to BGI company in shenzhen for Solexa deep sequencing.9,533,729 reads were generated,1,961,666 low quality re ads were removed. Finally,6,913,561 reads were left after removing adapters from 5’/3’and other contamination.4,527,258 reads were macthed in pig genome （gen bank\Rfam database in NCBI and genome\ repeat\ exon\ intron\ miRNA\ rRNA\ t RNA\ snRNA\ snoRNA\ scRNA databases of BGI）; 65 miRNAs reported in pig w hich 237,590 total reads（792 unique reads） represented were detected and 74 SNPs were detected in 14 miRNAs of them,104 novel miRNAs were discovered and t he secondary structures and formation of them were prediced,47 of them were m atched with other species in miRbase（http://www.mirbase.org/）,466,757 target gene of 95 novel miRNAs were predicted. piRNAs were predicted by piRNApredictor（ http://59.79.168.90/piRNA/analysis.php） and data were counted. Candidate piRNAs were chosen by aligning with piRNAs in pirbase （http://pirnabank.ibab.ac.in/） and t heir characters in length and chromosome distribution.2. The verification of Real-time quantitative PCR and semi-quantitative PCR in porcine testis7 piRNAs and 5 miRNAs were successfully cloned by stem-loop RT-PCR and sequenced, and then their expression was detect in porcine testis between 60 day and 180 day, The results showed that pi002142\ pi 004234\pi021297\pi017724\pi0 061545\pi004153\m0071₃p were up-regulated both in Large White pigs and Meish an pigs, relative expressions of pi021297\pi017724\pi004153\mi507\mi508₅p\m0102₃p\m-0071₃p were significantly different between Large White pigs and Meishan pigs with 60 day and relative expression of pi0022572 was significantly different between Large White pigs and Meishan pigs with 180 day. Semi-quantitative RT-PCR were used to detect 7 highly expressed piRNAs in porcine testis and piR021 297\piR017724 were shown to be exclusively expressed in porcine testis.3. PIWIL1 sequencing and polymorphism analysisSus_Scrofa PIWIL1 （2376bp） were amplified and sequenced with the pair prime rs from the alignments of Homo sapiens or Mus_musculus PIWIL1 and Sus Scrofa ESTs data in NCBI. The similarity was 91% between Sus_Scrofa PIWIL1 and Homo_sapiens PIWIL1. A T/C mutation in exon 7 was detected by comparative sequencin g and then a PCR-RFLP method was used to detect this polymorphism. SNP were genotyped in Large White （n=476）. DIV （n=131）, Pietrain （n=33）, Duroc （n=29）, W hite Duroc （n=31）, Meishan （n=20）, Huainan （n=26）, Tong Cheng （n=37）. Allele fre quence analysis results showed that T allele frequence ranged from0.73 to 1. Associ ation analysis between polymorphism and litter size traits results showed that there were no significant differences between different genotypes, however CC genotype h ad a tendency to have a larger litter size than TT or TC genotype.