Cloning And Expression Of PiRNAs In Chicken And Repression Of Piwi By RNA Interference In Vitro

Piwi-interacting RNAs (piRNAs) are endogenous small RNAs abundant in the germline, and Piwi, protein-coding gene discovered in germ cells that has been implicated in maintenance of germline stem cells and spermatogenesis across animal species. Since piRNAs are enriched in the germ cells and drastically reduced in Piwi-/-testes, which suggests a possible role for germline maintenance and gametogenesis. However, the structural commonality, biogenesis, expression profiles and function of piRNAs, as well as the mechanism of its association with Piwi are still not well understood. This paper was designed to explore characteristics of genetics, mechanism of occurrence and disappearance for poultry piRNAs, and accumulate basic materials for research about bird small RNA-binding protein and practical application. So, we took chicken as the model to obtain chicken piRNAs and explore genetic characteristics and expression profiles:we discovered19piRNAs sized23-39nt by constructing cDNA library of small RNAs, T-A cloning and sequencing in chicken testicular tissue, and according to size, homology and secondary structure, three different sequences were selected for analyzing temporal and spatial expression of piRNAs by using Q-PCR technology in different tissues at different growth and development stages of Chinese indigenous Rugao chicken and introduced recessive white feather chicken. Then, we first silenced Piwi in chicken PGCs by RNA interference technology in vitro and established method of Piwi-siRNAs transfection, and select siRNAs with the highest inhibitory efficacy:chicken PGCs were transiently transfected by LipofectamineTM2000. Then, siRNAs with the highest inhibitory efficacy were evaluated and simultaneously detected mRNA relative expression of three PGCs-specific marker genes CVH, Dazl and CDH in germline and three most important transcription factors of PouV. Nanog, Sox2for regulations of self-renewal and pluripotency of stem cells. The results were presented as follows:1. We found that consistent with other organisms, distribution of piRNA-encoding sequences in the chicken genome were found to be asymmetrically distributed on chromosomes, meanwhile, displayed a preference for intergenic regions across genome. Interestingly, unlike miRNAs with unique stem-loop structures, distinct secondary structures of piRNAs were predicted.2. In addition, chicken piRNAs were not only abundant in germline, but also existed in somatic tissues, and expression level was influenced mainly by different piRNAs, breed and gender. The expression level of gga-piR-1was the lowest in all tissues of two breeds and reached the same trend at week12, while gga-piR-4showed moderate expression levels. gga-piR-5revealed the highest and moderate levels in all tissues of the male Rugao chicken and recessive white feather chicken, respectively and they were up to peak at week8for the male Rugao chicken, whereas it expressed moderately in both female breeds.3. siRNAs targeting Piwi can block Piwi mRNA expression in PGCs efficiently and specifically: compared to negative control group, three Piwi-specific siRNAs inhibited Piwi mRNA expression distinctly at different time, and knock-down efficacy was influenced by position of target gene and transfection time, that is, Piwi mRNA expression was most obviously suppressed at24h and increased gradually at48h, and decreased at72h. The comparison between negative control group and blank control group was not significant (P>0.05), while negative control group and Piwi-specific siRNAs showed significant (P<0.05). Inhibitory efficacy of siR-3was higher than other two siRNAs. Therefore, siR-3at48h after transfection was selected for future experiments.4. In the Piwi knock-down PGCs, compared with negative control group, the mRNA levels of CVH、Dazl and Nanog raised obviously (P<0.05), whereas PouV and Sox2were significantly declined (P<0.05). No significant difference was measured for CDH (P>0.05). Thereby, In chicken PGCs, Piwi possibly up-regulated PouV and Sox2, by contrast, down-regulated CVH, Dazl and Nanog, and no interaction with CDH was existed.