The PIWI protein regulates gene expression at the epigenetic and post-transcriptional level with a variety of endogenous small non-coding RNAs. In poultry, the biological function of the PIWI protein and PIWI binding to small RNAs had not been determined. The present study cloned and analyzed the sequences of the PIWIL1 protein. We also characterized PIWIL1 binding to small RNAs from adult quail testis, where the PIWIL1 protein is specifically expressed. Small RNAs showed a strong peak at 24–27 nt in the testicular RNA library, mapped primarily to repeat sequences and were similar to rasiRNAs. MicroRNAs (miRNAs) were abundant in the ovarian RNA library at a peak of 22 nt.

Salmonella enterica Enteritidis (S. Enteritidis) and Salmonella enterica Pullorum (S. pullorum) are regarded as a threat to poultry production. This study's aim is to characterize the expression profiles in response to three different challenges and to identify infection-related genes in the chicken spleen and caecum. Groups of the Chinese chicken breed Langshan were challenged with either S. Enteritidis, S. pullorum, or poly(I:C). The concentrations of cytokines and antibodies and the Salmonella colonization level of the caecum and liver were detected in each group at 7 days postinfection. Expression microarray experiments were conducted using mRNA isolated from both spleen and caecum. Crucial differentially expressed genes (DEGs) associated with immunity were identified. Four DEGs were identified in spleen of all three challenge groups (RBM16, FAH, SOX5, and RBM9) and different four genes in caecum (SOUL, FCN2, ANLN, and ACSL1). Expression profiles were clearly different among the three challenged groups. Genes enriched in the spleen of birds infected with S. pullorum were enriched in lymphocyte proliferation related pathways, but the enriched genes in the caecum of the same group were primarily enriched in innate immunity or antibacterial responses. The DEGs that appear across all three challenge groups might represent global response factors for different pathogens.

Piwi gene involves in the germline stem cells self-renewal, transposon silencing and post-transcriptional gene regulation in the majority of organisms; however, the biological function of Piwi gene in poultry remains unclear. Here we cloned the Piwi-like 1 (Piwil1) gene and characterized its expression in the Langshan chickens during the development. The results showed that the PIWIL1 protein was the homolog of mice MIWI and human HIWI proteins (100 % identity), and encoded a cytoplasmic protein including the two conserved domains PAZ and PIWI. In males, the expression of Piwil1 gene showed a bimodal distribution in the gonads during embryogenesis with peaks at embryonic 14.5 and 17.5-18.5 days respectively. After puberty, the expression of Piwil1 gene increased sharply and reached a high level at the sexual maturity. The mRNA expression of Piwil1 gene at 27 weeks of age is 35-40 times that of 0 week of age, indicating that the high expression of Piwil1 gene was essential to maintain the spermatogenesis. In females, the expression of Piwil1 gene showed a unimodal distribution in the embryonic gonads. A strong peak appeared at E16.5-17.5d when the primary oocytes have entered the prophase I of meiosis. Subsequently, the expression of Piwil1 gene decreased gradually and kept at the low level during the embryogenesis. So Piwil1 gene was likely to play an important role during the meiosis I. This report filled in partly the gap of the Piwi gene researches in poultry and defined our research directions in future.

Piwi-interacting RNAs (piRNAs) are small RNAs abundant in the germline that have been implicated in germline development and maintenance of genomic integrity across several animal species including human, mouse, rat, zebrafish and drosophila. Tens of thousands of piRNAs have been discovered, yet abundant piRNAs have still not been detected in various eukaryotic organisms. This is a report on the characterization, cloning and expression profiling of piRNA-like chicken RNAs. Here, we identified 19 piRNAs, each 23-39 nucleotides long, from chicken testis using a small RNA cDNA library and T-A cloning methods. Three different pilRNAs were selected according to size, homology and secondary structure for temporal and spatial expression by Q-PCR technology in different tissues at five growth and four development stages of Chinese indigenous Rugao chickens (RG) and introduced recessive white feather chickens (RW). We found that, consistent to other organisms, pilRNA-encoding sequences within the chicken genome were asymmetrically distributed on the chromosomes while displaying a preference for intergenic regions across the genome. Interestingly, unlike miRNAs with unique stem-loop structures (mature miRNAs form stem section and the rest form loop section), distinct secondary structures of pilRNAs were predicted. In addition, chicken pilRNAs were not only abundant in the germline but also existed in somatic tissues, where, expression levels were influenced mainly by different pilRNAs, breed and gender. Taken together, our results suggest that two distinct secondary structures exist between pilRNAs and miRNAs, which may clarify the splicing and processing mechanisms of the two small RNAs are possible different. Moreover, our results suggest that pilRNAs may not only be confined to development and maintenance of the germline but may also play important roles in somatic tissues. Additionally, different pilRNAs may be involved in the unique regulatory machinery of complex biological processes.