Preliminary Study On The Biological Function Of Piwil1Gene And PiRNAs In Poultry

Piwi (P-element induced wimpy testis) gene involves in germline stem cell self-renewal, meiose, RNA silence and transcriptional regulation. In model organisms, it has been found that the epigenetic and transcriptional regulation of PIWI protein mediated by piRNAs (Piwi-interacting RNAs) was the key factor in the process of maintaining germline stem cells, gametogenesis and fertilization, but Piwi gene has rarely been reported in poultry. PIWI protein is widely present in a variety of animals, but its expression is mostly restricted in the gametogenesis and early embryonic development and the expression pattern is also inconsistent, which impedes the study on the mechanism of PIWI protein in the reproductive process. In this regard, the poultry has an incomparable superiority for that their gametes and embryos are easy to harvest and manual operation, providing a research platform for exploring the regulation mechanism of PIWI protein. As the major source of animal protein for most human populations, the poultry industry production is currently at a critical period of development and remains many problems to be solved. On the one hand, the reproductive capacity of poultry is declining and each year five to twelve percents of the azoospermia chicken were eliminated in commercial varieties. On the other hand, some RNA viruses, such as avian influenza virus, avian leukosis virus, brought huge losses to the poultry industry for that their frequent variation resulted in the failure of disease prevention and treatment. In this study, the Chinese indigenous chicken breed-Langshan chickens were selected as animal models and the relatively comprehensive and systematic research on the Piwill gene was carried out at the following four aspects of gene cloning, gene expression, transcriptional regulation, and function suppression respectively. Meanwhile, this study selected the Japanese quails as a reference group and characterized the expression profiles of quail Piwill gene and PIWI binding to piRNAs. This study will accumulate basic data for exploring the biological function of Piwi gene and the enhancement of reproductive capacity in poultry, as well as provide a new theoretical basis for the formation of germ cells and the development of novel RNA treatment method.Firstly, this study has successfully cloned the full-length cDNA sequence of chicken Piwill gene from the Langshan testicular tissue at12weeks of age with reference to the mRNA sequence of the red jungle fowl Piwill gene by RT-PCR and RACE technology. This sequence includes an ORF of2604bp,5’UTR of161bp and3’UTR of660bp. It is predicted that the ORF encoded867amino acid residues and contained the two conserved domain PAZ and PIWI that characterized by Ago family proteins. The result of sequence analysis on the promoter region of PIWIL1gene indicates that a long489bp CpG island exists. However, there is no a TATA box in the region of-25to-30bp upstream of the transcription initiation site that is a typical DNA sequence element in the eukaryotic gene promoter.To reveal the tissue and cellular expression patterns of Piwill gene, this study has detected the mRNA expression of Piwill gene in different tissues of Langshan chicken at12weeks of age (testis, brain, hypothalamus, kidney, adrenal, thyroid and ovary) and different germline cells (PGCs, SSCs, Spermatogonia/Spermatocytes and mature sperms) by Real-Time qPCR technology. The result shows that the mRNA expression of Piwill gene in testes was significantly higher than that of other tissues, and the mRNA expression of Piwill gene in spermatogonia/spermatocytes of adult testis and mature sperms was significantly higher than that of PGCs and SSCs. The higher mRNA expression of Piwill gene in mature sperm suggests that Piwill gene may play a role in the process of sperm insemination.To explore the role of Piwill gene in chicken spermiogenesis, this study has detected the mRNA expression of Piwill gene in six tissues (testis, brain, hypothalamus, kidney, adrenal, and thyroid) at five life stages (respectively0,5,10,12,27weeks of age) in Langshan chickens by Real-Time qPCR technology. The result shows that the mRNA expression of testicular Piwill gene increases with the growth of individuals, while the mRNA expression of Piwill gene in other tissues maintains the basic level. In testes, before10weeks of age, the mRNA expression of Piwill gene is lower and stable; after10weeks of age, the mRNA expression of Piwill gene rises sharply. The mRNA expression of Piwill gene at27weeks of age is35～40times that of0week of age. The mRNA expression of Piwill gene increased significantly in testis reaching sexual maturity and is higher in spermatogonia/spermatocytes and mature sperm, suggesting that Piwill gene expression should be essential to maintain chicken spermatogenesis. In addition, this study also detected the mRNA expression of Piwill gene in ovaries at above five life stages in Langshan chickens. The result shows that the mRNA expression of Piwill gene decreased with the growth of individuals, which is opposite to that in testes.To explore the role of Piwill gene in the gonad development of chick embryos, this study has detected the mRNA expression of Piwill gene in the male and female gonads during the embryonic period by Real-Time qPCR technology. In male gonads, the mRNA expression of Piwill gene showes a bimodal distribution with peaks at embryonic14.5day and17.5～18.5days respectively, suggesting that the high expression of Piwill gene in this point may be related to the "DNA re-methylation" and self-renewal of spermatogonia stem cells. In female gonads, the mRNA expression of Piwill gene shows a unimodal distribution with a strong peak at embryonic16.5-17.5days when the primary oocytes have entered into the prophase of meiosis I, suggesting that Piwill gene may play an important role in the process of oocyte meiosis I.Previous studies have shown that Piwi gene was mainly expressed in the germline cells with the specificity of the germline. Our preceding results also show that the mRNA expression of chicken Piwill gene differs among tissues and cells. To explore the specific expression mechanism of Piwill gene in chicken, this study firstly has searched out the core promoter region of Piwill gene using a series of deletion mutants of the promoter region. A series of deletion fragments of the promoter region of Piwill gene were inserted into the upstream of luciferase reporter gene5’-end and successfully constructed seven recombinant vectors containing the chicken promoter sequence. Then, these seven recombinant vectors were respectively co-transfected GC-1(germline cells) and COS-7cells (non-germline cells) with Renilla luciferase reporter gene vector (internal control). The result of luciferase activity assay shows that the5’flanking sequence from-90to-43is important for its transcriptional activity. The transcription regulation in eukaryotes depends largely on the interaction between cis-acting elements and trans-acting factors. Because the Piwill gene lacks a TATA box, putative transcription elements were identified using the TFSEARCH software. A CCAAT box (-56--52) and a TCCC box (-34to-31) was observed within the core promoter region from-90to-16, which are putative binding sites of transcription factors NF-Y and Ik-2respectively. To functionally determine the importance of these elements, site-directed mutagenesis was performed and three mutants were constructed respectively, containing a single mutated CCAAT box, a single mutated TCCC box and their double mutant. Subsequently, these three mutants were respectively co-transfected GC-1and COS-7cells with the internal control plasmid. The result of luciferase activity assay shows that the promoter activity of all mutants reduced, as compared to the wild type construction, suggesting that both the CCAAT box and TCCC box are important for the promoter activity of Piwill gene.Based on the previous results, the chicken Piwill gene is highly expressed in the adult testes. However, as compared to the mammals, the harvest of all levels of spermatogenic cells in the seminiferous epithelium of seminiferous tubules is under exploration. Considering that PGCs are progenitor cells in germline in which chicken Piwill gene is expressed and the technology of PGCs harvest and culture in vitro is relatively mature, this study choose PGCs as the cell model for exploring Piwill gene function. To explore the function of Piwill gene in the chicken germline, the mRNA expression of Piwill gene was down regulated in chicken PGCs using DNA vector-based RNAi technology-shRNA expression cassettes. It is found that different siRNA target sequences showed a different gene silencing effect on the mRNA expression of Piwill gene with a drop of9to36%. Subsequently, two ORFs of CR1-B and CR1-F subfamily were examined by Real-Time qPCR technology after most efficient knock-down in chicken PGCs. The result shows that only the mRNA expression of CR1-F ORF1increased in knock-down PGCs compared with control, to some extent suggesting that Piwill gene play a role in inhibiting the transposon activity.To accomplish more efficient delivery of siRNA in vitro, this study tested lentivirus vector-based RNAi technology to suppress the mRNA expression of Piwill gene in chicken PGCs. The over-expression cassette of chicken Piwill gene was firstly successfully constructed served as the target of miRNA interference sequences. The result of cellular localization shows that the over-expression cassette of chicken Piwill gene successfully encoded a cytoplasmic protein. Secondly, miRNA interference expression cassettes were respectively co-transfected HEK293cells with Piwill over-expression cassette. The most efficient miRNA interference cassette would be screened out using Real-Time qPCR technology. Then, the miRNA interference expression cassette was successfully subcloned into the destination vector via Gateway technology to be a Piwil1-miRNA lentiviral expression vector. Finally, the miRNA lentiviral expression vector was co-transfected293FT cells with lentiviral packaging vectors to produce a lentiviral stock. The lentivirus titer is1×108TU/ml. The result of cell transduction shows that the lentivirus construct could successfully reduce the expression of Piwill gene.To investigate the conservative fuction of Piwi gene in poultry, this study selected the Japanese quails as a reference group and has successfully cloned the full-length cDNA sequence of quail Piwill gene from the adult testicular tissue by RT-PCR and RACE technology. This sequence includes an ORF of2595bp,5’UTR of142bp and3’UTR of667bp. It is predicted that the ORF encoded864amino acid residues with deletion of N-terminal3acid residues compared to that of chicken Piwil1gene, and contained the two conserved domain PAZ and PIWI that characterized by Ago family proteins. In adult quails, the PIWIL1protein is also specifically expressed in testes, suggesting that it has a conserved fuction between chicken and quail. In order to further explore the regulation mechanism of PIWI protein in spermiogenesis and transposon activity, this study cloned the sequences of small RNAs from adult quail gonads by deep sequencing technology and characterized PIWIL1binding to small RNAs. Small RNAs show a strong peak at24～27nt in the testicular RNA library and map primarily to repeat sequences followed by encoded gene sequences, which are similar to the Drosophila piRNAs, suggesting that poultry piRNAs could play a role in maintaining the structure of heterochromatin and controlling the repetitive sequences transcription and tranposon transposition. PIWIL1protein binds to24～25nt piRNAs in the immunoprecipitation library, while miRNAs are abundant in the ovarian RNA library with a peak of22nt, suggesting that the biological fuction of PIWIL1protein is mediated by piRNAs in poultry.