【作者】 陈蓉；

【导师】 陈国宏； 常国斌；

【作者基本信息】 扬州大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 Piwi (P-element induced wimpy testis)基因参与生殖干细胞自我更新、减数分裂、RNA沉默以及转录调控过程。在模式生物上,研究发现piRNAs (Piwi-interacting RNAs)介导PIWI蛋白的表观遗传调控和转录初水平调控过程是生殖干细胞维持、配子形成以及受精的关键因素,但有关家禽Piwi基因的研究报道甚少。PIWI蛋白虽然广泛存在于各种动物中,但大部分只局限在配子发生和胚胎发育早期表达,而且表达模式也不一致,影响了其对生殖过程的具体作用机制的探讨。就此而言,禽类配子、胚胎易于获取且便于人工操作,具有其他生物无可比拟的优越性,可为探讨PIWI蛋白如何发挥调控作用的具体机制提供研究平台。另外,作为人类主要的动物蛋白来源食品,家禽业生产正处于发展的关键时期,仍有不少问题亟待解决：一是繁殖力持续下降,商业品种中每年有5～12%的无精症鸡遭淘汰；二是如禽流感、白血病等一些RNA病毒,因其容易变异造成预防、治疗失败,给家禽产业带来了巨大的损失。本研究以中国地方鸡种——狼山鸡为研究对象,分别从基因克隆、基因表达、转录调控和功能抑制等4个方面对Piwi like1(Piwil1)基因进行了较为全面系统的研究；并以日本鹌鹑为参照群体,分析了鹌鹑Piwil1基因和PIWI蛋白特异性结合piRNAs的表达谱,为探明Piwi基因在禽类中的生物学功能和家禽繁殖力的提高积累基础资料,也为解析生殖细胞发生机制和发展新型RNA治疗方法提供新的理论依据。本研究首先参考GenBank上红色原鸡Piwil1基因的mRNA序列,利用RT-PCR和RACE技术从12周龄狼山鸡睾丸组织中成功克隆了Piwil1基因的全长cDNA序列,包括一个2604bp的ORF,161bp的5’UTR和660bp的3’UTR。生物信息学分析结果表明,该ORF编码867个氨基酸残基,含有PAZ和PIWI两个Ago家族蛋白的保守结构域。启动子区的序列特征分析结果表明,Piwil1基因的5’调控区存在着一个长489bp的CpG岛,转录起始位点上游-25～-30bp区域没有真核基因启动子区典型的DNA序列元件TATA box。为了揭示鸡Piwill基因的组织、细胞表达模式,本研究利用Real-Time qPCR技术分别检测了12周龄狼山鸡不同组织(睾丸、大脑、下丘脑、肾脏、肾上腺、甲状腺和卵巢)和不同类型生殖系细胞(PGCs、SSCs、精原／母细胞和成熟精子)中Piwill基因的nRNA表达量。结果表明,Piwill基因在睾丸组织的表达量显著高于卵巢、肾脏等其他组织；在睾丸精原和精母细胞以及成熟精子中的表达量显著高于PGCs和SSCs细胞。Piwill基因在成熟精子的高表达,推测Piwill基因可能在精子授精过程中发挥作用。为了探索Piwill基因在鸡精子形成过程中的重要作用,本研究利用Real-Time qPCR技术检测了狼山鸡5个发育阶段(0、5、10、12、27周龄)6个组织(睾丸、大脑、下丘脑、肾脏、肾上腺和甲状腺)中Piwill基因的表达情况。结果表明,随着周龄的增加,睾丸中Piwill基因的表达量不断增加,而其他组织中Piwill基因的表达量基本不变。在睾丸组织中,10周龄之前,Piwill基因的表达量变化不大；10周龄之后,Piwill基因的表达量急剧上升；27周龄时,Piwill基因的表达量是0周龄时的35～40倍。性成熟后,Piwill基因在睾丸组织中的表达量急剧增加,而且主要在精原(母)细胞和成熟精子中表达,推测Piwill基因的高丰度表达应该是维持鸡精子发生所必须的。此外,本研究还检测了Piwill基因在狼山鸡上述5个发育阶段卵巢组织中的表达情况。结果表明,随着周龄的不断增加,Piwill基因在卵巢组织中的表达量逐渐降低。为了探索Piwill基因在鸡胚性腺发育过程的重要作用,本研究利用Real-Time qPCR技术检测了胚胎期雌雄鸡胚性腺中Piwill基因的表达情况。结果表明,在雄性睾丸组织中,Piwill基因的表达呈双峰分布,峰值分别位于第14.5天和第17.5～18.5天,推测此时Piwill基因的高表达可能与“DNA re-methylation"过程以及精原干细胞的自我更新有关。在雌性卵巢组织中,Piwill基因的表达呈单峰分布,峰值位于第16.5～17.5天,此时初级卵母细胞已进入减数分裂Ⅰ前期,推测Piwill基因可能在卵母细胞减数分裂Ⅰ过程发挥重要作用。前期研究表明,Piwi基因主要在生殖系细胞中表达,具有生殖系特异性。通过前面的研究结果,发现鸡Piwill基因mRNA在不同组织和细胞中差异表达。为了探索鸡Piwill基因选择性表达的调控机制,本研究首先从基因组DNA水平通过启动子区系列缺失法来寻找Piwill基因的核心启动子区。本研究将启动子区的系列缺失片断连接至荧光素酶报告基因5’端上游,成功构建了7个含有鸡Piwill基因启动子区序列的萤火虫荧光素酶报告基因载体。然后,分别将这7个重组载体与海肾荧光素酶报告基因载体(内参质粒)共转染GC-1细胞(生殖系细胞)和COS-7细胞(非生殖系细胞),荧光素酶活性检测结果均表明Piwill基因5’侧翼区-90--43区域具有重要的转录调控活性。真核基因的转录调控主要依赖于顺式作用元件和反式作用因子之间的相互作用。前面的结果已表明,Piwill基因没有真核基因启动子区典型的顺式作用元件TATA box。为了寻找核心启动子区域的重要转录元件,本研究利用TFSEARCH软件对Piwill基因5’侧翼区进行了预测分析。结果发现,-90--16区域含有CCAAT box (-56--52,转录因子NF-Y的结合位点)和TCCC box (-34--31,转录因子Ik-2的结合位点)两个转录元件。为了探讨这些转录元件的生物学功能,本研究利用定点突变技术构建了3个突变体,分别为CCAATbox单突变、TCCC box单突变和二者的双突变。随后,分别将这3个突变载体与内参质粒共转染GC-1和COS-7细胞,荧光素酶活性检测结果表明,与野生型相比,突变体的启动子活性均发生了降低,这进一步表明CCAAT box和TCCC box可能对Piwill基因启动子活性具有重要作用。前面的研究结果已表明,鸡Piwill基因在成年鸡睾丸组织中高表达。但是,与哺乳动物相比,获取鸡睾丸组织曲精细管生精上皮中各级生精细胞的方法还不成熟。鉴于PGCs细胞是生殖祖细胞,鸡PGCs中表达Piwill基因以及PGCs体外获得和培养相对成熟等优势特性,本研究选择PGCs作为Piwill基因功能研究的细胞模型。为了研究鸡Piwill基因在生殖系中的功能,本研究首先选择基于DNA载体的RNAi技术——shRNA干扰表达载体来抑制鸡PGCs细胞中Piwill基因的表达。结果表明,不同的siRNA靶序列具有不一样的Piwill基因沉默效果,Piwill基因的mRNA表达量相应地下降了9-36%。随后,本研究检测了在最佳干扰效果下CR1-B和CR1-F转座子的mRNA表达量变化。与对照组相比,鸡CRl-F转座子ORF1mRNA的表达量升高,这从一定程度上表明Piwill基因具有抑制转座子活性的功能。为了提高siRNA在体内的传递效率,本研究选择基于慢病毒载体的RNAi技术来抑制鸡PGCs细胞中Piwill基因的表达。首先,本研究通过分子克隆技术成功构建鸡Piwill基因过表达载体作为筛选miRNA干扰表达载体的靶质粒。细胞定位结果表明,该Piwill基因过表达载体能够成功表达一种细胞质蛋白。其次,本研究将miRNA干扰表达载体和Piwill基因过表达载体共转染HEK293细胞,利用Real-Time qPCR技术筛选出具有最佳干扰效果的miRNA干扰表达载体。接着,本研究将该miRNA干扰表达载体通过Gateway技术亚克隆至目的载体中成功构建了Piwill基因-miRNA曼病毒表达载体。最后,本研究将miRNA慢病毒表达载体与辅助质粒共转染293FT细胞生产慢病毒颗粒。慢病毒液滴度为1×108TU/ml。细胞侵染结果表明,该慢病毒液能够成功抑制Piwill基因的表达。为了检测Piwi基因在物种内的序列保守性,本研究以日本鹌鹑为参照群体,再次利用RT-PCR和RACE技术从成年鹌鹑睾丸组织中成功克隆了Piwil1基因的全长cDNA序列,包括一个2595bp的ORF,142bp的5’UTR和667bp的3’UTR。生物信息学分析结果表明,该ORF编码864个氨基酸残基,含有PAZ和PIWI两个Ago家族蛋白的保守结构域。与鸡Piwil1基因编码蛋白相比,N端缺失3个氨基酸残基。在成年鹌鹑中,PIWIL1蛋白在睾丸组织中特异性表达,表明PIWIL1蛋白在鸡和鹌鹑之间具有保守的功能。为了进一步探索禽类PIWI蛋白在精子发生和转座子调控中的作用机制,本研究利用深度测序技术获得成年鹌鹑性腺组织small RNAs表达谱。在睾丸组织中,small RNAs主要集中在24-27nt之间,主要定位于基因组中重复序列区域,其次是编码基因序列,与果蝇中piRNAs的分子遗传特征相类似,推测禽类piRNAs与果蝇piRNAs一样,在生殖系中参与维持异染色质结构,抑制重复序列转录和转座子转座。免疫沉淀的结果表明,与禽类PIWIL1蛋白相结合的主要是24～25nt之间的piRNAs,而在卵巢组织中,miRNAs的丰度最高,表明在禽类中PIWIL1蛋白也是通过piRNAs介导进行相关生物学功能调节的。更多还原

【Abstract】 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.更多还原