鸡pilRNAs分子的克隆及表达规律分析

旨在为禽类piRNAs的遗传特性及发生和消失机理提供基础依据,也为禽类小分子RNA及其结合蛋白的深入研究和实际应用积累基础资料。本研究以鸡作为研究对象,通过构建小RNAs的cDNA文库和TA克隆测序的方法从睾丸组织中获得了19个大小为23～39nt的pilRNAs序列。根据pilRNAs的大小、同源序列和二级结构特征,选择了3个不同序列并采用Q-PCR技术分析了中国地方鸡种如皋鸡和引进隐性白鸡的不同生长阶段不同组织中pilRNAs时空表达规律。结果表明,编码鸡pilRNAs基因序列在染色体和基因组中的分布同其它物种一样分别具有不均匀性和基因间区偏爱性;二级结构预测,发现pilRNAs具有与miRNAs不同的独特的茎-环结构;鸡pilRNAs不仅大量存在于生殖组织,还大量存在于其他组织,表达规律因pilRNAs种类、品种、性别的变化而不同,gga-piR-1在2个品种的公、母鸡所有组织中的表达量均是相对最低,且到12周龄时均趋向一致的水平;而gga-piR-4表达量相对较高;gga-piR-5在如皋公鸡中所检测的全部组织中表达量最高,且在8周龄达到最高峰,在隐性白公鸡中的表达量相对较高,而在如皋母鸡、隐性白母鸡中均呈现中度表达水平。二级结构的独特特征从不同的角度阐明了pilRNAs和miRNAs 2种成熟小RNAs的剪切方式和加工机制的完全不同;鸡pilRNAs的多组织表达表明它可能不仅局限于生殖系的发育和维持,在其他组织中也扮演了重要的角色,不同种类的pilRNAs可能在生命发育过程中参与不同的调控功能。

Cloning and Expression Profiling of piRNA-like RNAs in Chicken

This study was designed to explore characteristics of genetics,mechanism of occurrence and disappearance of poultry piRNAs,and accumulate basic materials for research about birds small RNA-binding protein and practical application.19 pilRNAs sized 23-39 nt were discovered by constructing cDNA library of small RNAs,TA cloning and sequencing in chicken testicular tissue.According to size,homology and secondary structure,3 different sequences were selected for analyzing temporal and spatial expression of pilRNAs by using Q-PCR technology in different tissues at different growth and development stages of Rugao chicken and Recessive White Feather chicken.The result showed that,consistent with other species,distribution of pilRNA-encoding sequences in the chicken genome was found to be asymmetrical on chromosomes,meanwhile,displayed a preference for intergenic regions across genome.Unlike the secondary structure of miRNAs,pilRNAs were predicted an unique stem-loop secondary structure.Chicken pilRNAs were not only abundant in germline tissue,but also abundant in other tissues,and expression at mRNA level was influenced mainly by different pilRNAs,breeds and gender.The expression level of gga-piR-1 was the lowest in all tissues of two breeds and reached the consistent trend at week 12,while gga-piR-4 showed moderate expression levels.gga-piR-5 revealed 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 week 8 for the male Rugao chicken,whereas it expressed moderately in female of both breeds.Therefore,the difference of the secondary structures between pilRNAs and miRNAs indicate that the difference in splicing and processing mechanisms of two small RNAs,pilRNAs may not only be confined to development and maintenance for germline tissue,but also play important roles in somatic tissues and different pilRNAs may be involved in different regulatory function in the complex biological processes.