Abstract:

MicroRNA (miRNA) is a class of endogenous and non-coding RNAs, which can regulate the growth and development of organism at post-transcriptional level. Direct cloning is one of the universal and efficient methods to identify miRNAs. In this study, total RNAs were extracted from ovules of 0~10 days post anthesis (DPA) and then 17~24 nt small RNAs were screened to create a single small RNA cDNA library, from which some positive clones were selected for sequencing. After blasted in miRBase, 4 conserved miRNAs were obtained. Further analysis was carried on their time-course and tissue-specific expression profile. It was found that all of them could express in different development stages and miR167a, miR172c and miR394b could express in roots, stems and leaves of cotton seedlings. On this basis, 55 target genes of the identified miRNAs were predicted and most of them encode transcription factors, metabolizing enzymes and proteins related to development. This further confirmed that miRNAs participated in regulating the morphogenesis and development, etc. physiological activities  of cotton fibers.

Key words: upland cotton, fiber development, miRNA, direct cloning, identification

摘要：

microRNA(miRNA)是生物中一类起负调控作用的非编码的小分子RNA,主要在转录后水平上调节生物体的生长与发育。直接克隆法是鉴定植物中miRNA最常用也是最有效的方法之一。利用直接克隆法分离了陆地棉开花后0~10 d(days post anthesis, DPA)胚珠中总RNA,从中筛选17~24 nt小分子RNA构建cDNA文库,对初筛得到的阳性克隆进行测序。通过与miRNA数据库比对,最终获得4个保守的miRNA。进一步对他们的转录表达进行分析,发现所有miRNA在棉花胚珠纤维不同发育时期均能表达。其中miR167a、miR172c和miR394b在棉花幼苗的根茎叶中有不同程度的表达。预测得到55个miRNA的靶基因,多数靶基因编码转录因子、代谢酶类以及发育相关蛋白,进一步证明miRNA参与调控棉花纤维的形态建成、发育等各项生理活动。

关键词: 陆地棉;纤维发育;miRNA;直接克隆法;鉴定