Cloning And Sequence Analysis Of Wheat Microrna

MicroRNAs (miRNAs), ranging in size from 20 to 25 nucleotides, are a class of endogenous regulatory noncoding RNAs that have been found in various eukaryotes. Mature miRNAs are processed from longer primary transcripts by a series of ribonuclease, and then incorporated into RNA-induced silencing complex (RISC), in which they recognize target mRNA by base-pairing and guide RISC to cleave the mRNA or inhibit the translation of the mRNA, depending on the complementarity between them. Recent advances have revealed that miRNAs participate in a variety of regulatory pathways, including development, virus prevention, hematopoiesis, organogenesis, cell proliferation and apoptosis, fat metabolism, etc.Compared with animal miRNAs, the discovery of plant miRNAs is much later. Moreover, plant miRNAs studies have been confined mainly to Arabidopsis and rice, two plant model species with fully sequenced genomes. Wheat is a heterogenous sixfold plant and has 42 chromosomes, whose genome is very huge compared with Arabidopsis and rice and has not been sequenced completely yet. Therefore, it is difficult to identify wheat miRNAs, and the related reports are quite few. However, wheat is one of the most important grain crops in the world, hence it is of great significance to investigate wheat miRNAs. The purpose of this study is to identify some wheat miRNAs by constructing a cDNA library of small-sized RNAs from wheat and lay some foundation for the experimental cloing and validation of more wheat miRNAs and the study of their physiological functions in the future.Firstly, extract total RNAs from 4-week old wheat leaves and enrich small-sized RNAs according to a protocol described in the main text. Secondly, small RNAs with a size from 16 to 28 nucleotides were recovered from denaturing 15% polyacrylamide gel fractionation, and then polyadenylated by using poly(A) polymerase. Thirdly, add a poly(dG) tail to the 3′end of the first strand cDNAs by using terminal deoxynucleotidyl transferase(TdT) after reverse transcription of poly(A)-tailed RNA, and amplify the cDNAs by polymerase chain reaction(PCR). Lastly, recover and purify the interested PCR products, insert them into cloning vectors, and construct a cDNA library of small-sized wheat RNAs by transformating competent cells.Select positive clones containing interested fragments from the cDNA library by clony PCR, randomly choose 60 from them for further sequencing, and get 17 single sequences. Search the wheat DNA bank for these sequences'origin and predict their secondary structures with Mfold. Finally, 7 candidate wheat miRNAs were obtained. Northern blot confirmed that these candidate miRNAs were stably expressed in wheat cells from at least one tissue. According to the principle of identification of miRNAs, the 7 candidates were novel wheat miRNAs.