Identification And Analysis Of MicroRNA In Common Wild Rice

MicroRNA(miRNA) are a class of non-coding RNAs involved in post-transcriptional control of gene expression, either via degradation or translational inhibition of target mRNAs. Common wild rice (Oryza rufipogon Griff.) is considered to be the ancestor of Asian cultivated rice species, Oryza sativa L. And it is also a important germplasm for cultivated rice modification. In order to reveal the variation of miRNA during the domestication of cultivated rice and, hopefully, clone common wild rice specific miRNA, we use Illumina sequencing approach to characterize, identify and do expression analysis of sRNA cDNA library. Over expression vector harboring precursor of miRNA which were found to be specific to common wild rice were constructed and transferred into cultivated rice variety Nipponbare via Agrobaterium-nediated method, setting the stage for studying the function of those miRNA. The main results were as follows:Dada analysis of small RNA library for Illumina sequencingUsing Illumina sequencing technology, we analyze the common wild rice sRNA cDNA library and obtain High quality reads 8558370 consisting of 2307484 unique sequences; Through Aligning small RNA to the miRNA precursor of Oryza sativa in miRBase14.0 we identify 104 miRNA belonging to 25 conserved miRNA families; According to the miRNA count in our study and previously reports the most highly expressed conserved miRNA in common wild rice ranked from high to low is miR156,miR168,miR528,miR166,miR167, while in cultivated rice is miR169, miR156, miR168, miR 172;Besides the conserved miRNAs, we also found 215 Rice specific miRNA belonging to 78 families; Using UNAFOLD we found 353 sRNA sequences seemed to be able to generate fold-back secondary structure, further analysis including the existence of miRNA* and the free energy of the harpin, we identified 23 novel miRNA; 17 target genes of 11 new miRNA were predicted by way of bioinformatics approach.Validation of new miRNAAfter extracted small RNA, we use Stem-loop RT-PCR for detecting the expression of 353 small RNA sequences which were predicted to be able to generate fold-back secondary structure. Among those, by sequencing, we got 32 75bp PCR product containing 21nt small RNA except for 23 new miRNA which have miRNA*. Suggesting those 32 sequences are expressed so they are candidate new miRNA. Using on line software--miRU: Plant miRNA Potential Target Find, 23 out of 32 candidate have predicted targets.Expression patterns of newly identified miRNA and vector construction and transformation of cultivated riceKnowledge about the expression of miRNA might provide clues about their functions. We did Semi-quantitative PCR for CWR-miR28 , CWR-miR61 , CWR-miR64 , CWR-miR68 ,CWR-miR153,CWR-miR200,CWR-miR264,CWR-miR308 and found all of them expressed in common wild rice, and all of them show different expression level in root, stem and leaf except for CWR-miR28,CWR-miR153,CWR-miR264,.In order for further study of functions of new miRNAs, we constructed 23 over expression vector harboring precursor of new miRNAs and transferred them into cultivated rice variety Nipponbare via Agrobaterium-mediated method. Now 2 of them are in the differentiation stage.