Studies On The Sequence Characteristics And Biological Functions Of Plant Small RNAs

This research project, focusing on plant small RNAs (sRNAs), employed both experimental approaches and bioinformatics methods to do a comprehensive and in-depth study on the sequence characteristics, regulatrory mechanisms, and biological roles of the sRNA species. The results are summarized as follows:1. microRNA (miRNA) microarray profiling and Northern blot were carried out for the rice mutant osaxr (Oryza sativa auxin resistant) to sort out the miRNA candidates potentially involved in auxin-mediated root development control. Both the promoters and the downstream targets were analyzed. Based on the miRNA overexpression experiment, the critical role of miR160in root cap formation was demonstrated to be highly conserved in plants. More interestingly, the expression of miR167was intensively repressed in three allelic osaxr mutants with different backgrounds. Overexpression of this miRNA gene in the mutant with Kasalath background could recover the defect of adventitious root (AR) development. The AR numbers of the miR167-overexpressed transgenic lines even exceed those of the wild type lines transduced with empty vectors. Target prediction and modified5'RACE (rapid amplification of cDNA ends; cDNA, complementary DNA)-based verification indicated that the auxin signal was tranduced by miR167through its targets OsARF12(Oryza sativa Auxin Response Factor12) and OsARF17, to modulate AR development.2. By utilizing the currently available sRNA high-throughput sequencing (HTS) data, the sequence characteristics and the chromosome-wide distribution patterns of the sRNAs were analyzed in26angiosperms, including16eudicots and10monocots. The scene of sRNA generation within the overlapping regions of natural antisense transcript pairs was investigated. Both the commonality and the divergence of sRNA features between the eudicots and the monocots were discussed.3. Based on the miRNA information retrieved from miRBase (http://www.mirbase.org/), the whole-genome SNP (single nucleotide polymorphism) data, and various kinds of HTS data, a plant microRNA knowledge base (PmiRKB; http://bis.zju.edu.cn/pmirkb/) was established. The five major functional modules,"MiR info","SNP","Pri-miR","MiR—Tar" and "Self-reg", provided by PmiRKB can assist the functional studies on plant miRNAs.4. T-plots (target plots) were generated by using plant degradome sequencing data in order to perform large-scale screening and validation of miRNA(*)—target pairs in planta. Based on the miRNA(*) target lists, comprehensive gene regulatory networks mediated by miRNAs and miRNA*s were constructed in both Arabidopsis (Arabidopsis thaliana) and rice. The regulatory effects of miRNA*s on their target transcripts through cleavages were unraveled for the first time in plants. Further analysis of certain subnetworks provided us with some novel biological hints, implying the value of the established networks for miRNA-related studies.5. During these studies, we made some timely summarizations of the current research status of the plant miRNA area. As a result, several review and opinion papers were published on the international journals, which served as the cornerstone for our research.By presenting these findings, I hope that my studies could partially expand the current understanding of plant sRNAs, both their sequence characteristics and the biological functions, and could inspire further in-depth studies on the huge sRNA population in plants.