Molecular Evolution Of Rice Resistance - Related Genes And MiRNAs And Cold Function Of OsCORA Gene

Rice (Oryza sativa) is one of the most important cereal crops in China. However, rice production is increasingly threatened by low temperature, drought and other environmental stresses. It’s believed that stress-related genes/microRNA(miRNA)s help plant survive under abiotic stresses. Thus, it is important to systematically investigate the function and evolution of stress-related genes/miRNAs. Rice has become an important model specie with complete genome sequence. Plant development and evolution were largely illustrated via using rice as a genetic model system. Recently, scientists started to uncover the regulatory function and mechanism of stress-related genes/miRNAs in rice. The rice genome sequence and the abundant expression data of rice provide tremendous opportunities to study the function and evolution of rice stress-related genes. In this study, we have constructed the Plant Tolerance Gene Database. We have extensively analyzed the molecular evolution of rice stress-related genes/miRNAs using comparative genomics and bioinformatics technologies. In addition, we cloned the cold resist gene OsCORA and OsCAP, constructed their expression vector, and studied the cold resist functions of OsCORA gene. These important results were summarized as follows:1. We have collected 308 experimentally verified stress-related genes through the literature retrieval from 51 plant species. The function annotation indicated that the 308 genes involved in 1248 GO terms and 203 regulatory pathways. Most genes involved in metabolic pathways. Plant Tolerance Gene Database with a visulized web interface was developed to ntegrate these above nformation (http://rich.yunda.org/test/rg01/index.php).2. Based on the above Plant Tolerance Gene Database, we have systematically investigated the evolution of the 22 rice stress-related genes using comparative genomics and bioinformatics methods. The results demonstrated that these rice stress-related genes have homologs with the lower plant such as Chlamydomonas reinhardtii. With the continuing evolution of plant and the change of survival environment, the number of the rice stress-related genes also incessantly increase via gene duplication. In plants, genes with similar function share similar gene structures and motif structures. Notedly, we found three-specific-motif stucture in GSK1 and RAN2 gene, i.e. GSK1 with P-rich motif, RAN2 with G-rich motif and E-rich motif. The three-specific-motif stucture is closely related with gene function. Finally, we discovered that the rice stress-related genes underwent strong purify selection. We also detected lots of positive selection site in the about 50% of these genes. These positive selection sites provide important mutation accumulating for genes to adapt to the external environment changes.3. Stress-related miRNAs play important roles in regulating stress responses in plants. However, the systematic investigation of stress-related miRNAs is limited in Rice(Oryza sativa) which is a typical abiotic stress-resistant crop specie. In the present work, via systematically investigating stress-related miRNAs in silico, first, we identified 177 putative stress-related miRNAs in O.sativa. Second, we found most stress-related miRNAs were driven by TATA-promoter and most stress-related miRNA promoter regions contained the stress-related elements. Third, we found many stress-related miRNA families were species/family specific and a set of miRNAs might derive from genomic repeat-sequences in O.sativa. Finally, we found the stress-related miRNAs in O.sativa target 553 genes with 1562 predicted target sites in which 89.50% sites have cleavage activity and 10.50% sites have translation inhibition activity.4. In the study, we cloned the cold resist gene OsCORA and OsCAP, constructed their expression vector, and studied the cold resist functions of OsCORA gene. The full-length cDNA of OsCORA is 885 bp with an ORF of 873 bp, encoding a 290 a.a. polypeptide, and the predicted molecular mass and isoelectric point of the putative protein are 54.41 kDa and 7.97, respectively. Whilst, the full-length cDNA of OsCAP is 645 bp with an ORF of 633 bp, encoding a 210 a.a. polypeptide, and the predicted molecular mass and isoelectric point of the putative protein are 54 kDa and 5.11, respectively. In addition, we constructed the pBI 121-OsCORA and pBI 121-OsCAP recombinant plasmid. We constructed also pET-28a-OsCORA recombinant plasmid, and studied the cold resist functions of OsCORA gene. The results indicated that the OsCORA protein expression significantly enhanced the survival rate of escherichia coli after high temperature and low temperature treatment.In this thesis, we built the Plant Tolerance Gene Database under the windows operation system. A web interface was developed for visualization of the gene function, GO terms, regulatory pathway and plant species. The constructed Plant Tolerance Gene Database may serve as an essential resource for future studies of the function and evolution of plant stess-related genes. Based on the Plant Tolerance Gene Database, we have systematically analyzed the molecular evolution of the rice stress-related genes, and our findings provide an insight into the adaptive evolution of the rice stress-related genes. Based on collecting and identifying the known stress-related miRNAs from O.sativa, we extensively analyzed these miRNAs, including their genome location and cluster pattern, promoter sequences and conserved cis-acting regulatory elements, CpG islands, target gene function annotations, and phylogenetic comparison of cross-species conservation miRNA families. Our studies provide valuable information on the stress-related miRNAs and improve our understanding on regulatory functions of the stress-related miRNAs in stress tolerances. In addition, the function study of OsCORA gene will be helpful for us to further study the transgenic rice cold resistance.