Comparative Transcriptome Analysis Of Drought Tolerance And Function Characterization Of OsDRAP1 In Rice

Drought stress has dominantly reduced rice production in the world. The physiological and biochemical response and phenotypic changes to drought stress are controlled by plant related genes, which are involved in all kinds of pathways, forming a series of drought regulation network in response to drought resistance. Due to the complicated nature of drought resistance, rice functional genomics (genomc, transcriptomc and protcomics) and bioinformatics largely benefit the drought resistance gene cloning via reverse genetics, since forward genetics method has a limit to explore rice drought resistance related genes.Rice (Oryza sativa. L) is more sensitive to drought stress than other cereals. There exists large genotypic variation in drought tolerance (DT) within the cultivated rice gene pool and its wild relatives. In past decades, selective introgression of DT donor segments into a drought-sensitive (DS) elite recurrent parent by backcrossing has efficiently improved drought stress tolerance in rice. To dissect the molecular mechanisms underlying DT in rice, deep transcriptome sequencing was used to investigate transcriptome differences among a DT introgression line H471, the DT donor P28, and the drought-sensitive, recurrent parent HHZ under drought stress. The results revealed constitutively differential gene expression before stress and distinct global transcriptome reprogramming among the three genotypes under a time series of drought stress, consistent with their different genotypes and DT phenotypes. A set of genes with higher basal expression in both H471 and P28 compared with HHZ were functionally enriched in oxidoreductase and lyase activities, implying their positive role in intrinsic DT. Gene Ontology (GO) analysis indicated that common up-regulated genes in all three genotypes under mild drought stress were enriched in signaling transduction and transcription regulation. Meanwhile, diverse functional categories were characterized for the commonly drought-induced genes in response to severe drought stress. Further comparative transcriptome analysis between H471 and HHZ under drought stress found that introgression caused wide-range gene expression changes; most of the differentially expressed genes (DEGs) in H471 relative to HHZ under drought were beyond the identified introgressed regions, implying that introgression resulted in novel changes in expression. Co-expression analysis of these DEGs represented a complex regulatory network, including the jasmonic acid and gibberellin pathway, involved in drought stress tolerance in H471. Comprehensive gene expression profiles revealed that genotype-specific drought induced genes and genes with higher expression in the DT genotype under normal and drought conditions contribute jointly to DT improvement. The molecular genetic pathways of drought stress tolerance uncovered in this study, as well as the DEGs co-localized with DT-related QTLs and introgressed intervals, will serve as useful resources for further functional dissection of the molecular mechanisms of drought stress response in rice.Explanation and utilization of rice DT molecular mechanism largely depends on the function identification of DT related genes. DT candidate gene OsDRAP1 was further characterized by over-expression and RNAi technology. Results revealed that OsDRAPl belongs to the DREB2 subfamily of AP2/EREBP, which expressed in all organs (mainly expressed in different organs of vascular bundle group) of rice, and can respond to a variety of environmental stresses and hormones. Subcellullar localization indicated that OsDRAPl dominantly localized in the nucleus. Further analysis showed that OsDRAP1 has transcription activation activity and could activate the downstream DT related genes and also maintain normal growth and development of rice plant under drought stress. Yeast two-hybrid.. BiFC and CoIP analysis showed that OsDRAP1 can interact with OsCBS1 to response drought stress. And cytological results supported the OsDRAP1 might participate in the regulation of development of vascular bundle system and play important roles in against environmental stresses.Novel promoters that confer root-specific expression would be useful for diminishing the negative effects of over-expressed OsDRAP1 in rice, engineering resistance against problems with nutrient and water absorption by roots. In this study, we used reverse transcriptase polymerase chain reaction to identify seven genes with root-specific expression in rice. The isolation and characterization of upstream promoter regions of five selected genes (rRSP1 to rRSP5) and A2P (the promoter of OsAct2) revealed that rRSP1, rRSP3 and rRSP5 are particularly important with respect to root-specific activities and rRSP2 and rRSP4 dominantly activated in root tissues. Furthermore, rRSP1, rRSP3 and rRSP5 were observed to make different contributions to root activities in various species. These three promoters could be used for root-specific enhancement of target gene(s). A2P::OsDRAP1 and rRSP2::OsDRAP1 double T-DNA expression vectors were constructed and transformed into the rice genome. However, it is necessary to detect the effects of over-expression of OsDRAP1 and root-specific expression in rice promoted by A2P and rRSP2 in future. Especially, the application of root-specific promoters and drought related genes can provide genetic resource for DT rice molecular breeding.