Identification of Functional Candidate Genes for Drought Tolerance and Proteomic Analysis in Rice

Abstract: Drought tolerance (DT) in rice is known to be controlled by many quantitative trait loci (QTLs) and involved differential expression of large numbers of genes, but linking QTLs with their underlying genes remains the most challenging issue in plant molecular biology. To shed some light on this issue, differential gene expression in response to PEG simulated drought in 3 unique genetic materials (a lowland rice, IR64 and its derived line, PD86 which has 11 introgressed DT QTLs, and a upland rice IRAT109) was investigated using a PCR-based subtractive hybridization strategy. More than 300 unique subtracted cDNA sequences, covering genes of diverse cellular activities and functions, were identified and confirmed by semi-quantitative and quantitative RT-PCR. Detailed bioinformatics analyses of the data revealed two interesting results. First, the levels and mechanisms of DT of the three rice lines were associated with the number and types of differentially expressed genes, suggesting different DT mechanisms in rice are controlled by different sets of genes and different metabolic pathways, and most differentially expressed genes under drought were able to contribute to DT. Second, there appeared a high correspondence in genomic location between DT QTLs and clusters of differentially expressed genes in rice, suggesting some DT QTLs may represent clusters of co-regulated and functionally related genes. Thus, differential gene expression analysised using genetically characterized materials can provide additional insights into the molecular basis of QTLs and convergent evidence to shortlist the candidate genes for target QTLs.17 genes were selected to analysis the expression parttern of differentially expressed genes under differential stresses and hormone treatments by real-time PCR. Most (14 of 17) had the same expression parttern under drought, salt and cold conditions, which suggested there were the same pathways to induce these genes. About half of genes were found to be up-regulated by ABA, and a putative protein kinase only induced by dought indicated some independent pathway for drought stress. The expression analyses of the genes in roots under drought revealed the method of DT was different from leaf.Furthermore, in order to investigate the initial response of rice to drought stress, changes in protein expression were analyzed in rice using a proteomic approach. Proteins were extracted from leaf, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. After drought stress for 8 days, 23 proteins increased in abundance and the level of 5 proteins decreased. Mass spectrometry helped to identify 12 of the drought-responsive proteins, and the method of aligning to the maps in the rice proteomic database helped to identify 19 proteins. These proteins are involved in redox metabolism, photosynthesis, cytoskeleton stability, defense, nucleic acid metabolism, protein metabolism, cell structure, ion transportion and signal transduction. Several drought-responsive pathways were analysised by this work:ABA in osmotic stress signaling, protein degradation, photosynthesis, redox metabolism, Programmed cell death and cytoskeleton organization. there also appeared a high correspondence in genomic location between DT QTLs and differentially expressed proteins in rice These findings may contribute to further understanding of the mechanisms that lead to drought tolerant of rice and other plants as well…
Key words: drought tolerance; candidate genes; QTL; real-time PCR; two-dimensional gel electrophoresis; mass spectrometry; rice