Gene Expression Analysis In A Rice Introgression Line With Enhanced Alkali Tolerance And Preliminary Functional Characterization Of Two Candidate Genes

Rice is a staple food for more than half of human population and to grow it,farmers largely depend on soil. Land salinization and alkalization is a majorenvironmental factor limiting plant growth and productivity. Alkalization is oftenassociated with salinization, alkaline stress not only included salt stress, but also withthe added influence of high-pH stress, so it affects plant growth badly, but so far onplant alkali resistance mechanism is not clear.One of the most effective measures to improve land salinization and alkalizationis to develop new salt/alkali-tolerant varieties. The introgression by backcross methodis the most effective method in the development of improved crop varieties，andcompared with traditional hybridization, it has a much shorter time and moreeffectively usually. Gene expression microarray makes it possible to high-throughputanalysis of the mRNA expression, finding key genes, understanding metabolicpathways. Using gene expression microarray to detect the function of genes havebecome very popular.To investigate the possible genetic basis of alkali tolerance in rice, we generatedan introgressed rice line （K83） with significantly greater alkali stress tolerance thanthe parental cultivar （Jijing88）. Seedling treated with sodium bicarbonate （NaHCO₃）and sodium carbonate （Na₂CO₃）, then we used microarray analysis to examine theglobal gene expression profiles of K83and Jijing88, and we found that more than1,200genes were constitutively and differentially expressed in K83in comparison toJijing88with572genes up-and654down-regulated. Upon alkali treatment, a total of347genes were found up-and156down-regulated in K83compared to591and187,respectively, in Jijing88. Among the up-regulated genes in both K83and Jijing88,only34were constitutively up-regulated in K83, suggesting that both the constitutivedifferentially expressed genes in K83and those induced by alkali treatment are mostlikely responsible for enhanced alkali tolerance. A gene ontology analysis based on allannotated, differentially expressed genes revealed that genes with expressionalterations were enriched in pathways involved in metabolic processes, catalyticactivity, and transport and transcription factor activities, suggesting that thesepathways are associated with alkali stress tolerance in rice. Further analysis of relativegene expression data using real-time quantitative PCR, real-time quantitative PCRresults basically agrees well with microarray results, it means microarray data arereliable. Our study results might have provided useful clues to gene cloning andfunctional studies of alkali tolerance in rice.Based on the microarray data, we identified some candidate genes. Theexpression patterns of candidate genes were examined in rice under various abioticstress and hormone treatments. The results showed that two candidate genes （Os01g0871600and Os12g0282000） induced by abiotic stress and hormone, andsuggested these candidate genes may play a role in plant alkali stress. The two geneswere cloned, and their over-expression recombinant plasmids were constructed.Transformation of rice was conducted by agrobacterium tumefaciens infection. Alarge number of positive transgenic plants were obtained, and which showedhigh-expression of the candidate genes. Transgenic plants showed significantly moretolerance than the non-transgenic control based on seedling resistance assays. Theresults of this study suggest that genes identified from microarray analysis may serveas a potential gene pool to identify functional genes that can be used for rice geneticimprovement towards enhanced tolerance to alkalization.