| Rice (Oryza sativa L.) is one of the most important food crops in the world. The cultivars Zhonghan3(tolerance) and Aizaizhan(sensitive) were used as materials in the research. Under drgouht stress, the expression levels of different types of genes were analyzed by gene chip and real-time PCR, and numbers of drought-tolerant key genes were found. Amoung these genes, SalT presented a high expression level when facing drought stress. After cloning coding region, SalT over-expressing and RNAi transgenic rice seedings were obtained. In order to get more information about protein structure and function of SalT, its recombination protein was isolated from the Escherichia coli prokaryotic expression system. The research provided materials and basis for exploration and functional verification of drought-tolerant key genes in rice. The main results were summarized as follows:1. Water content of rice seedling leaves was reduced under drought stressWithin three days, the leaf water content of two cultivars had varying degrees of decline under drought stress. Water lose speed in Aizaizhan was quicker than that in Zhonghan3. After the third day, the changes had tended to be flat. During the period of drought stress, leaf water content of Zhonghan3was obviously higher than that of Aizaizhan, which indicated the former has a stronger water-holding capacity compared with the latter.2. Drought stress affected the expression levels of drought-tolerant key genesFirst, we investigated the differential expression genes in rice leaves by Affymetrix gene chip after being treated for3days.1034differently expressed genes, including892up-regulated genes and242down-regulated genes, were identified in Zhonghan3. Meanwhile,213differently expressed genes were found in Aizaizhan, which contained132up-regulated genes and81down-regulated genes. Then, according to the transcriptional levels and potential function,15possible drought-tolerant key ones were screened out. They were related to anti-dehydration and water channel protein, transcription factor and signal transduction, respectively. The relative quantitative expression of these genes was verified by real-time PCR after being treated with10%PEG6000for6days. The results revealed that14ones in Zhonghan3were significantly up-regulated. Amoung these genes, late embryogenesis abundant protein(LEA3-1), Dehydrin(RAB16B), β-glucosidase aggregating factor(BGAF) and salt stress-induced protein(SalT) related genes were strongly induced; At the same time, only7ones were significantly up-regulated in Aizaizhan, where LEA3-1, RAB16B and SalT related genes were induced at high levels.Compared with the results from gene chip, numbers of genes in Aizaizhan leaves were obviously induced after been treated for6days. It revealed that these possible drought-tolerant key genes were just begun to express in a large amount. However, those14ones in Zhonghan3maintained induced expression at high levels. Under drought stress, the response of these genes in Aizaizhan was slower than that in Zhonghan3. So when facing water shortage, the adjustment of gene expression in Zhonghan3may be one of its main drought-tolerant mechanisms at the earlier stage of drought stress.3. Obtaining transgenic rice and prokaryotic expression of SalTBased on the vector pTCK303, SalT over-expressing and RNAi vectors were constructed and transferred into Zhonghan3callus by Agrobacterium-mediated transformation method. Finally, the transgenic rice seedings were obtained. It provided the materials for the further research, In addition, the purified SalT recombinant protein has agglutination activity and mannose specificity. But in the presence of mannose, the agglutination activity was obviously inhibited. |
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