Cloning And Functional Analysis Of OsGST Gene In Cold Tolerant Candidate Gene Of 131 Bud In Rice

Rice is a typical temperature-sensitive crops. Low-temperature and freezing injury are significant limiting factors of rice quality and yield. Heilongjiang Province occupies an important position in China’s rice production areas. However, Heilongjiang suffers from frequent low-temperature and freezing injury which cause sharp reduction of rice. Meanwhile, the grow of rice direct seeding area in Heilongjiang demands the rice seeds higher cold-tolerance capacity during their germination periods. To investigate the cold tolerance capacities of individual rice varieties, and then to select the rice varieties with high cold tolerance property is the most effective approach to resist the low-temperature injury and to guarantee the risk management in cold region rice cultivation.This research perform rice bud cold tolerance identification to screen out cold tolerance and sensitive seeds. We apply Solexa sequencing chip to analyze their expression profiles of these sorted seeds, and then screen out and clone hypothermia induced upregulation expression Os GST genes. Meanwhile, the connection between Os GST genes and plant resistance is studied. It is also expected to illustrate the molecular mechanisms that cold-induced expression are applied to improve the low temperature tolerance of cold-sensitive crops, as well as some available regulatory solutions. This research could service for crop cold-tolerance genetic engineering by providing with theory and gene resources.The main contributions are listed as follows:1. Cold-tolerance identification test on twelve individual rice varieties is conducted: Heilongjiang No.1 accumulated temperature zone(Songjing 9, Daohuaxiang), Heilongjiang No.2 accumulated temperature zone(Songjing 10, Longjing 11, Longjing 21, Longjing 26, Suijing 10), and Heilongjiang No.3 accumulated temperature zone(Kongyu131, Kongyu 131(pi2-7), Kongyu 131(pi2-9), Longjing 31, Xueguang). The result indicates their cold tolerance capacities exhibits obvious differences. Kongyu131 shows the best cold tolerance property, and Longjing 11 is the most cold sensitive rice variety.2. In each accumulated zone of Heilongjiang, the individual rice varieties shows similar feature, that is, their cold tolerance properties in bud period approximatively represent their cold tolerance properties in seeding and booting stages. Selected high cold tolerance rice variety in bud period has more likelihoods to be cold resistant in their seeding and booting stages.3. Determination experiment employs the real-time PCR technology to analyze thecold-tolerance performance of Os GST gene at different low temperature processing time points. The experiment shows Os GST genes resistant to cold, cold-sensitive rice varieties expression differences are upregulated, but Cold varieties Kongyu131 expression was significantly higher than that of cold-sensitive varieties of Longjing 11. Under chilling stress, the cold tolerance differences of rice varieties are caused by the different expressions of Os GST genes.4. Os GST full-length clone and sequence analysis of genes, the full-length c DNA sequence of Os GST has 1292 bp, in which its CDS has 696 bp, encoding 231 amino acids and other information.5. By gateway cloning technology and Agrobacterium-mediated,the gene Os GST of callus in cold-sensitivity Longjing 11 is transformed, and have the transgenic positive rice plants of callus. To prepare verification the gene function Os GST.