Cloning And Functional Analysis Of OsGH18 Candidate Gene For 131 Bud In Early Japonica Rice

The low-temperature stress is the most important environmental factors for plants growth, and it is also the most common factors leading to significant crop production cuts in the all the country, especially in Northeast China. By the continuous research on the molecular mechanisms of stress tolerance, the gene has been found, whose expression has a significant change in low temperature. In order to improve the stress tolerance of plants, the transgenic technology has been studied, so that improving plants resistance to abiotic stress has important research value.This study identifies that Konyu131 is the cold-tolerance rise, but Longjing11 is the cold-sensitive rise. Researching them, analyzing their gene expression profiles and screening amino sugars and nucleotide sugar involved in glucose metabolism genes-chitinase(GH18), the genes occur significant changes in the low-temperature induction. Using real-time quantitative PCR to low-temperature stress, analyze OsGH18 genes expression at different time points by Gateway cloning technology, respectively clone OsGH18 gene in the cold-tolerance varieties Kongyu131 and the cold-sensitive varieties Longjing11, and carry out the genetic transformation of Longjing 11. The main findings are as follow:1. Identify the genes of rice Kongyu 131 bud responding to low-temperature stressAnalyzing the gene expression profiles, find that a total of 229 differentially expressed genes in four low-temperature treatment time points are raised. Pathway analysis reveals that the low-temperature stress is involved in amino sugars and nucleotide sugar metabolism, osmotic adjustment, photosynthesis and other metabolic pathways.2. Identify the candidate genes of Kongyu131 involved in cold tolerance at the bud burst(CTBP)By real-time PCR experiments technology, taking 0h as a reference, for cold treatment at different time points, OsGH18 gene of cold tolerance expression analysis showed that cold-tolerance varieties Kongyu131 is highly expressed. At each treatment time points the expression level of cold-tolerance varieties Kongyu131 and cold-sensitive varieties Longjing 11 increases, but Kongyu131 expression is significantly higher than Longjing 11.3. Clone and analyze OsGH18 geneTake the cDNA of cold treatment for 12 h as a template to the cold-tolerance varieties Kongyu131 and the cold-sensitive varieties Longjing11 PCR amplification. Clone the cold stress-related OsGH18 gene, By Blast comparison, ORF Finder analysis, find that the nucleotide sequence exists codon degeneracy.4. Construct the plant expression vector of OsGH18 gene and identify the plant of genetic transformationUsing Gateway cloning technology, connect OsGH18 gene and plant expression vector pH7WG2, mediated by agrobacterium, the cold-sensitive varieties Longjing11 callus is transformed. By molecular biology detecting, identify that OsGH18 is the stress tolerance-related genes.