Cloning And Functional Analysis Of OsTPP1 Gene Related To Cold Stress In Rice

Abiotic stresses such as salinity, drought and low temperatures are main factors leading to crop production, decline in quality and limit crop geographical distribution, especially there is a a common problem that low temperature stress can cause a significant reduction of the crop in world. With the deepening of the stress resistance molecular mechanism, the researchers found that the expression of some genes undergone significant changes in the face of adversity stress conditions, genetic transformation of these genes can improve plant stress tolerance, and thus more significant improved crop resistance effect.Kongyu 131 is a wide range of rice varieties grown in the northeast region, with cold-resistant and high-yield characteristics. In this study, based on the result of DGE(Digital gene expression), we screened out an obvious change in the amount of gene expression-trehalose- 6- phosphate the phosphatase, we do OsTPPl gene expression analysis by half quantitative RT-PCR and Real time-PCR experiments to cold stress different times, clone the gene and genetic transformation of Arabidopsis thaliana, ultimately, we get the transgenic plants. There is the important research value to improve plant stress tolerance and provide a theoretical basis for crop resistance to cold application of genetic engineering and genetic resources. The main findings are as follows:1. The gene expression analysis of OsTPPl:The use of semi-quantitative RT-PCR and Real Time-PCR method to detect the cold stress treatment of rice material target gene expression level changes, The results show that the OsTPPl genes under cold stress conditions are transient expression, the expression of Kongyu 131 within cold stress 24 hours is sustained, the expression of Longjing 11 is regularity, the difference of the two rice cold resistance is likely due to the TPP1 gene expression differences in transcriptional regulation of the level.2. OsTPPl full-length genes cloning and sequence analysis:We clone OsTPPl gene by PCR amplification, the clone template is kongyu 131, the full-length cDNA sequence of the gene 1478bp, the CDS region has 1113bp,encoding 371 amino acids.3. Plant expression vector and genetic transformation:The fragment of OsTPP1 (full ORF) was recombinanted to the expression vector pBI121.Arabidopsis plant was transformed by Agrobacterium which incorporated the recombinant vectorUsing floral dip.The transformation efficiency was 2% when putative homozygotes were examined in T2 by molecular biology detection. As a result, the degree of inhibition for transformed plants was lower than that of WT plants when they were under cold stress. It is proved that overexpression of TPP1 could improve the resistance of low temperature in transformed Arabidopsis, and this confirmed that the TPP1 is important stress-inducible gene involved in plant osmotic stress.