Study On The Mechanism Of Tomato SIMPK1 Under High Temperature Stress

High temperature is a common stress factor in plant growth process which seriously affects plant growth, development, yield and quality. The process of plants subjected to high temperature stress is the cells sensing, transfering temperature signalling, and changing cell metabolism, to initiate various defense mechanisms to maintain its life. Mitogen activated protein kinase (MAPK) cascade, considered to be an important component of extracellular signal to intracellular signaling pathways in cells, were involved in responses to a variety of abiotic stress. The cascades always consist of MAPKKK-MAPKK-MAPK. In recent years, many MAPKs have been found and the functions of MAPKs are explored in-depth, but the mechanism of MAPK response to high temperature stress is poorly understood.At present, the tomato genome sequencing has been completed and it is possible to analyze the tomato MAPK family information at the genomic level. The results show that SlMPK1is involved in high-temperature response, but the physiological and molecular mechanisms of this process are unclear. In this study, SlMPK1interference vector was constructed and transformed into tomato, and the transgenic tomato were obtained. The transgenic tomato and wild type were used as meterials to investigate the phenotype and related physiological reaction under high temperature stress, to analyze functions of SlMPK1response to high temperature stress. The yeast two-hybrid system was also used to screen a high-temperature-induced tomato cDNA library to detect hybrid proteins that could interact with SlMPK1. The major results are as follows:1. Transgenic tomato plants were constructed.The201bp3’UTR DNA fragment of SlMPK1was cloned and was linked to the binary vector pGSA1285to construct interference (RNAi) vector. Then, through Agrobacterium-mediated leaf disc transformation in tomato and50‰kanamycin to screen transgenic tomato, the positive transgenic lines were obtained. Further detection of genetically modified plants with Real-time PCR, we received a total of10independent transgenic lines.2. Analysis of thermotolerance of transgenic tomato plants(SlMPK1-RNAi).T1generation of transgenic plants and wild type were treated with short-term high temperature (40℃-45℃) or long-term high temperature (38℃/28℃), and then were restored under normal growth conditions. We found the transgenic tomato plants had high survival rate, and the degree of inhibition was significantly lower than that of wild-type plants, indicating transgenic tomato plants having stronger thermotolerance than wild-type plants.3. Analysis of antioxidant system of transgenic tomato (SlMPK1-RNAi).Under high temperature stress, Accumulation of ROS in transgenic tomato was less than the wild-type tomato, and the degree of membrane lipid peroxidation (MDA) showed the same trend. However, the antioxidant enzyme (SOD, POD, CAT, APX, GR) activity and non enzymes (AsA and GSH) content of transgenic tomato were significantly enhanced. In addition,the results of real-time PCR showed that the expression levels of antioxidant genes (CAT1,APX2,FeSODl, POD) and relative resistant genes (PPO、DGD1、MSRB5、TPS11) in leaves of transgenic tomato were higher than that of wild-type plants.4. Screening SlMPK1-interactive proteins by Yeast Two Hybrid.The yeast expression vector bait pGBKT7-SlMPK1was constructed, and was confirmed to be no ill behave and self activation in yeast cell. Then high-temperature-induced tomato cDNA library (Pgadt7-Tomato cDNA library) was screened and seven protins were found during initial screening processes.