Lempk1/2 Gene Expression Vector Of The Building And The Preliminary Research Of The Transformation Of Tomato

Tomato (Lycopersicon esculentum Mill.) is one of main cultivated vegetables in greenhouse. Heat, cold, high salt and other abiotic stress factors seriously affect the growth, yield and quality of tomato.To improve the ability of resistance to abiotic stresses of tomato is being one of the important content of the tomato breeding and cultivation studies. Investigate the molecular basis and physiological and biochemical mechanism of tomato resistance to abiotic stress is of great significance for improving the efficiency of the heat tolerance of tomato cultivation and breeding.Mitogen-activated protein kinase (MAPK) cascade system, considered to be one of the main ways in plant cells converted extracellular stimuli signals into intracellular reactions, plays an important role in response to various environmental stress and gene expression. In recent years, a large number of MAPKs have been isolated in plants, and have made great progress in terms of its mechanism. Relative to Arabidopsis, tobacco and other terms of the mode crops, the study of the role of MAPK in stress signal transduction in vegetable has only just begun. It is speculated that there are18kinds of the MAPK in tomato, but the most functions are unknown. The studies have focused on biotic stresses, but the study of biological stress is little.So. tomato was used as materials in the experiment, including cloning the gene LeMPK1and LeMPK2of tomato, expression characteristics and constraction of the vector of expression (overexpression, transient expression), transformation of tomato with Agrobacterium-mediated, and to get the valuable transgenic plants, which will lay the foundation for future of tomato MAPK function in abiotic stress. The main findings are as follows:1.LeMPK1/2cloningDesign the full-length primers according the sequence of LeMPK1/2gene cds from NCBI genebank. cloned the full-length fragment of gene LeMPK1/2from tomato1479.2. Expression characteristics of LeMPK1/2Analysis the spatial and temporal expression characteristics of LeMPK1/2on tomato by RT-PCR. The expression levels of the two genes have no obvious changes with the passage of time. LeMPKl expression is significantly in flowers and greenfruits, higher than that of other organizations (root, stem, leaf, redfruit) expression. The amount of expression of LeMPK1in flowers and greenfruit, followed by redfruit. The expression of LeMPK1is significantly higher than LeMPK2at the same level of RNA.3. Construction of expression vector Cloning the full-length gene of LeMPK1/2to the called pBI121which constitutive of promoter35S so that get the plant expression vector called pBI121-LeMPK1and pBI121-LeMPK2. then put them into Agrobacterium EHA105preparing for the transformation of protoplasts.4. Genetic transformation of tomatoThe system of genetic transformation of tomato was established. The pBI121-LeMPKl and pBI121-LeMPK2were transformed into cotyledon of tomato1479with Agrobacterium tumefaciens EHA105.35S promoter as supra, cited in the target gene as examined by PCR to detection of positive plants. The number of pBI121-LeMPK1positive plants is42, and the number of pBI121-LeMPK2positive plants is36. By the detection of RT-PCR. the overexpression plant of pBI121-leMPK1and pBI121-LeMPK2is1. respectively.