Studies On The Molecular Physiological And Cytological Techniques Of Stress Signal Transduction In Arabidopsis Thaliana

The model plant Arabidopsis has many advantages for the genetic analysis including a small genome, short life cycle, small stature, prolific seed production, and easy transformation. Because of Arabidopsis completely sequenced resources and a near saturation insertion mutant collection, many mutants related to environment stresses are provided for functional gemomics and many genetics allels regulated by environment stresses are revealed. However, to completely understand the function and the roles of these genes in environment stresses signal transduction, multidisciplinary technologies are required to analyse the position of the intermediates in signal transduction casscade and the possible regulative pathway. Here we try to establish the technology of electrophysiological measuring K+ channel activity in Arabidopsis root cells. We also establish the technology of intracellular calcium concentration measurement by combined molecular genetics and cytology and establish the technological foundations for the functional gemomics research. We can get much valuable information about the salt stress through studying the characterization of channels in plasma membrane by patch clamp technology. In patch clamp recording especially adding reagents for pharmacological analysis, there are many defects including the lower percent of successful sealing, the shorter recording time and unstable sealing. In order to get the Arabidopsis root protoplasts that are high quality and suitable for patch clamp recording, the cultural methods and the digested conditions are studied. We analyzed the effects of different cultural methods — sand, vermiculite, nutritive soil, water and Murashige-Skoog medium — on the development of root protoplasts isolated from Arabidopsis thaliana. The digested conditions including the digesting models, temperature and time were discussed. In patch clamp recording we compared the qualities of the root protoplasts from different cultural methods. Consequently it provides the technical support for understanding the mechanism of salt stress in electrophysiological research. Cold shock is accompanied by a large transient rise in cytosolic free calcium concentration ([Ca²⁺]cyt). The increase in [Ca²⁺]cyt resulting from cold treatment can induce a number of genes expression. But it is not known the base of especial Ca2+ oscillation or signature in plant cells and many components in the process of calcium signals transducing into nucleus and their genetics spatio-temporal positions do not make certain. The transcription factor, CBF1,CBF2 and CBF3, was found as the key factor in cold acclimation. So the transgenic Arabidopsis expressing the calcium sensitive photoprotein aequorin are crossed to CBF1,CBF2 and CBF3 overexpression lines. At the same time the plasmid containing the apoaequorin gene was transformed Agrobacterium GV3101. The positive clones are checked by PCR. Transformed the plant by Floral dip, and obtained the transgene seeds later. Screening the T2 transgene seedlings with hygromycin, we have obtained potential transgene plants. From the change of intracellular calcium concentrotion in response to cold stress in the transgeneic Arabidopsis, the real time supervising of cytosolic calcium concentration was carried out. The firm cytological and genetic foundations of the mechanism of cold stress are established by analysising the calcium signal system in the position and function of cold signal transduction pathway.