Research On Characterstics Of Intrcellular Calcium Signal During The Process Of Sugar Beet(Beta Vulgaris L.)Resisitance To Rhizomania

Sugar beet (Beta vulgar is L), an important sugar crop in the world, is another major sugar source in addition to Tropical cane. However, sugar beet is threatened by many diseases in the production process, among which Rhizomania has become one of the most serious diseases. Rhizomania which is a virus disease has happened in the world scale and the damage is great to the production of sugar beet. For example, the disease happened fields normally reduce the yield by40%-60%and decrease the sugar containing by4-9levels. Sometimes, the disease was so serious that sugar beet can completely loss the yields. Rhizomania has become the main important factors that inhibiting sugar beet production and its industry development. Increasing the ability of sugar beet resistance to Rhizomania has become an urgent problem that needs to be solved. Therefore, intense research on mechanism of sugar beet resistance to Rhizomania has the great significance in accelerating disease resistance breeding of beet, expanding the sugar beet planting area, improving the farmer income, and promoting rural economic development. In this dissertation, using Rhizomania infected sugar beet as materials, we adopted the laser confocal microscopy techniques and isotope labeling techniques together with calcium ion fluorescence probe to observe concentration and distribution changes of calcium ion, which is the second intracellular messenger in signal transduction pathways. At the same time, the activity of calcium ion related protein kinase was also measured. Based on the results, the function of calcium ion and related protein kinase in Rhizomania resistance signal transduction pathways was revealed, which provide the theoretical foundation and experiment basis for studying the resistance signal transduction pathways in sugar beet after infecting with Rhizomania. Results are as follows:1. Establish the technical system to detect the intracellular calcium ion. Load calcium ions probe Fluo-3/AM in sugar beet leaves at40C and incubated for90min, and then room temperature incubated for90min, which will effectively avoid the incompletely esterified fluorescent dye gathering in the cell or the organelles. Also, in the experimental process there was no fluorescence destroy. These methods provide the technical foundation in studying the function of intracellular calcium ions after sugar beet were infected with BNYVV. 2. According to the laser confocal microscope results, the concentration of Ca2+was obviously increased in resistant sugar beet leaves’protoplast cell after infected with BNYVV virus, whereas it stayed constantly in susceptible cultivars. Experiments indicated that the increases of intracellular calcium ions, which were induced by infecting with BNYVV, mainly coming from the intercellular calcium ions. Ca2+, as the second messenger during the interaction of sugar beet and Rhizomania, plays very important role in activating sugar beet resistance to Rhizomania.3. SA, JA plays an important role in plant resistance signal transduction pathways. After exogenous JA and SA were supplied on the leaves, intercellular calcium ions fluorescence intensity were measured in sugar beet leaf cells by using laser confocal microscopy. Results suggested that SA, JA can induce intercellular calcium ions in sugar beet leaf cells, which matches the increase pattern of intercellular calcium ions when the leaves were challenged with BNYVV. This result revealed that JA and SA are the upstream signal molecules for intercellular calcium ions, which provide direct evidence for the involvement of JA and SA in the process of sugar beet resistance to Rhizomania.4. Several factors like exogenous substrate, ATP concentration, reaction time and metal ions concentration were checked to determine the best way of measuring the protein kinase activity in sugar beet leaf cells. The results indicate that Histon-Ⅲ was the best exogenous substrate to measure the protein kinase activity in cells after sugar beet leaf infected by BNYVV virus. When the concentration of Histon-Ⅲ and ATP were0.5mg/mL, and50u,mol/L respectively, the reation started. After10min, when Mg2+concentration is5mmol/L, protein kinase activity reached to its peak. Several comparative experiments were completed to check the activity of the protein kinase by adding Ca2+or not adding, adding Ca2+and CaM or adding Ca2+and TFP. Comparative experiments preliminarily show that CDPK is the protein kinase which is the receptor of Ca2+signaling in BNYVV infected sugar beet leaves.