Studies On Resistance Of CsPLDα-Transgenic Tobacco Seedlings To Salt And Drought Stress

Plant phospholipase D(PLD), which can hydrolyze membrane phospholipids to produce phosphatidic acid(PA), a secondary signaling molecule, has been proposed to function in diverse plant stress responses, which can increase plant stress tolerance to a certain extent(Mishra et al.,2006; Hong et al.,2008a;Li et al.,2004).CsPLDα was cloned from cucumber leaves, the full-length of CsPLDα c DNA consisted of 2,756 bps which contains an open reading frame of 2,427 nucleotides coding for a 808-amino acid(Accession number in Gen Bank is EF363796). Based on existing studies, we investigated the effects of over-expression(OE) CsPLDα on tobacco stress tolerance to reveal the biological mechanism of CsPLDα gene involved in plant stress responses, and provide scientific basis for cultivating drought-and salt-resistant cucumber varieties by genetic engineering means. The main results are shown as follows:1. When treated with NaCl and PEG, the transcript levels of CsPLDα in leaves and roots of cucumber increased at first and then decreased. Under NaCl treatment,the expression of CsPLDα peaked at 1 h in leaves and 3 h in roots; Under PEG treatment, the expression of CsPLDα peaked at 3 h in leaves and 1 h in roots,suggested that CsPLDα was involved in salt and drought stress reaponses.2. Seeds of CsPLDα OE and WT tobacco plants were sown into plastic pots filled with nursery substrate(peat:vermiculite:perlite = 2:1:1), and we found that the seeds of OE plants germinated neatly, and the germination time was remarkably shorter. At day 30, the root length, lateral root number, fresh weight and dry weight of transgenic tobacco seedlings was significantly higher than that of WT.3. When treated with different concentrations of NaCl, PEG and ABA, germination rates and seedling sizes of the transgenic lines were significantly greater than WT. In addition, the germination times for transgenic lines were also remarkably shorter. Further studies indicated that transgenic lines had longer primary roots and more biomass accumulation than WT plants. The electrolyte leakage rate and water loss in transgenic lines was also much lower than in WT.4. Under drought stress condition, compared with WT, the fresh weight and dry weight of transgenic tobacco seedlinfgs shoot and root were significantly higher, the content of osmoprotectants such as proline、soluble protein and soluble sugar were also significantly higher too, the solute potential、electrolyte leakage rate and MDA content in transgenic lines were significantly lower.In addition, the relative water content of transgenic tobacco leaves was significantly higher than that of WT, and the water loss from detached leaves has been lower during treatment times, suggested that transgenic lines has a stronger water retaining capacity, and that CsPLDα gene was involved in the regulation of stomatal closure.5. Under salinity stress condition, the root fresh weight and dry weight of transgenic tobacco lines was significantly higher than that of WT. Besides, salt stress promoted the rapid accumulation of proline, soluble sugar, soluble protein, electrolyte leakage rate, O2.- and H2O2 content in different tobacco genotypes, and they increased gradually with the increasing level of stress, but the proline, soluble sugar and soluble protein content in transgenic lines was always higher than that of WT, the electrolyte leakage rate, O2.- and H2O2 content was always lower. In addition, the activity of antioxidant enzymes SOD, POD, CAT and APX in the leaves of transgenic tobacco and WT increased firstly and then decreased, but the variation of transgenic tobacco was significantly higher than that of WT. These results suggested that CsPLDα has the potential to regulate plant osmotic adjustment ability and removal reactive oxygen species to some extent to reduce lipid peroxidation, maintain the normal physiological and biochemical functions of cells so as to reduce salinity stress damage.