The Studies On The Responses Of NCED Gene Expression And ABA Accumulation To Stresses In Chorispora Bungeana

In our experiments, we used Chorispora bungeana Fisch et.Mey (Chorispora bungean) as material, by doing ABA-ELISA, measuring the physiological parameters as the proline, MDA content, REC and TTC activity and constructing semi-quantitative RT-PCR of NCED (9-cis-epoxycarotenoid dioxygenase) gene, in order to investigate the relationship between cold hardiness of regenerated plants and accumulation of endogenous abscisic acid (ABA) after low temperature treatment(4°C) and found the mechanism of cold tolerance in alpine subnival plants. We got results as follow:1. Cloned the reading frame of NCED (9-cis-epoxycarotenoid dioxygenase) gene in Chorispora bungean.2. Consructed the optimized semi-quantitative RT-PCR to detect NCED gene expression difference after different time of exposed to cold, using P-Actin as inner control. The results showed that the NCED gene expression increased with lengthening the exposure time of cold stress, and there lay in different expression mechanisms of NCED gene family. In the same time, we found during other stresses such as heat stress, osmotic stress and SNP treatment, the NCED expression increased differently. These observations were consistent with a conclusion that it was the cold that triggered the chilling acclimation of Chorispora bungean, not the indirect factors such as the loss of cell turgor or decreased cell bulk.3. When exposed to low temperature(4°C), the regenerated plant of Chorispora bungean had an increased level of proline and remained a high TTC activity, meanwhile it had an obvious accumulation of endogenous ABA. When added 1mM LNNA (an inhibitor of nitric oxide synthesis) or 1mM Flu (an inhibitor of ABA biosynthesis), respectively, this cold tolerance decreased even vanished. These results indicated that the endogenous ABA accumulation played a majorrole in inducing cold tolerance of Chorispora bungean, and this induction may be mediated by the signal molecular NO.4. We also measured other physiological parameter and endogenous ABA changes during other stress treatments, such as osmosis stress, using 20%PEG (polyethylene glycol), heating(42°C) and lmM SNP. From the different changing tendency of physiological parameters, we believed that the osmosis tolerance, cold tolerance and heat tolerance had their own gene expression mechanisms and signal transduction, respectively. And we delightly found that Chorispora bungean could be a typical model plant for cold tolerance research, and could be an appropriate object for correlative studies on osmosis, heat and other stress as well.5. The cDNA library of Chorispora bungean was constructed through SMART method, and was amplified in order to increase the titer to 1.1 X 108 pfu/mL.From what has been discussed above, we may finally draw the conclusion that the phytohormone abscisic acid (ABA) played an important role in enhancing the cold hardiness of the alpine subnival plant. The regulatory mechanisms for cold tolerance in plants may involve in the increased expression of NCED gene, No and NOS acting as assistant factors, and correlated endogenous ABA accumulation. Moreover, there existed different expression mechanisms of NCED family members during different time and stress treatments. These results certainly helped to do further studies on establishing stress resistance and ABA-independent signal transduction in plants.