Investigation Of The Salt Tolerance And Its Mechanisms In Bcl-2/Ced-9/PpBI-1Gene Transgenic Rices

NaCl stress is the major factor limiting plant growth and development, which seriously affects the yields and qualities of crops. Using transgenic technique has been an important strategy to produce agricultural crops to improve stress resistance. It has been shown that apoptotic suppressors such as Bcl-xL and Ced-9can enhance the resistance of plant cells to biotic or abiotic stresses, but detailed mechanisms remain still largely unclear. Here, we choose two rice strains(Oryza sativa L.subsp.Japonica cultivar Zhonghuall; Oryza sativa cv. Nipponbare) as an excellent model for studying various aspects of salt induced programmed cell death (PCD). And our studies were carried out to elucidate the mechanisms and enhance the resistant to salt stress by overexpressing Bcl-2/Ced-9/PpB1-1in rice. Results obtaind in our research are summarized as follows:1Bcl-2improves the tolerance to salt stress via suppressing cytosolic Ca2+increase and PCD caused by salt stress in rice.1.1Bcl-2transgenic rice possessed the ability to overcome NaCl stress. Under saline conditions, it can obviously improve the seed germination, root elongation and cell viability.1.2The profile of DNA laddering indicates that NaCl induces cell death accompanied by typical hallmarks of PCD, while Bcl-2could promote NaCl tolerance in transgenic rice via inhibition of PCD.1.3Investigation with Fluo-3/AM CONFOCAL shows that salt stress lead to increase cytosolic Ca2+in WT line, contrast to in Bcl-2line, suggesting that Bcl-2mediate closely with Ca2+signaling pathway induced upon salt stress.1.4La3+has the distinct ability to improve the seed germination and root elongation, reduce cytosolic Ca2+and inhibit DNA laddering in WT line, but not in Bcl-2line.2Using the Non-invasive Micro-test Technique (NMT), we confirm that under salt stress, Bcl-2can suppress NaCI-induced PCD by means of the regulation of ion fluxesin in rice cell.2.1The main cellular events under salt stress involve K+efflux from rice tip cells, which may be effectively suppressed by Bcl-2, so that salt-induced PCD can be inhibited. Otherwise, H+efflux does not change in such condition.2.2La3+can contribute to inhibition of K+efflux under salt stress in WT rice. However, its potential indicates unclearly in Bcl-2line.2.3Ca2+efflux is a common phenomenon under salt stress, which undertakes to much more extent in Bcl-2than in WT line.3K+efflux in cytoplasm under salt stress lead to transcriptional activation of OsVPE-2and OsVPE-3, furthermore, results in PCD. Such activation can be remarkably down regulated by La3+in WT rice, but not in Bcl-2line.3.1Q-RT-PCR detection showed that salt stress significantly increase expression of OsVPE-2and OsVPE-3in WT, but the expressions were inhibited in Bcl-2line.3.2Under salt sress, La3+treatment inhibited expression of OsVPE-2and OsVPE-3in WT, but not in in Bcl-2line.4Ced-9and PpBI-1gene were tansformed into rice calli by Agrobacterium tumefaciens-mediated transformation. Two transgenic lines, which possessed tolerance to salt, were successfully obtained in rice.4.1The incorporation of pCAMBIA13011-Ced-9/PpB1-1into rice genome was detected/identified by hygromycin selection, and PCR relative to target genes and Southern blotting. The expression of Ced-9/PpBl-1were confirmed by RT-PCR analysis in transgenic lines, and as a result, homozygotic lines of transgenic rice were obtained in two lines.4.2As with the investigations of pCAMBIA13011-Ced-9/PpBI-1transgenic T3lines, Ced-9line shows normal developmental phase, but not in PpBI-1line, suggesting that it would be probably caused by the different locus of T-DNA insertion.4.3Ced-9/PpBI-1transgenic T3lines possessed tolerance to salt, with respect to the seed germination and root elongation.4.4Through detailed analysis of DNA laddering, Ced-9was recognized as an effective factor to suppress NaCl-induced PCD, in rice.