Cloning And Characteration Of Two Salt-induced Genes MsPBL And MsALD In Medicago Sativa L.

Salinization has become a global problem and The impaction to corps has become more serious. 6% of the world's total land area are salt affected, 2% of the land farmed by dryland agriculture and 20% of the irrigated land are salt affected. Most of this salt affected land has arisen from natural causes, from the accumulation of salts over long periods of time in arid and semiarid zones. Apart from natural salinity, a significant proportion of recently cultivated agricultural land has become saline owing to land clearing or irrigation, both of which cause water tables to rise and concentrate the salts in the plant root zone. One method to solve this problem is to raise new Varieties that can not only withstand high levels of salt but can also maintain optimum yield levels.Plants differ greatly in their tolerance of salinity, as reflected in their different growth responses. In the simplest analysis of the response of a plant to salinity stress, the reduction in shoot growth occurs in two phases: a rapid response to the increase in external osmotic pressure, and a slower response due to the accumulation of Na+ in leaves. In the first, osmotic phase, which starts immediately after the salt concentration around the roots increases to a threshold level, the rate of shoot growth falls significantly. The second, ion-specific, phase of plant response to salinity starts when salt accumulates to toxic concentrations in the old leaves, and they die. The mechanisms of salinity tolerance fall into three categories: Tolerance to osmotic stress, Na+ exclusion from leaf blades, Tissue tolerance.Salt stresses affect virtually every aspect of plant physiology and metabolism. Numerous changes that occur under these stresses have been documented. Although some of the changes are clearly adaptive, many may simply be pathological consequences of stress injury. True, there may be active signaling to activate pathological responses. Knowledge of them is just as important because they represent suitable targets for genetic suppression to improve salt and drought stress tolerance. In nature, for a plant to sacrifice a part of its structure constitutes an adaptive strategy to survive a stress episode. For adaptive or presumed adaptive responses, it may be helpful to conceptually group them into three aspects: homeostasis that includes ion homeostasis, stress damage control and repair or detoxification, growth control. Accordingly, salt and drought stress signaling can be divided into three functional categories: ionic and osmotic stress signaling for the reestablishment of cellular homeostasis under stress conditions, detoxification signaling to control and repair stress damages, and signaling to coordinate cell division and expansion to levels suitable for the particular stress conditions.Obtaining salt-torlanrance mutants and finding out the key genes in the mouts or some genes induced by salt is an effective and common method to studuy the mechanism of plant salt-torlanrance. Alfalfa (Medicago sativa L.) is one of the most important forage crops in the world. Generally, alfalfa is a salt-torlance plant, but the different varieties of alfalfa have different salt-torlance ability. At present, there are several salt-torlance culture varieties of alfalfa at home and abroad, 'Zhongmu NO.1' alfalfa selected and bred by IAS-CAAS (Institute of Animal Science, Chinese Academy of Agricultural Sciences) is one of them. The yield of 'Zhongmu NO.1' alfalfa growing in saline land was increased obviously compared with many other varieties of alfala.It's important to study the salt-torlance mechanism of 'Zhongmu NO.1' alfalfa. Because we may get some important imformation about how plant adapt the salt environment, and it also may be of great significance for us to breed new salt-torlanrace corps. In order to study about the salt-resistance molecular mechanism of'Zhongmu NO.1'alfalfa, we constructed a suppression subtraction hybridization (SSH) library induced by salt and got ESTs of salt inducee genes. In this study, based on the ESTs we cloned two new genes of alfalfa using RACE method. One gene encoded a protein which contains a PB1 (Phox and Bem1) domain, and we predict it as a component in cell signalling. The putative amino acid sequence of another new gene was highly similar to ALD from some other plants, such as Pisum sativum and Trifolium pratense. We analysis the two genes in bioinformatics, and predict the subcellular localization of them. We also use RT-PCR and transgentic methods to analysis the two genes.When fused to the green fluorescent protein, we found the gene (MsPBL) containing a PB1 domain to be localized in the nucleus of onion epidermal cells. We cloned the 1500 bp Upstream region of MsPBL. The Transcripts of this gene rase obviously when 'Zhongmu NO.1' alfalfa was treated with 300 mM NaCl, 100 uM ABA or 20% polyethylene glycol (PEG6000), respectively. These results indicated that this gene may be functional within the nucleus as a signal transduction protein, but uncertainly. was inserted into the transgenic vector PBI121 and expression in Tobacco, The result showed that there were some differences between transgenic tobacco leaves and wild type tobacco leaves. Another gene may be the fructose-1,6-bisphosphate aldolase gene (MsALD) of alfalfa. subcellular localization prediction indicated MsALD may localized in chloroplast. RT-PCR analysis result indicated MsALD can be induced in 300 mM NaCl after being treated for 2 h. MsALD may participate in the salt-torlanrance of alfalfa. We hope the results of this study could make a contribution to the salt-torlanrance mechanism of alfalfa and breeeding new variety of salt-torlanrance.