Molecular Cloning And Function Analysis Of A Betaine Aldehyde Dehydrogenase Gene In Sesuvium Portulacastrum L.

Under stresses, plant establishes osmotic and ionic homeostasis and maintains physiological and biochemical steady states to sustain growth. Among many osmotic materials, glycine betaine is a best micro-molecular organics, and functionally work for osmotic regulation in plants, as its non-toxics to plant growth. During the process of plant growth and development under natural conditions, few of glycine betaine is accumulated in vivo plant; however, its accumulation is rapidly increased under the environmental stresses (e.g.drought, low temperature and salt stresses). Presently, the molecular mechanism of the glycine betaine functions in plant stress is a hot topic on researching the salt-tolerance and improving p salt stress of plants. Sesuvium portulacastrum, which has a strong tolerant ability to salt, drought, heavy metal and so on, can be regarded as an ideal material to study the salt-tolerance mechanism of euhalophytes. BADH is a key enzyme during the synthesis of glycine betaine. In this research, a BADH gene has been isolated from S. portulacastrum, and named as SpBADH. The functions, enzymic and gene expressional characterizations, molecular regulation of the BADH gene named as SpBADH have been studied. The aims of this research are to analyze the mechanism of the SpBADH to stress tolerance. The main results of this thesis are as follows:1. A SpBADH gene has been successfully isolated from S. portulacastrum (SpBADH accession number:JN192464). The full-length of the SpBADH gene is1947bp, with an open reading frame of1503bp and a5' non-coding region of57bp and a3' non-coding region of387bp. It encodes a SpBADH protein with500aa, molecular weight55KDa. Bioinformatic analysis showed that the SpBADH protein has a conserved catalytic domain of (VTLELGGKSP) and C residues for emzymic activity of aldehyde dehydrogenase. The SpBADH protein has a high similarity with AhBADH (Amaranthus hypochondriacus), SoBADH (Spinacia oleracea), KfBADH (Kalidium foliatum)and AtBADH (Arabidopsis thaliana), which is90%,81%,87%and79%, espectively.2. The real-time PCR results indicate that, the expression of the SpBADH in vivo S. portulacastrum is found in roots, stems, leaves, which has no tissue specificity, and with higher expression level in leaves, lower expression level in stems. Moreover, the expression of the SpBADH in response to NaCl stress is more sensitive in leaves, then followed by stems; also it's expression in response to PEG, ABA and H2O2after48h stresses, which the gene expression in stems to ABA and H2O2is more sensitive than that in leaves and roots, whereas, to PEG,, it is more sensitive in roots than in leaves and stems. The expression of the SpBADH in response to low temperature is increased in leaves, stems and roots after48h, while during0-24h its expression is almost no changed, whereas, to higher temperature, it is more sensitive in roots and leaves than in roots. Meanwhile, the level of glycine betaine was increased in tissues of S. portulacastrum under600mmol/L NaCl, seawater,20%PEG, H2O2and lower temperature4?, but it was no difference under the stresses of high temperature and ABA.3. The enzymic analysis showed that the activity of the betaine aldehyde dehydrogenase in vivo S. portulacastrum is highest in chloroplasts, followed by cytoplasms, and lower activity is also checked in peroxysomes and microsomes. Thus it is deduced that there are more BADH genes in S. portulacastrum. The expression of the SpBADH gene fused with GFP in Arabidopsis protoplasts showed that the SpBADH location is in chloroplast.4. The prokaryotic expression of SpBADH based on pET vector and BL21(DE3) is high-efficiency. The optimal and efficiency conditions in E. coli is:the culture temperature is37?; the gene expression at cell concentration OD600-0.6, IPTG0.2mmol/L,4h, which the expressed SpBADH protein is as high as301? g/mL.5. The enzymes characteristic analysis showed that the SpBADH protein purified from the expressed full-length SpBADH in E. coli has catalytic activity to aldehyde dehydrogenase, which arrives as high as87U/mg, however, no activity is founded in its mutants. Thus, the SpBADH gene isolated from S. portulacastrum is one of betaine aldehyde dehydrogenase, and the conserved ten polypeptides and the Cys amino acid play an important role on its activity. The optimal conditions for SpBADH catalytic activity are37?, pH=7.2. Na+and K+can increase SpBADH activity, and Zn2+and Cu2+have competitive inhibition for SpBADH, especially Cu2+, while, small molecule alcohols have a protective effect to maintain the microenvironment of catalytic activity.6. The tolerance of the expressed SpBADH E. coli is Na+>K+>Mn2+>Zn2+and Cu2+; and it also tolerant to lower temperature. The recombinant yeast pYES-SpBADH showed some tolerance to Na+?K+and Li+. 7. The SpBADH has been transformed into Arabidopsis thaliana by Agrobacterium-mediated method and experimented under salt and drought stresses. The results showed that over-expression SpBADH in Arabidopsis could increase the activity of BADH and level of glycine betaine (3-6folds) under natural and stressed conditions. Under natural conditions, the levels of SOD, POD, CAT, Pro, MDA and H2O2) is similar in SpBADH transgenic and wild type Arabidopsis, however, under salt and dought stresses, the levels of POD, SOD, CAT and Pro are higher in SpBADH transgenic Arabidopsis than in wild type; while, the levels of MDA and H2O2are higher in the wild type than in SpBADH transgenic Arabidopsis.8. The microarray analysis of the differential expression genes in SpBADH transgenic and wild type Arabidopsis under salt and natural conditions, the results showed that under salt stress, most of the up-regulation genes are included in stress response pathways and enzymes for removing ROS damage, such as redox reaction system, Oxygen stress response system, response to ions for stresses, so on. Further analysis was found that most of these differential genes are located on plasma membrane as functional and structural proteins, which relate to ion channel and transcriptional facts.9. In summary, the SpBADH gene play a role in plant tolerance to salt and drough stresses and works for removing ROS damage, and ion osmosis.