| Drought and salinity are the most important abiotic stresses that affect the normal growth and development of plants. Many important crop plants, such as rice, potato and tobacco, are sensitive to drought and salinity. Drought and salinity frequently cause the severe production losses of crops due to lack of effective tolerance mechanism. Improvement of crops and breeding of new varieties to increase their drought and salinity tolerance is the most economic and effective ways for using arid and salty soils. With the rapid development of modern molecular biology, the great progress has been achieved in improving crop drought and salinity tolerance by genetic engineering. One of the fundamental physiological mechanisms of high plant to cope with environmental stresses is osmotic adjustment, and glycine betaine is one of the most important of osmolytes. Glycine betaine was regarded as one of the most promising osmolytes because it possessed simple biosynthesis pathways, non-toxic to cells, and non-osmotic functions of stabilizing enzyme activities and membrane structures, reduction the peroxidation of membrane lipids.Some crop plants, such as rice, potato, tobacco and tomato, are unable to synthesize glycine betaine. Introduction of genes related to glycine betaine synthesis and establishing a glycine betaine biosynthetic pathway in glycine betaine-deficient crop plants through genetic engineering, which could be enhanced drought and salinity tolerance of crop plants. Until now, many transgenic plants were obtained by introducing the related genes of glycine betaine synthesis and increased the ability of plants at the various levels to tolerate drought and salinity stresses.The main purpose of the studies is to isolate a betaine aldehyde dehydrogenase (BADH) gene which is the key enzyme for glycine betaine synthesis from spinach (Spinacia oleracea L.) and a drought, salt, low temperature and ABA-induced rd29A gene promoter from Arabidopsis thaliana, construct the expression vectors of BADH gene under the control of the promoters CaMV 35S and rd29A, and introduce them into potato cultivars and increase their drought and salt tolerance. The molecular mechanism of effective expression of exogenous genes under the control of different promoters in abiotic stress was also studied, which could enrich transgenic theory of plant and provide theoretic basis to breed new stress-resistant crop varieties. The major results as following:1. A cDNA of BADH gene was isolated from spinach leaves. The sequence analysis showed that the BADH cDNA contains 1494 bp open reading frame (ORF), which encoded a protein of 497 amino acids. The nucleotide sequence of BADH cDNA shared 99.87% identity with BADH gene firstly cloned from spinach and more than 80% identity with other plant BADH genes. Northern blotting analysis indicated that BADH mRNA levels increased with the raise of salt stress concentration. The levels of BADH transcripts under continuous 500 mmol/L NaCl stress for 4 d increased 4-fold when compared to the control plants. Drought was promoted by withholding water, and the levels of BADH mRNA in leaves increased with the stress time. Those results demonstrated that expression levels of spinach BADH was induced by salt and drought stress.2. The 824 bp upstream regulatory region of rd29A gene was amplified from Arabidopsis thaliana genome by PCR technique. Sequence analysis showed that this cloned fragment shared 99.39% identity with the reported rd29A promoter and contained several cis-acting elements including dehydration responsive elements (DRE) and ABA responsive elements (ABRE). The plant expression vector pBIrd was constructed with GUS gene controlled by rd29A promoter. Transgenic potato plants were obtained by Agrobacterium tumefaciens system. Both Northern blotting and histochemical analysis of GUS activity revealed the very similar results, that untreated transgenic potato expressed GUS activity weakly, but the level of GUS mRNA increased steadily after 24 h exposure to 4℃ low temperature, 100 μmol/L ABA,...
|
PDF Full Text Request