| The problems of drought seriously affect the growth and the production of plants, it is very important to breeding new varieties of plants that with excellent drought and salt tolerance. When exposed to various adverse environmental stresses, the organism is able to accumulate some osmoprotectants to response the changes of environment. Glycerol is an extremely desirable agent to protect cells against osmotic stress. 3-glycerophosphate dehydrogenase (GPDH), as the key enzyme in the metabolic pathway of glycerol, plays a main role in the distribution of material flow direction and the glycerol synthesis level in the catabolism of glucose to glycerol. As an important model plant, tobacco is important to the researchers of genetics, physiology and metabolism. The study of over-expressing GPDH which comes from Candida glycerinogenes WL2002-5 in tobacco to improve the resistance has not yet been reported, this study aimed at breeding a new transgenic tobacco that increases the glycerol synthesis level to strongthen the ability of osmotic stress tolerance.To analyze and understand the function of the upstream sequence of GPD1 from C. glycerinogenes, a series of promoters PCggpd with different lengths and the gene gfp encoding green fluorescent protein were cloned, and then ligated into the expression vector pYX212 to form the recombinant plasmids pYX212-PCggpd-gfp, then the plasmids were transformed into S. cerevisiae W303-1A by electroporation, respectively. The expression of gene gfp in recombinant strains S. cerevisiae W303-1A-gfp were examined by using fluorescent microscopy when the recombinant strains were cultured in the YEPD medium and the YEPD medium containing 6 % NaCl, respectively. The results showed that the gene gfp was expressed under the control of PCggpd longer than 800 bp in S. cerevisiae. Furthermore, there is an apparent difference of the expression among the different recombinant strains when cultured under 6 % NaCl. The green fluorescence was much more intensive than the others when the length of the promoter in the recombinant strain is 1000 bp. This proved that the PCggpd of 1000 bp was most affected by the osmotic stress. This may be related with the existence of the stress response element in the upstream sequence of GPD1.After we got the strongest promoter PCggpd(1000), the recombinant plant vector of p3300-zeocin-PCggpd(1000)-gfp was constructed and transferred into Agrobacterium tumefaciens, then the transgenic tobacco was obtained by the way of leaf-disc with the help of A. tumefaciens-mediated transformation. According to the expression of GFP in the leaf of transgenic tobacco, we found that the promoter PCggpd starts successfully, and to a certain extent, the intensity of GFP increased with the increasing of osmotic stress.Finally, the key enzyme gene GPD1 in glycerol metabolism of C. glycerinogenes was cloned and transferred into tobacco by the way of A. tumefaciens-mediated transformation. The glycerol content and GPDH activity of transgenic tobacco increased after treated with different NaCl concentration osmotic stress, and in a certain range, both of them increased with the NaCl concentration increases, to improve the ability against osmotic stress.
|
PDF Full Text Request