Cloning Of GSH Synthetase Genes From Saccharomyces Cerevisiae And Determing Of GshⅠ Gene Functions Of Drought Tolerance In Arobidopsis Thaliana

When plant is subjected to environmental stress, drying, salinity, extrem high or low temperature, ect., it produces reactive oxygen species (ROS), such as ultra-oxygen ion, singlet oxygen, H₂O₂and OH. The ROS production and accumulation in the body inevitably results in overoxidation of lipid membrane or degreases and production of malondialdehyde (MDA), finally, destruction of the membrane system in cells. However, glutathione (GSH), one of the important antioxidants, efforts its reactions to eliminate or reduce ROS damages on the memberane system through its glutathione-ascorbic acid (GSH-AsA) reductive system.GSH synthesises in organisms in the way of two steps in the presence of ATP: (i) synthesis of γ-glutamylcysteine from L-glutamate and L-cysteine, catalyzed by γ-glutamylcysteine synthetase (γ-GCS or: GSH â… ) (ii) addition of glycine to the C-terminus of γ-glutamylcysteine to yield GSH, catalyzed by glutathione synthetase (GC or: GSH â…¡). The genes coding GSH â…  ,GSH â…¡ ,ATP synthetases regulate GSH synthesis and are important in drought tolerance in plants.In the dissertation, GSH synthetase genes, gsh â…  and gsh â…¡, were cloned from Saccharomyces cerevisiae strain (ScHL5). Gsh â…  was constructed prokaryotic expression vector and transferred into E.coli BL21, plant expression vector was constructed and transferred into EHA105. Then, gsh â…  gene was transferred into arobidopsis through Agrobacterium-mediated transformation. The recombinants were screened by PCR, Southern blotting, Northern blotting. Then, the transgenic plants were measured their physiological criteria of drought stress. Exploring the influence of ATPase activity on the synthesis of GSH, activities of ATP synthase in mitochondria, contents of ATP and GSH were tested in different cultivation conditions, temperature, time and pH values. Important results of the dissertation were summarized as below:1 ,The gsh â…  and gsh â…¡ genes were achieved Based on the open reading frames of gsh â…  gene released in GenBank by Lisowsky (1993) and gsh â…  gene by Yoshiharu (1998) , primers were designed and, the GSH synthetase genes gsh â…  and gsh â…¡ were cloned from genome of yeast strain (ScHL5) , selected from high active dry yeast supplied by Anqi Biology Group in Hubei province of China. The gene sequences showed that the sequence homologies of gsh â…  gene and gsh â…¡ gene in strain ScHL5 are 98% and 99% compared with those of Lisowsky and Yoshiharu, respectively. Analysis of copy number of gsh â…  gene indicated that the gene is one copy. The Genes of gsh â…  and gsh â…¡ cloned from the strain ScHL5 were registered in GenBank, and accession number are EF633694 and EF633695, respectively.2,E.coli BL21 transferred gsh â…  gene expressed specific proteinGsh â…  gene, the rate-limiting enzyme of GSH synthesis, was constructed the prokaryotic expression vector pRSET-B. The recombined plasmid was transferred into E.coli BL21. A fusion protein was produced under the induction of IPTG and its molecular weight of 81kDa which was consistent with that analyzed with the software method of DNAStar. pRSET-B expressed in E.coli BL21 and its quantities of expression are positively as the culture time increasing at 37 ℃. The vector is suitable for gsh â…  gene expression in the prokaryotic organism.3, The gsh â…  gene transgenic plants of Arabidopsis thaliana were achievedThe gsh â…  gene was constructed into vector pBI121 and transferred into Arabidopsis thaliana by Agrobacterium tumefaciens EHA105. The seedlings of the recombinants were analised with PCR-Southern blotting, Southern blotting and Northern blotting. As the results, the target gene was integrated into the genome of Arabidopsis thaliana. The gsh â…  gene was expressed in the transgenic plant.4,The transgenic plants reduce the increasing rate of MDA and electrolyte leakage (EL) under drought stressThe contents of GSH increased in both transgenic and control plants when the plants subjected to drying treatment, however, the GSH specific production rate was higher in transgenic than that in control plants. The activity of GR in transgenic plants was higher than that in control plants, which lead to a higher ratios of GSH to GSSG in transgenic plants. The leaves' water content, SOD and CAT did not show obvious differencs between the transgenic and control plants in changes under drougth stress. However, the MDA contents and electrolyte leakage (EL) (conductivity) increased in both transgenic and control, but those of the transgenic plant was slower than those of control. It is concluded that the GSH is different mechanism in eliminating ROS to increase plant drought tolerance from that peroxydase system.5 ,ATP synthase in yest mitochondria influence on GSH synthesis The ATP was a factor in GSH biosynthesis, however there have not been seen the report about ATPase and GSH synthesis. The research showed that content of GSH content was closely related to the activity of ATP synthase. After 60h fermentation, the GSH content reached the highest on the yeast culture at 30℃, with pH adjusted at 6.0. On the conditions, the activity of ATP synthase was 21.922 μmol/(mg.protein.h) and the ATP in yeast cells accumulated at the highest value of 14.755 nmol/g.DW.