Comparative Analysis On Molecular Mechanism Of Glutathione Synthetase For Heavy Metal Pollution Bioremediation

With development of social, economy and industry, heavy metal pollution becomes a worldwide environment problem. It not only affects the growth and development of plants and animals, the decline in the quality of agricultural products, but also threats to human health through the food chain. Thus, it is time to solve such problem. Because of being high efficiency, economy, green, clean and environment-friendly, bioremediation technology raises more concern in heavy metal pollution treatment, as an important topic in pollution ecology. Microorganisms and hyper-accumulated plants naturally occurring with heavy metal bioremediation roles grew slowly and were with low biomass in heavy metal stress conditions. These defects often affected the efficiency of bioremediation. In this study, by using methods with genetic engineering, genetic transformation, medium analog, screening of transgenic resistant microorganisms and plant, the experiments were carried out by using glutathione synthetase to enhance the heavy metal remediation effects of E. coli and energy sugar beet, and explored the heavy metal stress responsive mechanism in these glutathione synthetase broadly, and their expression characteristics in transgenic E. coli, Arabidopsis and energy sugar beet. The research also focused on the comparison of phenotype, physiological and biochemical differences and absorption of Cd, Zn and Cu in these different genotypes of recombinant E. coli and transgenic plants under heavy metal stresses, and the functions of these glutathione synthetase in enhancing the tolerance and accumulation of heavy metal ions of E. coli, Arabidopsis and energy sugar beet, and the roles of recombinant E. coli and transgenic plants with resistance of heavy metal in bioremediation. The results will provide new ideas to the variety of bioremediation. The main contents included the following aspects:1. The three genes of AtGCS （Genbank accession no. NM₁18439）, AtGS （Genbank accession no. NM₁22620） and StECS-GS （Genbank accession no. GQ848551） were cloned by using RT-PCR, and they encoded three glutathione synthetase of Arabidopsis γ-GCS, GS and Streptococcus thermophilus StECS-GS separately. The CDS length of such three genes was1569bp,1620bp and2265bp, respectively; the molecular weight of protein was58.56kDa,60.27kDa and85.12kDa, respectively; the isoelectric point was6.52,6.45and4.9, respectively; according to PSORT prediction, such three enzymes were located in peroxisome, chloroplast membrane and cytoplasme with indentity of56%,94%and45%, respectively; meanwhile, there were cis-acting elements related to environmental stress such as drought in5’ untranslated region of AtGCS and AtGS, there may be certain responsive relationship between these two genes and certain environmental stresses.2. This study compared the transcriptional expression characteristics of AtGCS and AtGS under Cd, Zn and Cu heavy metal stresses comprehensively. The results showed that the two genes were induced by200μM Cd2+, Zn2+and Cu2+stress in their transcriptional level. Compared with the control plants without any heavy metal treatment, the expression level of AtGCS and AtGS were increased in shoots and roots, and there were some differences and changes in genes’expression under different processing time of heavy metal stresses. In Cd treatment, the expression of AtGS was higher than AtGCS in Arabidopsis shoots, and they were opposite in roots; in Zn and Cu stresses, the response of AtGCS was more obvious in Zn stress, and AtGS was more sensitive to Cu; in such three heavy metal stresses, Cd could enhance the inductive expression of the two genes more evidently, and there was no difference in Zn and Cu. These results exhibited that AtGCS and AtGS were responsive to heavy metal stresses, and they might participate in physiological activity of Arabidopsis resistance to heavy metal stress in different degrees.3. By using exogenous protein recombinant technology in E. coli, this study compared the heavy metal tolerance and accumulation of E. coli over-expressing TrxA-AtGCS, TrxA-AtGS and TrxA-StECS-GS under1mM CdCl2, ZnCl2and CuCl2stress, respectively. The results showed that the growth of control cells over-expressing TrxA was damaged severely, while E. coli over-expressing TrxA-StECS-GS exhibited strongest heavy metal tolerance, and the survival rate of recombinant strains performed a trend of StECS-GS>AtGCS>AtGS. Meanwhile, in heavy metal stresses, there were obvious advantages in heavy metal accumulation ability of recombinant bacteria than control cells:the Cd, Zn and Cu accumulation of E. coli over-expressing TrxA-StECS-GS were about15,8and9-fold of control strains; cells over-expressing TrxA-AtGCS could accumulate Cd, Zn and Cu with10,5and7-fold of control cells; while Cd, Zn and Cu accumulation of stains over-expressing TrxA-AtGS was relatively low, and it was5,4and5-fold of control cells, respectively. The trend in GSH content of （TrxA-StECS-GS>TrxA-AtGCS>TrxA-AtGS>TrxA） could explain the phenomenon of high heavy metal ions accumulation in recombinant bacteria. Meanwhile, the functional proteins involving in heavy metal resistance mechanisms of OsHSP90and OsCATb were introduced as comparative experiments. It was showed that, although the growth of E. coli over-expressing GST-OsHSP90and GST-OsCATb was better than control cells over-expressing GST, and between cells over-expressing TrxA-AtGCS and TrxA-AtGS, their ability of heavy metal accumulation and GSH content were not improved significantly. These results indicated that there were differences among glutathione synthetase, molecular chaperone and antioxidant enzyme in heavy metal resistance mechanisms, and glutathione synthetase had more obvious advantages in exercising the functions of heavy metal bioremediation. The role of StECS-GS was more prominent.4. AtGCS, AtGS and StECS-GS genes were separatively transformed into model plants-- Arabidopsis thaliana by transgenic technology, and were analyzed their tolerance capacity of heavy metal stresses and the potential and the molecular mechanisms of heavy metal bioremediation comparatively. The results showed that, under normal growth conditions, there were almost no differences among transgenic Arabidopsis （gcs2, gs2and ecs-gs4） over-expressing AtGCS, AtGS and StECS-GS, and wild-type （wt）. As increase of concentrations of CdCl2, ZnCl2and CuCl2stresses, the growth of wt received serious inhibition, and its root length and fresh weight were only1/4-1/3of ecs-gs4; the tolerance ability of transgenic Arabidopsis over-expressing AtGCS, AtGS and StECS-GS genes was enhanced in different degree under Cd and Cu stresses, and StECS-GS lines were most obviously. While in Zn stress, only StECS-GS transgenic plants exhibited improved heavy metal tolerance. In the aspect of heavy metal accumulation in transgenic plants, it was much higher in shoots than in roots, and it was showed the trend of StECS-GS genotype>AtGCS、AtGS genotypes>wt. The accumulation of Cd in ecs-gs4was highest, and the level was more than4.5-fold of control plants. ecs-gs4accumulated less Zn and Cu than Cd. GSH and PC contents exhibited the similar tendency, and with treatment of heavy metal stresses, the level of GSH was declined, while the PC content was increased. These results declared that the three glutathione synthetases released the feedback inhibition by their over-expression, and thus could produce more GSH and PC. GSH and PC could chelate heavy metal ions and functioned as antioxidant. These features made the transgenic Arabidopsis acquire a higher heavy metal accumulation and tolerance. The unique advantages of StECS-GS resistance to heavy metal stress made it a dominant gene in artificial hyper-accumulated plants by using genetic engineering, and it had a potential advantage in heavy metal phytoremediation.5. On the basis of the above study, and as the bioremediation basic materials of energy sugar beet which could produce bio-fuel ethanol, the experiment studied on functional role of StECS-GS in transgenic sugar beet tolerance and bio-accumulation under single or multiplex heavy metal stresses. The results showed that transgenic sugar beet over-expressing StECS-GS （s2, s4and s5） exhibited significant growth advantage on root length and fresh weight compared with control plants （wt） under different concentrations of50,100,200μM Cd, Zn and Cu. This advantage was proportional to the amount of StECS-GS expression. Meanwhile, the chlorophyll contents of transgenic sugar beet were higher than control plants, but the differences between the transgenic plants were slight. In100μM heavy metal stresses, the Cd, Zn and Cu accumulation concentrations in transgenic sugar beet s2, s4and s5shoots were5-4-fold of the control sugar beet with the trend of s2>s4>s5>wt. In roots, although heavy metal contents of s2were lower than s4and s5, they were about2-3-fold of control plants. In general, the heavy metal accumulations of shoots transgenic plants were more than3-fold of roots. These indicated that the accumulation of heavy metal in transgenic plant were mainly concentrated in shoots. Under normal growth conditions, GSH contents of s2, s4and s5were5,4and3.8-fold of wt, respectively, and it could be deduced that over-expression of StECS-GS catalyzed and formed large amount of GSH in transgenic sugar beet. Under Cd, Zn and Cu stresses, GSH concentrations decreased, and PC contents increased in s2, s4, s5and wt, but the GSH and PC level of transgenic plants were always higher than that of control. This phenomenon could be explained that some GSH was used for synthesis of PC to chelate heavy metal ions, and some GSH might play an important role in heavy metal anti-oxidative stress. Under double Cd-Zn, Cd-Cu and Zn-Cu and multiple Cd-Zn-Cu multiplex heavy metal stressful conditions, StECS-GS transgenic sugar beet exhibited obvious advantages on phenotype, biomass and heavy metal accumulation, compared with the control sugar beet, and these heavy metals were accumulated in transgenic plants as the manner of addition. It could be concluded that sugar beet over-expressing StECS-GS had characteristics of heavy metal tolerance, high biomass, non-specific accumulation of heavy metal ions, and potential of heavy metal pollution phytoremediation. There will be a great development value by using transgenic energy sugar beet in heavy metal pollution bioremediation.