| Abiotic stresses are serious threats to plant growth and development, and result in a series of changes on physiological, morphological, biochemical and molecular levels. Since traditional breeding limits the diversity of plants, it is important to carry out researches on the molecular biology of stress-resistant plants and to gain the gene resources from the stress-resistant plants. Plants are born with certain mechanisms of resistance,such as carotenoids and GSH.Carotenoids are organic pigments that protect plants against UV-B radiation, and it is of great importance to clone the genes related to the process of biosynthesis of carotenoids. Geranylgeranyl diphosphate(GGPP) is a precursor of carotenoid biosynthesis, and the synthesis is catalyzed by geranylgeranyl diphosphate synthase(GGPS). Two full-length nucleotide sequences of GGPS(named Lc GGPS1 and Lc GGPS2) were cloned from Lycium chinense. Nucleotide sequence analysis revealed that the Lc GGPS1/2 genes contain open reading frames respectively encoding protein of 358/365 amino acid residues with calculated molecular mass of 38.79/39.91 k D.They contain 7 highly conserved domains. Plant expression vectors were constructed containing the ORF of the Lc GGPS1/2 genes, and then the target genes were successfully transferred into the genome of tobacco. Total carotenoids were extracted and HPLC tests showed that there were no significant differences between Lc GGPS1 transgenic line and the wide type tobacco. However, the total carotenoid content and the composition zeaxanthin/xanthophyll/neoxanthin of the Lc GGPS1 transgenics line were all enhanced. Upon exposure to UV-B radiation, the tested tobacco of net photosynthetic rate, transpiration, Fv/Fm, chlorophyll a and chlorophyll b content are basically on a downward trend. The Lc GGPS1 transgenic tobacco and wild type dropped more seriously than the Lc GGPS2 transgenic tobacco. Upon exposure to UV-B radiation, the leaves of the tested tobacco wilted and shrieked, the vein around turned thiner, and some local cells were dead. By the use of 3,3'-Diaminobenzidine and Nitrotetrazolium blue chloride as the chromogenic substrate, we described the histochemical detection of hydrogen peroxide and superoxide anion. The Lc GGPS1 transgenic tobacco was stained more deeply than the Lc GGPS2 transgenic tobacco,and this indicated that the former was damaged more seriously by UV-B radiation than the latter. In conclusion, over-expression of Lc GGPS2 in tobacco not onlyimproved the carotenoid content, but also improved their tolerance against UV-B radiation.As a result of industrial development, soil become highly damaged by heavy metal pollution. Soil remediation through plant genetic engineering is a widely used as a method to tackle this problem. The basic concept of this study is to provide a new genetic resource for heavy metal phytoremediation through the study and manipulation of heavy-metal-responsive rate-limiting enzymes in plants. In this study,the gene Lc GS, coding glutathion synthetase(GS) of Lycium chinense was cloned.Expression of Lc GS in L.chinense under Cd Cl2 stress over a period of 24 h was monitored and analyzed using semi-quantitative RT-PCR. The expression of Lc GS gene was up-regulated following the increase of Cd2+stress period initially, and then remained at a relatively high level from 9 h till the end of the monitoring period. The recombination expression vector containing p CAMBIA-2300 vector and gene GS was also constructed. Then the Lc GS expression cassette was transformed into tobacco and was confirmed by PCR. Under the stress of Cd Cl2, transgenic plants carrying Lc GS gene contain higher contents of Glutathione(GSH), Phytochelatins(PCs) and Chlorophyll than wild-type plants, indicating their higher tolerance to Cd Cl2.Therefore, over-expression of glutathione synthetase genes through genetic engineering can be a promising strategy for heavy metal phytoremediation. |
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