Cloning And Analysis Of Function Of γ-glutamylcysteine Synthetase Gene With Heavy Metal Accumulation In Phragmites Communis Trirn.

The application of modern molecular biology methods in phytoremediation is presently a major concern in the field of environment protection. Most hyperaccumulators known are low-grown and relatively low biomass plants, and restrained by client and soil. Therefore, using the transgenic technology, introducing novel genes responsible for (hyper) accumulating heavy metal into high biomass plants is a promising strategy for the development of effective techniques of phytoremediation.The key mechanisms responsible for heavy metal hyperaccumulation andresistance in plants is detoxification or homeostasis of heavy metals by Cys-richpeptides, such as glutathione(GSH) and phytochelatins(PCs), which can combineheavy metal with Cys. GSH also is a pre of PCs. γ- glutamylcysteine synthetase(γ-ECS ) is an key enzyme of the biosynthesis of GSH.In this study, we close Phragmites communis Trirn., a model plant used for phytoremediation, with ability of accumulating Cd, Zn, Cu, Fe, Se, as our experimental material. Here we describe the molecular cloning, characterization and functional analysis of a novel γ-ECS gene designated as PcGCS, from P. communis. A yeast expressing vector and a plant expressing vector were constructed. Plant regeneration system for Agrostis palustris Huds. was also established, and PcGCS was transformed into A. s palustris.Main results obtained in this study are summarized as follows:By using RT-PCR and RACE techniques, we have identified and cloned a novel gene, designated as PcGCS. Sequence analysis showed that the nucleotide length of ORF region of PcGCS is 1125 bp; and its putative proteins contain 374 amino acid residues. The molecular masses of the PcGCS proteins are predicted to Mr 43032, which are smaller than predicted molecular masses of the proteins cloned in oryza sativa and zea mays, and similar to the predicted molecular masses of the protein from Triticum aestivum. The isoelectric point of the PcGCS protein is 5.7, which are acidity, the same as the most γ-ECS proteins.The yeast expression vector pDR195 with PcGCS gene cloned, named pDR-GCS, was constructed. The plant expression vector pROK2 carrying nptII gene, PcGCS gene and 35S promoter, pROK-GCS, was also obtained.The recombinant plasmid pDR-GCS was used to transfor into the strain of S.cerevisiae. The result of GSH concentration showed that the concent of GSH in transgenic yeast is 30% higher than empty vector transformed control, indicated that the biosynthesis of GSH was increased by the expression of PcGCS in yeast.The hypocotyl-derived embyogenic calli from A. palustris subcultured for 7 d were transformed with the Agrobactrium tumefaciens strain AGL1 (with 0.1mmol/L AS) harboring plasmid pROK-GCS and co-cultured. The embyogenic calli were selected with 5 mg/L kanamycin and 132 resistant plants regenerated from the resistant calli. Five transgenic plants were identified by PCR amplification with specific primer of PcGCS gene. The positive frequency of PCR was 3.8%.