| Heavy metal pollution has become a world focus problem along with industry development. Nowadays, there are thousands acres soil contaminated by heavy metal in China, and bring serious damage to human. Therefore, remove heavy metal from soil efficiently has become a very urgent task.Molecular biology research of phytoremediation is the hot spot in the field of environmental protection. Traditional phytoremediation depends on finding hyper tolerance or hyper accumulation plant from the polluted environment, and it requires a long time. The hyper accumulation plants were found usually small, slow-growing and lack of the ability of transport the heavy metal through the roots to shoot, which greatly limits the use and development of phytoremediation. An effective way of repairing heavy metal contaminated soil by phytoremediation is to enhance the heavy metal accumulation of high biomass-producing species by transgenic approach..Genetic approach enhance heavy metal accumulation in plants has made progress in a number of related research. A large number of genes has been identified and cloned, and these genes were transferred into plants for remediation of heavy metal contaminated soils. Many experiments showed that heavy metal detoxification related genes from animal and plant itself were transferred into high biomass plants, their heterologous expression can mediate high tolerant and accumulating of heavy metal in transgenic plants.Phytochelatins (Phytochelatins, PCs) are induced by heavy metals. It is a class of small peptides that could reduce the toxicity of heavy metals on plants by chelate heavy metals. Thereby, PCs are the most important mechanism for plant response to heavy metal stress. Limiting enzyme in the synthesis PCs are phytochelatin synthase (PCS) andγ-glutamyl cysteine synthetase gene (y-ECS). Their expression in small biomass plant Arabidopsis and Indian mustard had confirmed its function of accumulation of heavy metals.Reed (Phragmites australi (Cav.) Trin. Ex Steud) is a hyper-accumulation plant for heavy metals (Cd2+, Pb2+, Zn2+). It has a strong ability to clean contamination, and had been used as the main sewage treatment wetland species. The cloning of key genes for heavy metal accumulation has not been reported. Bentgrass (Agrostis palustris Huds) and tall fescue (Festuca arundinacea Tall Fescue) are world-wide planted, large biomass, fast growth turf grass. Heavy metals accumulation related genes engineering has not been reported yet. Using genetic engineering to obtain turfgrass with high resistance and accumulation to heavy metal can achieve the purpose of remediate heavy metal contaminated soils by continuously cutting, recycling, reduce the amount of heavy metals. Therefore, to genetically modified turf grass to enhance the accumulation of heavy metal is of great significance.In previous experiments, our laboratory cloned the y-glutamyl cysteine synthetase gene (PaGCS) from the reeds and transferred into a high biomass, fast-growing lawn grass bentgrass.5 transgenic Agrostis with PaGCS were abtained. On this basis, this paper first cloned full-length cDNA of reed phytochelatin synthase gene (PaPCS), and verified its function by express it in Zn, Cd-sensitive yeast YK44; We achieved a single gene (PaPCS, PaGCS) and series two-gene (PaPCS/PaGCS) transgenic plant of tall fescue grass by embryogenic callus transformation. We analyzed the biological effects of transgenic plants (accumulation/resistance to heavy metal), and the relationship between gene expression and cadmium resistance/accumulation. We also obtained transgenic tall fescue with high cadmium resistance/accumulation. Meanwhile, we analyzed the PaGCS transgenic bentgrass for the molecular biological identification, growth characteristics, physiological and biochemical analysis, Cd tolerance/accumulation test, resistance to heavy metal.The main course and results of the study: 1 Cloning and Identification of Phytochelatin Synthase Gene1.1 Cloning of PaPCS full-length cDNAA phytochelatin synthase gene full-length cDNA, was isolated from the reed by RACE-PCR technology, named as PaPCS. Sequence analysis showed that the ORF nucleotide length is1497bp. It is encoding 498 amino acids, and its protein molecular weight is 54.9kDa.1.2 Sequence Analysis of PaPCSPhylogenetic analysis showed that the nucleotide sequence of the gene has a high similarity with wheat (84.3%), bermudagrass (84.29%), and rice (70.94%). Cluster analysis of protein found that the gene encoded protein has the highest homology with bermudagrass PCS.1.3 The function verification of PaPCS in the Zn, Cd-sensitive yeastWe construct a yeast expression vector pYES2-PaPCS, and transferred into Zn, Cd-sensitive yeast strain YK44. The results of the yeast complementation experiment showed that, under 100μM CdCl2 stress, the growth condition of the yeast with PaPCS better than the control (non-transgenic yeast), indicating that the overexpression of PaPCS in deficient yeast cells increased its resistance to Cd.1.4 The function verification of PaPCS and PaGCS in tall fescue1.4.1 Construction of plant over-expression vectorwe constructed a single gene (PaPCS) and series two genes (PaPCS/PaGCS) over-expression vector pROK/PaPCS and p3301/PG).1.4.2 Induction and transformation of tall fescue callusThe callus of tall fescue were induced from following axes, then were sub cultured, and yellow, granular embryogenic callus were filter out (high differentiation rate, reaching 90%). Vectors above were transferred into embryogenic callus cultured for 7d via d Agrobacterium-mediated method. After cultured in dark for 3 days, resistant calli were screened under high concentration (50 mg/L) Kan. Resistant callus differentiate into postemergence on differentiation medium IB. Then postemergence were further filtered with 25 mg/L Kan,197 resistant plants (95 plants were transferred with PaPCS,87 with PaGCS plants,15 with PaPCS/PaGCS plants) were obtained.1.4.3 Obtained transgenic plantsTotal DNA of the resistance plants were analzed by PCR using gene specific primers.7 PaPCS transgenic plants, turn PaPCS identified seven plants,11 PaGCS transgenic plants,3 PaPCS/PaGCS transgenic plants were filtered out. Semi-quantitative RT-PCR analysis showed that the target gene in transgenic plants (PaPCS or PaGCS, two genes PaPCS/PaGCS) were higher than those in control. Southern hybridization showed that, a single copy of PaPCS was integrated into the two of PaPCS transgenic plants. Single and double copy of PaGCS was integrated into the two of PaGCS transgenic plants respectively. Single copy of PaPCS and PaGCS were integrated into the two of PaPCS/PaGCS transgenic plants.1.4.4 SDS-PAGE analysis of PaPCS and PaGCS expression in transgenic plantsTotal protein of transgenic plants and wild-type tall fescue leaf were analyzed by SDS-PAGE. The result showed that new band with the same size of predicted PaPCS and PaGCS protein were respectively found in PaPCS transgenic plants (TP1 and TP2) and PaGCS transgenic plants (TG1 and TG2). Both the new band were found in total protein of PaPCS/PaGCS transgenic plants (T(GP)-1 and T(GP)-2). It showed that the gene (PaPCS and PaGCS) can be heterologous expressed in transgenic plants.1.4.5 Growth and Cd accumulation of wild-type and transgenic tall fescue under cadmium StressWild-type and transgenic tall fescue were treated with 0.15 mM CdCl2 for 5 days. The biomass of transgenic lines were higher than that of wild-type tall fescue, and the PaPCS/PaGCS transgenic plants was the highest, followed by PaGCS transgenic plants. Cadmium content in roots, leaves and whole plants was measured by ICP-MS after treated with 0.15 mM CdCl2 for 5 days. Whether in transgenic plants or in wild-type plants, the cadmium content in roots was higher than the leaves. A comparison of the cadmium content of three transgenic plants and wild-type plants found:PaGCSIPaPCS transgenic plants>PaGCS transgenic plants>PaPCS transgenic plants>WT (wild-type). The ratio of cadmium content in leaves/cadmium content in root was higher in transgenic plants than that of wild-type, in which T (GP)-2 the cadmium content of leaves almost equal to that of roots, indicating that the transgenic plants resulted in cadmium accumulated inside plant was able to transfer into leaf.1.4.6 GSH, PC, TNP-SH content of transgenic tall fescue under cadmium stressGSH, TNP-SH and PC content of transgenic plants and wild-type plants were measured after 0h,6h,12h,24h,48h,72h,120h treated in 0.15 mM CdCl2. At 0h, when the GSH content of the three transgenic plants were similar to the wild-type, while the PC and TNP-SH were higher than those of wild-type plants. With the Cd treatment continues, the GSH content of transgenic plants and wild-type did not change significantly, PC and TNP-SH content continued to increase. At 120h, PC and TNP-SH content reached the highest, the overall trend is:PaPCS/PaGCS transgenic plants> PaGCS transgenic plants>PaPCS transgenic plants>WT (wild-type), indicating that the double gene (PaPCS and PaGCS) transgenic plants may accumulate more phytochelatins compared to single gene (PaGCS or PaPCS) transgenic plants.1.4.7 MDA content, POD and SOD activity of transgenic tall fescue under cadmium stressMDA content and SOD and POD activity of transgenic plants and wild-type plants were measured after 0h,6h,12h,24h,48h,72h,120h treated in 0.15 mM CdCl2. At 6h, MDA content in transgenic plants and wild-type control begin to increase, and MDA content in wild-type increased the fastest. With the highest biomass, T (GP)-1, and T (GP)-2 MDA showed the slowest growth trends, followed by TG-1 and TG-2, TP-1 and TP-2 were the fastest among transgenic plants, The MDA content of wild-type was increased faster than all the transgenic plants, showed that the transgenic plants lipid peroxidation to a lesser extent, and transgenic plant cells were in a certain degree of protection by heterologous expression of PaPCS, but the effect is not as good as heterologous expression of PaGCS. SOD and POD activity of three types of transgenic plants and wild-type under Cd stress all has a peak in the early time, SOD activity peaked at 6 hours after cadmium treatment, SOD activity at this time were PaGCS/PaPCS transgenic plants> PaGCS transgenic plants> PaPCS transgenic plants> WT (wild-type). POD activity peaked at 12 hours after cadmium treatment, POD activity at this time were PaGCS/PaPCS transgenic plants> PaGCS transgenic plants> PaPCS transgenic plants> WT (wild-type). Then SOD and POD activity has a rapid decrease. Thus, after 24h Cd treatment, SOD and POD activity were lower than the initial value. All transgenic SOD and POD activity was lower than wild-type plants, the activity of the two both were PaGCS/PaPCS transgenic plants> PaGCS transgenic plants> PaPCS transgenic plants > WT (wild-type).This article also performed a series analysis on PaGCS transgenic bentgrass for identification of molecular biology, growth characteristics, physiological and biochemical analysis, accumulation and tolerance of Cd.2 The effect of the heterologous expression of Phragmites australis y-glutamylcysteine synthetase on the Cd accumulation of Agrostis palustris2.1 Transgene screeningThe PCR profile of DNA from five transgenic plants showed the presence of the PaGCS amplicon in all five of them.The Southern hybridization profile of the five PCR-positive regenerants showed that a single copy of PaGCS had been stably integrated into individuals T1, T2, T4 and T5, and two copies into T3.2.2 Gene expression profilingUsing semi-quantitative RT-PCR analyze the expression of GCS in transgenic and wild-type bentgrass, the results showed that expression levels of GCS in all the transgenic lines were higher than that of the wild-type, but T4 and T5 were significantly higher. The three enzymes GCS, GS and PCS are all involved in PC biosynthesis. The expression of both GCS and PCS was enhanced in the root and leaf of the transgenic line in the presence of imposed Cd stress, suggesting GCS and PCS was induced by CdCl2. The expression of both GS and GST was not markedly affected by Cd stress. The abundance of PaGCS transcript was highly accumulated in T4.2.3 Analysis of total protein by SDS-PAGELeaf extracts from transgenic line T2 and T5 selected for SDS-PAGE showed a new band which gives a calculated molecular weight for the protein of～43 kDa, in good agreement with the size predicted for the PaGCS from the gene sequence, and indicated that the transgenic plants were successfully synthesised protein encoded by PaGCS.2.4 The growth of transgenic A. palustris lines under Cd stressAll the lines suffered some colour lost in the leaves after five days exposure to 0.15 mM CdCl2, while the wild-type was affected by a severe colour loss. The biomass accumulated by the transgenic lines in the presence of 0.15 mM CdCl2 was compared to the performance of the wild-type after five and 21 days of stress treatment. After 5 days, the biomass of lines T1-T4 was at the same level of the wild-type, but that of T5 was significantly greater. After 21 d, the ranking of the transgenic lines was T1=T3 |
PDF Full Text Request