Overexpression Of Secreted Plant Metallothionein Enhances The Enrichment And Resistance Of Heavy Metals In Transgenic Tobacco

The Saccharomyces cerevisiae MF-?signal peptide,enhanced green fluorescent protein?EGFP?and human metallothionein?MT3?coding sequences were used to construct a fusion gene,and the recombinant protein MF-?-EGFP-MT3 was expressed in the transgenic plants.Previous studies from our laboratory have obtained that MF-?can mediate EGFP-MT3 secretion into plants and increase the resistance of transgenic plants to some heavy metals.Plants and animals have different metallothionein structures,and rice has a large family of metallothionein genes?encoding 11metallothioneins,OsMTs?.The Salicornia bigelivii Torr?Salicornia europaea L.?has the ability to resist salt and alkali,and is resistant to metals.Therefore,this paper attempts to use MF-?to mediate the overexpression of secreted rice metallothionein gene OsMT-I-2b and Salicornia bigelivii Torr metallothionein gene SbMT-2 in transgenic plants.In order to facilitate the detection of expressed proteins,the coding regions of these two protein genes were fused to the reporter gene EGFP.The SbMT2and OsMT2 and EGFP fusion gene sequences were synthesized by a biotechnology company.Then we got two gene sequences:EGFP-SbMT2 and EGFP-OsMT2.We used Gateway technology to construct two plant expression vectors pK-MF-?-EGFP-SbMT2 and pK-MF-?-EGFP-OsMT2 containing?-factor signal peptide?MF-??.Then the tobacco and geranium were transformed to obtain the corresponding transgenic plants.The enrichment ability and resistance of the transgenic plants to heavy metals were investigated,and the mechanism of excessive secreted metallothionein was analyzed.The following results were obtained:1.We selected the genomic PCR to detect successful transgenic tobacco plants,and detect the transgene transcription level and protein expression level.Subsequently,we found that SbMT2 and OsMT2 were expressed in the leaves of transgenic plants,and EGFP-SbMT2 and EGFP-OsMT2 fusion proteins were expressed in roots,stems and leaves.The roots of the transgenic plants were treated with Cd²⁺.The results showed that more Cd²⁺accumulated in the transgenic plants,and the aboveground part was larger than the lower part.The expression level of secreted metallothionein has a good correlation with the accumulation of Cd²⁺in different tissues of transgenic plants.The root treatment solution and the surface water washing solution were collected.Electrochemical analysis showed that EGFP-SbMT2,EGFP-OsMT2 protein and Cd²⁺were found in the transgenic root treatment solution and the surface water washing solution.It indicated that MF-?successfully mediates the secretion of metallothionein from roots and leaves,and the secreted metallothionein binds to Cd²⁺.The subcellular localization analysis of the expressed protein by laser confocal scanning microscopy revealed that GFP fluorescence was observed in the root hairs and leaf hairs of the transgenic plants.In addition,GFP fluorescence was also observed in the apoplastic space of stem and leaf cortical parenchyma cells,which confirmed that the overexpressed EGFP-SbMT2 and EGFP-OsMT2 proteins were secreted into the plant through the leaf hair and root hair,and the other part was secreted to the cortical thin wall.The apoplastic space of the cell.2.The roots of WT and SbMT2,OsMT2 transgenic tobacco plants were treated with Cd²⁺?100?mol/L?,Pb²⁺?15 mg/L?and Cu²⁺?500?mol/L?,respectively.It was then investigated whether overexpression of secreted metallothionein could enhance the enrichment and absorption capacity of transgenic plants for other heavy metals.After one week of treatment,the growth of three heavy metal-treated SbMT2 and OsMT2 transgenic tobacco plants was better than that of the WT plants,indicating that overexpression of secreted metallothionein SbMT2 and OsMT2 could enhance the resistance of transgenic plants to various heavy metals.Electrochemical analysis of the absorption of three heavy metals showed that the Pb²⁺absorbed by OsMT2 transgenic tobacco plants was greater than that of SbMT2 plants,while the SbMT2 transgenic plants had better absorption of Cd²⁺and Cu²⁺.This result indicates that different metallothioneins have different binding abilities to different heavy metal ions,so that the transgenic plants have different absorption capacities for different heavy metals.Apoptosis of root tip cells was analyzed by PI and FDA complex staining.The results showed that the WT plants had more dead cells than SbMT2 cells after Cu²⁺treatment.Although there are more dead cells in the root cap and meristematic zone of the root tip of transgenic SbMT2,the number of dead cells in the apical elongation zone is significantly less than that of the WT plants,which confirms that the overexpression of secreted metallothionein SbMT2 and OsMT2 can be Enhance plant resistance to heavy metal-induced apoptosis in root tip cells.3.In order to study the over-expression of secreted metallothionein to enhance the enrichment and absorption mechanism of heavy metals in transgenic plants,the roots of WT,MT3,SbMT2 and OsMT2 transgenic tobacco plants were treated with Cd²⁺,Pb²⁺and Cu²⁺,respectively.Changes in photosynthetic related indicators were analyzed after 7 days.The results showed that after treatment of three transgenic plants with Cd²⁺,net photosynthetic rate?Pn?,transpiration rate?E?,stomatal conductance?C1?and intercellular CO₂ concentration?CO₂int?were significantly increased in the WT plants,and Pn of transgenic plants reached the WT plants were1.5² times,while the transpiration rate?E?of SbMT2 is 0.2 mmol/m2·s,which was4 times that of the WT plants.After one week of Pb²⁺treatment,the Pn and CO₂int of the transgenic plants were higher than those of the WT plants,but both C1 and CO₂int were significantly lower than the WT plants.The changes of Pn,E,C1 and CO₂int of the three transgenic plants after Cu²⁺treatment were similar to those of Cd²⁺ treatment,which were significantly higher than those of the WT plants.The content of malondialdehyde?MDA?and H₂O₂ in different tissues was determined by collecting roots and stems.The results showed that the content of MDA in different tissues of transgenic plants was 2³ times lower than that of the WT plants,and the content of H₂O₂ in leaves of transgenic plants was also significantly lower than that of the WT plants.The changes of different antioxidant enzyme activities were analyzed.The results showed that the activities of POD,SOD,APX and CAT of transgenic plants were significantly higher than those of the WT plants after treatment with Cd²⁺,Pb²⁺and Cu²⁺.The increase of the activities of SOD,APX and CAT was particularly significant.These results indicated that plants absorb Cd²⁺,Pb²⁺,and Cu²⁺in different tissues,resulting in a large amount of harmful substances such as H₂O₂ and MDA in the cells.This result affected the conductance of the stomata,the net photosynthetic rate and transpiration,and produces oxidative stress and oxidative damage,resulting in chlorosis,blackening of the stem,and death of the apical cells.However,the over-expression of secreted metallothionein could regulate the activity of POD,SOD,APX and CAT,and timely removed the active oxygen accumulated by heavy metals such as Cd²⁺,Pb²⁺and Cu²⁺,improved the photosynthetic characteristics of transgenic plants,thereby significantly increased the enrichment ability and resistance of heavy metals in transgenic plants.