Heterologous Expressing Nahc/ScPDI Enhance Tolerance To 2,4,6-TCP In Malus Robusta

In China,long-term abuse of organic pesticides has led to serious soil pollution in orchards,especially chlorophenol pesticides(such as 2,4,6-TCP).Phytoremediation of polluted soil is a green and environmentally friendly process for removing pollutants from the environment.Many studies demonstrate that phytoremediation has a broad application prospect in remediation of environment.As M.robusta has a strong tolerance to many biotic and abiotic stress and a wide range of adaptability,we speculate that M.robusta can remediation of chlorophenol pesticide contaminated soil in the orchard.However,there are no reports about the remediation of 2,4,6-TCP contaminated soil by M.robusta.Therefore,in this experiment,we took M.robusta as the research material,and selected 2,4,6-TCP as the representative of chlorophenol pesticides to investigate the remediation ability of M.robusta on 2,4,6-TCP contaminated soil.The purpose of this work is to examine the feasibility that enhancing phytoremediation of the 2,4,6-TCP by plants overexpressing 1,2dihydroxynaphthalene dioxygenase gene(Nahc)and S.cerevisiae protein disulfide isomerase(PDI)gene(ScPDI)were selected to examining the assumption in this study.The experimental results are as follows:1.The effect of different concentration gradients of hygromycin on cotyledon differentiation was studied by using the cotyledon as explants.The results showed that the optimal medium for inducing cotyledon callus was Sc+2.0mg.11TDZ+0.2mg.l-1 NAA+0.5mg.l-1 GA;,the optimal medium for bud regeneration was Sc+2.0mg.l-1 TDZ+0.2mg.l-1 NAA,and the optimal medium for rooting was 1/2Ms+0.25mg.l-1 IBA.Therefore,through this experiment,we determined that the critical concentration of the element screening for differentiation of M.Robusta was 2.0mg.l-1-4.0mg.l-1,and the screening concentration of the rooting hygromycin was 2.0mg.l-1-3.0mg.l-1.This study established the basis for the genetic transformation of M.robusta.2.The 1,2-dihydroxynaphthalene dioxygenase gene(Nahc,J04994.1)from Pseudomonas putida and protein disulfide isomerase gene(ScPDI,M62815.1)from Saccharomyces cerevisiae were chemically synthesized according to the bias codons of the plant using PCR-based two-step DN A synthesis(PTDS)method.The synthesized fragment of the Nahc gene and ScPDI gene were introduced into Agrobacterium tumefaciens EHA105 by electroporation and then transferred into M.robusta and A.thaliana through the leaf disc method and floral dip method,respectively.Four independently transformed hygromycin-resistant M.robusta and A.thaliana were isolated.3.Under 2,4,6-TCP exposure condition,transgenic plants had higher tolerance to 2,4,6-TCP than the wild type.And the contents of H2O2 and MDA were significantly lower than those of wild-type plants.The transgenic M.robusta exhibit high tolerance to 2,4,6-TCP.The content of H2O2 and MDA in transgenic plants was lower than that of wild type.H2O2 content in the leaf of transformed M.robusta are lower than wild type under 2,4,6-TCP.Nahc-ScPDI transformed M.robusta are lower than Nahc transformed and ScPDI transformed M.robusta;MDA content in the leaf of Nahc transformed and Nahc-ScPDI transformed M.robusta are lower than wild type under 2,4,6-TCP.However,there was no significant difference between the ScPDI transgenic M.robusta and the wild type.All of the transgenic A.thaliana displayed a higher longer root length and fresh weight than WT.This result indicate that Nahc gene and ScPDI gene enhance tolerance to 2,4,6-TCP in A.thaliana.H2O2 content in the leaf of transformed A.thaliana are lower than wild type under 2,4,6-TCP.Nahc-ScPDI transformed A.thaliana are lower than Nahc transformed A.thaliana and ScPDI transformed A.thaliana.4.Transgenic plants have higher absorption capacity of 2,4,6-TCP than wild type plants.The absorptive capacity of transgenic M.robusta was higher than that of wild type by HPLC.However,there was no significant difference in the absorptive capacity between the Nahc-ScPDI transgenic M.robusta and the Nahc transgenic M.robusta and ScPDI transgenic M.robusta.The absorptive capacity of transgenic A.thaliana was higher than that of wild type by HPLC.The absorptive capacity of transgenic A.thaliana with Nahc-ScPDI gene was significantly higher than that of transgenic A.thaliana with single gene.5.Under 2,4,6-TCP treatment,transcriptome analysis of the Nahc-ScPDI transgenic lines of M.robusta and wild-type,939 differentially expressed genes(DEGs)were identified,containing 478 up-regulated(p-value<0.05)and 461 down-regulated(p-value≤0.05).GO enrichment analysis showed that there were significant differences among 6 GO terms in the biological process,4 GO terms in the cellular component and 5 GO terms in the molecular function.According to biological process,the GO term "phenol-containing compound metabolic process,GO:0018958" and "oxoacid metabolic process,GO:0043436" were enriched.The expression of NFXL1 and HGD was up-regulated in these GO terms.The NFXL1 and HGD are involved in the metabolism of ABA and hydroquinone,which can improve the tolerance of plants to abiotic stress.KEGG enrichment analysis revealed that there were 16 pathways with significant enrichment.6 genes were upregulated and 8 genes were down-regulated in the glutathione metabolism pathway(ko00480).The down-regulated genes contain a glutathione reductase gene,which was involved in glutathione redox.