Function Of BnPCS1 Gene In Ramie Under Cadmium Stress

Phytochelatins（PCs）catalyzed the synthesis of Glutathione（GSH）by phytochelatin synthase（PCs）,which played an important role in alleviating Cd toxicity in plants.Ramie（Boehmeria Nivea L.）is a fiber crop unique to China.It has strong tolerance and enrichment ability to cadmium（Cd）,arsenic（As）,lead（Pb）and other heavy metals,and is an alternative crop for planting heavy metal contaminated soils in southern China.Currently,the molecular mechanism of ramie tolerance to Cd is poorly understood.This severely restricted the process of ramie stress tolerance genetic breeding.Based on the analysis of ramie transcriptome data under cadmium stress in the previous stage of our laboratory,it was found that the plant chelating peptide synthase gene PCS1 showed increased transcription.In this study,the effects of different concentrations of Cd on the growth and physiological characteristics of ramie and the response characteristics of PCs were studied by pot experiment.Secondly,by constructing Bn PCS1yeast expression vector and Bn PCS1 overexpression vector,the Cd resistance function of Bn PCS1 was analyzed.The main research results are as follows:1.With the increase of Cd stress concentration,the growth of ramie showed a phenomenon of low promotion and high inhibition.Cd mainly accumulates in the ground,and the transport coefficients under different Cd stresses are all less than 1.The Cd accumulation and transport coefficient are the highest under 25 mg/L Cd stress;The enzyme（SOD）activity was significantly increased,the malondialdehyde（MDA）activity was significantly decreased,and the hydrogen peroxide（H₂O₂）content was significantly increased（p<0.05）,increased under 50 mg/L Cd stress;GSH content in aerial and underground parts decreased under Cd stress.2.Under low concentration（≤5 mg/L）stress,Cd content was positively correlated with SOD activity（p<0.05）;Under high concentration（25 mg/L and 50 mg/L）of Cd,Cd content was negatively correlated with SOD activity in underground part（p<0.05）,and was positively correlated with PCs content in underground part（p<0.05）.There was a significant positive correlation between Cd content and the relative expression of Bn PCS1in shoot（p<0.05）,and there was a positive correlation between Cd content and the relative expression of Bn GCL1 in shoot at high concentrations（p<0.05）.3.Yeast expression vector Bn PCS1-p426 GDP was constructed,Bn PCS1-p426 GDP、EV-p426 GDP were transferred into wild-type yeast Y252 and CD-sensitive yeastΔyap1,respectively.EV-Y252、Bn PCS1-Y252、EV-Δyap1、Bn PCS1-Δyap1 were tested for Cd stress at 0,50,75 and 100μmol/L,respectively.By observation of the growth of yeast,it was found that Bn PCS1 overexpressed yeast had stronger tolerance to Cd stress.4.The plant overexpression vector Pcambi A1300-35S-Bn PCS1-MYC was construct-ed and transferred into Agrobacterium tumefaciens by heat shock method.Then,Bn PCS1was transferred into arabidopsis genome by inflorescence infection method.Under the stress of 100μmol/L Cd,the root length and fresh weight of Bn PCS1 overexpressed arabidopsis seedlings were significantly higher than those of the wild type.Overexpression of Bn PCS1 increased the activities of POD,CAT and SOD,and decreased the contents of MDA and H₂O₂.Overexpression of Bn PCS1 increased the content of PCs and GSH in transgenic Arabidopsis thaliana,as well as the transcription levels of cadmium-tolerant genes（At GCL1,At GS1,At HMA1 and At ATM1）.