Stress Response Of VsCCoAOMT Gene To Heavy Metals And Its Potential In Cd Contaminated Soil Remediation

Cadmium(Cd)is a non-essential element in the plant,which is considered to be one of the most toxic heavy metals.Excessive heavy metals in the soil not only affect the crop yield and quality,but also harm the health of the human body by the food chain.Enhancing plant heavy metal tolerance can reduce the negative impact of heavy metal pollution.The plant cell wall plays a role of fixing and adsorbing the plants against cadmium stress.Lignin is one of the components of the cell wall,which is synthesised by the lignin monomer catalyzed by enzymes such as Caffeoyl-CoA-O methyltransferase(CCoAOMT).The synthesis of lignin can be involved in the response of plants to environmental stress.In this study,we focused on the response of CCoAOMT to Cd stress and studied its role in heavy metal tolerance and the ability to play a role in Cd-contaminated soil by overexpression of CCoAOMT in Arabidopsis thaliana.Caffeyl coenzyme A-O methyltransferase(VsCCoAOMT)gene sequence was cloned from the Vicia sativa L.,and its amino acid sequence and phylogenetic tree were analyzed.The results showed that VsCCoAOMT was the same as that of the monocotyledonous plant CCoAOMT homology is higher,and the amino acid sequence of CCoAOMT gene is highly conserved.Semi-quantitative RT-PCR analysis showed that VsCCoAOA4T was expressed in the roots,stems and leaves of the Vicia sativa L.and had higher expression in the roots.The results of subcellular localization showed that the expression product of VsCCoAOMT was located in the cytoplasm and nucleus.Real-time RT-PCR results showed that VsCCoAOMT was responsive to Cu,Cd,H2O2,NaCl and PEG.The expression of VsCCoAOMT was increased in the Cd-resistant peanut 3(L3)Vicia sativa L.after Cd treatment for 24 h,while the expression level of Cd-sensitive cultivar Zhangye(ZM)was decreased;Under Cu treatment,The expression levels of VsCCoAOMT were decreased.After H2O2,NaCl and PEG treatment,and the expression of VsCCoAOMT was increased.The expression of VsCCoAOMT was increased in the L3 before 6 hours after the treatment with 25 ?M Cd for 0,6,12 and 24 h,and decreased after 6 hours;and in ZM the expression level of Cd was not obvious before 12 h,This suggests that L3 responds to Cd stress earlier.From the above results,it can be seen that the expression of VsCCoAOMT is induced by abiotic stress such as Cu,Cd,H2O2,NaCl and PEG,and belongs to the gene of abiotic stress response.In order to further study the effect of VsCCoAOMT on the accumulation and distribution of Cd,we constructed the plant expression vector of VsCCoAOMT and transferred it into the model plant Arabidopsis to overexpression.The results showed that the growth and root length of VsCCoAOMT overexpressing plants and wild-type plants were inhibited after two weeks on 1/2 MS medium containing 70 ?M Cd,but the root length and dryness of VsCCoAOMT overexpressed plants should be significantly higher than wild type;In the hydroponics experiment,10?M Cd treatment for 5 days,VsCCoAOMT gene overexpression of Arabidopsis thaliana at he shoots and the root of the accumulation of Cd are higher than the wild,and the translocation coefficient of VsCCoAOMT Arabidopsis thaliana was higher than that of wild type;the accumulation of Cd in the cell wall is also the same trend,while the accumulation of Cd in the cytoplasm of shoots and root was lower than that of wild type;and the accumulation of Cd in shoots'cellulose,roots' pectin,hemicellulose 1 and cellulose is higher than that in wild type.On the other hand,the histochemical staining of stem and root of Arabidopsis thaliana showed that the staining of VsCCoAOMT Arabidopsis was stronger than that of wild type,and lignin accumulation was more than that of wild type.The results showed that the lignin content of VsCCoAOMT Arabidopsis thaliana was higher than that of wild type plants after 10?M Cd treatment for 5 days,which was consistent with the results of pyrogallol staining.In the Cd-contaminated soil,the growth of VsCCoAOMT gene overexpression of Arabidopsis thaliana was better than that of wild type and the accumulation of Cd is higher than wild.It can be seen that the VsCCoAOMT gene is tolerant to Cd,which can increase the accumulation of Cd in the shoots;Cd contaminated soil may have some potential for repair.