| Copper is one of the necessary trace element in plant growth and development process,in the process of plant growth and development plays a very important role,but if too much copper can cause toxic to plants,affect plant,plant height,yield,plant biochemical processes in the body,severe cases will be lethal,and through the food chain will accumulate in the body,which cause serious damage to human health.Cell walls play an integral role in plant resistance to heavy metal stress and often act as the first line of defense against heavy metals.However,the role of lignin,one of the main components of cell wall,in plant resistance to heavy metal stress is rarely studied.Lignin is usually synthesized by the catalysis ofCCoAOMT,LAC,POD and so on.It is A kind of substance with complex structure.In this study,we focused on the response of rice lignin biosynthesis related genes OsCCOAOMT,OsLAC and OsPOD to heavy metal copper stress,and investigated their roles in heavy metal copper tolerance by overexpression of rice OsCCOAOMT,OsLAC and OsPOD genes in Arabidopsis thaliana.The following research results are obtained in this paper:1.The rice OsCCOAOMT gene mutants osccoa1 and osccoa20 obtained by CRISPR/Cas 9 showed a copper sensitive phenotype under copper stress,and their root length was shorter than that of the wild type at the same time,and the growth and development of rice seedlings were weaker than that of the wild type.The results of subcellular localization showed that both OsCOAOMT1 and OsCOAOMT20 were localized on the cell membrane and might be involved in the biosynthesis of cell wall and resistance to various stresses.2.The lignin biosynthesis related genes OsCCOAOMT1,OsCCOAOMT20,OsCLAC11,OsCLAC30 and OsPrx134 were overexpressed in Arabidopsis thaliana,respectively,and positive transgenic lines were obtained.The results of hydroponic experiments showed that the growth of wild-type and transgenic Arabidopsis plants was inhibited after 10 days of culture on 1/2 MS medium containing 50 μM Cu,but the root length of each transgenic Arabidopsis plant was significantly higher than that of the wild-type.After 14 days of culture on 1/2 MS medium containing 50 μM Cu,OsCCOAOMT1 and OsCCOAOMT20 gene transfection reduced Cu accumulation in shoots and roots of Arabidopsis but did not affect Cu transport to shoots of Arabidopsis.Similarly,overexpression of the OsLAC gene increased Cu uptake and accumulationin Arabidopsis,but did not alter Cu transport in Arabidopsis.Overexpres-sion of OsPrx134 decreased Cu uptake in Arabidopsis roots,but increased Cu transport from root to shoot.3.The phloroglucinol histochemical staining of roots of Arabidopsis thaliana showed that transgenic Arabidopsis thaliana had deeper staining than wild type,that is,more lignin accumulation than wild type.The lignin content of transgenic Arabidopsis thaliana was higher than that of wild-type plants,which was consistent with the histochemical staining results of lignin.These results indicate that the overexpression of lignin biosynthesis related genes increases the lignin content and deposition,and alleviates the toxic effect of excessive Cu on plants.4.Online prediction software(PLACE,Plant Care)was used to analyze cis acting elements of rice OsCCOAOMT1 and OsCCOAOMT20 gene promoters,and the analysis results showed that the sequence of OsCCOAOMT1 and OsCCOAOMT20 gene promoters contained plant hormone responding elements such as methyl jasmonate,abscisic acid and gibberellin,as well as Cu responding elements.Therefore,the upstream promoter sequences of OsCCOAOMT1 and OsCCOAOMT20 were deleted by 100 bp segment,and a zinc finger protein Zn P was screened and identified by yeast single hybridization technology,which could interact with OsCCOAOMT20-13.Further studies are needed in the future.Overexpression of lignin biosynthesis related genes OsCCOAOMT,OsLAC and OsPOD increased the tolerance of Arabidopsis mutants under copper stress,and increased the lignin accumulation in roots and the content of lignin in cell walls. |
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