Overexpression Of Rice OsGLPs Genes Enhance The Tolerance To Heavy Metal Toxicity In Arabidopsis Thaliana And Its Mechanisms

Plants were suffering from a variety of biological and abiotic stress in the process of growth,plants have evolved a variety of strategies against these stress in the long-term adaptation process,germin-like proteins（GLPs）plays an important role in these process.The superoxide dismutase（SOD）activity of GLP promotes the disproportionation of O2·-into H₂O₂ in plants and plays an important role in the antioxidant system of plant.In the study,we focus on the response of GLP to the tolerance of Cu or Cd,and research its function in heavy metal tolerance relying on the overexpression of Oryza sativa in the Arabidopsis thaliana.The following results are obtained:1.Rice cDNA was used as template,The CDS sequences of OsGLP8-7,OsGLP 4-1 and OsGLP 8-11 were successfully cloned,The complete coding regions of the three genes were 678,765 and 678bp,respectively,encoding 225,254 and 225 amino acids,amino acid sequence alignment were showed,OsGLP has one typical Cupin domain.The results of evolutionary tree analysis showed that rice OsGLP and wild rice GLP had the highest similarity,up to 99%.2.In order to clarify the functions of three OsGLP family members OsGLP 8-7,OsGLP 4-1 and OsGLP 8-11 under the stress of heavy metals Cu and Cd,The above three gene CDS sequences were recombined with the Gateway expression bodies with flag labels,And it was overexpressed in model plant A.thaliana,Four lines of arabidopsis thaliana were successfully obtained which overexpressed OsGLP8-7,OsGLP1-1 and OsGLP8-11 genes;two strains with high OsGLP expression were selected as the following materials.The research results show,two weeks after culture on 1/2 MS solid medium containing 70-80μM CdCl2 and 50 μM CuSO4,the overexpression of OsGLP gene in A.thaliana and wild-type was significantly inhibited,However,the root length of A.thaliana plants with overexpressed OsGLP gene was significantly higher than that of wild type.Under the condition of water culture,10 μM CuSO4 treatment for 5 days,the overexpression of OsGLP gene in arabidopsis thaliana was lower than that in the wild type.Under the treatment of 10 μM CdCl2,the overexpressed OsGLP gene had no significant effect on the Cd content of A.thaliana’s overgrowth and root system compared with the wild type.However,Cd distribution in leaf protoplasts of arabidopsis thaliana was decreased.3.To investigate the role of OsGLP in oxidative stress induced by heavy metal poisoning.We studied the effects of Cu and Cd treatment on hydrogen peroxide（H₂O₂）accumulation and antioxidant enzyme activity or gene expression in arabidopsis thaliana plants under hydroponics.The results showed that compared with the wild type,the overexpression of OsGLPs gene decreased the accumulation of H₂O₂ in A.thaliana plants under the treatment of 10 μM Cu.Under the treatment of 10 μM Cd,there was no significant difference in H₂O₂ content between arabidopsis thaliana with OsGLP gene and wild type.Meanwhile,H₂O₂ histochemical staining of A.thaliana leaves was performed,Leaves of A.thaliana overexpressed OsGLP gene were significantly darker than wild type under Cu treatment.However,there was no significant difference in the accumulation of H₂O₂ in leaves treated with 10 μM Cd.Further analysis showed that the enzyme activity of POD and APX in arabidopsis thaliana strains with overexpressed OsGLP8-7 and OsGLP4-1 genes was higher than that of wild type under the treatment of Cu and Cd.Cd treatment increased POD activity in OsGLP4-1 and OsGLP8-7 A.thaliana lines.Cu treatment significantly increased the expression of SOD1 gene in three transgenic lines.4.Heavy metal stress often causes plant cell wall lignification,so the accumulation of lignin in the cell wall of arabidopsis thaliana under Cu and Cd treatment was further analyzed.The results showed that the over-expression of OsGLPs gene significantly increased the lignin accumulation in A.thaliana root and shoot compared with the wild type when treated with 5 μM Cu and Cd.These results were also confirmed by root lignin staining and cell wall microscopic observations.This may be related to the up-regulated expression of lignin-related genes COMT1 and CCR1.Under the treatment of 5 μM Cu and Cd,the over-expression of OsGLPs gene increased the content of heavy metals in A.thaliana’s overgrowth and root system to different degrees,which may be related to the improvement of cell wall lignification.