Genetic Transformation And Antioxidant Function Analysis Of PnCCH7 And PnCCH17 Genes In Populus Simonii×populus Nigra

Copper is an essential microelement for plant growth and development and plays a key role in plant physiological activities.Plants have produced a complete set of copper homeostasis regulatory networks during long-term evolution,and copper chaperone protein is an important participant.It can bind copper ions and transport them to target protein.CCH(copper chaperone)in Arabidopsis is the first copper chaperone protein identified in plant.Based on the previous research,transient expression vectors and two kinds of plant expression vectors of PnCCH6,PnCCH7.PnCCH15 and PnCCH17 genes were constructed in this study.The subcellular localization of PnCCH6,PnCCH7,PnCCH15 and PnCCH17 proteins were studied by transient transformation of Populus trichocarpa protoplasts.Using the constructed plant expression vectors of PnCCH7 and PnCCH17 genes,wild type plants,cch mutant and atxl mutant plants of Arabidopsis thaliana were transformed by floral-dip method,T3 generation transgenic plants were obtained and physiological and biochemical indexes were measured.After transformed to Populus simonii × Populus nigra by leaf disc method,the resistant plants of overexpressing PnCCH7 and PnCCH17 genes were obtained.The main findings are as follows:(1)The transient expression vectors pBS-PnCCHs-GFP were constructed by enzymatic digestion and ligation method using the cloned genes PnCCH6,PnCCH7,PnCCH15 and PnCCH17,and transfected into stem protoplasts of P.trichocarpa.Fluorescence microscope observation results showed that PnCCH6 and PnCCH7 proteins localized in the nucleus,membrane and cytoplasm.PnCCH15 and PnCCH17 protein localized in membrane and nucleus.(2)The Arabidopsis overexpression vectors pCAMBIA1302-PnCCHs of PnCCH6.PnCCH7,PnCCH15 and PnCCHl 7 genes were constructed,and the Agrobacterium strain GV3101 containing recombinant plasmids were obtained.Using the constructed Arabidopsis overexpression vectors of PnCCH7 and PnCCH17 gene,the wild type plants,cch mutant and atxl mutant plants of Arabidopsis thaliana were genetically transformed.The Ti generation seeds of WT-PnCCH7,WT-PnCCH17 and T3 generation seeds of cch-PnCCH7,atxl-PnCCH7.cch-PnCCH17,and atxl-PnCCH17 were obtained.The detection of the T3 generation plants of cch-PnCCH7,atxl-PnCCH7,cch-PnCCH17,and atxl-PnCCH17 were conducted,and the Arabidopsis transgenic lines with higher expression level of PnCCH7 and PnCCH17 genes were selected(line 3,5,and 9 of cch-PnCCH7,line 3,4,and 7 of atx]-PnCCH7,line 4,9,and 10 of cch-PnCCHl 7,line 2,6,and 8 of atxl-PnCCH17).(3)The seedlings of Arabidopsis wild type(WT),cch mutant atxl mutant and transgenic lines were cultured hydroponically in half strength Hoagland nutrient solution containing different concentrations of copper ions(0,0.5 ?mol/L,35 ?mol/L CuSO4)for 5 d.And then,the antioxidant enzyme activities and malondialdehyde(MDA)contents of Arabidopsis thaliana leaves after copper treatments were detected.The results showed that the MDA content of cch mutants and atx1 mutants was significantly higher than that of WT plants after treated with three concentrations of copper ions,indicating that these two mutants suffered more oxidative damage than WT plants under copper treatments.Under the copper deficiency condition,the superoxide dismutase(SOD)activity of cch-PnCCH17 transformed lines were significantly higher than that of cch mutants,and the peroxidase(POD)activity of cch-PnCCH7,cch-PnCCH17 and atxl-PnCCH17 transformed lines were significantly lower than that of corresponding mutants,and the catalase(CAT)activity of WT plants and cch-PnCCH17 transformed lines were significantly higher than that of the cch mutant plants.After treatment with high concentration of copper ions,the POD activity of WT plants and cch-PnCCH7 transformed lines were significantly higher than that of cch mutants,and the CAT activity of cch-PnCCH7,atxl-PnCCH7 and atxl-PnCCH17 transformed lines were significantly higher than that of corresponding mutants.In addition,the MDA content of cch-PnCCH7,atxl-PnCCH7,cch-PnCCH17,atxl-PnCCH17 transformed lines were significantly lower than that of the corresponding mutant plants after treated with three concentrations of copper ions.The above results indicated that PnCCH7 and PnCCH17 increased the antioxidant capacity of Arabidopsis cch and atxl mutants to a certain extent.(4)The P.simonii × P.nigra overexpression vectors pROKII-PnCCHs of PnCCH6,PnCCH7,PnCCH15 and PnCCH17 genes were constructed,strains containing recombinant plasmids were obtained after electroporation transformed into Agrobacterium strain EHA105.Using the constructed overexpression vectors of PnCCH7 and PnCCHl 7 genes,the P.simonii×P.nigra wide type plants were transformed by Agrobacterium-mediated leaf disc method,and resistance screening were performed using the medium containing kanamycin.10 resistant lines overexpressing PnCCH7 gene,as well as 8 resistant lines overexpressing PnCCH17 gene,were obtained.