Antioxidant Function Identification Of PnATXs And Genetic Transformation Of PnATX1 Gene

Populus simonii×Populus nigra belongs to Populus of Salicaceae with the excellent characteristics of drought,low temperature and barren resistant.Based on the above qualities,Populus simonii×Populus nigra is a kind of afforestation tree species of north area.ATX1(Anti-oxidant)is a kind of protein with antioxidant function and it plays an important role in the process of copper homeostasis.In this study,the PnATXs gene was used to construct the yeast expression vectors.By yeast functional complementary experiments,the antioxidant and copper chaperone functions of PnATXs were verified.The promoters of PnATXs genes were cloned,and the results of bioinformatics analysis showed that there were copper response elements CuRE located in the promoters.Besides,the bioactivities of PnATX1 and PnATX2 full-length promoters were verified preliminary by CUS staining.And then,the plant expression vector of PnATX1 gene was constructed and transformed into Populus simonii×Populus nigra.Finally,the transgenic plants have been got.The main results obtained from these studies are as follows:(1)The yeast expression vectors of ATXs genes were been constructed,and then transformed into yeast sodl ? and atxl ? mutant cells.The results showed that the transformed sod]? cells could grow on the SC-Lys medium in aerobic state,which indicated that PnATX proteins had the function of antioxidant.The transformed atxl ? cells could grow on the medium containing iron chelator,and this result showed that PnATX proteins had the function of copper chaperone.(2)Genomic sequence of Populus trichocarpa was used to design the primers and then the full sequences of PnATX.s promoters were cloned by PCR.Bioinformatics analysis showed that the PnATXs promoters contained light response elements such as ATCT-motif,hormone-responsive element such as GARE-motif and TGACG-motif,fungal inducing element Box-W1.It is worth noting that both PnATXs promoters contained the copper response element CuRE.The 35S promoter of the pBI 121 vector was replaced by the PnATXs promoters.After that,the vectors were transformed into Arabidopsis thaliana by Agrobacterium mediated method.Then the homozygous were screened.The results of GUS staining showed that the full-length promoters of PnATX1 and PnATX2 had bioactivity.However,further study would be carried out to detect the mechanisms of how the PnATXs promoters been regulated by copper.(3)The plant expression vector pROKI I-PnATX1 was successfully constructed by using the method of enzyme digestion and ligation method.After transformed into P.simonii×P.nigra via agrobacterium infestation,ten transgenic lines were obtained by PCR analysis which showed that the PnATXl gene has integrated into the genome of P.simonii×P.nigra.The results of qRT-PCR showed that PnATX1 gene has expressed successfully in seven lines.This study laid the experiment foundations for further illuminate the copper chaperone function of poplar PnATXs proteins and its important roles in maintaining the intercellular copper homeostasis in woody plants.