Physiological And Biochemical Indexes Determination And Promoter Deletion Analysis Of PnATXs Transgenic P.Simonii×P.Nigra

Copper ions play important roles in organisms.Copper deficiency would affect the growth and development of organisms.However,excessive copper ions are also potentially toxic.In the process of evolution,organisms have formed complex regulatory mechanisms to ensure intracellular copper ion homeostasis.In addition,copper ions entering the cell are chelated by proteins and small molecular ligands,and the transport of these copper ions is accomplished through the copper-chaperone protein.In this study,the cloned copper chaperone protein genes PnATX1 and PnATX2 were used to construct RNAi expression vectors using Gateway method,and transgenic lines were obtained after genetic transformation of P.simonii×R nigra.The PnATXs promoter deletion sequences of P.simonii×P.nigra were obtained by PCR method,and the fusion expression vectors of the deletion sequences and GUS gene were constructed and genetically transformed into Arabidopsis thaliana.GUS staining preliminarily determined the biological activity of PnATX1 and PnATX2 promoter deletion sequences.The physiological and biochemical indexes of PnATXs overexpression transgenic plants and wild-type plants under copper stress conditions were detected.The main results are as follows.(1)The RNAi expression vectors of PnATXs genes of P.simonii×P.nigra were successfully constructed by using Gateway method through BP and LR reaction,and the agrobacterium strains containing recombinant plasmids were obtained after electrotransformation of agrobacterium competent cells.Ten PnATX1-RNAi and six PnATX2-RNAi resistant lines were obtained after genetic transformation of P.simonii×P.nigra through leaf disc method.The identification results of PCR and qRT-PCR showed that the expression levels of PnATXl and PnATX2 genes in PnATX1-RNAi and PnTAX2-RNAi lines were suppressed and the suppression degree was different.Compared with wild-type plants,in the PnATX1-RNAi lines,the expression level of PnATXl gene in shoots of no.3,5 and 9 transgenic lines was significantly decreased,and the expression level of this gene in both roots and shoots in no.10 transgenic lines was significantly decreased.The expression level of PnATXl gene in shoots of no.2,3,4,5,and 6 PnTX2-RNAi lines was significantly decreased.(2)Primers were designed using the full-length promoter sequences of PnATXs,and the three deletion segments of PnATX1 and PnATX2 promoters(PnATX1-1,PnATX1-2,PnATX1-3,PnATX2-1,PnATX2-2 and PnATX2-3)were obtained by PCR method.After replacing the 35S promoter of plant expression vector pBI121 with the promoter deletion segments,the fusion expression vectors containing the GUS gene were constructed.A.thaliana wild-type plants were genetically transformed by agrobactcrium-mediatcd dipping method.GUS staining results of T2 generation transformed plants showed that PnATX1-3 and PnATX2-3 promoter deletion sequences had the promoter function,while PnATX1-1,PnATX1-2,PnATX2-1 and PnATX2-2 promoter deletion sequences could not start the expression of GUS reporter gene.The T2 generation transformed plants of A.thaliana were cultured with different medium containing null and different concentrations of copper ions(5?moL/L and 50?moL/L).Histochemical staining results showed that the expression of GUS gene in the transgenic A.thaliana contianing PnATX1-3 and PnATX2-3 promoter deletion sequences was not affected by copper ion concentration.The specific mechanism of PnATX1 and PnATX2 promoter deletion sequences of P.simonii×P.nigra responding to copper ion stress needs further experimental verification.(3)The wild type,PnATX1 overexpression lines no.2,3,5 and PnATX2 overexpression lines no.1,2,5 of P.simonii×P.nigra were treated under conditions of copper deficiency,normal copper ion concentration and high concentration of copper ions.The root length,plant height,leaf area,chlorophyll content,peroxidase(POD),superoxide dismutase(SOD),catalase(CAT)enzyme activity and malondialdehyde(MDA)content of the treated plants were determined.There was no significant difference in plant height and leaf area between wild type and PnATXs overexpression plants after treated with copper deficiency,normal copper concentration and high concentration of copper ions.Compared with wild-type plants,the root length of PnATXs overexpression plants did not change significantly after treatment with copper deficiency and normal copper concentration.The root length of PnATXl overexpression lines no.3 and 5 increased significantly after 10 ?mol/L Cu2+treatment,and the root length of PnATX1 overexpression lines no.3,5 and PnATX2 overexpression no.5 was significantly increased after treated with 15 ?mol/L Cu2+.The chlorophyll content of PnATX1 and PnATX2 overexpression plants was significantly higher than that of wild-type plants after treatment with high concentration of copper ions.The determination results showed that the MDA content in young leaves of PnATX1 overexpression lines no.3 and PnATX2 overexpression lines no.1 responded to copper deficiency treatment was significantly lower than that of wild type plants,while other transgenic lines did not change significantly after normal copper concentration and copper deficiency treatments in young leaves and mature leaves.The SOD activity of young and mature leaves of PnATX1 and PnATX2 overexpression plants of P.simonii×P.nigra was significantly higher than that of wild-type plants after high concentration copper ion treatment,especially 15?mol/L copper ion treatments.In addition,after treatment with 10 and 15?mol/L CuSO4,the POD activity in mature leaves of PnATX1 overexpression no.2 transgenic lines,and the CAT activity in young leaves of PnATX1 overexpression no.5 transgenic lines and PnATX2 overexpression no.5 transgenic lines were no significant dificrcnces from those of wild-type plants.In addition to this,the activities of peroxidase and catalase in young leaves and mature leaves of other transgenic.simoniixR nigra plants were significantly higher than those of wild-type plants.These work laid an experimental foundation for further elucidation the biological functions of copper chaperone proteins PnATX1 and PnATX2 and their specific functions in maintaining copper homeostasis in poplar cells.