Cloning And Functional Characterization Of PnATX Gene Of P.simonii×P.nigra

Copper?Cu?is one of the trace elements necessary for plant growth,but excess copper ion will produce toxicity.Copper chaperone protein with a copper ion binding region,can be transferred copper ion transport to the target cell,thereby maintaining the concentration of copper ion in the cell,to avoid the toxic damage.The ATX-like family is a class of important copper chaperone protein family in plants,divided of ATX-type and CCH-type.In this paper,results are listed as follows:?1?The specific primers were designed from the "Xiaoheiyang" to clone the ATX-type genes,named PnATX1 and PnATX2,NCBI accession number is KM434243 and KM434244,the full-length ORF were 255 bp and 258 bp.The PCR showed that in senescent leaves gained the highest transcripts,followed by stems and young leaves,lowest in the roots.We prediction the PnATXl and PnATX2 code proteins,and motif-based sequence analysis found two genes having a heavy metal binding domain HMA_domain,conserved sites for MXCXXC;Isoelectric point analysis,two genes were 5.35 and 5.34,is acidic;Signal peptide prediction found that they have no signal peptide gene exist;Hydrophilicity analysis found that the two genes encoding proteins are hydrophobic proteins.And the establishment of the system to be identified with other ATX-type gene phylogenetic analysis and modules.?2?0 mmol/L and 50 mmol/L concentrations of Cu2+ treated with "Xiaoheiyang",quantitative real-time PCR showed that two genes expression in 50 mmol/L is higher then 0 mmol/L in roots,young leaves and old leaves.?3?We successfully constructed pEASY-PnATXl and pEASY-PnATX2 vector,and transformed into saccharomyces cerevisiae.Copper resistance analysis found,the saccharomyces cerevisiae contain pEASY-PnATX1 and pEASY-PnATX2 vectors obviously enhanced the copper resistant then contain pEASY vector.?4?We successfully constructed pCAMBIA1302-PnATX1 and pCAMBIA1302-PnA TX2 vector.And we identified Arabidopsis atxl homozygous mutant.The mutant SALK₀26221.Through the stained flower method infection of Arabidopsis wild type and mutant of Arabidopsis thaliana,obtain homozygous transgenic lines.And these transformed lines were expression analysis,to obtain the high expression of plant.We detected high expression transgene lines of root length,fresh weight,CAT activity,SOD activity,POD activity and MDA found,compared with the control group,the transgene lines in fresh weight and root length is significantly increased;CAT enzyme activity,SOD enzyme activity,POD enzyme activity was significantly enhanced;the MDA was reduced in transgene lines.To certify that PnA TXl and PnA TX2 genes have a role in the resistance of copper ion stress.Other functions for the further study of ATX-type gene family members to provide the theoretical support.