Cloning And Functional Divergence Of Betaine Aldehyde Dehydrogenase Genes In Populus Euphratica

Plant betaine aldehyde dehydrogenase (BADH) is a physiologically important enzyme in response to salt or drought stress. In this study, two BADH genes (PeBADHl and PeBADH1) were cloned from Populus euphratica. The gene structure,expression pattern, protein structure and function of the two genes were studied in detail.1、The full length cDNA of PeBADH1contained a1512bp open reading frame,21bp upstream non-coding sequences and20bp downstream non-coding sequences. The full length cDNA of PeBADH2contained a1512bp open reading frame,93bp upstream non-coding sequences and34bp downstream non-coding sequences. The PeBADH1and PeBADH2genes encoded two protein of503amino acid residues, with a calculated molecular mass of54.93and54.90kDa, respectively. The genomic sequence analysis showed PeBADH1and PeBADH2contain14introns, respectively. The full length of DNA of PeBADH1and PeBADH2is5707bp and5430bp, respectively.2、By structure modelling of the two proteins,both PeBADH1and PeBADH2contain3conserve domains:NAD-binding domain, catalytic domain and oligomerization domain.1-132and151-230amino acids were NAD-binding domain, binding with NAD+specificly;231-480amino acids were catalytic domain, binding with different substrates;481-490and133-150amino acids were oligomerization domain including two long (3-sheet and one short p-sheet.3、Reverse transcription-polymerase chain reaction revealed that PeBADH2were not expressed in the phloem under normal condation, salt and H2O2stress. The PeBADH1and PeBADH2were expressed in the stem, leaf, shoot tissues. The PeBADH1were not expressed in the root, but salt stress induced PeBADHl expression in the root, but inhibited the PeBADH1and PeBADH2expression in other tissues. H2O2stress inhibited the PeBADHl and PeBADH2expression in the stem and bud. These results indicated expression patterns divergence between the duplicate genes PeBADH1and PeBADH2.4、The PeBADH1and PeBADH proteins were overexpressed in E. coli following by purification with affinity chromatography. The enzyme activity assays revealed that the recombinant PeBADH1and PeBADH2had enzymatic activity towards the substrate aldehyde. The enzymatic activity were0.073μmol/(min·mg) and0.107μmol/(min·mg). The PeBADH1protein revealed higher thermal stability than PeBADH2protein. At40℃, PeBADH1maintained85%of the maximum activity PeBADH2only maintained25%of the maximum activity. At50℃, the PeBADH1maintained60%of the maximum activity, PeBADH2only maintained10%of the highest activity.Therefore, based on differences in gene expression patterns and enzymatic properties of proteins, these results indicated obvious functional divergence between the PeBADH1and PeBADH2genes.