Study On The Interaction Between Arabidopsis Thaliana AtRD22 Protein And Heavy Metal Ions

Abiotic stress are the main factors that influencing the growth and development of plants and it is also the main cause for decreased yield of crops. To adapt to diverse stress environments, plants have developed different kinds of mechanisms in the long-term evolution, responding to different adverse conditions. It is important to understand the molecular mechanism of plant in response to stresses and improve plant stress tolerance through molecular breeding.RD22 was known as a gene which can be induced by drought or ABA. AtRD22 protein was a typical member of BURP domain family. Our former results demonstrated that AtRD22 could be located in the apoplast. Several copper response elements presented in the promoter of AtRD22.Overexpression of AtRD22 could enhance the tolerance of Arabidopsis to excessive copper stress. These studies suggested that AtRD22 was involved in the process of plant responsing to heavy metal stress, but the related functional mechanisms remain unclear. In this study, The expression of special eukaryotes proteins in prokaryotic cells and following purification were studied. Based on this, the soluble AtRD22 protein was purified and the binding ability of AtRD22 protein with metal ions in vitro was analyzed, and further the role of –CH motifs in BURP domain of AtRD22 in binding metal ions was tested. The results are as follows:The fusion expression vectors pSUMO-RD22,pSUMO-RD22?-cys? and pSUMO-RD22?-his? were constructed for expressing the SUMO-RD22, SUMO-RD22?-cys? and pSUMO-RD22?-his? protein in Escherichia Coli?E. Coli?, respectively. Results showed that the production of the soluble AtRD22 expressed in E. Coli can be elevated by the SUMO tag when E. Coli transformants was inducted under low temperature?< 28 ??. The Ni-NTA agarose was further used to purify the SUMO-RD22, SUMO-RD22?-cys? and pSUMO-RD22?-his? protein. And the AtRD22, RD22?-cys? and RD22?-his? protein were further obtained by cleavage off the SUMO tag using enzyme ULP1. The metal binding properties of AtRD22, RD22?-cys? and RD22?-his? protein were analyzed using immobilized affinity chromatography?IMAC?. Results showed that Cu²⁺ and Zn²⁺ could be bound to AtRD22 and RD22?-cys? completely, Cd²⁺ were only bound to AtRD22 and RD22?-cys? partially, whereas Mg²⁺ could not be bound. The protein RD22?-his? with the His mutated to Gln could bind Cu²⁺ and Zn²⁺ completely and not bind Cd²⁺and Mg²⁺. The results suggested that the-CH motif in the BURP-domain, especially the His, is important for binding some metal ions, e.g. Cd²⁺, but it is not responsible for binding Cu²⁺ and Zn²⁺. Results imply that the function of AtRD22 in plant in tolerance to excessive copper stress maybe not dependent on the –CH motifs in BURP domain.