Expression And Role Of AtRD22 In Arabidopsis Thaliana Under Cu²⁺ Stress

BURP protein family is comprised of a number of plant-specific proteins that share conserved primary structures such as the hydrophobic signal peptide at the N-terminus and the BURP domain at the C-terminus,and a specific repetitive sequence or variable domain in the middle.These proteins are involved in various biological processes and play important roles in the stress response of plants.AtRD22 is a member of BURP family,which induced by drought stress,salt stress and abscisic acid treatment in Arabidopsis thaliana.The AtRD22 protein contain a classic BURP primary structure that have four conserved CH motif(CHX₁₀-CHX?23-27?-CHX_23-26-CHX₈-W),suggesting that domain might be metal-binding site and play an important role in resisting heavy mental ions stress.Our preliminary study found that AtRD22 protein could bind to CuZ²⁺,Zn²⁺ and Cd²⁺,but the molecular functions is still unclear.Analysis of the sequences of AtRD22 promoter,showed that some tissue specific cis-elements such as endosperm specific expression related element?-300ELEMENT?,gene specific expressed in mesophyll expression related element?CACTFTPPCA1?,sequences in promoter lead to distinctive pattern of expression in roots?ROOTMOTIFTAPOX1?,and several copper response elements?CURECORECR?were presented in its promoter.It suggests that the expression of AtRD22 gene might be tissue-specific or induced by copper stress.Semi-quantitative PCR analysis indicated that AtRD22 expressed restrictly in shoot,and induced in a short period by relatively high concentration or lack of copper ion.We cloned the promoter and constructed vector p CAMBIA1301-RDpro-GUS and transferred Arabidopsis to study the expression model of AtRD22 deeply under abiotic stress.Results showed that the AtRD22 gene could induce by lack of copper and Cd²⁺ stressIn order to analysis the roles of protein domain of AtRD22 in resistance to Cu in plant,the vector p CAMBIA1302-RD22?for expression of the full length of AtRD22?,p CAMBIA1302-RD-SC?for expression of middle conserved domain,named RD-SC?,p CAMBIA1302-RD-SB?for expression of the BURP domain,named RD-SB?were constructed.Three vectors were transformed into Arabidopsis thaliana and screened for the transgenic plants,and obtained the transgenic plants named RDoe,RDoe-SC and RDoe-SB,respectively.The resistance to Cu²⁺ of transgenic plants RDoe,RDoe-SC,RDoe-SB and RDi were analyzed under the stress of Cu²⁺.Results showed that transgenic plants RDoe and RDoe-SB had higher ability of resistance to Cu²⁺ than the wild type plants,while the RDi plants were less resistance to the wild type.And the concentration of Cu²⁺ in the shoot of RDi is lower than WT after Cu²⁺ treated.It indicates that deletion of AtRD22 could decrease the resistance of Arabidopsis thaliana to Cu²⁺ stress.Overexpression of AtRD22 or its BURP domain could enhance the resistance of plants to copper stress.In order to understand whether the function of AtRD22 plays a crucial role in the interaction with other proteins,the purified AtRD22 protein was used to capture the interaction protein from Arabidopsis thaliana through pull-down assay.We constructed the vector of fusion protein GST-NSP-RD22 without the signal peptide and transformed into Ecoli.BL21?DE3?.Protein was induced with 0.6 mmol/L IPTG at 16 ? for overnight.The purified protein was used to pull down its potential interaction proteins through incubating with total proteins extracted from Arabidopsis thaliana leaves.As the results,we found that AtRD22 may interact with itself.This result needs further verification.In conclusion,AtRD22 is tissue-specific gene expressed strictly in shoot in Arabidopsis thaliana seedlings.It could be also induced in shoot by relatively high concentration or lack of copper within a short period.Deletion of AtRD22 could decrease the resistance of Arabidopsis thaliana to Cu²⁺ stress.Overexpress of AtRD22 or its BURP domain could enhance the resistance of Arabidopsis thaliana to copper stress.It indicates that BURP domain is a key function domain for AtRD22 toward copper stress in plant.Thus,Arabidopsis thaliana can improve the ability to resist copper stress by up-regulating the expression of AtRD22 gene.