Functional Characterization Of AtDTX18, A Member Of The MATE Family From Arabidopsis

Just like other organisms,plants synthesize and accumulate a diverse range of secondary metabolites,which are very important in the processes of growth and development of plants,or in participating defense responses to various insects and microbes ect.However,excessive secondary metabolites accumulated in cytoplasm are toxic for plants.Therefore,disposal and detoxification of these toxic compounds are necessary for plants.There is a multidrug and toxic compound extrusion family in the model plant Arabidopsis,which consist of 56 members.Some of the family members have been identified as multidrug and toxic compound transporters.In this paper,AtDTXI8 which belongs to the MATE family,was chosen as a target gene for functional characterization.1.The cDNA of AtDTX18 was cloned by RT-PCR and ligated to pMD18-T vector.The sequencing result shows that it is identical with the data as predicted by AGI.This gene locates on the third chromosome encoding a 469 aa protein.Structural analysis reveals AtDTXI8 has the general MATE characteristic,such as 12 transmembrane domains.2.Then,the cDNA fragment of AtDTX18 was subcloned to pTrc99A vector to construct the recombinant plasmid pTrc99A-DTXlS.Then the construct was transformed into the mutant bacterial KAM3 to do the functional complementation.After being induced by IPTG,the transformed strain grew weaker than the negative controls on the culture medium containing 100μg/rnL berberine,or 0.45～0.75 mol/LTMACI or 3 mmol/L IAA.This result implies AtDTX18 maybe an influx transporter of berberine,TMACI and IAA or their analogues.3.The promoter fragment of AtDTX18 was amplified by PCR from genomic DNA of Arabidopsis, and then ligated into binary plasmid pBIlO1.2 to construct pBIIO1-DTX18-GUS,which was,subsequently transformed into Arabidopsis by agrobacterium mediated method.The histochemical GUS staining result shows hat AtDTX18 expresses in leaves specifically,and its expression was induced by wound.After 0.5 hour stimulation of wound,the transcripts of AtDTX18 start to increase.4.Semi-quantities RT-PCRanalysis confirms that under wound stress,the expression level of AtDTX18 increases in the leaves of wild type plants.5.Bioinformatics analysis showed that AtDTX18 may be involved in MJ dependent signaling pathway to participate defense responses as some secondary metabolite transporter.