Functional Analysis Of A Cation Diffusion Facilitator (CDF) Family Gene AtMTP6 In Arabidopsis

Cation diffusion facilitator (CDF) proteins are a recently discovered family of cation efflux transporters that might play an essential role in metal homeostasis and tolerance. After Arabidopsis genomic sequencing, no less than 12 genes of CDF family are identified and named AtMTP1-12. This study cloned AtMTP6 and primarily characterized the function. AtMTP6 locates on the second chromosome encoding a 471aa protein. Structural analysis reveals this protein has the general CDF characteristics, such as a C-terminal cation efflux domain and an N-terminal signature segment, but it has only five transmembrane(TM) domains. This study describes the cloning, characterization of the biochemical and physiological function, localization of this gene. We have got the follow results.1. The cDNA cloning was obtained by RT-PCR, and then sequenced. It showed the identical sequence with that of the prediction by AGI. Then cDNA was subcloned to pTrc99A and transformed to the E. coli mutant KAM3. The results on the solid media showed that the expression of AtMTP6 made the cells more sensitive to the excessive Zn²⁺, EtBr.2. Meanwhile, the transgenic plants transformed with the AtMTP6::GFP fusion construction were detected with the confocal fluorescence microscope. The result indicated that AtMTP6 located in the membrane of intracellular organelle, identical with the prediction of AtMTP6 locating in the membrane of mitochondria by the PSORT internet programme.3. To investigate the physiological function of this gene, overexpression and complementation transgenic plants were also constrcted. Phenotypes of these transgenic plants, together with T-DNA knockout mutant and wild type arabidopsis, were observed simultaneously under normal or stress conditions. The Arabidopsis overexpression line was more sensitive to EtBr. It seems that AtMTP6 could transport EtBr from cytosol to the mitochondria result in the accumulation of EtBr in the mitochondria, and finally caused damage of the cells. But the four lines growing in the various concentrations of Zn²⁺ did not showed any phenotype. The seeds of overexpression line are smaller in size, and the color of the seed coats are brighter suggesting AtMTP6 is related to the transporting of pigments. When the four lines are grown at the various concentrations of mannitol, the seed germination of the overexpression line was obvious inhibited indicating AtMTP6 is related to the osmotic stress. At normal growth conditions, the overexpression lines flowers much earlier, suggesting this gene takes part in the regulation or identity of flowering time.