Cloning And Functional Characterization Of OsMTP1,a Metal Transporter From Rice (Oryza Sativa L.)

Metal elements such as zinc, iron, and copper etc., are necessary nutrients for all organisms and involve in a lot of biological chemical events through composing the active positions of enzymes, participating transmit of electron in body, and regulating the expression of genes. On the other hand, excess accumulation of these metal ions can cause poisonous effects to plants, which like the results led by heavy metals including cadmium and mercury. Therefore, organisms forme a set of exact regulating system in evolution course in order to control their absorption, spread, store and to reduce the toxicity. With the swift development of genetics and experiment methods in molecular biology, many genes involved in the transport of metal ions had been cloned, such as CDF family (cation diffusion facilitator family), ZIP family(ZRT,IRT-like protein). It has become a new hot point in the research field of trace elements that the structure, function, regulation mechanism and their potential value of metal ion transporters concerning.Using the amino acid sequence of rat zinc transporter (ZnT-2, GenBank accession number: NP₀37022.1) as a seed sequence to search the rice genome sequence in chromosome 5. A putative full-length rice metal transporter gene was obtained by assembling genome sequences and ESTs database, then cloned by RT-PCR. It has been designed as the OsMTP1(Oryza sativa Metal tolerance protein 1).The OsMTP1 was the first report of CDF-type metal transporters from the monocots. It has no introns and encodes for 418 amino acid with the MW=45.5kDa and pI=5.87. The sequence was deposited in GenBank with accession number AY266290. The alignment of OsMTP1 with other CDF family members shows that it shares 62%⁶7% of identity and 70%⁷7% of similarity with the amino acid sequence from AtMTP1, AsMTP1, TaMTP1, TgMTP1t1, TgMTP1t2, TmMTP1 and PtdMTP1. All of proteins have six transmembrane (TM) domains, and most of them including OsMTP1 own the histidine-variable regions. The prediction of TM regions by TMHMM program revealed that there was very similar TM structures between OsMTP1 and AtMTP1, suggesting similar transporting ability of them.Expression patterns of the OsMTP1 indicated that it was constitutively expressed intested rice tissues, with relatively higher level in leaves than that in other tissues. The expression of OsMTP1 was significantly induced in the seedlings in response to Zn and Cd treatments, with very similar induced model between cultivar Nipponbare (japonica subspecies) and IR26 (indica subspecies). Meanwhile when exposure to excess other metals such as Cu, Fe, Mg, the expression of OsMTPl was also slightly increased. Transient expression analysis indicated the OsMTP1 ::GUS fusion protein was localized to plasma membrane in onion epidermal cells. It was suggested that OsMTP1 might participate in Zn efflux.In order to understand the function of OsMTP1, the vector pFL61-OsMTP1 was constructed to make expression analysis in yeast. It was shown that wild type yeast carrying pFL61-OsMTP1 could grow better than the yeast just carrying pFL61 in the culture medium which containingl5mM ZnSO4. Therefore, OsMTP1 may be involved in zinc tolerance. Now we had obtained six transgenic tobacco seedlings, which were confirmed by PCR. The expression of OsMTP1 can be detected by RT-PCR in two of them. It will make good base to the further understanding of OsMTP1 functions.