Subcellular Localization Of MxIRT1, Cloning And Functional Identification Of The Two Sections Of Signal Peptide And The Variable Region Deletion Mutant In MxIRT1

Using PCR technology, we deleted the signal peptide and the variable region of MxIRT1 respectively, and named them DsIRT and MIRT. Different expression vectors constructed, and transformed into the yeast mutants which were defective for a kind of bivalent metal ion transport. Using the method of functional complementation of yeast mutant strains, combined with inductively coupled plasma mass spectrometry (ICP-MS), the ion concentration in yeast cells was measured. The result was that MxIRT1 is a bivalent metal ion transporter which could transfer a few kinds of ion. It could transfer Cd, strongly; Ni,Co slightly and could take up Zn, Fe partly; but little effect on Cu and Mn. In our experiment, heavy metals were all poisonous more or less. The absorptivity and endurance of cells were different between different heavy metals. The poison of Cd was most serious. Only trace of Cd could restrain the growth. It is implied that the signal peptide in the forepart of MxIRT1, almost 105bp, was very important for absorption. The absent of this region immediately impacted the function of MxIRT1. The variable region with histidine-rich motif between TM III and TM IV was believed to have some function during the transport of Zn,Fe. The absent of this region restrained spots, and sharply reduced the absorptivity of Zn, when there was no Zn supplied. The same result could be found in Fe experiments. But, it seems that there were little effects on the transport of Cd,Co,Ni.According to structural forecast, IRT protein was expected to be an integral membrane. In order to further confirm its subcellular localization , pYES2.0-MxIRT1::GFP with a inducible promoter was constructed and then transformed into the yeast strain by LiAc method. We observed the cells under the fluoroscope and laser confocal microscope through a method of Fm4-64 (a lipophilic styryl dye), red and green fluorescent co-localized in plasma membrane. It was elementarily deduced that MxIRT1 should be localized in the plasma membrane; the MxIRT1 was restricted in some specific region in PM as same as raft-like structures. This vector could be induced by Galactose as carbonaceous. It could be proved that 6-20 hr's inducing was the best time for observation.The expression vector of plant was constructed and transformed into tobaccos, and we gained seeds of TO. Following work of functional detection can be ongoing.