Functional Analysis Of OsSEC27p And Cloning, Subcellular Localization Of OsYSL1

In previous research, our lab acquired a group of high -Fe/EDTA-Fe ratio genes responded to iron deficiency in rice roots based on the Microarray analysis. In this thesis, we cloned two of them aimed to determine their functions.The mRNA of OsSec27p expressed highly both through cDNA microarray and realtime-PCR studied at the 5^th day under Fe-deficient condition in rice roots. Blast results indicated that it exhibited a little bit like Sec27p in yeast, so named OsSec27p temporarily. Full length cDNA of OsSec27p from rice roots was cloned and submitted for a GenBank accession no. DQ434847. The subcellular localization experiment with GFP and FM4-64 respectively referred that OsSEC27p may be localized in vesicles on plasma membrane and endomembrane. Then pCAMBIA1302-OsSEC27p::GFP recombinant was transformed into tobacco (Nicotiana tabacum var. Samsun NN). T₁ and T₂ germs of transgenic tobaccos were acquired and identified. Using Scanning Ion-selective Electrode Technique (SIET) of non-invasive microsensing system to measure H⁺ fluxes, we found that the fluxes of H⁺ around transgenic tobaccos' roots had an out-secretion trend.In the other hand, full length sequence of OsYSL1 was acquired and plant expression vectors pCAMBIA1302-OsYSL1-gfp and pGPTV-AGPp-BAR-OsYSL1 were constructed. To observing the subcellualar location, recombinant plant expression vector pCAMBIA1302-OsYSL1-gfp had been transformed into onion epidermic cells and BY-2 cells, then localizated object gene through observation of GFP instant expression. The results indicated that OsYSL1 was localized in the plasma membrane. At the same time, expression vector pGPTV-AGPp-BAR-OsYSL1 had been transformed into the callus of rice. Sequence alignment analysis showed that OsYSL1 had 43.43% identity with ZmYS1. So, it can be preliminary considered as an iron-phytosiderophore transporter protein.