Structure Analysis And Function Identification Of Fe-deficient Related 3.3kb CDNA Fragment

FDR is one of the differential screened clones from cDNA expression library of Fe-deficient induced maize root. The fdr is a 3.3kb fragment containing a l,068bp ORF1 and a 648bp ORF2, called fdrl and fdr2 respectively. Based on the analysis of hydrophobicity, transmembrane domain and cell localization sites of their amino acid sequence, we suppose that fdrl and fdr2 encode members of membrane-bound proteins, fdrl is likely a chloroplast protein and fdr2 is likely an endoplasmic reticulum membrane protein. Having homology searched in GenBank indicated that fdr2 has 82% homology with hrp(Hypersensitivity Response Secretion Protein) gene.In order to acquire corresponding antibody to facilitate the following research of the cellar localization and expression, fdlr and fdr2 were first cloned into the prokaryotic expression vector pGEX-2T and pET-32a respectively. Then we cleavaged fdrl and fdr2 fragments from pGEX-fdrl and pET32a-fdr2 and subcloned them into the plant expression vector pBI121. Two sense expression constructions named pBF and pBH were further constructed. The two constructions were then transformed into tobacco through Agrobacterium-mediatQd leaf disc transformation method. 58 lines of pBF and 34 lines of pBH Km resistance transgenic tobaccos were collected after growing on the medium containing antibiotics. PCR reaction using NPTII as primer and Southern hybridization using fdr2 fragment as probe were performed, The results showed that we have obtained the transgenic tobacco plants. At the same time, we performed Southern blotting using the fdr2 fragment as probe with the genomic DNA of Physcometrella patens, Triticum aestivum, Oryza sativa, Arabidopsis thaliana, Nicotiana tabacum , and xiaojinensis mslus.