Cloning And Expression Analysis Of Related Genes Under Fe-deficiency Stress In Malus Xiaojinensis

It is very important to investigate and clone related genes under Fe-deficiency stress in Malus xiaojinensis, which not only provides the probability for exploring molecular mechanism of the absorbability and transport of Fe, but also may enhance the resistance under Fe-deficiency stress of plants by inserting related genes into chromosome genome.The study was undertaken to isolate Fe (II)- transporter cDNA and related binding protein cDNA under Fe-deficiency stress from a Fe-deficiency root cDNA expression library of Malus xiaojinensis by screening library using maize Fe (II)- transporter cDNA and wheat TaMRE-BP cDNA as a probe. The main results as follows:1 Out of approximately 120000 plaques, four positive cDNA clones encoding Fe (II)- transporter proteins, designated pFTl -. pFT2, pFT3 and pFT4 respectively, were isolated from a Fe-deficiency root cDNA expression library of Malus xiaojinensis by screening library for three times using Fe (II)-transporter of maize as a probe, then two representative clones (pTFl and pTF2) were sequenced.2 Sequences were analysed and compared in GenBank: pTFl is 494bp and pTF2 is 763bp in longth, the deduced proteins consist of 88 and 172 amino acids respectively, these cDNA-clones encode the same gene-Fe (II)- transporter.3 Southern blotting showed that Fe (II)- transporter was a single copy in Malus xiaojinensis genome and it was also existed in Malus baccata and Malus zumi .4 Northern blotting showed that the Fe (II)- transporter mRNA was expressed lower in Fe-sufficiency roots and was strengthened expression by the treatment of Fe-deficiency, particularly in the sixth day of the treatment in roots.5 Out of approximately 120000 plaques, ten positive cDNA clones were isolated from a Fe-deficiency root cDNA expression library of Malus xiaojinensis by screening library for three times using TaMRE-BP cDNA as a probe, then two representative clones were 5' sequenced. Sequences analysis showed that these clones had a highly homology to MYB-related protein of Arabidopsis. the longest cDNA-clone, designated MxMybl for Malus xiaojinensis Mybl, was sequenced entirely.6, Sequence analysis showed that MxMybl was a full-length MYB transcription factor possessing 1134bp in longth and a complete open reading frame capable of encoding a polypeptide of 302 amino acids. MxMybl contains a highly conserved single DNA binding domain. The IR sequence of MxMybl is most homologous with that of AtMyb (86.9%) and somewhat homologous with that of StMyb 1(77.0%); There is very low homology among N- and C-terminal regions outside of the IR regions of all of the MYBs; the protein MxMybl contains a proline-rich region as well as a serine-rich region near the C-terminus, such structure motifs are implicated in transcriptional activation.7 As indicated myb-like genes are members of families of related genes. We therefore performed a Southern blot experiment to determine the approximate copy number of the MxMybl gene. To avoid cross hybridzation due to the conservative myb-domain a fragment specific only for the C-terminal part of the gene was used as a probe. Independent of the restrictase used for digestion single band was detected. This suggests that MxMybl represents a single copy gene in the genome of Mains xiaojinensis.8 Expression pattern was analysed by Northern blot and RT-PCR. The results showed that MxMybl mRNA was expressed in root and leaf and it was strengthened expression by the treatment of Fe-deficiency for three days in roots especially.9 Expression of MxMybl gene in E.coli was experimented. The structure of its recombinant expression vectors had not base mutant and drift. SDS-PAGE of pET-MxMybl proteins showed that there was a strengthened protein band in expectant 38kDa protein Marker.