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| Cloning of a Mannose-binding Protein 2 Gene (MdMBP2) from Malus domestica and Identification of Its Biological Activity |
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Abstract Mannose binding protein (MBP) is a subgroup of plant lectin and thought to play a role in various biological processes such as cell-to-cell and host-pathogen interactions. In our previous experiments, an apple MBP gene was found among the highly expressed genes induced by inoculation with Botryosphaeria dothidea, a causal agent of a disastrous disease, apple ring rot. To investigate the role of the MBP gene in the defense responses of apple(Malus domestica) against B. dothidea, we cloned the full-length cDNA sequence of the MBP gene and designated as MdMBP2(GenBank accession No. JX126857). The full-length cDNA was of 1 553 bp with a 1 368 bp of open read frame (ORF) encoding a protein of 455 amino acid residues. The calculated molecular weight and isoelectric point of the MdMBP2 protein were 50.4 kD and 8.68, respectively. Sequence and structure analysis indicated that MdMBP2 possessed a B-lectin domain and PAN-apple domain. There was a predicted signal peptide of 27 amino acids in the N terminus. BLAST analysis revealed that MdMBP2 had high identity to epidermis-specific secreted glycoprotein from Vitis vinifera (66%) and Glycine max (42%), and mannose binding protein from Arabidopsis thaliana (57%). Phylogenetic analysis revealed that all B-lectin domain containing proteins could be grouped into two classes and MdMBP2 belonged to the ClassⅠwhich lacked SLP domain and PKc (protein kinase) domain compared with the proteins in ClassⅡ. The expression of MdMBP2 gene was observed in all tissues examined here, and the highest expression of MdMBP2 gene was found in bark and leaf, and the lowest in root. This expression pattern suggested that MdMBP2 might be involved in many physiological pathways. The responses of MdMBP2 expression to B. dothidea infection were also examined using quantitative Real-time PCR. The results indicated that MdMBP2 expression was significantly enhanced by B. dothidea infection, and the temporal expression was related with the course of disease, which suggested that MdMBP2 might be involved in the defense responses against B. dothidea infection. The recombinant protein of MdMBP2 was prepared with prokaryotic expression system and used to test carbohydrate binding activity. The results indicated that MdMBP2 exhibited strong affinity toward oligomannosides and high-Man N-glycans, but interacted weakly with D-Mannose. The results indicated that MdMBP2 gene might be involved in the recognition of fungal pathogen. All together, in the present study, we identified a new apple MBP gene and analyzed its mRNA expression and biological activity of recombinant protein. The results revealed that the gene is involved in the defense responses against B. dothidea and may play a role in recognition of the pathogen.
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Received: 28 May 2012
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