Abstract
The recently reported Dendrobium findleyanum agglutinin (DFA) was identified and determined in different parts of D. findleyanum pseudobulbs by using Western blot analysis, LC–MS/MS, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and histochemical procedure. Western blot analysis of crude protein extract with horseradish peroxidase (HRP), a mannose-rich glycoprotein, showed only one band at 14.5 kDa, which had the same molecular mass as DFA. This band was a major band when the membrane was stained with Coomassie Brilliant Blue. The protein profiles from SDS-PAGE showed higher band intensity of the 14.5 kDa mannose-binding protein in nearly mature and mature stages, compared to very young and young stages of the orchid. In addition, the band intensity was to a great extent different between the swollen and the non-swollen internode of the pseudobulb. Using LC–MS/MS, the sequence tags of the 14.5-kDa protein bands from the node, swollen internode and non-swollen internode revealed that the protein was DFA. Histochemical procedure in the transverse section of the pseudobulbs demonstrated major HRP binding sites, which reflected the location of DFA, in periphery of parenchymal cells. The purified DFA showed anti-fungal activity against Alternaria alternata and Collectotrichum sp. Using reverse transcription polymerase chain reaction and DNA sequencing, the deduced amino acid sequence of the DFA precursor revealed 94% homology with a lectin precursor from D. officinale. N-terminal sequencing demonstrated the processing site between residues 24 and 25 of the DFA precursor.
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Acknowledgments
The authors are thankful for the financial support provided by the Thailand Research Fund through the Royal Golden Jubilee Ph.D. Program (Grant No. PHD/0015/2548) to R. Sudmoon and N. Sattayasai, and the Khon Kaen University’s Graduate Research Fund Academic year 2004. We also thank the staff of Bioservice Unit (BSU), National Science and Technology Development Agency (NSTDA), Thailand for protein identification using LC–MS/MS with database search and DNA sequencing, and the staff of Scientific Equipment Center, Prince of Songkla University, Thailand for N-terminal sequencing of protein.
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Communicated by P. Kumar.
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Sattayasai, N., Sudmoon, R., Nuchadomrong, S. et al. Dendrobium findleyanum agglutinin: production, localization, anti-fungal activity and gene characterization. Plant Cell Rep 28, 1243–1252 (2009). https://doi.org/10.1007/s00299-009-0724-0
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DOI: https://doi.org/10.1007/s00299-009-0724-0