J Integr Plant Biol. ›› 2006, Vol. 48 ›› Issue (2): -.

• Research Articles •    

Purification, Biological Activities, and Molecular Cloning of a Novel Mannose-Binding Lectin from Bulbs of Zephyranthes candida Herb (Amaryllidaceae)

Chuan-Fang Wu, Jian Li, Jie An, Li-Qing Chang, Fang Chen and Jin-Ku Bao   

Abstract: A novel mannose-binding agglutinin was purified from bulbs of Zephyranthes candida Herb by extraction, precipitation with 80% (NH4)2SO4, and ion-exchange chromatography on DEAE-Sepharose followed by gel filtration on Sephacryl S-100. The purified Z. candida agglutinin (ZCA) migrated as a single band of 12 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing and non-reducing conditions. The apparent molecular mass of the lectin, as determined by gel filtration chromatography, was 48 kDa. The results indicated that ZCA was composed of four identical subunits of 12 kDa each (homotetrameric nature). The ZCA agglutinated rabbit erythrocytes, Escherichia coli and Saccharomyces cerevisiae cells at concentrations of 0.95, 1.90, and 31.30 μg/mL, respectively. Bioassays indicated that ZCA has a significant effect on wheat aphid survival. Mortality after 7 d was > 90% at 0.26%. A degenerate primer was designed in accordance with the N-terminal partial sequence of purified ZCA. The full-length cDNA was cloned by 3''- and 5''-rapid amplification of cDNA ends. The full-length cDNA had 661 bp and the sequence encoded an open reading frame of 168 amino acids. The mature protein of ZCA includes 109 amino acid residues and the molecular weight of the protein was 12.1 kDa. The result show that the zca gene encodes a protein precursor with a signal peptide, a mature protein, and a C-terminal cleavage amino acids sequence. Molecular modeling of ZCA indicated that its three-dimensional structure strongly resembles that of the snowdrop agglutinin. Blocks’ analysis revealed that the deduced amino acid sequence of ZCA has three functional domains specific for agglutination and three carbohydrate binding boxes (QDNY).(Author for correspondence.E-mails: scuchenfang@hotmail.com and jinkubao@yahoo.com)

Key words: agglutinating activity, insecticidal activity, molecular clone, sequence analysis, Zephyranthes candida agglutinin.

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