Cloning and functional characterization of a GNA-like lectin from Chinese Narcissus (Narcissus tazetta var. Chinensis Roem).
A full-length cDNA encoding Narcissus tazetta lectin (NTL) was isolated from Chinese narcissus (N. tazetta var. Chinensis Roem). The open reading frame (ORF) was 519 bp long and encoded 172 amino acids with a theoretical isoelectric point of 5.27 and a calculated molecular mass of 18.6 kDa. Conserved domain analysis indicated that it possessed three D-(+)-mannose-binding sites, presumed to be similar to those of Galanthus nivalis agglutinin (GNA)-like lectins. A recombinant (glutathione S-transferase) GST-NTL fusion protein of around 40 kDa was successfully synthesized in vitro. Lysates of cells expressing this recombinant protein exhibited significant hemagglutinating activity [418 hemagglutinating units (HU)], as did the purified protein (265 HU). Sugar specificity assays suggested that mannose is the only sugar that significantly inhibits this hemagglutinating activity, confirming that NTL is a member of the GNA-like lectin family. NTL is highly transcribed in flowers, leaves and roots, but less so in scales. However, similar levels of the NTL protein were observed in all four of these organs by western blotting. A fluorescent NTL-GFP (green fluorescent protein) fusion protein was found to be primarily localized in the vacuole of transformed onion epidermal cells, indicating that NTL may be a vacuolar storage protein. This is the first study in which the function of NTL has been examined and provides a considerable body of data concerning its physiological role in Chinese narcissus. The results obtained may be useful in the molecular engineering of plants with enhanced tolerance of biotic and abiotic stresses. Moreover, they may be relevant to medical applications of lectins.
International Center for Bamboo and Rattan, SFA Key Open Laboratory on Bamboo and Rattan Science and Technology, Beijing 100102, China.
Zhi M
ZM
Gao
Bo
B
Zheng
Wen Y
WY
Wang
Qiang
Q
Li
Qi P
QP
Yuan
D000222:Adaptation, Physiological; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005982:Glutathione Transferase; D049452:Green Fluorescent Proteins; D006386:Hemagglutination Tests; D008358:Mannose / Q000378:metabolism*; D037241:Mannose-Binding Lectins / Q000378:metabolism; D031427:Narcissus / Q000235:genetics / Q000378:metabolism*; D019697:Onions / Q000235:genetics / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D037121:Plant Lectins / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D018749:RNA, Plant / Q000235:genetics; D011993:Recombinant Fusion Proteins; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D018076:DNA, Complementary; D037241:Mannose-Binding Lectins; D037121:Plant Lectins; D012333:RNA, Messenger; D018749:RNA, Plant; D011993:Recombinant Fusion Proteins; C063117:snowdrop lectin; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; D008358:Mannose