Molecular cloning and characterization of multiple isoforms of the snowdrop (Galanthus nivalis L.) lectin

Abstract

Screening of a copy-DNA (cDNA) library constructed from RNA isolated from young developing ovaries of snowdrop (Galanthus nivalis) resulted in the isolation of five lectin clones which clearly differed from each other with regard to their nucleotide sequence and deduced amino-acid sequence. Sequence comparison between the coding regions of different lectin cDNAs revealed the highest homology between lectin clones LECGNA 3 and LECGNA 5, showing 96.4% and 93.6% similarity at the nucleotide level and at the deduced amino-acid level, respectively, whereas lectin clones LECGNA 1 and LECGNA 3 showed the lowest homology of 81.6% and 68.6% for the nucleotide sequence and the amino-acid sequence, respectively. Only very few lectin cDNA clones containing a polyadenylated tail could be isolated. Moreover all these cDNA clones were derived from isolectin 3 and showed some variability within the length of the 3' untranslated region. The major transcription initiation site was located 30 bases upstream from the AUG codon as could be deduced from primer-extension analysis. Taking into account the small 5' untranslated region of the lectin clones, the size of the lectin mRNA, which is approx. 780 nucleotides as determined by Northern blot analysis, is in good agreement with the length of the cDNA clones isolated. Besides the ovary tissue, both the leaf and the flower tissue were also shown to express the lectin mRNA in a flowering snowdrop plant.

The closely related homomeric and heterodimeric mannose‐binding lectins from garlic are encoded by one‐domain and two‐domain lectin genes, respectively

“…4). Intercstingly the second signal corresponds in size to the length of the mRNA encoding the Amaryllidaceae lectins (Van Damme et al, 1991 b). This result was identical when a lectin cDNA insert from snowdrop was used.…”

“…Furthermore, the overall similarity between the different lectin clones is much higher for the garlic lectin clones, being 95% or more. Similar to the Amaryllidaceae lectin clones, some of the differences in the sequence result in different charges along the polypeptide which can account for the occurrence of multiple lectin isoforms in garlic (Van Damme et al, 1991~). Taken together with the results from Southern blot analysis, this indicates that the garlic lectin is also encoded by a family of closely related genes.…”

“…Poly(A)-rich RNA was translated in a wheat-germ cellfree extract as described previously (Van Damme et al, 1991 b). 1 ml incubation mixture for cell-free translation contained 0.4 ml extract, 20 mM Hepes KOH, pH 8.0,90 mM potassium acetate, 2.5 mM magnesium acetate, 1 mM ATP, 20 pM GTP, 25 pg/ml creatine kinase, 8 mM creatine phosphate, 2 mM dithiothreitol, 0.4 mM spermidine, 19 unlabeled amino acids (100 pM each) and 50 pCi [35S]methionine.…”

“…Cleavage at this site is consistent with the N-terminal amino acid sequence of the mature protein (Kaku et al, 1991) and will result in a precursor lectin with an apparent molecular mass of 30112 Da. However, since the mature lectin was found to be composed of two different subunits of approximately 11.5 kDa and 12.5 kDa (Van Damme et al 1991 b), determined by SDSjPAGE of the purified lectin, further processing of the lectin precursor is likely to occur. Since at that time it was already evident that the garlic lectin is a mixture of two different molecular forms encoded by mRNA of different size, both ASAI and ASAlI were purified and the N-termini of their polypeptides determined.…”

Transgenic Rice Expressing Snowdrop Lectin (Galanthus nivalis agglutinin; GNA) Shows Resistance to Rice Brown Planthopper (Nilaparvata lugens Stål)

“…The gene construct containing the GNA coding sequence from pLECGNA2 was previously described [31]. To achieve tissue-specific expression of GNA in the photosynthetic tissue of rice plants, the GNA coding sequence was fused to the rice Rubisco small subunit (rbcS) promoter and nos terminator in pBluescript-SK.…”