全 文 ：Received 27 Apr. 2004 Accepted 15 Aug. 2004
Supported by the National Natural Science Foundation of China (30000032).
* Author for correspondence. E-mail: .
** Author for correspondence. E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (11): 1301-1306
Molecular Cloning of a Novel Mannose-binding Lectin Gene from Bulbs
of Amaryllis vittata (Amaryllidaceae)
WU Chuan-Fang, AN Jie, HE Xiao-Jia, DENG Jie, HONG Zhi-Xia, LIU Chao, LU Hong-Zhou,
LI Yi-Jin, WANG Chen-Ji, CHEN Fang* , BAO Jin-Ku**
(College of Life Sciences, Sichuan University, Chengdu 610064, China)
Abstract: A new mannose-binding agglutinin gene was cloned from bulbs of Amaryllis vittata Ait. The full-
length cDNA of A. vittata agglutinin (AVA) was 686 bp. The start codon of ava cDNA was at 41-43 bp and the
stop codon was at 515-517 bp. Analysis in the BLAST of GenBank showed that ava gene encodes a protein
precursor composed of a signal peptide, mature protein and C-terminal amino acid cleavage sequence. The
mature protein of AVA includes 109 amino acid residues and the molecular weight is 11.9 kD. The homolo-
gous analysis showed that the identity between AVA and Galanthus nivalis agglutinin, Narcissus hybrid
cultivar agglutinin, Lycoris radiata agglutinin, Clivia miniata agglutinin are 73.4%, 85.3%, 80.7%, 83.5%,
respectively. Molecular modeling of AVA indicated that its three-dimensional structure strongly resembles
that of the snowdrop agglutinin. Blocks’ analysis revealed that the deduced amino acid sequence of AVA has
three functional domains specific for agglutination and three carbohydrate-binding boxes (QDNY).
Key words: Amaryllis vittata agglutinin (AVA); monocot mannose-binding lectin; degenerate primers;
3 and 5 RACE; homology alignment
The term “monocot mannose-binding lectins (MBL)”
refers to a superfamily of strictly mannose-specific lectins,
which up to now has been found exclusively in a subgroup
of the monocotyledonous plants (van Damme et al., 1998).
All monocot MBL consist of subunits with a similar se-
quence and overall three-dimensional structure. The first
description of a monocot MBL dates back to 1987 when a
lectin with an exclusive specificity toward mannose was
isolated from snowdrop bulbs. Later, more and more mono-
cot MBL have been isolated and cloned. During the last
few years evidence has been accumulated for the occur-
rence of a large superfamily of related mannose-binding
lectins in representatives of the plant family Alliaceae,
Amaryllidaceae, Araceae, Bromeliaceae, Liliaceae, and
Orchidaceae (van Damme et al., 1995). Despite their recent
discovery, monocot MBL already attract a lot of attention
in different scientific disciplines. Initially, most of the inter-
ests is in biomedical research in an attempt to exploit the
potent anti-(retro) viral activity of GNA and some other
monocot MBL (Balzarini et al.,1991; 1992). The lectins from
Galanthus nivalis, Hippeastrum hybrid, Narcissus
pseudonarcissus, and Listera ovata inhibit infection of MT-
4 cells by human immunodeficiency virus types 1 and 2
(HIV-1 and HIV-2) and simian immunodeficiency virus at
concentrations comparable to the concentrations at which
dextran sulfate inhibits these viruses. The lectins from the
orchid species Cymbidium hybrid (CA), Epipactis helle-
borine (EHA) and Listera ovata (LOA) are also highly in-
hibitory to human immunodeficiency virus type 1 (HIV-1)
and type 2 (HIV-2) in MT-4, and show a marked anti-human
cytomegalovirus (CMV), respiratory syncytial virus (RSV)
and influenza a virus activity in HEL, HeLa and MDCK
cells, respectively. Later the attention shifted toward the
exploitation of the anti-insect properties of GNA in plant
protection (Hilder et al., 1995). Up to now, most of the lec-
tin genes cloned from species of Amaryllidaceae show more
or less inhibition to sap-sucking insects including aphids
and planthopper by artificial diet assays. As transgenic
plants expressing the snowdrop lectin also exhibit an in-
creased resistance against sucking insects and nematodes,
we can assume in reason that at least this lectin is involved
in the plant’s defense against invertebrates (Hilder et al.,
1995). In this study, we cloned the lectin gene from A. vittata,
which enables us to test its effect on antiviral and antiinsect
in the future.
1 Materials and Methods
1.1 Amaryllis vittata plants
The young bulbs of A. vittata Ait collected from Sichuan
University, China were instantly frozen in liquid nitrogen
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041302
and stored at -70 ℃ before use.
1.2 RNA isolation
The total RNA was extracted using the RNA extraction
kit (TaKaRa Biotech Co. Ltd.) according to the
manufacturer’s instructions.
1.3 3 RACE of ava gene
cDNA synthesis was performed with the 3 RACE kit
(TaKaRa Biotech Co. Ltd.). Firstly, RNA was reversely
transcribed, primer 1 (5- TTYATCATGCARGAAG-ACTG-
3) was designed according to the conserved mannose-bind-
ing amino acid sequence MQEDCNL presented in mono-
cot lectins. The 3 RACE was performed essentially accord-
ing to the manufacturer’s instructions. PCR was performed
under the following condition: cDNA was denatured at 94
℃ for 30 s, followed by 30 cycles of amplification (94 ℃ for
30 s, 55 ℃ for 30 s and 72 ℃ for 2 min) and 7 min at 72 ℃.
The PCR product was purified and cloned into pUC18-T
vector (TaKaRa Biotech company) for sequencing.
1.4 5 RACE of ava gene
Based on the sequence of the 3 RACE product, the
specific primers 2, 3 and 4 were designed to amplify the 5
end of AVA. RNA was reversely transcribed with primer 2
(5-CTTAGCTGACTTGTCGGTCAC-3) followed by tailing
cDNA with oligA. The first round of PCR was performed
with primer 3 (5-AACGGTTCCGGTGTAAGTTCC-3) and
abridged anchor primer (AAP). PCR was carried out as pre-
vious steps. The PCR product was diluted 50 folds for the
third round of amplification with primer 4 (5 -CCCAGATCG
GCTGTCAACATC-3) and abridged universal anchor primer
(AUAP). PCR was performed as before. The PCR product
was purified and cloned into pUC18-T vector for
sequencing.
1.5 Generation of AVA full-length cDNA sequence
Based on the nucleotide sequence of the 3 and 5 RACE
products, 5 and 3 gene-specific primers 5 (5-AACTGCAAA
ATGGCTAAGACAAGCT-3) and 6 (5 -AAAGCTCGCTTG
GGGCATTACTTAG-3) were designed for the amplification
of full-length cDNA of AVA. The thermal cycling program
was the same as that utilized for 3 and 5 RACE.
1.6 Bioinformatic analysis of AVA sequence and struc-
ture
DNA sequence and the associated molecular informa-
tion were analyzed by DNA Tools 5.1, Vector NTI 6.0,
HCAdraw 2 and Swiss-Model.
Fig.1. Nucletide acid sequence and deduced amino acid sequence of Amaryllis vittata. The start codon and stop codon are underlined,
the immature proteins consist of 158 amino acids and the mature ones consist of 109 amino acids. The arrows indicate cleaving sites.
WU Chuan-Fang et al.: Molecular Cloning of a Novel Mannose-binding Lectin Gene from Bulbs of Amaryllis vittata (Amaryllidaceae)1303
2 Results
2.1 The cloning of full-length cDNA of AVA
Based on the primer 1 designed for the amplification of
3 end of AVA cDNA, a 500 bp fragment was obtained. Three
specific primers (primer 2.3.4) designed according to the 3
RACE fragment were used for the amplification of 5 AVA
cDNA and a 250 bp fragment was obtained. Finally the full-
length cDNA sequence of AVA was obtained through RT-
PCR reaction using the gene-specific primers 5 and 6. The
full-length cDNA of AVA (Fig.1) was 686 bp and contained
a 474 bp open reading frame (ORF) encoding a 158 amino
acids protein. According to the rules of predicting lectin
signal peptide (Von Heijine, 1986), a 24-amino acid signal
peptide with the signal peptide cleavage site between S24
and D25 was identified from the AVA full-length cDNA
sequence, which was in good agreement with those of other
Amaryllidaceae species such as Lycoris radiata and
Zephyranthes candida. The cleavage site of C-terminal was
between T133 and G134. Translated with DNA Tool 5.1, the
mature protein was 109 aa in length with a molecular weight
of 11.9 kD and an isoelectric point of 4.45. The richest amino
Fig.2. Multi-alignment of the predicted Amaryllis vittata agglutinin (AVA, AAP57409) amino acid sequence with other monocot
mannose-binding lectins from family Amaryllidaceae. Identical amino acid residues are indicated with black background and white
foreground. The conservative amino acids are indicated with grey background and white foreground. The amino acid residues that show
weak similarity are indicated with grey background and black foreground. Mannose-binding sites (QDNY) are boxed. GNA, Galanthus
nivalis agglutinin (AAL07474); LRA, Lycoris radiata agglutinin (AAP20877); NHA, Narcissus hybrid cultivar agglutinin (AAA33552);
ZCA, Zephyranthes Candida Agglutinin (AAM27447).
Acta Botanica Sinica 植物学报 Vol.46 No.11 20041304
Figs.3-4. 3. Predicted comparison of the Hydrophobic Cluster Analysis (HCA) plots of Galanthus nivalis agglutinin (GNA) (A) and
Amaryllis vittata agglutinin (AVA) (B). The twelve strands of ¦Âsheet delineated on the HCA plot of GNA were reported on the HCA
plot of the AVA. Some structurally conserved hydrophobic residues common to both sequences are indicated by delineations to facilitate
the identification of the structurally conserved regions common to both lectins monomer. These structurally conserved regions, Which
mainly respond to hydrophobic cluster, are circled in bold. 4. Side view of the three-dimensional models of Galanthus nivalis agglutinin
(GNA) (a) and Amaryllis vittata agglutinin (AVA) (b). The strands of b-sheet (indicated by gray arrows) associate in three four-stranded
bundles and form the b-prism fold. The amino acids constituting mannose-binding sites are indicated as ball and stick.
WU Chuan-Fang et al.: Molecular Cloning of a Novel Mannose-binding Lectin Gene from Bulbs of Amaryllis vittata (Amaryllidaceae)1305
acid residues of AVA was Gly (10.7%), followed by Asp
(10%) and Thr (8.2%). Acidic and basic residues consti-
tuted 7.6% and 10.5% of the lectin respectively.
2.2 Bioinformatic analysis of A.vittata agglutinin
The program AlignX (the InforMax Company, Maryland,
USA) and PSI-BLAST (Altschul et al.,1997) were used to
compare the amino acid sequence of AVA with sequences
of other monocot MBL from family Amaryllidaceae. Data-
base retrieval showed that AVA belonged to the monocot
MBL superfamily. Homologous analysis showed the iden-
tity between AVA and Galanthus nivalis agglutinin , Nar-
cissus hybrid cultivar agglutinin , Lycoris radiate aggluti-
nin and Clivia miniata agglutinin were 73.4％,85.3％,
80.7％,83.5％ respectively (Fig.2). AVA also had three man-
nose-binding sites (QDNY) like L. radiate and Zephyranthes
candida, which binded mannose through a network of four
hydrogen bonds interconnecting the hydroxyls C2-OH, C3-
OH and C4-OH of mannose to the four residues. In addition,
a valine residue, which was absolutely conserved in each
subdomains of all selected monocot mannose-binding
lectins, was also conserved in AVA, interacting with C3 and
C4 of mannose by van der waals. The deduced amino acid
sequence of AVA precursor shared a reasonable sequence
identity with GNA. To determine whether the overall fold-
ing of AVA and the structure of the carbohydrate-binding
sites also resembled to those of GNA, Hydrophobic Clus-
ter Analysis (HCA) (Gaboriaud et al., 1987; Lemesle-Varloot
et al., 1990) and molecular modeling were carried out using
GNA as a model protein. Molecular modeling of AVA was
carried out using the program Swiss-Model (Schwede
et al., 2003).
HCA was performed to delineate the structurally con-
served b-sheets along the amino-acid sequences of the two
domains of AVA using the GNA as a model. HCA plots were
generated using the program HCAdraw2 (Doriane, Paris,
France). The HCA plots indicated that the structural orga-
nization in three subdomains found in GNA was conserved
in AVA (Fig.3). Building of the three-dimensional models of
AVA from the X-ray coordinates of GNA confirmed that the
overall folding AVA was very similar with GNA and exhib-
ited the same b-prism structure as the GNA monomer. The
three tandem subdomains were connected by loops to form
a 12-stranded b-barrel containing three putative mannose-
binding sites located in the clefts formed by the three
bundles of b-sheet.
The comparison of the amino acid sequence of AVA with
those of GNA and other monocot mannose-binding lectins
indicated that these amino acid residues within mannose-
binding sites were totally conserved. Molecular modeling
showed that the three-dimensional structure of the binding
sites of AVA also resembled that of the active binding sites
of GNA (Fig.4). Therefore it was presumed that AVA might
have similar functions to many other mannose-binding
lectins such as anti-viral and anti-insect properties.
3 Discussion
Using degenerate primers specifically designed accord-
ing to conserved sequences of Amaryllidaceae lectins and
RACE-PCR technique, a new mannose-binding lectin gene
was cloned from bulbs of A. vittata. Bioinformatics analy-
sis of ava gene and its deduced amino acid sequence with
those of other Amaryllidaceae species indicated that AVA
full-length cDNA was 686 bp, encoding a protein precursor
with signal peptide, mature protein and C-terminal amino
acid cleavage sequence. The mature peptide included 109
amino acid residues with a calculated molecular mass of
11.9 kD. AVA was a mannose-binding lectin with three man-
nose-binding boxes (QDNY) like lectins from other
Amaryllidaceae species and the molecular modeling indi-
cated that the three-dimensional structure strongly re-
sembled that of the snowdrop lectin.
Today, the structure and function of MBL have been
most extensively studied, especially mannose binding site
and the progress on anti-insect and anti-virus have at-
tracted a lot of attention in different scientific fields. Now
there are many reports on this aspect in our country, such
as Pang transferred the lra gene into tobacco and obtained
transgenic tobacco plants with higher level of LRA expres-
sion and resistance to aphids (Pang et al., 2004). Wu and
Yuan transferred the GNA gene into tomato and tobacco,
and obtained resistance plants (Wu et al., 2000; Yuan et al.,
2001). The results indicated that MBL could have similar
properties on resistance. AVA belongs to monocot MBL, it
is speculated that AVA may also have a similar inhibitory
effect on sap-sucking insects like other Amaryllidaceae spe-
cies lectins and may play a role in controlling sap-sucking
insects’ genetic engineer. The cloning of A. vittata agglu-
tinin gene will enable us to test its potential role in viral
diseases and controlling pests by transferring the gene
into other plants in the future.
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