Molecular cloning and characterization of a mannose-binding lectin gene from Crinum asiaticum

Full-length cDNA of a mannose-binding lectin or agglutinin gene was cloned from a traditional Chinese medicinal herb Crinum asiaticum var. sinicum through RACE-PCR cloning. The full-length cDNA of C. asiaticum agglutinin (caa) was 820 bp and contained a 528 bp open reading frame encoding a lectin precursor (preproprotein) of 175 amino acid residues with a 22 aa signal peptide. The coding region of the caa gene was high in G/C content. The first 20 bp of the 5' UTR had a dC content of 50%, which was a typical feature of the leader sequence. By cutting away the signal peptide, the CAA proprotein was 15.79 kDa with a pl of 9.27 and contained 3 mannose-binding sites (QDNY). Random coil and extended strand constituted interlaced domination of the main part of the secondary structure. B-lectin conserved domain existed within N24 to G130. Predicted three-dimensional structure of CAA proprotein was very similar to that of GNA (Galanthus nivalis agglutinin). It is significant that besides certain homologies to known monocot mannose-binding lectins from Amaryllidaceae, Orchidaceae, Alliaceae and Liliaceae, caa also showed high similarity to gastrodianin type antifungal proteins. No intron was detected within the region of genomic sequence corresponding to the caa full-length cDNA. Southern blot analysis indicated that the caa gene belonged to a low-copy gene family. Northern blot analysis demonstrated that caa mRNA was constitutively expressed in all the tested tissue types including the root, bulb, leaf, rachise, flower and fruit tissues.     

Molecular cloning of mannose-binding lectins from Clivia miniata

Screening of a cDNA library constructed from total RNA isolated from young developing ovaries of Clivia miniata Regel with the amaryllis lectin cDNA clone resulted in the isolation of four different isolectin clones which clearly differ from each other in their nucleotide sequences and hence also in their deduced amino acid sequences. Apparently the lectin is translated from an mRNA of ca. 800 nucleotides encoding a precursor polypeptide of 163 amino acids. Northern blot analysis of total RNA isolated from different tissues of Clivia miniata has shown that the lectin is expressed in most plant tissues with very high lectin mRNA concentrations in the ovary and the seed endosperm.     

Expression and purification of a novel mannose-binding lectin from Pinellia ternata

Pinellia ternata agglutinin (PTA) from the tubers of P. ternata is a monocot mannose-binding lectin that catalytically agglutinated rabbit erythrocytes. The potential effect of PTA has gained considerable interest in recent years owing to clinical use of native PTA as the preparation against cancer and for plant protection against insect pests. Here we report a successful strategy to allow high-level expression of PTA as inclusion bodies in Escherichia coli M15. Purification of refolded recombinant protein from solubilized inclusion bodies by Ni-NTA agarose affinity chromatography yielded biological activity recombinant PTA (final yield of about 10 mg/L). The recombinant PTA agglutinated rabbit erythrocytes to a dilution similar to that determined for “native” lectin purified from P. ternata. The expression and purification system makes it possible to obtain sufficient quantities of biologically active and homogenous recombinant PTA sufficient to carry out advanced clinical trials. This is the first report on the large-scale expression and purification of biologically active recombinant PTA from E. coli.     

Molecular characterization and expression analysis of a gene encoding mannose-binding lectin from bulb of Zephyranthes grandiflora

In this paper we report on the molecular cloning, sequencing and partially characterisation of a lectin from bulb of the Chinese medicinal plant Zephyranthes grandiflora. The full-length cDNA of Z. grandiflora bulb lectin (ZGBL) consisted of 986 bp and contained a 576 bp ORF encoding a 191 amino acid protein. Bioinformatics analysis results clearly indicate that ZGBL belongs to the monocot mannose-binding lectin family, which contains 3 putative mannose-binding sites per subunit. RT-PCR analysis results indicate that ZGBL is constitutively expressed in all the tested tissue types including root, bulb, leaf and flower. Interestingly, ZGBL is more closely related to the Orchidaceae rather than the Amaryllidaceae family on molecular evolution.     

cDNA cloning and expression analysis of a mannose-binding lectin from <Emphasis Type="Italic">Pinellia pedatisecta</Emphasis>

Pinellia pedatisecta agglutinin (PPA) is a very basic protein that accumulates in the tuber of P. pedatisecta. PPA is a hetero-tetramer protein of 40 kDa, composed of two polypeptide chains A (about 12 kDa) and two polypeptides chains B (about 12 kDa). The full-length cDNA of PPA was cloned from P. pedatisecta using SMART RACE-PCR technology; it was 1146 bp and contained a 771 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acid residues with a 24 amino acid signal peptide. The PPA precursor contained 3 mannose-binding sites (QXDXNXVXY) and two conserved domains of 43% identity, PPA-DOM₁ (polypeptides A) and PPA-DOM₂ (polypeptides B). PPA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families such as Araceae, Alliaceae, Iridaceae, Liliaceae, Amaryllidaceae and Bromeliaceae. Southern blot analysis indicated that ppa belonged to a multi-copy gene family. Expression pattern analysis revealed that ppa expressed in most tested tissues, with high expression being found in spadix, spathe and tuber. Cloning of the ppa gene not only provides a basis for further investigation of its structure, expression and regulatory mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.     

Molecular cloning, characterization and expression of a novel jasmonate-dependent defensin gene from Ginkgo biloba

A novel defensin gene was isolated from Ginkgo biloba. The full-length cDNA of G. biloba defensin (designated as Gbd) was 534bp. The cDNA contained a 240-bp open reading frame encoding an 80-amino acid protein of 5.68 kDa with a potential 30 aa signal peptide. The putative GbD mature protein showed striking similarity to other plant defensins, representing low molecular size antimicrobial polypeptides. Eight cysteine sites conserved in plant defensins were also found in GbD at similar positions. Three-dimensional structure modeling showed that GbD strongly resembled defensin from tobacco (NaD1) and consisted of an alpha-helix and a triple-strand antiparallel beta-sheet that were stabilized by four intramolecular disulfide bonds, implying GbD may have functions similar to NaD1. The genomic DNA gel blot indicated that Gbd belonged to a multigene family. Expression analysis revealed that Gbd was up-regulated by wounding and methyl jasmonate treatments, suggesting that Gbd is potentially involved in plant resistance or tolerance to pathogens during wounding.     

Isolation and characterization of a new mannose-binding lectin gene from<Emphasis Type="Italic">Taxus media</Emphasis>

In this paper, we report the cloning and characterization of the first mannose-binding lectin gene from a gymnosperm plant species,Taxus media. The full-length cDNA ofT. media agglutinin (TMA) consisted of 676 bp and contained a 432 bp open reading frame (ORF) encoding a 144 amino acid protein. Comparative analysis showed that TMA had high homology with many previously reported plant mannose-binding lectins and thattma encoded a precursor lectin with a 26-aa signal peptide. Molecular modelling revealed that TMA was a new mannosebinding lectin with three typical mannose-binding boxes like lectins from species of angiosperms. Tissue expression pattern analyses revealed thattma is expressed in a tissue-specific manner in leaves and stems, but not in fruits and roots. Phylogenetic tree analyses showed that TMA belonged to the structurally and evolutionarily closely related monocot mannose-binding lectin superfamily. This study provides useful information to understand the molecular evolution of plant lectins.     

Molecular cloning and characterization of a mannose-binding lectin gene from <Emphasis Type="Italic">Pinellia cordata</Emphasis>

Using RNA extracted from Pinellia cordata young leaves and primers designed according to the conserved regions of Araceae lectins, the full-length cDNA of Pinellia cordata agglutinin (PCL) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of pcl was 1,182 bp and contained a 768 bp open reading frame (ORF) encoding a lectin precursor of 256 amino acids. Through comparative analysis of pcl gene and its deduced amino acid sequence with those of other Araceae species, it was found that pcl encoded a precursor lectin with signal peptide. PCL is a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that pcl is expressed in all tested tissues including leaf, stem and bulbil, but with the highest expression in bulbil. PCL protein was successfully expressed in Escherichia coli with the molecular weight expected.     

Cloning and molecular characterization of a novel lectin gene from Pinellia ternata

The full-length cDNA of Pinellia ternata agglutinin (PTA) was cloned from inflorescences using RACE-PCR. Through comparative analysis of PTA gene (pta) and its deduced amino acid sequence with those of other Araceae species, pta was found to encode a precursor lectin with signal peptide and to have extensive homology with those of other Araceae species. PTA was a heterotetrameric mannose-binding lectin with three mannose-binding boxes like lectins from other Araceae and Amaryllidaceae species. Southern blot analysis of the genomic DNA revealed that pta belonged to a low-copy gene family. Northern blot analysis demonstrated that pta constitutively expressed in various plant tissues including root, leaf, stem and inflorescence. The pta cDNA sequence encoding for mature PTA protein was cloned into pET-32a plasmid and the resulting plasmid, pET-32a-PTA containing Trx-PTA fusion protein, was investigated for the expression in E. coli BL21. SDS-PAGE gel analysis showed that the Trx-PTA fusion protein was successfully expressed in E. coli BL21 when induced by IPTG. Artificial diet assay revealed that PTA fusion protein had significant levels of resistance against peach potato aphids when incorporated into artificial diet at 0.1% (w/v). The cloning of the pta gene will enable us to further test its effect in depth on aphids by transferring the gene into crop plants.     

Molecular cloning of the lectin and a lectin-related protein from common Solomon's seal (Polygonatum multiflorum)

The most prominent protein ofPolygonatum multiflorum (common Solomon's seal) rhizomes has been identified as a mannose-binding lectin. Analysis of the purified lectin demonstrated that it is a tetramer of four identical subunits of 14 kDa. Molecular cloning further revealed that the lectin from this typical Liliaceae species belongs to the superfamily of monocot mannose-binding proteins. Screening of cDNA libraries constructed with RNA isolated from buds, leaves and flowers ofP. multiflorum also yielded cDNA clones encoding a protein, which contains two tandemly arranged domains with an obvious sequence homology to the mannose-binding lectins. Molecular modelling of thePolygonatum lectin and lectin-related protein indicated that the three-dimensional structure of both proteins strongly resembles that of the snowdrop lectin. In addition, this approach suggested that the presumed carbohydrate-binding sites of the lectin can accommodate a mannose residue whereas most of the carbohydratebinding sites of the lectin-related protein cannot.Abbreviations GNA Galanthus nivalis agglutinin - HCA hydrophobic cluster analysis - LECPMA cDNA clone encoding PMA - PM30 30 kDa protein isolated fromPolygonatum multiforum - PMA Polygonatum multiflorum agglutinin - PMLRP Polygonatum multiflorum lectin-related protein     

cDNA cloning and characterization of a mannose-binding lectin fromZingiber officinaleRoscoe (ginger) rhizomes

Using RNA extracted fromZingiber officinale rhizomes and primers designed according to the conservative regions of monocot mannose-binding lectins, the full-length cDNA ofZ. officinale agglutinin (ZOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA ofzoa was 746 bp and contained a 510 bp open reading frame (ORF) encoding a lectin precursor of 169 amino acids with a signal peptide. ZOA was a mannose-binding lectin with three typical mannose-binding sites (QDNY). Semi-quantitative RT-PCR analysis revealed thatzoa expressed in all the tested tissues ofZ. officinale including leaf, root and rhizome, suggesting it to be a constitutively expressing form. ZOA protein was successfully expressed inEscherichia coli with the molecular weight expected. To our knowledge, this is the first mannose-binding lectin cDNA cloned from the family Zingiberaceae. Our results demonstrate that monocot mannose-binding lectins also occur within the family Zingiberaceae     

Molecular cloning and characterization of a novel WRKY gene from Brassica chinensis

A new WRKY gene was cloned from Brassica chinensis by rapid amplification of cDNA ends (RACE). The full-length cDNA of BcWRKY was 1175 bp long and contained a 924-bp open reading frame (ORF) encoding a putative W-box-binding protein of 308 amino acids. The predicted BcWRKY protein was found to have a potential bipartite nuclear localization sequence (NLS-PB) in its N-terminal region followed by a WRKY DNA-binding domain. Bioinformatic analysis revealed that BcWRKY resembled other WRKY domain-containing proteins from Arabidopsis thaliana (AtWRKY18), tobacco (WIZZ), parsley (PcWRKY4), and wild oat (ABF2). Expression of the BcWRKY gene could be induced by salicylic acid (SA) and influenced by Pseudomonas syringae pv. tomato strain DC3000 infection and wounding treatment. Our study implies that BcWRKY might have similar functions possessed by other WRKY genes, such as inducing the expression of some defense-related genes and increasing plants’ disease resistance ability. Published in Russian in Molekulyarnaya Biologiya, 2006, Vol. 40, No. 5, pp. 816–824. The text was submitted by the authors in English.     

The monomeric and dimeric mannose-binding proteins from the Orchidaceae speciesListera ovata andEpipactis helleborine: sequence homologies and differences in biological activities

The Orchidaceae speciesListera ovata andEpipactis helleborine contain two types of mannose-binding proteins. Using a combination of affinity chromatography on mannose-Sepharose-4B and ion exchange chromatography on a Mono-S column eight different mannose-binding proteins were isolated from the leaves ofListera ovata. Whereas seven of these mannose-binding proteins have agglutination activity and occur as dimers composed of lectin subunits of 11–13 kDa, the eighth mannose-binding protein is a monomer of 14 kDa devoid of agglutination activity. Moreover, the monomeric mannose-binding protein does not react with an antiserum raised against the dimeric lectin and, in contrast to the lectins, is completely inactive when tested for antiretroviral activity against human immunodeficiency virus type 1 and type 2. Mannose-binding proteins with similar properties were also found in the leaves ofEpipactis helleborine. However, in contrast toListera only one lectin was found inEpipactis. Despite the obvious differences in molecular structure and biological activities molecular cloning of different mannose-binding proteins fromListera andEpipactis has shown that these proteins are related and some parts of the sequences show a high degree of sequence homology indicating that they have been conserved through evolution.Abbreviations EHMBP Epipactis helleborine mannose-binding protein - LOMBP Listera ovata mannose-binding protein Note: The nucleotide sequences reported in this paper will appear in the Genbank/EMBL Data library with the accession numbers L18894, L18895 and U07787.     

Cloning and characterization of the lectin cDNA clones from onion,shallot and leek

Characterization of the lectins from onion (Allium cepa), shallot (A. ascalonicum) and leek (A. porrum) has shown that these lectins differ from previously isolated Alliaceae lectins not only in their molecular structure but also in their ability to inhibit retrovirus infection of target cells.cDNA libraries constructed from poly(A)-rich RNA isolated from young shoots of onion, shallot and leek were screened for lectin cDNA clones using colony hybridization. Sequence analysis of the lectin cDNA clones from these three species revealed a high degree of sequence similarity both at the nucleotide and at the amino acid level.Apparently the onion, shallot and leek lectins are translated from mRNAs of ca. 800 nucleotides. The primary translation products are preproproteins (ca. 19 kDa) which are converted into the mature lectin polypeptides (12.5–13 kDa) after post-translational modifications.Southern blot analysis of genomic DNA has shown that the lectins are most probably encoded by a family of closely related genes which is in good agreement with the sequence heterogeneity found between different lectin cDNA clones of one species.     

Molecular cloning and the cDNA-derived amino acid sequence of Narcissus tazetta isolectins

Recently several complete cDNAs encoding the Narcissus tazetta lectins (NTL) were cloned. The sequence analyses of the cloned DNAs reveal that there are at least three unidentical positive clones for NTLs. The primary structure of the three NTL clones contains a mature polypeptide consisting of 105 amino acids and a C-terminal peptide extension beyond the C-terminal amino acids Thr-Gly. There are two fixed-position cysteines within the protein domain (amino acids 29 and 52), which are probably involved in the disulfide-bond linkage within the molecules to confer the secondary structure of the mature lectin. One third of the deduced amino acid composition consisted of glycine, leucine, and asparagine. From the cDNA-derived amino acid sequences the three NTL clones are not identical and are suggested to be isolectins present in N. tazetta var. chinensis. This study further confirms the previous isolation of mannose-specific isolectins from Chinese daffodil leaves [Ooi et al. (2000), J. Protein Chem. 19, 163-168].     

Purification and cDNA cloning of a lectin and a lectin-like protein from Apios americana Medikus tubers

An Apios americana lectin (AAL) and a lectin-like protein (AALP) were purified from tubers by chromatography on Butyl-Cellulofine, ovomucoid-Cellulofine, and DEAE-Cellulofine columns. AAL showed strong hemagglutinating activity toward chicken and goose erythrocytes, but AALP showed no such activity toward any of the erythrocytes tested. The hemagglutinating activity of AAL was not inhibited by mono- or disaccharides, but was inhibited by glycoproteins, such as asialofetuin and ovomucoid, suggesting that AAL is an oligosaccharide-specific lectin. The cDNAs of AAL and AALP consist of 1,093 and 1,104 nucleotides and encode proteins of 302 and 274 amino acid residues, respectively. Both amino acid sequences showed high similarity to known legume lectins, and those of their amino acids involved in carbohydrate and metal binding were conserved.     

Purification and characterization of a new mannose-specific lectin from Sternbergia lutea bulbs

A new mannose-binding lectin was isolated from Sternbergia lutea bulbs by affinity chromatography on an α(1-2)mannobiose-Synsorb column and purified further by gel filtration. This lectin (S. lutea agglutinin; SLA) appeared homogeneous by native-gel electrophoresis at pH 4.3, gel filtration chromatography on a Sephadex G-75 column, and SDS-polyacrylamide gel electrophoresis, These data indicate that SLA is a dimeric protein (20 kDa) composed of two identical subunits of 10 kDa which are linked by non-covalent interactions. The carbohydrate binding specificity of the lectin was investigated by quantitative precipitation and hapten inhibition assays. It is an α-D-mannose-specific lectin that interacts to form precipitates with various α-mannans, galactomannan and asialo-thyroglobulin, but not with α-glucans and thyroglobulin. Of the monosaccharides tested only D-mannose was a hapten inhibitor of the SLA-asialothryroglobulin precipitation system, whereas D-glucose, D-galactose and L-arabinose were not. The lectin appears to be highly specific for terminal α(1-3)-mannooligosaccharides. The primary structure of SLA appears to be quite similar to that of the snow drop (Galanthus nivalis) bulb lectin which is a mannose-binding lectin from the same plant family Amaryllidaceae. The N-terminal 46 amino acid sequence SLA showed 7% homology with that of GNA. Abbreviations: AAA, Allium ascalonicum agglutinin (shallot lectin); ASA, Allium sativum agglutinin (garlic lectin); AUA, Allium ursinum agglutinin (ramsons lectin); DAP, 1,3-diaminopropane; GNA, Galanthus nivalis agglutinin (snowdrop lectin); HHA, Hippeastrum hybr. agglutinin (amaryllis lectin); LOA, Listera ovata agglutinin (orchid twayblade lectin); NPA, Narcissus pseudonarcissus agglutinin (daffodil lectin); PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline, SLA, Sternbergia lutea agglutinin; SDS, sodium dodecyl sulfate; Me, methyl; Bn, benzyl; PNP, p-nitrophenyl. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular cloning and characterization of O-methyltransferases from the flower buds of Iris hollandica

In plants, O-methyltransferases (OMTs) play an important role in methylation of secondary metabolites, especially flavonoids and other phenylpropanoids, and two cDNA clones, IhOMT1 and IhOMT2 (Iris hollandica OMT), encoding OMTs were successfully isolated from a cDNA library of flower buds of I. hollandica. IhOMT1 encodes an open reading frame (ORF) of 365 amino acids with calculated molecular mass of 40,193Da and isoelectric point (pI) of 5.54, while IhOMT2, which shares 31.5% amino acid sequence identity with IhOMT1, encodes 369 amino acids with calculated molecular mass of 40,385Da and pI of 5.50. In addition, the molecular masses of both recombinant IhOMT1 and IhOMT2 proteins were estimated to be about 40kDa by protein gel blot analysis. Characterization of the enzymatic properties using the recombinant IhOMT1 protein confirmed that IhOMT1 cDNA encodes a S-adenosyl-l-methionine (SAM)-dependent caffeic acid 3-OMT, which catalyzes the transfer of the methyl moiety from SAM to caffeic acid to form ferulic acid. Its optimum activity was observed at pH 7.5-8.0 and at 35 degrees C. This is the first report of the isolation and characterization of a COMT cDNA clone involved in the phenylpropanoid biosynthesis of Iridaceae plants. In contrast, IhOMT2 showed no activity in SAM-dependent assays for various phenylpropanoids.