Genomic Cloning and Characterization of a Novel Lectin Gene from <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis>

A new lectin gene was isolated by using genomic walker technology and revealed to encode a mannose-binding lectin. Analysis of a 2233 bp segment revealed a gene including a 1169 bp 5′ flanking region, a 417 bp open reading frame (ORF) and a 649 bp 3′ flanking region. There are two putative TATA boxes and eight possible CAAT boxes lie in the 5′ flanking region. The ORF encodes a 15.1 kDa precursor, which contains a 24-amino acid signal peptide. One possible polyadenylation signal is found in the 3′-flanking region. No intron was detected within the region of genomic sequence corresponding to zaa (Zantedeschia aethiopica agglutinin) full-length cDNA, which is typical of other mannose-binding lectin gene that have been reported. The deduced amino acid sequence of the lectin gene coding region shares 49–54% homology with other known lectins. The cloning of this new lectin gene will allow us to further study its structure, expression and regulation mechanisms.     

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.     

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     

Cloning and Characterization of an Agglutinin Gene from Arisaema lobatum

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5′-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation 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.     

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 novel mannose-binding lectin gene from Amorphophallus konjac

A new lectin gene was cloned from Amorphophallus konjac. The full-length cDNA of Amorphophallus konjac agglutinin (aka) was 736 bp and contained a 474 bp open reading frame encoding a 158 amino acid protein. Homology analysis revealed that the lectin from this Araceae species belonged to the superfamily of monocot mannose-binding proteins. Molecular modeling of AKA indicated that the three-dimensional structure of AKA strongly resembles that of the snowdrop lectin. Southern blot analysis of the genomic DNA revealed that aka belonged to a low-copy gene family. Northern blot analysis demonstrated that aka expression was tissue-specific with the strongest expression being found in root.     

Molecular Cloning and Characterization of a Mannose-binding Lectin Gene from Dendrobium officinale

Using RNA extracted from Dendrobium officinale young leaves and primers designed according to the conservative regions of Orchidaceae lectins, the full-length cDNA of Dendrobium officinale agglutinin (DOA) was cloned by rapid amplification of cDNA ends (RACE). The full-length cDNA of doa was 768 bp and contained a 498 bp open reading frame (ORF) encoding a lectin precursor of 165 amino acids. Through comparative analysis of doa gene and its deduced amino acid sequence with those of other Orchidaceae species, it was found that doa encoded a precursor lectin with signal peptide. DOA was a mannose-binding lectin with three mannose-binding sites. Semi-quantitative RT-PCR analysis revealed that doa mRNA expression was detected in all tested tissues including root, stem and leaf, however, the expression was higher in stem, lower in leaf. As the doa mRNA was detected in all the tested plant tissues, the doa was considered to be a constitutively expressed gene.     

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.     

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.     

Anti-HIV Ⅰ/Ⅱ Activity and Molecular Cloning of a Novel Mannose/Sialic Acidbinding Lectin from Rhizome of Polygonatum cyrtonema Hua

The anti-human immunodeficiency virus （HIV） Ⅰ/Ⅱ activity of a mannose and sialic acid binding lectin isolated from rhizomes of Polygonatum cyrtonema Hua was elucidated by comparing its HIV infection inhibitory activity in MT-4 and CEM cells with that of other mannose-binding lectins （MBLs）. The anti-HIV activity of Polygonatum cyrtonema Hua lectin （PCL） was 10- to 100-fold more potent than other tested MBLs, but without significant cytotoxicity towards MT-4 or CEM cells. To amplify cDNA of PCL by 3＇/5＇-rapid amplification of cDNA ends （RACE）, the 30 amino acids of N-terminal were determined by sequencing and the degenerate oligonucleotide primers were designed. The full-length cDNA of PCL contained 693 bp with an open reading frame encoding a precursor protein of 160 amino acid residues, consisting of a 28-residue signal peptide, a 22-residue C-terminal cleavage peptide and a 110-residue mature polypeptide which contained three tandemly arranged subdomains with an obvious sequence homology to the monocot MBL. However, only one active mannose-binding site （QDNVY） was found in subdomain Ⅰ of PCL, that of subdomain Ⅱ and Ⅲ changed to HNNVY and PDNVY, respectively. There was no intron in PCL, which was in good agreement with other monocot MBLs. Molecular modeling of PCL indicated that its three-dimensional structure resembles that of the snowdrop agglutinin. By docking, an active sialic acid-binding site was found in PCL. The instabilization of translation initiation region （TIR） in mRNA of PCL benefits its high expression in rhizomes.     

一种新的甘露糖结合凝集素--朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列.该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517 bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白.成熟蛋白由109个氨基酸残基组成,分子量为11.9kD.成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4%、85.3%、80.7%和83.5%的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY).     

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.     

A novel serine protease with clip domain from scallop Chlamys farreri

The serine proteases with clip domain are involved in various innate immune functions in invertebrate such as antimicrobial activity, cell adhesion, pattern recognition and regulation of the prophenoloxidase system. A serine protease with clip-domain cDNA (Cf SP) was obtained by Expressed sequence taggings (ESTs) method and rapid amplification of cDNA ends (RACE). The Cf SP full-length cDNA was of 1,152 bp, including a 5'-terminal untranslated region (UTR) of 63 bp, a 3'-terminal UTR of 81 bp with a canonical polyadenylation signal sequence AATAAA and a poly(A) tail, and an open reading frame of 1,008 bp encoding a polypeptide of 336 amino acids with a putative signal peptide of 19 amino acids. The deduced amino acid sequence of Cf SP contained an amino-terminal clip domain with three disulfide bonds formed six conserved Cys residues, a carboxyl-terminal trypsin-like domain with the conserved His-Asp-Ser catalytic triad, and a low complexity linker sequence. The Cf SP was strongly expressed in hemocytes and the mRNA expression of Cf SP was up-regulated and increased 3.2-fold and 2.6-fold at 16 h after injection of Vibrio anguillarum and Micrococcus luteus. The results suggested that Cf SP gene might be involved in immune response of Gram-negative and Gram-positive microbial infection in scallop.     

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.     

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     

一种新的甘露糖结合凝集素——朱顶兰凝集素基因的克隆及序列分析

运用同源克隆的方法设计简并引物,通过3′和5′RACE技术,从石蒜科植物朱顶兰(Amaryllis vittata Ait)总RNA中克隆了编码此凝集素(AVA)的全长cDNA序列。该基因全长686 bp,起始密码子位于第41～43 bp,终止密码子位于515～517bp处,开放阅读框长474 bp,编码158个氨基酸,包含信号肽序列、成熟蛋白序列和C-末端剪切序列的前体蛋白。成熟蛋白由109个氨基酸残基组成,分子量为11.9kD。成熟蛋白在氨基酸水平上与雪花莲凝集素、水仙凝集素、石蒜凝集素和君子兰凝集素分别有73.4％、85.3％、80.7％和83.5％的同源性;朱顶兰凝集素的分子模式显示其与雪花莲凝集素有极其相似的三维结构;在Blocks数据库中检索AVA蛋白氨基酸序列的结构域,发现有3个凝集素功能结构域,并具有3个典型的甘露糖专一结合位点盒(QDNY)。     

Cloning of a lectin cDNA and seasonal changes in levels of the lectin and its mRNA in the inner bark ofRobinia pseudoacacia

A cDNA clone encoding a lectin was isolated by immunological screening of an expression library prepared from poly(A)⁺ RNA from the inner bark ofRobinia pseudoacacia. The cDNA clone (RBL104) had an open reading frame of 858 bp that encoded a polypeptide with a predicted molecular weight of 31210. This molecular weight corresponded closely to that of a polypeptide immunoprecipitated from products of translationin vitro of the poly(A)⁺ RNA. Thus, RBL104 appeared to be a full-length cDNA. The N-terminal amino acid sequence of the purified lectin protein matched a portion of the predicted amino acid sequence. It appeared that the lectin was synthesized as a precursor that consisted of a putative signal peptide of 31 amino acids and a mature polypeptide of 255 amino acids. Southern blot analysis of the genomic DNA revealed that the lectin was encoded by a small multigene family. The lectin was mostly localized in the axial and ray parenchymal cells of the inner bark. A small amount of lectin was also found in the axial and ray parenchymal cells of the xylem. The lectin accumulated in the inner bark in September, remained at high levels during the winter and disappeared in May. The mRNA for the lectin was detected from August to the following March. The appearance and disappearance of the mRNA were observed prior to those of the lectin protein.     

毛尖紫萼藓抗旱相关基因Gp-LEA的克隆与表达分析

以毛尖紫萼藓干旱cDNA文库中获得的一段与LEA基因同源性较高的EST序列为基础,采用RACE技术分离该基因cDNA全长序列,命名为Gp-LEA。Gp-LEA基因的cDNA全长814bp,开放阅读框456bp,编码含151个氨基酸蛋白质。生物信息学分析结果显示,Gp-LEA蛋白为稳定蛋白,分子质量为16.612kD,理论等电点（pI）为5.06,含有LEA₂功能结构域,不属于跨膜蛋白且不存在信号肽。系统发生分析表明,Gp-LEA基因编码蛋白与花旗松LEA蛋白亲缘关系最近。荧光定量PCR分析显示,Gp-LEA基因在复水和快速干旱模式下均能表达。推测Gp-LEA基因在毛尖紫萼藓的复水和干旱过程中起着重要作用。