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.     

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     

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.     

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.     

滴水珠珠芽发育过程研究

珠芽是滴水珠(Pinellia cordata)的营养繁殖结构,为揭示滴水珠珠芽发育过程中的特征,该研究以野外采集材料进行盆栽观察试验,通过形态观察法和石蜡切片方法,探索滴水珠叶片和叶柄处珠芽发育过程中的形态学和解剖学结构特征。结果表明:滴水珠珠芽发育过程在形态上分为叶柄无现象时期、早期珠芽结构分化和珠芽膨大成熟时期三个时期;相应的解剖学发育时期为珠芽原基启动期和形成期、原基分化期和膨大成熟期四个阶段。叶片和叶柄珠芽起始细胞均由叶柄腹面表皮下层薄壁细胞组织脱分化形成,起始细胞不断分裂形成细胞团最后发育成珠芽原基;在原基分化期的特征是生长点形成,最终分化形成芽原基和鳞片叶;膨大成熟期的特征是珠芽结构不断生长,生长点侧生分化鳞片叶增多。叶片和叶柄处珠芽成熟脱落时期为灰色椭圆形结构,测得平均直径分别为(2.79±0.40)mm和(2.69±0.36)mm,外部有鳞片包裹,内部含大量营养物质。在环境适宜条件下,珠芽遇土萌发,萌发率达75%。这表明滴水珠珠芽与同属植物半夏珠芽发育过程差别不大,都是无性克隆营养繁殖体结构。     

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.     

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.     

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.     

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 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 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.     

Molecular cloning and characterization of a 2C-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-erythritol 2,4-cyclodiphosphate synthase gene from <Emphasis Type="Italic">Cephalotaxus harringtonia</Emphasis>

The full-length MECPS cDNA sequence (designated as Chmecps, GenBank Accession No.: DQ415658) was isolated by rapid amplification of cDNA ends (RACE) for the first time from Cephalotaxus harringtonia. The full-length cDNA of Chmecps was 1,146 bp containing a 753 bp open reading frame (ORF) encoding a polypeptide of 250 amino acids with a calculated mass of 26.67 kDa and an isoelectric point of 9.35. Comparative and bioinformatics analyses revealed that ChMECPS showed extensive homology with MECPSs from other plant species. Phylogenetic analysis indicated ChMECPS was more ancient than other plant MECPSs. Southern hybridization analysis of the genomic DNA showed that Chmecps was a single copy gene. Tissue expression pattern analysis revealed that ChMECPS expressed strongly in root and leaf, weakly in stem.     

Sequencing and characterization of asclepain f: the first cysteine peptidase cDNA cloned and expressed from <Emphasis Type="Italic">Asclepias fruticosa</Emphasis> latex

Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.     

Molecular characterization and mRNA transcript profile of vitellogenin in Chinese shrimp, Fenneropenaeus chinensis

A full-length cDNA encoding vitellogenin (Vg) was cloned from Chinese shrimp, Fenneropenaeus chinensis using RACE method. The full-length cDNA consist of 7,942 nucleotides including a 7,761 bp open reading frame, which encodes 2,587 amino acid residues. The deduced amino acid sequence showed high (from 94% to 37%) identity with other known crustacean Vgs. In addition, a consensus cleavage site (R-X-K/R-R) recognized by an endopeptidase and a member of subtilisin family of serine protease were identified in the deduced Vg precursor. RT-PCR analysis shown that Vg mRNA can be detected in both ovary and hepatopancreas of vitellogenic females but not in other experimental tissues including muscle, heart, lymph organ, gill, haemocytes and intestine. These results suggest that the Vg gene may be expressed exclusively in mature females, and both ovary and hepatopancreas are the possible tissues for Vg synthesis in F. chinensis. In addition, Vg gene is detected in genomic DNA of both females and males.     

Molecular cloning,characterization and expression analysis of a new gene encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase from <Emphasis Type="Italic">Salvia miltiorrhiza</Emphasis>

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate (MVA), which is the first committed step in MVA pathway for isoprenoid biosynthesis in plants. In this study, a full-length cDNA encoding HMGR was isolated from Salvia miltiorrhiza by rapid amplification of cDNA ends (RACE) for the first time, which was designated as SmHMGR (GenBank Accession No.EU680958). The full-length cDNA of SmHMGR was 2,115 bp containing a 1,695 bp open reading frame (ORF) encoding a polypeptide of 565 amino acids. Bioinformatic analyzes revealed that the deduced SmHMGR had extensive homology with other plant HMGRs contained two transmembrane domains and a catalytic domain. Molecular modeling showed that SmHMGR is a new HMGR with a spatial structure similar to other plant HMGRs. Phylogenetic tree analysis indicated that SmHMGR belongs to the plant HMGR super-family and has the closest relationship with HMGR from Picrorhiza kurrooa. Expression pattern analysis implied that SmHMGR expressed highest in root, followed by stem and leaf. The expression of SmHMGR could be up-regulated by salicylic acid (SA) and methyl jasmonate (MeJA), suggesting that SmHMGR was elicitor-responsive. This work will be helpful to understand more about the role of HMGR involved in the tanshinones biosynthesis at the molecular level.     

Cloning and heterologous expression of nitrate reductase genes from <Emphasis Type="Italic">Dunaliella salina</Emphasis>

A cDNA corresponding to the nitrate reductase (NR) gene from Dunaliella salina was isolated by RT-PCR and (5′/3′)-RACE techniques. The full-length cDNA sequence of 3,694 bp contained an open reading frame of 2,703 bp encoding 900 amino acids, a 5′-untranslated region of 151 bp and a 3′-untranslated sequence of 840 bp with a poly (A) tail. The putative gene product exhibited 78%, 65%, 59% and 50% identity in amino acid sequence to the corresponding genes of Dunaliella tertiolecta, Volvox carteri, Chlamydomonas reinhardtii, and Chlorella vulgaris, respectively. Phylogenetic analysis showed that D. salina NR clusters together with known NR proteins of the green algae. The molecular mass of the encoded protein was predicted to be 99.5 kDa, with an isoelectric point of 8.31. This protein shares common structural features with NRs from higher plants and green algae. The full-length cDNA was heterologously expressed in Escherichia coli as a fusion protein, and accumulated to up to 21% of total bacteria protein. Recombinant NR protein was active in an enzyme assay, confirming that the cloned gene from D. salina is indeed NR.     

半夏珠芽发育过程的形态学和解剖学研究

通过形态学观察和石蜡切片方法研究了半夏[Pinellia ternata(Thunb.)Breit.]的珠芽发育过程,结果显示:半夏珠芽着生于叶柄的下部,起始于幼嫩叶柄的腹面最外轮维管束外周薄壁细胞;恢复分裂的薄壁细胞分裂形成珠芽原基细胞团,在原基生长突破叶柄表皮后分化形成具有生长点的珠芽结构,发育中的珠芽无根分化;珠芽的生长被动地终止于叶片衰老(倒苗),无明显的成熟发育过程。研究表明,半夏的珠芽是不定芽性质的无性繁殖结构,但在发育过程上明显区别于其它植物的珠芽发育。     

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.     

Cloning,characterization and TBT exposure response of CuZn superoxide dismutase from Haliotis diversicolor supertexta

The full-length cDNA and genomic DNA of a cytoplasmic copper, zinc superoxide dismutase (CuZn-sod) were cloned from the hepatopancreas of small abalone Haliotis diversicolor supertexta by RT-PCR, RACE and TAIL PCR. The full-length cytoplasmic CuZn-sod cDNA (designated sasod) comprises 984 bp. Its ORF encodes a polypeptide of 154 amino acids with a predicted molecular mass of 15.7 kDa and theoretical isoelectric point of 6.30. The deduced amino acid (designated saSOD) shares a common consensus pattern with the SODs of vertebrate and invertebrate animals. The full-length sasod genomic DNA comprises 5,574 bp, containing five exons and four introns. The splice donor and acceptor sequence of the four introns is 5′GT-AG3′. Real time quantitative PCR analysis revealed that sasod expression level in hepatopancreas of small abalone was no significant difference at 2, 6, 48 and 192 h post TBT exposure (P > 0.05). However, the sasod expression level at 12 and 24 h post TBT exposure was decreased significantly (P < 0.05).