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.     

Cloning and Characterization of a Root-specific Expressing Gene Encoding 3-hydroxy-3-methylglutaryl Coenzyme a Reductase from Ginkgo biloba

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34) catalyzes the first committed step in mevalonic acid (MVA) pathway for biosynthesis of isoprenoids. The full-length cDNA encoding HMGR was isolated from Ginkgo biloba for the first time (designated as GbHMGR, GenBank accession number AY741133), which contained a 1713 bp ORF encoding 571 amino acids. The GbHMGR genomic DNA sequence was also obtained, revealing GbHMGR had four exons and three introns. The deduced GbHMGR protein showed high identity to other plant HMGRs and contained two trans-membrane domains and a catalytic domain. The three dimensional model of GbHMGR represented a typical spatial structure of HMGRs. The Southern blot and RT-PCR assay results indicated that GbHMGR belonged to a small gene family, and expressed in a tissue-specific manner with a low level expression being only found in root. The potential significance of GbHMGR gene was also discussed.     

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 cloning, characterization and expression of atpA and atpB genes from Ginkgo biloba

The chloroplast ATP synthase (ATPase) utilizes the energy of a transmembrane electrochemical proton gradient to drive the synthesis of ATP from ADP and phosphate. The chloroplast ATPase α and β subunits are the essential components of multisubunit protein complex. In this paper, the full-length cDNA and genomic DNA of ATPase α (designated as GbatpA) and β (designated as GbatpB) subunit genes were isolated from Ginkgo biloba. The GbatpA and GbatpB genes were both intronless. The coding regions of GbatpA and GbatpB were 1530 bp and 1497 bp long, respectively, and their deduced amino acid sequences showed high degrees of identity to those of other plant ATPase α and β proteins, respectively. The expression analysis by RT-PCR revealed that GbatpA and GbatpB both expressed in tissue-specific manners in G. biloba and might involve in leaf development. The recombinant GbATPB protein was successfully expressed in E. coli strain using pET28a vector with ATPase activity as three times high as the control, and the results showed that the molecular weight of the recombinant protein was about 54 kDa, a size that was in agreement with that predicted by bioinformatics analysis. This study provides useful information for further studying on overall structure, function and regulation of the chloroplast ATPase in G. biloba, the so-called “living fossil” plant as one of the oldest gymnosperm species. These authors contributed equally to this work     

Molecular Cloning of a Novel Gene ZAhi-1 and its Expression Analysis during Zebrafish Gametogenesis

Proto-oncogen Ahi-1 is closely related to a lot of human and mouse diseases. Ahi-1 mutation will lead to leukemia in mice and Joubert syndrome in human beings. We have cloned the full cDNA sequence of Ahi-1 homologous in zebrafish, and RT-PCR results of ZAhi-1 in different tissues reveal that ZAhi-1 expressed highest in the mature gonad. In situ hybridization results of zebrafish gonad show that ZAhi-1 only expressed in the early stages’ gamete cells. RT-PCR analyses of mouse Ahi-1 in different stages of spermatogenesis have been done according to the published Ahi-1 sequence, and the findings reveal that Ahi-1 is expressed in gamete of pachytene stage. It can then be safely concluded that Ahi-1 might take place in the spermatocyte from the early pachytene stage to the late pachytene stage.     

Embryogenesis from microspores of Ginkgo biloba L., a medicinal woody species

Summary The present work establishes that isolated microspores of Ginkgo biloba L. cultured at densities of 1.5 to 5·10⁴ per milliliter in Bourgin and Nitsch (1967) liquid medium are able to divide, both in the presence and in the absence of exogenous growth regulators, and to germinate by growing a pollen tube. In all experiments the microspores exhibited various modes of division leading to embryo formation in the liquid medium. Four weeks later, the microspores which had been previously submitted to various electrical stresses showed pro-embryo development earlier than those which had not. After ten weeks the number of embryos was found to be 300 to 5300 ml^–1 following the experiments. When the embryos exhibited a slower growth in liquid medium, they were transferred onto various solid media for maturation. Two months later, embryos had proliferated visibly.Abbreviations BN Bourgin and Nitsch (1967) medium - IAA Indole-3-acetic acid - KIN Kinetin - GS Growth substances     

Direct embryogenesis from female haploid protoplasts of Ginkgo biloba L., a medicinal woody species

Summary Haploid protoplasts isolated from prothallus (i.e. female gametophyte) of Ginkgo biloba, at densities ranging from 5×10⁴ to 10⁵ protoplasts per milliliter, were able to divide and form microclones which directly evolved into embryos, when they were cultured in two different liquid media. These were: the Murashige and Tucker medium (1969) modified by omitting ammonium ions and supplementing with glutamine, benzyladenine and various levels of naphthaleneacetic acid; or the Bourgin and Nitsch medium (1967) without growth regulators, supplemented with coconut milk. Three months later, the number of embryos ranged from 165 to 1900 embryos ml^–1 depending on the culture medium. After four months, embryos at whatever stage (globular, oblong or heart) exhibited a slow growth, which delayed the transfer onto solid media.Abbreviations BA 6-benzyladenine - BN Bourgin and Nitsch (1967) medium - MT Murashige and Tucker (1969) medium - NAA naphthaleneacetic acid     

荠菜LOS2基因的克隆与分析

利用RACE-PCR方法从荠菜(Capsella bursa-pastoris)中克隆到了新的LOS2全长基因(Cblos2)。序列分析表明,该基因的全长cDNA为1694bp,拥有一个由444个氨基酸组成的开放读码框,预测的CbLOS2蛋白包括一个烯醇酶N-端结构域、烯醇酶结构域以及与拟南芥的LOS2高度保守的DNA结合域和基因功能抑制域。生物信息学分析表明,Cblos2与LOS2极为相似。冷胁迫适应实验表明,Cblos2基因在荠菜中组成性表达,且其表达与胁迫适应过程密切相关。该研究表明Cblos2是具双重功能的植物烯醇酶基因家族的新成员。     

孔石莼(Ulva pertusa)质体蓝素基因的克隆及基因特征分析(英文)

采用cDNA末端快速扩增的办法,从孔石莼(Ulva pertusa)中克隆获得质体蓝素基因。该基因完整的cDNA为787bp,包括40 bp 5’端非编码区和327 bp的3’端非编码区,以及一个420 bp的开放阅读框架,编码139个氨基酸的蛋白质。该基因编码质体蓝素的前体肽,其N端41个氨基酸残基为信号肽,后面为98个氨基酸残基的成熟肽。从Genbank中选择了13个质体蓝素的前体肽基因进行序列比对分析和构建进化树。孔石莼质体蓝素基因与其它质体蓝素基因的同源性为48.2%至78.8%。该进化树将来源于6种藻类植物的7个质体蓝素基因聚类在一起,显示出它们较近的进化关系。同样,也表现出11种生物的分子进化关系。序列比对结果显示,在质体蓝素的基因序列中存在两个高度保守的基序,它编码质体蓝素蛋白的铜结合活性位点。     

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 calreticulin cDNA from the pinewood nematode Bursaphelenchus xylophilus

The cloning and characterization of a cDNA encoding a calreticulin from the pinewood nematode Bursaphelenchus xylophilus is described herein. The full-length cDNA (Bx-crt-1) contained a 1200 bp open reading frame that could be translated to a 399 amino acid polypeptide. The deduced protein contained highly conserved regions of a calreticulin gene and had 66.2–70.1% amino acid sequence identity to other calreticulin sequences from nematodes. RNAi, RT-PCR amplification, and southern blot suggest that Bx-crt-1 may be important for the development of B. xylophilus.     

Deterrents extracted from the leaves of Ginkgo biloba: effects on feeding and contact chemoreceptors

Powdered dried ginkgo tree leaves were subjected to various chemical extractions and successive extracts were monitored for antifeedant activity against larvae of Pieris brassicae. Many fractions moderately inhibited food intake, and some were deterrent at levels as low as 25–50 ppm. Some behaviourally highly active fractions were tested electrophysiologically for neural responses in the maxillary taste sensilla. These extracts appeared to stimulate deterrent receptors. There were distinct differences in responses between Pieris brassicae and P. rapae. Ginkgolide A, B, and C each strongly stimulated deterrent receptors in P. rapae, which corresponds with the observation (Matsumoto & Sei, 1987) that these compounds are effective antifeedants for this species. No toxic effects were observed in insects after feeding for 24 h on diets containing ginkgo extracts.

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Résumé Des feuilles de Gingko séchées et broyées ont été soumises à différentes extractions. On a testé l'activité déterrante (antiapprétant) des extraits successifs sur les larves de Pieris brassicae. Un grand nombre de fractions inhibent modérément la consommation, et certaines sont déterrantes à des concentrations aussi faibles que 25–50 ppm. Par électrophysiologie, on a testé la réponse nerveuse des sensilles gustatives maxillaires à certaines fractions qui montraient une forte action sur le comportement. Il s'avère que ces extraits stimulent les récepteurs déterrants. Les réponses diffèrent pour P. brassicae et P. rapae. Les gingkolides A, B et C stimulent chacun fortement les récepteurs déterrants chez P. rapae, ce qui correspond aux observations (Matsumoto & Sei, 1987) selon lesquelles ces composés sont des antiappétants efficaces pour cette espèce. On n'a pas observé d'effet toxique sur les insectes nourris durant 24 heures par des diètes contenant des extraits de Gingko.

     

Cloning, Expression and Characterization of a Thiolase Gene from Clostridium pasteurianum

A thl gene encoding the thiolase (EC 2.3.1.9) of Clostridium pasteurianum was cloned by thermal asymmetric interlaced (TAIL) PCR. It consists of 1179 bp with 36.8% GC content and encodes 392 amino acids with a deduced molecular mass of 40,954 Da and shows 77% identity and 88% similarity to that of Clostridium tetani E88 and should be classified as a biosynthetic thiolase with three conserved residues Cys89, Cys382 and His352. The gene was over-expressed in Escherichia coli and the thiolase was purified with Ni-NTA agarose column to homogeneity. The K _m of this thiolase for acetoacetyl-CoA is 0.13 mM with 0.06 mM CoASH at pH 8.2, 25°C and a V _max value of 46 μmol min⁻¹ mg⁻¹.     

The secretory apparatus of Ginkgo biloba: structure,differentiation and analysis of the secretory product

Summary The differentiation of the secretory cavities of Ginkgo stem and the structural organization of the epithelial cells were followed by light and electron microscopy. The mode of formation of the cavities is schizo-lysigeneous. Functional complexes of leucoplasts and associated endoplasmic reticulum (ER) membranes are assumed to be the site of synthesis and translocation of the lipophilic secretory product. Most of the endoplasmic reticulum membranes are paired. The content of the cavities was directly collected and analysed by low- and high-resolution mass spectrometry. The cavities contain anacardic acids and cardanols, which are long-chain phenol lipids not characteristic of Ginkgo. The relationship between the plastid/ER complexes and the production of these secondary metabolites is discussed.     

Contribution of a phytotoxic compound to the allelopathy of Ginkgo biloba

Ginkgo (Ginkgo biloba L.) has not changed over 121 million years. There may be unknown special strategy for the survival. Gingko litter inhibited the growth of weed species ryegrass (Lolium multiflorum L.). The inhibition was greater with the litter of the close position than that of the far position from the gingko tree. A phytotoxic substance, 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid (HHPEBA) was isolated in the litter. HHPEBA concentration was greater in the litter of the close position than that of the far position from the tree. HHPEBA inhibited the ryegrass growth at concentrations greater than 3 μM. HHPEBA was estimated to be able to cause 47–62% of the observed growth inhibition of ryegrass by the gingko litter. Therefore, HHPEBA may contribute to the inhibitory effect caused by ginkgo litter and may provide a competitive advantage for gingko to survive through the growth inhibition of the neighboring plants.     

Somatic embryogenesis from immature zygotic embryos ofGinkgo biloba

Immature zygotic embryos ofG. biloba were taken, at various developmental stages, from ovules harvested in November 1993. Zygotic embryos showing the beginning of the cotyledonary development cultured on modified Murashige & Tücker (1969) media proliferated intensely. In fact, 98.5% of the immature zygotic embryos produced embryogenic and undifferentiated tissues (calluses), in proportions varying depending on the hormonal composition of the induction media. After two weeks of culture, direct embryogenesis was observed on the hypertrophic cotyledons when benzyladenine 10 M was used as the sole plant growth regulator in the induction media. The addition of different concentrations of NAA (5–10–20 M) and of BA (5 M) to the induction media led to an indirect embryogenesis after two months, when the calluses were transferred to the development media without auxin. The highest frequency of embryogenic tissues (90–95%) and the highest number of somatic embryos per explant (9.6) were obtained with benzyladenine (10 M) as the sole exogenous growth factor. Some embryos isolated mechanically or in situ on the callus developed as far as the later cotyledonary stage.Abbreviations AUX Auxin - BA Benzyladenine - CYT Cytokinin - IZE Immature zygotic embryo - MT Murashige & Tücker (1969) medium - NAA Naphtaleneacetic acid     

一个大鼠心肌缺血-再灌诱导表达上调的新基因Mip1的克隆和特性分析

采用生物信息学方法和5′cDNA末端快速扩增法(RACE)技术相结合,克隆了一个大鼠心肌缺血-再灌诱导表达上调的新基因Mip1,并经RT-PCR测序及多组织膜RNA印迹证实.生物信息学分析显示,MIP-1定位于大鼠染色体1q12区,含5个外显子和4个内含子,开放阅读框(ORF)为1 827 bp,编码608个氨基酸,其编码蛋白N端含KRAB结构域,C端含14个连续的C2H2型锌指蛋白结构域,氨基酸第277位至293位为双向的核定位信号,多组织膜RNA印迹显示该基因在脑组织表达最高,其次是心脏,在其他组织表达较低或无表达.进一步深入研究该基因的功能具有重要生物学意义.