烟草坏死病毒A诱导的基因沉默及其条件优化

以烟草坏死病毒A中国分离物(Tobacco necrosis virus A Chinese isolate,TNV-AC)侵染性cDNA克隆为基础,通过基因替换、基因插入策略构建获得多种重组TNV-AC,比较了外源基因片段插入位置、插入形式及接种植物培养温度对TNV-AC诱导的基因沉默的影响.外源基因片段替换CP基因19～828 nt的重组TNV-AC丧失了在本生烟中的系统移动能力,也不能有效诱导相应基因发生明显的沉默,说明替换策略不适合于TNV-AC.向CP基因终止密码子UAG附近插入外源基因片段后,TNV-AC仍可进行复制,但最适的插入位点位于UAG之后,且容纳外源片段的长度约为120 nt.当外源片段以反向重复的形式插入UAG之后,诱导基因沉默的效率较高.接种植物的培养温度也会显著影响基因沉默的效率以及插入片段的稳定性,低温(18℃)条件下诱导NbPDS基因沉默的效率明显高于高温(24℃)条件,且沉默表型可持续110天以上.除了本生烟PDS基因,TNV-AC沉默载体还可诱导本生烟sulfur基因Su和镁离子螯合酶H亚基基因ChlH发生沉默,以上结果说明,TNV-AC具有开发为本生烟基因功能鉴定的新VIGS载体的潜力.     

Production of monoclonal antibodies against the FimA protein of Porphyromonas gingivalis in Nicotiana benthamiana

Porphyromonas gingivalis, a gram-negative anaerobic oral bacterium, causes periodontal disease by binding to saliva-coated oral surfaces. The FimA protein from P. gingivalis is a crucial pathogenic component of the bacterium and a target for vaccine development against periodontal disease. Complementary DNAs encoding the heavy and light chains of two monoclonal antibodies that bind specifically to the FimA protein were cloned into a plant expression vector under the control of the duplicated Cauliflower Mosaic Virus 35S promoter, and agroinfiltration was used to allow the vectors to infiltrate tobacco plants. The expressions of the heavy and light chains in the leaf tissue were detected using antibodies specific to each antibody chain. Western blot analysis showed the specific binding of the plant-derived monoclonal antibodies to the native FimA protein purified from P. gingivalis. Our finding that plant-derived monoclonal antibodies bound specifically to the native FimA protein indicates that plantderived monoclonal antibodies can protect against P. gingivalis invasion.     

Physical mapping of Nicotiana tabacum chloroplast DNA

Summary Nicotiana tabacum chloroplast DNA was digested with several restriction endonucleases chosen for their potential usefulness in distinguishing between species of the genus, Nicotiana. The resulting fragments were ordered into a circular configuration of about 160 kilobase pairs, equalling about 100x10⁶ daltons. The physical map features an inverted, repeated unit of about 24 kilobase pairs separated by a unique sequence region with a mean size of 13 kilobase pairs on the short side. The cistrons coding for chloroplast ribosomal RNA are contained within the inverted repeat and have the arrangement: 16S, spacer, 23S, 4.5S/5S. Restriction endonuclease maps obtained with the enzymes PvuII, XhoI, and BglI are presented.     

Intertrubal chloroplast transfer by protoplast fusion between Nicotiana tabacum and Salpiglossis sinuata

Summary Chloroplast tranfer was achieved by protoplast fusion between Nicotiana tobacum (Cestreae, Cestroideae) and Salpiglossis sinuata (Salpiglossideae, Cestroideae) in the family Solanaceae. Isolation of cybrid clones was facilitated by irradiation of the cytoplasm donor protoplasts, and the use of appropriate plastid mutants, streptomycin-resistant as donor, or light-sensitive as recipient. Cybrid colonies were selected by their green colour against the background of bleached (light-sensitive or streptomycin-sensitive) colonies. In the Nicotiana (Salpiglossis) cybrid plants possessing normal tobacco morphology and chromsome number, the presence of Salpiglossis, plastids was verified by restriction analysis of the chloroplast DNA. A similar analysis of the mitochondrial DNA of these lines revealed unique, recombinant patterns in the case of both fertile and sterile plants. Progeny showed no appearance of chlorophyll-deficiency in F₁ and an additional back-cross generation. Attempts at transfer of entire chloroplasts between Nicotiana tabacum and Solanum nigrum (Solaneae, Solanoideae) did not result in any cybrid cell lines in a medium suitable for green colony formation of both species. These results suggest that fusion-mediated chloroplast transfer can surmount a considerable taxonomical distance, but might be hampered by a plastome-genome incompatibility in more remote combinations.     

Genetic modification of alternative respiration in Nicotiana benthamiana affects basal and salicylic acid-induced resistance to potato virus X

Background  

Salicylic acid (SA) regulates multiple anti-viral mechanisms, including mechanism(s) that may be negatively regulated by the mitochondrial enzyme, alternative oxidase (AOX), the sole component of the alternative respiratory pathway. However, studies of this mechanism can be confounded by SA-mediated induction of RNA-dependent RNA polymerase 1, a component of the antiviral RNA silencing pathway. We made transgenic Nicotiana benthamiana plants in which alternative respiratory pathway capacity was either increased by constitutive expression of AOX, or decreased by expression of a dominant-negative mutant protein (AOX-E). N. benthamiana was used because it is a natural mutant that does not express a functional RNA-dependent RNA polymerase 1.     

Genetic instability in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana

Summary Somatic hybrids of Nicotiana knightiana (2n=2X=24) and an albino mutant of Nicotiana tabacum (2n=4X=48) were selected after polyethylene glycol induced protoplast fusion. Three lines were selected on the basis of the simultaneous expression of shoot inducibility and green pigmentation, traits originally separated in the parental species.The hybrid nature of the lines was confirmed by their characteristic isoenzyme patterns, the morphology of the regenerated plants, and by the appearance of heterochromatic blocks in the interphase nuclei.Chromosome numbers in the somatic hybrids varied greatly within individual plants. Variegation in leaf and flower colour and segregation for morphological traits in vegetatively multiplied plants are attributed to segregation of chromosomes in the somatic cells, a consequence of the numerical instability. Hybridity, caryotypic changes induced by tissue culture, and high chromosome numbers, are discussed as possible reasons for the observed genetic instability.     

基于烟草脆裂病毒构建同时表达2种外源蛋白的载体及其应用

目的 构建在整个寄主植物中能同时表达两个非融合蛋白的病毒载体。方法 以烟草脆裂病毒（tobacco rattle virus，TRV）基因组RNA2的农杆菌侵染性克隆pYL156为材料，缺失TRV RNA2的2b基因5"端279 bp并将其起始密码子ATG改变为AGG、同时引入豌豆早枯病毒（pea early-browning virus，PEBV）外壳蛋白（coat protein，cp）基因启动子，获得pTRV2e²载体；在pTRV2e²载体的2b和PEBV的cp启动子下游插入不同的外源基因，测定病毒TRVe²表达外源蛋白的能力、携带外源基因后重组病毒的稳定性以及分析蛋白质的生物学功能。结果 病毒TRVe²能快速同时表达2个非融合的外源蛋白，且至少能表达70 ku外源蛋白，且该病毒携带~2.0 kb外源基因能稳定存活于寄主植物中；病毒TRVe²可用于分析蛋白质的生物学功能以及2个蛋白质间的相互作用。结论 本文构建的重组病毒TRVe²为快速有效地表达2个外源蛋白以及分析2个蛋白质间的相互作用提供技术工具。     

High-level HIV-1 Nef transient expression in Nicotiana benthamiana using the P19 gene silencing suppressor protein of Artichoke Mottled Crinckle Virus

Background  

In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef.     

Morphological characteristics and chloroplast DNA distribution in different cytoplasmic parasexual hybrids of Nicotiana tabacum

Summary Protoplast fusion makes possible the fusion of two different cytoplasms, allowing genetical analysis of cytoplasmic factors. Two varieties of Nicotiana tabacum differing by their cytoplasms have been used. Techne, the first variety, obtained by an interspecific cross between N. debneyi (female) and N. tabacum (male) is characterized by the nuclear tabacum genome inside the debneyi cytoplasm. Techne plants present abnormal flowers with cytoplasmic male sterility (cytoplasmic marker) and sessile leave (nuclear marker). Techne leaf protoplasts were fused with leaf protoplasts of N. tabacum var. Samsun (or Xanthi). The last variety is characterized by petiolated leaves and normal flowers, because it possesses the nuclear tabacum genome associated with the tabacum cytoplasm. The nuclear marker (leaf shape) and the cytoplasmic one (flower shape inducing male sterility or fertility) have been used to distinguish among the whole regenerated plants the somatic nuclear hybrids and the cytoplasmic hybrids (cybrids) displaying the nuclear phenotype of one of the two parents associated with a modified flower type, intermediate between the parental ones.The chloroplastic (cp) DNA contained in each parent has been specifically identified by using EcoRI restriction nuclease and gel electrophoresis. EcoRI fragment patterns of cp DNA isolated from the first progeny of the regenerated cytoplasmic hybrids revealed that only one of the two parental cp DNAs is present in all cases; neither mixture of both parental cp DNAs nor recombinant cp DNA molecules were observed. This indicates that a specific elimination of one or the other parental cp DNAs occurs after the initial mixing of the cytoplasms. The study of the association of the modified flower type with the cp DNA isolated from the corresponding plant showed that cp DNA seems independent from the mechanism of cytoplasmic male sterility in tobacco.     

Extensive rearrangements in the mitochondrial DNA in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana

Summary Mitochondrial DNA (mtDNA) was studied in the leaves of Nicotiana tabacum+Nicotiana knightiana somatic hybrids previously described. Restriction patterns generated by the SalI and BamHI restriction endonucleases were different from both parents in the eight hybrids, and made up of parental and non-parental fragments. Rearrangements in the mtDNAs have been confirmed by DNA-DNA hybridization using, as a probe, labelled 2/12 plasmid DNA which contains the E. coli 16S and 23S rRNA genes. Novel patterns can be explained by new combinations of unaltered parental mtDNA molecules, and by genetic recombination.     

An auxin-auxotrophic mutant of Nicotiana plumbaginifolia

Summary Auxin (indole-3-acetic acid) is considered to be an important signalling molecule in the regulation of plant growth and development but neither auxin synthesis nor its mode of action is clearly understood. To identify genes involved in these processes, mutations were sought that altered the auxin requirement of plant tissues for growth. For the first time mutant plants were obtained that carry a recessive mutation at a single nuclear locus (auxl) which results in an absolute requirement for exogenous auxin for normal growth. In the absence of auxin treatment, mutant plants undergo premature senescence and die.Abbreviations BAP 6-benzylaminopurine - BUdR 5-bromodeoxyuridine - 2,4-D 2,4-dichlorophenoxyacetic acid - FUdR 5-fluorodeoxyuridine - IAA-EE indole-3-acetic acid ethyl ester - IMS indole-3-methanesulfonic acid     

A membrane-bound matrix-metalloproteinase from Nicotiana tabacum cv. BY-2 is induced by bacterial pathogens

Background  

Plant matrix metalloproteinases (MMP) are conserved proteolytic enzymes found in a wide range of monocotyledonous and dicotyledonous plant species. Acting on the plant extracellular matrix, they play crucial roles in many aspects of plant physiology including growth, development and the response to stresses such as pathogen attack.     

Somatic hybridization of Nicotiana tabacum and Petunia hybrida

Summary Leaf mesophyll protoplasts of a nitrate reductase deficient streptomycin resistant mutant of Nicotiana tabacum were fused with cell suspension protoplasts of wild type Petunia hybrida. Somatic hybrid cell colonies were selected for streptomycin resistance and nitrate reductase proficiency. Six independent cell lines, capable of growth in selection medium, were analysed by electrophoresis of callus peroxidases and leucine aminopeptidases and also by hybridization with rDNA and a chloroplast encoded gene as molecular probes. The results show that all six lines represented nuclear somatic hybrids, possessing the chloroplast of N. tabacum, at an early stage of development. However, after 6–12 months in culture, genomic incompatibility was observed resulting in the loss of most of the tobacco nuclear genome in the majority of the cell lines. One of the latter cell lines regenerated plants which possessed the chloroplast of N. tabacum in a predominantly P. hybrida nuclear background.     

Mining the soluble chloroplast proteome by affinity chromatography

Chloroplasts are fundamental organelles enabling plant photoautotrophy. Besides their outstanding physiological role in fixation of atmospheric CO(2), they harbor many important metabolic processes such as biosynthesis of amino acids, vitamins or hormones. Technical advances in MS allowed the recent identification of most chloroplast proteins. However, for a deeper understanding of chloroplast function it is important to obtain a complete list of constituents, which is so far limited by the detection of low-abundant proteins. Therefore, we developed a two-step strategy for the enrichment of low-abundant soluble chloroplast proteins from Pisum sativum and their subsequent identification by MS. First, chloroplast protein extracts were depleted from the most abundant protein ribulose-1,5-bisphosphate carboxylase/oxygenase by SEC or heating. Further purification was carried out by affinity chromatography, using ligands specific for ATP- or metal-binding proteins. By these means, we were able to identify a total of 448 proteins including 43 putative novel chloroplast proteins. Additionally, the chloroplast localization of 13 selected proteins was confirmed using yellow fluorescent protein fusion analyses. The selected proteins included a phosphoglycerate mutase, a cysteine protease, a putative protein kinase and an EF-hand containing substrate carrier protein, which are expected to exhibit important metabolic or regulatory functions.     

Changes in the Gallus gallus proteome induced by Marek's disease virus

Marek's disease virus (MDV) is a highly oncogenic avian herpesvirus. We have used a modified MudPIT analysis to examine the effect of MDV infection on the chicken proteome. We identified 3561 unique nonphosphorylated peptides, representing 1460 chicken proteins, in a mock-infected sample versus 4240 unique nonphosphorylated peptides, representing 1676 proteins, in an MDV-infected sample. Of these unique peptides, 59.1% from the mock- and 49.6% from the MDV-infected samples were detected in both samples, and for the represented proteins, 69.1% from the mock- and 60.2% from the MDV-infected samples were common to both samples. In terms of phosphorylation, 357 and 506 phosphopeptides, representing 342 and 483 proteins, were detected in the mock- and MDV-infected samples, respectively. At the phosphopeptide level, 10.1% from the mock- and 7.1% from the MDV-infected samples overlapped, and for the represented phosphoproteins, 12.0% from the mock- and 8.5% from the MDV-infected samples were common to both samples. There were no significant differences in the hydropathicity values and number of transmembrane domains of the identified protein sets. Subtle differences were observed for subcellular localizations of the identified proteins. These results suggest that MDV infection may alter host cell biochemistry by perturbing the host's proteomic composition.     

Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics

Nicotiana tabacum (2n=48) is a natural amphidiploid with component genomes S and T. We used non-radioactive in situ hybridization to provide physical chromosome markers for N. tabacum, and to determine the extant species most similar to the S and T genomes. Chromosomes of the S genome hybridized strongly to biotinylated total DNA from N. sylvestris, and showed the same physical localization of a tandemly repeated DNA sequence, HRS 60.1, confirming the close relationship between the S genome and N. sylvesfris. Results of dot blot and in situ hybridizations of N. tabacum DNA to biotinylated total genomic DNA from N. tomentosiformis and N. otophora suggested that the T genome may derive from an introgressive hybrid between these two species. Moreover, a comparison of nucleolus-organizing chromosomes revealed that the nucleolus organizer region (NOR) most strongly expressed in N. tabacum had a very similar counterpart in N. otophora. Three different N. tabacum genotypes each had up to 9 homozygous translocations between chromosomes of the S and T genomes. Such translocations, which were either unilateral or reciprocal, demonstrate that intergenomic transfer of DNA has occurred in the amphidiploid, possibly accounting for some results of previous genetic and molecular analyses. Molecular cytogenetics of N. tabacum has identified new chromosome markers, providing a basis for physical gene mapping and showing that the amphidiploid genome has diverged structurally from its ancestral components.     

In vivo reorganization of the actin cytoskeleton in leaves of Nicotiana tabacum L. transformed with plastin-GFP. Correlation with light-activated chloroplast responses

Background  

The actin cytoskeleton is involved in the responses of plants to environmental signals. Actin bundles play the role of tracks in chloroplast movements activated by light. Chloroplasts redistribute in response to blue light in the mesophyll cells of Nicotiana tabacum. The aim of this work was to study the relationship between chloroplast responses and the organization of actin cytoskeleton in living tobacco cells. Chloroplast movements were measured photometrically as changes in light transmission through the leaves. The actin cytoskeleton, labeled with plastin-GFP, was visualised by confocal microscopy.     

Induced proteome of Trichoderma harzianum by Botrytis cinerea

As a notable biocontrol agent, Trichoderma harzianum can antagonize a diverse array of phytopathogenic fungi, including Botrytis cinerea, Rhizoctonia solani and Fusarium oxysporum. Elucidating the biocontrol mechanism of T. harzianum in response to the pathogens enables it to be exploited in the control of plant diseases. Two-dimensional gel electrophoresis (2-DE) was performed to obtain secreted protein patterns of T. harzianum ETS 323, grown in media that contained glucose, a mixture of glucose and deactivated B. cinerea mycelia, deactivated B. cinerea mycelia or deactivated T. harzianum mycelia. Selected protein spots were identified using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Ninety one out of 100 excised protein spots were analyzed and some proteins were sequence identified. Of these, one l-amino acid oxidase (LAAO) and two endochitinases were uniquely induced in the media that contained deactivated B. cinerea mycelia as the sole carbon source. Activities of the cell wall-degrading enzymes (CWDEs), including β-1,3-glucanases, β-1,6-glucanases, chitinases, proteases and xylanases, were significantly higher in media with deactivated B. cinerea mycelia than in other media. This finding suggests that the cell wall of B. cinerea is indeed the primary target of T. harzianum ETS 323 in the biocontrol mechanism. The possible roles of LAAO and xylanase were also discussed.     

温度对斯托克通氏烟草雄配子体形成和发育的影响

为探究低温对斯托克通氏烟草(Nicotiana stocktonii)花粉母细胞(PMC)减数分裂及其雄配子体发育过程的影响,采用卡宝品红染色法,研究不同温度条件下该材料雄配子体形成和发育的过程。结果表明:种植于昼温(31±0.5)℃、夜温(11±0.5)℃人工气候箱中的Nicotiana stocktonii花粉母细胞减数分裂过程异常现象较少,出现微核的比率较低,用新鲜成熟的花粉做萌发实验花粉萌发率较高,为(71±3)%; 而种植于昼温(25±0.5)℃、夜温(3±0.5)℃条件下的Nicotiana stocktonii开花后花药大多干瘪,用新鲜成熟花粉做萌发实验花粉萌发率低,为(13.67±3)%,花粉母细胞减数分裂过程出现染色体桥、染色体不同步、染色体断片、落后染色体等现象,存在微核的细胞比率较高。因此,Nicotiana stocktonii花粉母细胞减数分裂与小孢子发育过程易受温度影响,从而影响花粉的可育性。     

Chlorate-resistant variants of Nicotiana tabacum L.

Summary In somatic hybrids of each of four chlorate-resistant cell lines of Nicotiana tabacum L., two of which were also nitrate-nonutilizing, and a common chlorate-sensitive parent, nitrate utilization and/or chlorate resistance were found to be recessive traits. Complementation for nitrate utilization was observed in somatic hybrids of the two nitrate-nonutilizing cell lines. No chlorate-resistant or nitrate-nonutilizing seedlings were detected among a large number of progeny resulting from the self-fertilization of a somatic hybrid plant one of whose parents was one of the nitrate-nonutilizing cell lines.