Transgenic tobacco cultivars resistant to Pseudomonas syringae pv. tabaci

 Six oriental cultivars of tobacco (Nicotiana tabacum L.) were evaluated for transformation and foreign gene expression. Leaf-disc explant tissue was transformed with Agrobacterium tumefaciens strain LBA4404 carrying the plasmid pARK21, which contains NPTII gene and ttr (tabtoxin resistance) gene conferring the resistance to Pseudomonas syringae pv. tabaci. The disease resistance of regenerated plants and segregation of this trait up to R₇ progeny were investigated in a greenhouse and under field conditions. Our results indicated that the resistance to Pseudomonas syringae pv. tabaci introduced by transformation is heritable. Received: 10 June 1997 / Accepted: 31 March 1998     

Transgenic tobaccos transformed with a gene encoding a truncated form of the coat protein of tomato black ring nepovirus are resistant to viral infection

 Tomato black ring virus (TBRV) belongs to the nepoviruses, an important genus of phytoviruses characterized by isometric particles and by their transmission by longidorid nematodes. As for all other nepoviruses, the coat protein (CP) of TBRV is encoded by the 3′ terminal part of the viral RNA2 (positions 2801–4334). A hybrid gene driving the expression of a truncated form of the TBRV CP was constructed. It contains a frameshift deletion at position T₄₀₆₅ so that in the encoded protein the last 90 amino acids of the wild-type CP are replaced by 52 amino acids encoded by a different reading frame of the viral RNA. This hybrid gene was introduced into the genome of Nicotiana tabacum cv 'Xanthi' plants. When compared to control plants, progeny of some transformants expressing the mutated CP gene (CPm+ plants) showed resistance against TBRV infection. This resistance is characterized by a delay in the appearance of symptoms, a reduction in the number of infected plants and a reduction in virus accumulation. Received: 28 February 1997 / Revision received: 1 August 1997 / Accepted: 24 March 1999     

Expression of the blasticidin S deaminase gene (bsr) in tobacco: Fungicide tolerance and a new selective marker for transgenic plants

Summary Blasticidin S (BS), a fungicide of microbial origin, is used for the practical control of rice blast disease. It has broad antimicrobial activity but occasionally exhibits adverse phytotoxic effects on some dicot plants. An inactivating enzyme, BS deaminase, was discovered in the BS resistant strain, Bacillus cereus K55-S1, and the structural gene, bsr, for the enzyme has been cloned. We introduced the bsr gene into tobacco plants using the Ti plasmid vector system and demonstrated that the bsr gene conferred a BS resistant phenotype to the plants. Thus the bsr gene could be useful as a selective marker for plant transformation and provides an example for a new approach to the solution of phytotoxicity problems associated with the use of some types of fungicide.     

Transgenic tobacco plants expressing the bacterial mc gene resist virus infection

A bacterial rnc gene coding for a double-stranded RNA-dependent RNase III endoribonuclease and a mutant, rnc70, were expressed in tobacco plants. The RNase III protein produced in the transgenic plants was the same size as the bacterial protein. Expression of the wild-type gene could cause stunting in some plant lines, but not in others. Expression of the mutant protein did not affect normal growth and development of the transgenic plants. Transgenic plants of the R1 and R2 generations, expressing the wild type, as well as a mutant protein, were resistant to infection by three disparate RNA plant viruses with a divided genome but not against two viruses with a single-stranded RNA genome. Introduction of the rnc gene in crop plants may provide resistance to economically important virus diseases.     

Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Transgenic tobacco expressing fungal laccase promotes the detoxification of environmental pollutants

The phytoremediation of soils contaminated with organic pollutants offers a low-cost method for removal of such pollutants. We have attempted to enhance the environmental decontamination functions of plants by introducing appropriate enzymatic activities from microorganisms. In the present study, we introduced an extracellular fungal enzyme, the laccase of Coriolus versicolor, into tobacco plants. One transgenic plant, designated FL4, produced laccase that was secreted into the rhizosphere. FL4 was able to remove 20 mol bisphenol A or pentachlorophenol per gram dry weight. The efficiency of this removal was apparently greater than that of control lines. Our results should stimulate efforts to develop plant-based technologies for the removal of environmental pollutants from contaminated environments.     

Transgenic sweet orange plants expressing a dermaseptin coding sequence show reduced symptoms of citrus canker disease

Citrus canker provoked by Xanthomonas axonopodis pv. citri is a bacterial disease causing severe losses in all citrus-producing areas around the world. Xanthomonas infection is considered as an endemic disease in Northeast and Northwest Argentina, affecting as much as 10% of commercial citrus plantations. There is not known natural resistance neither in orange varieties nor in rootstocks used for grafting of commercial cultivars. To introduce resistance to this disease, plants of Pineapple sweet orange were transformed with a genetic construct allowing constitutive accumulation of dermaseptin. In comparison with non-transformed plants, transgenic plants showed symptom reduction levels of up to 50% in in planta assays performed under controlled conditions.     

转PvPGIP2基因小麦的获得与纹枯病抗性鉴定

多聚半乳糖醛酸酶抑制蛋白(PGIP)是一种植物防卫蛋白,可阻止一些病原真菌的侵害。本研究克隆出扁豆PvP-GIP2基因编码序列,构建了受玉米泛素(ubiquitin)启动子控制的PvPGIP2基因表达载体pA25-PvPGIP2;采用基因枪法将pA25-PvPGIP2转化小麦推广品种扬麦18幼胚愈伤组织4000块,获得了203株再生植株。PCR检测出阳性植株65株,转化率为1.625%。对转PvPGIP2基因小麦T1～T2植株,进行外源基因的PCR、RT-PCR、荧光定量RT-PCR(Q-RT-PCR)分析和小麦纹枯病抗性鉴定。结果表明,转入的PvPGIP2能够在转基因小麦中遗传、转录与表达;PvPGIP2基因的表达提高了转基因植株对小麦纹枯病的抗性。     

烟草抗CMV突变体的抗病性分析

在突变体植株“Ea201主、Ea201侧、Aa201侧”的5～6叶期接种CMV汁液,并按优选法去除病症最严重的病株,6周后将剩余植株分别移栽到田间露地和防虫网内,到盛花期分别统计植株的感病情况。其结果为：在田间露地和防虫网内,①感病的对照品种的植株高度只及健康的对照植株高度的30％和40％,突变体的则可达到70％和80％;②对照品种的植株感CMV的病情指数可达90％和50％,突变体的则只有30％和3％;③对照品种植株感TMV的病情指数可达60％和30％,突变体植株感TMV的病情指数只有30％和10％;④突变体植株群体中有症状回复现象,同时其育性不受任何影响。从而表明所筛选到的烟草突变体“Ea201主、Ea201侧、Aa201侧”对CMV、TMV的相对抗性较好,具有较高的抗病性。     

Transgenic tobacco plants expressing a coat protein gene of tobacco mosaic virus are resistant to some other tobamoviruses

Transgenic tobacco plants expressing the coat protein (CP) gene of tobacco mosaic virus were tested for resistance against infection by five other tobamoviruses sharing 45-82% homology in CP amino acid sequence with the CP of tobacco mosaic virus. The transgenic plants (CP+) showed significant delays in systemic disease development after inoculation with tomato mosaic virus or tobacco mild green mosaic virus compared to the control (CP-) plants, but showed no resistance against infection by ribgrass mosaic virus. On a transgenic local lesion host, the CP+ plants showed greatly reduced numbers of necrotic lesions compared to the CP- plants after inoculation with tomato mosaic virus, pepper mild mottle virus, tobacco mild green mosaic virus, and Odontoglossum ringspot virus but not ribgrass mosaic virus. The implications of these results are discussed in relation to the possible mechanism(s) of CP-mediated protection.     

Phytodegradation of organophosphorus compounds by transgenic plants expressing a bacterial organophosphorus hydrolase

Organophosphorus (OP) compounds are widely used as pesticides in agriculture but cause broad-area environmental pollution. In this work, we have expressed a bacterial organophosphorus hydrolase (OPH) gene in tobacco plants. An assay of enzyme activity showed that transgenic plants could secrete OPH into the growth medium. The transgenic plants were resistant to methyl parathion (Mep), an OP pesticide, as evidenced by a toxicity test showing that the transgenic plants produced greater shoot and root biomass than did the wild-type plants. Furthermore, at 0.02% (v/v) Mep, the transgenic plants degraded more than 99% of Mep after 14 days of growth. Our work indicates that transgenic plants expressing an OPH gene may provide a new strategy for decontaminating OP pollutants.     

用转基因植物修复重金属污染的土壤

将新的性状转入高生物量植物中,以此开发高效的转基因植物修复系统,用于重金属污染的土壤修复是一项具有广阔应用前景的技术.大量实验表明,将细菌、真菌、动物、人类及植物本身与重金属脱毒相关的基因转入高生物量植物,异源表达产物可介导转基因植物耐受和高积累重金属及类似物.综述了这方面的研究进展.     

Transgenic tobacco cell cultures expressing a Trichoderma harzianum endochitinase gene release the enzyme into the medium

 Calli and cell suspensions were obtained from tobacco plants transformed with an endochitinase-encoding cDNA from the biocontrol fungus Trichoderma harzianum. Calli from four primary transformants had high levels of endochitinase activity, like the plants from which they were derived. Endochitinase activity was also detected in the medium surrounding the calli and in the medium from transgenic cell suspensions. Western blots demonstrated the presence of the expected 40-kDa T. harzianum protein in transgenic samples but not in controls. These results indicate that the fungal enzyme is secreted and that the fungal signal peptide in the cDNA construct functions in plant cells. A cell suspension medium in which the protein concentration was increased up to 34-fold by ammonium sulfate precipitation inhibited germination of Penicillium digitatum spores. Some inhibition of spore germination was also observed in concentrated medium from control suspensions, probably due to the secretion and concentration of endogenous enzymes. Received: 6 May 2000 / Revision received: 6 September 2000 · Accepted: 14 September 2000     

转基因烟草的大田抗病性分析

为了尽快地将抗病转基因烟草品种应用于生产,在选育抗病优良株系的同时,进行了转基因株系的大田抗病性鉴定。结果表明：（１）在田间自然发病情况下,转基因烟草的ＮＣ８９各株系的发病率及病情指数显著低于对照ＮＣ８９,对ＣＭＶ的相对防治效果为５５％－７０％,表现较强的抗病性;同时对ＴＭＶ也有一定的抗病力;（２）转基因烟草的产量、产值也明显高于对照。     

Mice transgenic for the Huntington's disease mutation are resistant to chronic 3-nitropropionic acid-induced striatal toxicity

Neuronal loss in Huntington's disease (HD) is seen first in the neostriatum. It has been suggested that impaired metabolism underlies this degeneration, as striatal vulnerability to excitotoxicity is increased by metabolic compromise. At 12 weeks of age, a transgenic mouse carrying the HD mutation (R6/2 line) has been shown to have an increased vulnerability to the mitochondrial toxin 3-nitropropionic acid (3-NP). However, in contrast, younger R6/2 mice appear to be less vulnerable than wild-type (WT) mice to the excitotoxins kainic acid and quinolinic acid (QA). In this study, we examine the possibility that the sensitivity of R6/2 mice to 3-NP might be age dependent. We treated young, symptomatic R6/2 mice with 3-NP and found that despite their progressive neurological phenotype, they were not more susceptible to 3-NP intoxication than their WT littermates. Further, fewer R6/2 than WT mice developed striatal lesions. We suggest that compensatory mechanisms exist in the R6/2 mouse brain that protect it against the toxic effect of the transgene and coincidentally protect against exogenous toxins such as 3-NP, QA, and kainic acid. The existence of similar compensatory mechanisms may explain why, in humans, HD is a late-onset disorder, despite early expression of the genetic mutation.     

外源基因在转基因植物中的失活

进行植物基因工程研究首先需要获得外源基因稳定表达的转基因植株。近年来,有大量文献报道,转基因植株中外源基因的失活是一种普遍发生的现象。引起外源基因失活的原因是多方面的,外源基因失活在ＤＮＡ修饰水平,基因转录水平和转录后调节水平均可能发生。深入地了解外源基因失活的原因以及寻找出有效的防范对策,对顺利开展植物基因工程育种是非常必要的。     

转WMV-2外壳蛋白基因西瓜植株的病毒抗性

西瓜是夏季的重要水果,病毒病是影响其品质和产量的重要原因之一。植物基因工程的发展为抗病育种提供了新途径。利用外壳蛋白(coat protein)基因转化高等植物,赋予转基因植物以相应抗病性的成功例子已很多。本文报道WMV-2CP基因在自交子一代的分离符合孟德尔3：1的分离比。经过连续4代的选择鉴定,已从T7、T11和T323个独立转化子的后代中筛选获得8个转基因纯合株系,性状表现整齐一致。Western blot结果表明,R4T7-1、T4T11-3以及R4T32-73个不同来源的株系均能表达产生外壳蛋白。转基因纯合株系WMV-2感染后的病毒抗性实验表明,与未转基因对照相比,转基因株系可以推迟发病时间,减轻发病程度。实验筛选获得的转基因株系R4T32-7表现出对WMV-2的高度抗性,为利用植物转基因技术选育抗病新品种奠定了基础。     

Cross protection in transgenic tobacco plants expressing a mild strain of tobacco mosaic virus

Summary Cross protection of plant viruses is a phenomenon in which plants infected with one strain of a virus are protected from the effects of superinfection by other related strains. Recently, we have succeeded in the introduction and expression of a cDNA copy of the tobacco mosaic virus (TMV) genomic RNA in transgenic tobacco plants. Using this system, we introduced a cDNA copy of a mild strain of TMV into tobacco plants. The transgenic plants did not develop any severe symptoms upon inoculation with a virulent TMV strain, indicating that these transgenic plants were cross protected against TMV infection. The system described here can be a useful model system to study the mechanism(s) of cross protection.     

Transgenic fertile japonica rice plants expressing a modified cryIA(b) gene resistant to yellow stem borer

The japonica rice variety Taipei 309 was cotransformed by particle bombardment of immature embryo-derived embryogenic calli with a modified δ-endotoxin gene cryIA(b) of Bacillus thuringiensis (Bt) under the control of the rice Actin1 promoter, and the hygromycin resistance gene, hph driven by the CaMV35S promoter. Selected transgenic rice plants showed enhanced insecticidal activity against yellow stem borer (Scirpophaga incertulas), with mortality rates reaching up to 100% in a bioassay with cut stems. Introduction and expression of the Actin1 promoter-Bt gene into rice provides japonica rice germplasm resistant to insect attack. Received: 21 March 1997 / Revision received: 23 June 1997 / Accepted: 5 July 1997