麻疯树逆境蛋白(curcin 2)基因在烟草中的表达

麻疯树（Jatropha curcas）幼苗在干旱、高低温胁迫和真菌浸染下,其叶片中诱导产生了一种新的毒蛋白curcin 2。这意味着curcin 2在其它植物中的异源表达可能会增强植物对外界胁迫的抵抗。curcin 2 cDNA的两个片断：cur2p片断（编码前成熟蛋白）和cur2m片断（编码成熟蛋白）,通过农杆菌的介导分别转化烟草并获得转基因植株。但是,只有在插入了cur2p片断的烟草中检测到了curcin 2蛋白的表达。同时,curcin 2在烟草中的表达增强了植株对烟草花叶病毒（TMV）的抗性。     

A ribozyme gene and an antisense gene are equally effective in conferring resistance to tobacco mosaic virus on transgenic tobacco

Ribozymes of the hammerhead class can be designed to cleave a target RNA in a sequence-specific manner and can potentially be used to specifically modulate gene activity. We have targeted the tobacco mosaic virus (TMV) genome with a ribozyme containing three catalytic hammerhead domains embedded within a 1 kb antisense RNA. The ribozyme was able to cleave TMV RNA at all three target sites in vitro at 25°C. Transgenic tobacco plants were generated which expressed the ribozyme or the corresponding antisense constructs directed at the TMV genome. Six of 38 independent transgenic plant lines expressing the ribozyme and 6 of 39 plant lines expressing the antisense gene showed some level of protection against TMV infection. Homozygous progeny of some lines were highly resistant to TMV; at least 50% of the plants remained asymptomatic even when challenged with high levels of TMV. These plants also displayed resistance to infection with TMV RNA or the related tomato mosaic virus (ToMV). In contrast, hemizygous plants of the same lines displayed only very weak resistance when inoculated with low amounts of TMV and no resistance against high inoculation levels. Resistance in homozygous plants was not overcome by a TMV strain which was altered at the three target sites to abolish ribozyme-mediated cleavage, suggesting that the ribozyme conferred resistance primarily by an antisense mechanism.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).     

High-level expression of the neutralizing epitope of porcine epidemic diarrhea virus by a tobacco mosaic virus-based vector

Porcine epidemic diarrhea virus (PEDV) causes acute enteritis in pigs of all ages and is often fatal for neonates. A tobacco mosaic virus (TMV)-based vector was utilized for the expression of a core neutralizing epitope of PEDV (COE) for the development of a plant-based vaccine. In this study, the coding sequence of a COE gene was optimized based on the modification of codon usage in tobacco plant genes and the removal of mRNA-destabilizing sequences. The native and synthetic COE genes were cloned into TMV-based vectors and expressed in tobacco plants. The recombinant COE protein constituted up to 5.0% of the total soluble protein in the leaves of tobacco plants infected with the TMV-based vector containing synthetic COE gene, which was approximately 30-fold higher than that in tobacco plants infected with TMV-based vector containing a native COE gene. Therefore, this result indicates that the plant viral expression system with a synthetic gene optimized for plant expression is suitable to produce a large amount of antigen for the development of plant-based vaccine rapidly.     

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.     

杆状病毒p35基因诱导烟草产生广谱抗病机理分析

来源于昆虫病毒和动物的抗细胞凋亡基因能够诱导植物对生物或者非生物胁迫产生抗性.但其抗性机理有不同甚至相反的报道.本研究将来源于苜蓿银纹夜蛾核多角体病毒的p35基因转化烟草,T1代转化烟草Western blotting检测P35蛋白的表达,转化烟草接种烟草花叶病毒(Tobacco mosaic virus,TMV)抗病效果增强.进一步的抗病机理研究表明,转化和野生型烟草感染TMV后诱导过氧化氢积累无明显区别,野生型烟草感染24 h后出现DNA Laddering而转化烟草则没有;Western blotting结果显示PR-1蛋白表达没有显著差异.但接种另外一种病原真菌核盘茵(Sclerotiniasclerotiorum)后的RT-PCR分析结果表明,表达P35蛋白的烟草可增强感染核盘菌后PR-1基因的转录.而且表达时间提前.以上结果说明p35基因介导的广谱抗病反应的机理与接种的不同病原有关,对不同病原物的抗病机理存在差异,除抑制细胞凋亡外,还可能通过激活PR基因的表达提高对病原物的抗病能力.     

Salicylic acid has cell-specific effects on tobacco mosaic virus replication and cell-to-cell movement

Tobacco mosaic virus (TMV) and Cucumber mosaic virus expressing green fluorescent protein (GFP) were used to probe the effects of salicylic acid (SA) on the cell biology of viral infection. Treatment of tobacco with SA restricted TMV.GFP to single-epidermal cell infection sites for at least 6 d post inoculation but did not affect infection sites of Cucumber mosaic virus expressing GFP. Microinjection experiments, using size-specific dextrans, showed that SA cannot inhibit TMV movement by decreasing the plasmodesmatal size exclusion limit. In SA-treated transgenic plants expressing TMV movement protein, TMV.GFP infection sites were larger, but they still consisted overwhelmingly of epidermal cells. TMV replication was strongly inhibited in mesophyll protoplasts isolated from SA-treated nontransgenic tobacco plants. Therefore, it appears that SA has distinct cell type-specific effects on virus replication and movement in the mesophyll and epidermal cell layers, respectively. Thus, SA can have fundamentally different effects on the same pathogen in different cell types.     

Intracellular expression of TMV-specific single-chain Fv fragments leads to improved virus resistance in shape Nicotiana tabacum

We evaluated the concept for protection of plants against virus infection based on the expression of single-chain Fv (scFv) fragments in the apoplasm or cytosol of transgenic plants. Cloned cDNA of a tobacco mosaic virus (TMV)-specific scFv antibody, which binds to intact virions, was integrated into the plant expression vector pSS and used for Agrobacterium-mediated transformation of Nicotiana tabacum cv. Xanthi-nc. Regenerated transgenic tobacco plants were analysed by northern blot, western blot and ELISA to assess expression and functionality of recombinant antibody (rAb) fragments. A significant increase of scFv levels in T₁ progeny was obtained for plants secreting apoplastic scFv antibodies but not for scFvs expressed in the cytosol. Bioassays revealed that T₁ progeny producing scFvs in different plant cell compartments showed different levels of resistance upon inoculation with TMV. The most dramatic reduction of necrotic local lesion numbers upon virus infection was observed in T₁ plants expressing scFv fragments in the cytosol. Infectivity could be reduced by more than 90%, despite the observation that protein expression levels for functional scFv antibodies were very low. Furthermore, upon inactivation of the N-resistance gene at elevated temperature, a significant portion of the T₁ progenies inhibited systemic virus spread, indicating that expression of TMV-specific cytosolic scFvs confers virus resistance in these transgenic plants. Moreover, inoculation of protoplasts isolated from transgenic and non-transgenic tobacco plants with TMV-RNA demonstrated that accumulation of virus particles is affected by cytosolic scFv expression.     

Expression of active hBMP2 in transgenic tobacco plants

Bone morphogenetic protein 2 (BMP2) is important for bone tissue repair. The goal of this research is to construct a high level human BMP2 (hBMP2) expression system using transgenic tobacco plants as a bioreactor. Cauliflower mosaic virus (CaMV) 35S promoter, alfalfa mosaic virus (AMV) enhancer, tobacco mosaic virus (TMV) enhancer, matrix attachment regions (MARs) sequence, and “Kozak” sequence were used to construct recombinant expression vectors and the high-expression vectors were screened out through GUS-fusions assay. The promoter is the most important factor; double-CaMV 35S promoter is more effective than single promoter. The AMV or TMV enhancer is able to promote the foreign protein expression. After four-step purification, the activated hBMP2 (0.02% total soluble protein) was obtained. Our results suggested that the transgenic tobacco has great potential to be used as a bioreactor to produce hBMP2.     

Analysis ofcis-regulatory elements involved in induction of a tobacco PR-5 gene by virus infection

cis-Regulatory elements involved in tobacco mosaic virus (TMV)-inducible expression were indentified in a tobacco PR-5 gene, encoding an acidic thaumatin-like protein. By fusing upstream sequences of the PR-5 gene to the GUS reporter gene and analysing transgenic plants containing these fusions for local and systemic induction of GUS activity by TMV, it was found that sequences between-1364 and-718 are involved in TMV induction of PR-5 gene expression.     

Purified cholera toxin B subunit from transgenic tobacco plants possesses authentic antigenicity

Cholera toxin B subunit (CTB) mature protein was stably expressed in transgenic tobacco plants under the control of the CaMV 35S promoter and TMV Omega fragment. Fusion of the PR1b signal peptide coding sequence to the CTB mature protein gene increased the expression level by 24-fold. The tobacco-synthesized CTB (tCTB) was purified to homogeneity by a single step of immunoaffinity chromatography. The purified tCTB is predominantly in the form of pentamers with molecular weight identical to the native pentameric CTB, indicating that the PR1b-CTB fusion protein has been properly processed in tobacco cells. Furthermore, by immunodiffusion and immunoelectrophoresis, we have shown that the antigenicity of the purified tCTB is indistinguishable from that of the native CTB protein.     

Novel N gene-associated,temperature-independent resistance to the movement of tobacco mosaic virus vectors neutralized by a cucumber mosaic virus RNA1 transgene

The N gene conditions for resistance to Tobacco mosaic virus (TMV) but only below 28 degrees C. However, a TMV-based vector expressing green fluorescent protein (TMV-GFP) showed only limited movement at 33 degrees C in tobacco plants harboring the N gene and other genes cointrogressed from Nicotiana glutinosa. TMV-GFP moved efficiently in tobacco plants that either lacked these genes or that contained the N gene but were transgenic for RNA1 of Cucumber mosaic virus. These findings identified novel temperature-independent resistance to the movement of TMV-GFP which could be neutralized by a different viral transgene. Using the N gene and nahG gene-transgenic tobacco, we show that this novel resistance is manifested specifically by the N gene itself and operates via a pathway independent of salicylic acid.     

A dysfunctional movement protein of tobacco mosaic virus that partially modifies the plasmodesmata and limits virus spread in transgenic plants

A chimeric gene encoding a dysfunctional tobacco mosaic virus (TMV) movement protein (MP) mutant lacking amino acids 3, 4 and 5 (MPΔ3–5), was expressed in transgenic Nicotiana tabacum Xanthi and Xanthi NN plants. Immunogold labeling studies of tissues from transgenic plants indicated that while wild-type MP accumulated in the plasmodesmata, MPΔ3–5 did not. Tissue fractionation studies confirmed that only a low level of the mutant MP accumulated in the cell wall-enriched fraction compared with the accumulation of the wild-type MP. Dye coupling studies showed that MPΔ3–5 enabled the movement between leaf mesophyll cells of a fluorescently labeled dextran of 3 kDa, while 9.4 kDa molecules failed to move. In contrast, in transgenic plants expressing the wild-type MP gene the 9.4 kDa probe did move from cell to cell. Seedlings from self-fertilized transgenic plants were inoculated with TMV and observed for disease symptoms. Transgenic Xanthi NN plants that expressed the MPΔ3–5 gene developed fewer and smaller necrotic local lesions compared with control plants following inoculation with TMV. Transgenic Xanthi nn plants were delayed in the development of systemic symptoms. Inoculating the transgenic plants with TMV-RNA, and the tobamo-viruses TMGMV and SHMV, essentially produced the same results, i.e. inhibition of disease development. These results demonstrate that transgenic plants expressing an inactive MP can inhibit virus disease spread presumably by interfering with its cell-to-cell movement.     

Expression of modified gene encoding functional human α-1-antitrypsin protein in transgenic tomato plants

Transgenic plants offer promising alternative for large scale, sustainable production of safe, functional, recombinant proteins of therapeutic and industrial importance. Here, we report the expression of biologically active human alpha-1-antitrypsin in transgenic tomato plants. The 1,182 bp cDNA sequence of human AAT was strategically designed, modified and synthesized to adopt codon usage pattern of dicot plants, elimination of mRNA destabilizing sequences and modifications around 5' and 3' flanking regions of the gene to achieve high-level regulated expression in dicot plants. The native signal peptide sequence was substituted with modified signal peptide sequence of tobacco (Nicotiana tabacum) pathogenesis related protein PR1a, sweet potato (Ipomoea batatas) sporamineA and with dicot-preferred native signal peptide sequence of AAT gene. A dicot preferred translation initiation context sequence, 38 bp alfalfa mosaic virus untranslated region were incorporated at 5' while an endoplasmic reticulum retention signal (KDEL) was incorporated at 3' end of the gene. The modified gene was synthesized by PCR based method using overlapping oligonucleotides. Tomato plants were genetically engineered by nuclear transformation with Agrobacterium tumefaciens harbouring three different constructs pPAK, pSAK and pNAK having modified AAT gene with different signal peptide sequences under the control of CaMV35S duplicated enhancer promoter. Promising transgenic plants expressing recombinant AAT protein upto 1.55% of total soluble leaf protein has been developed and characterized. Plant-expressed recombinant AAT protein with molecular mass of around approximately 50 kDa was biologically active, showing high specific activity and efficient inhibition of elastase activity. The enzymatic deglycosylation established proper glycosylation of the plant-expressed recombinant AAT protein in contrast to unglycosylated rAAT expressed in E. coli ( approximately 45 kDa). Our results demonstrate feasibility for high-level expression of biologically active, glycosylated human alpha-1-antitrypsin in transgenic tomato plants.     

A role for inositol hexakisphosphate in the maintenance of basal resistance to plant pathogens

Phytic acid (myo-inositol hexakisphosphate, InsP6) is an important phosphate store and signal molecule in plants. However, low-phytate plants are being developed to minimize the negative health effects of dietary InsP6 and pollution caused by undigested InsP6 in animal waste. InsP6 levels were diminished in transgenic potato plants constitutively expressing an antisense gene sequence for myo-inositol 3-phosphate synthase (IPS, catalysing the first step in InsP6 biosynthesis) or Escherichia coli polyphosphate kinase. These plants were less resistant to the avirulent pathogen potato virus Y and the virulent pathogen tobacco mosaic virus (TMV). In Arabidopsis thaliana, mutation of the gene for the enzyme catalysing the final step of InsP6 biosynthesis (InsP5 2-kinase) also diminished InsP6 levels and enhanced susceptibility to TMV and to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae. Arabidopsis thaliana has three IPS genes (AtIPS1-3). Mutant atips2 plants were depleted in InsP6 and were hypersusceptible to TMV, turnip mosaic virus, cucumber mosaic virus and cauliflower mosaic virus as well as to the fungus Botrytis cinerea and to P. syringae. Mutant atips2 and atipk1 plants were as hypersusceptible to infection as plants unable to accumulate salicylic acid (SA) but their increased susceptibility was not due to reduced levels of SA. In contrast, mutant atips1 plants, which were also depleted in InsP6, were not compromised in resistance to pathogens, suggesting that a specific pool of InsP6 regulates defence against phytopathogens.