Over-expression of <Emphasis Type="Italic">CsGSTU</Emphasis> promotes tolerance to the herbicide alachlor and resistance to <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> pv. <Emphasis Type="Italic">tabaci</Emphasis> in transgenic tobacco

Glutathione transferases (GSTs) mainly catalyze the nucleophilic addition of glutathione to a large variety of hydrophobic molecules participating to the vacuole compartmentalization of many toxic compounds. In this work, the putative tolerance of transgenic tobacco plants over-expressing CsGSTU genes towards the chloroacetanilide herbicide alachlor was investigated. Our results show that the treatment with 0.0075 mg cm^-3 of alachlor strongly affects the growth of both wild type and transformed tobacco seedlings with the sole exception of the transgenic lines overexpressing CsGSTU2 isoform that are barely influenced by herbicide treatment. In order to correlate the in planta studies with enzyme properties, recombinant CsGSTs were in vitro expressed and tested for GST activity using alachlor as substrate. The recombinant GSTU2 enzyme was twice more active than GSTU1 in conjugating alachlor to GSH thus indicating that CsGSTU2 might play a crucial role in the plant defense against the herbicide. Moreover, as a consequence of the infiltration with a bacterial suspension of the P. syringae pv. tabaci, transgenic tobacco plants but not wild type plants bestowed the capability to limit toxic metabolite diffusion through plant tissues as indicated by the absence of chlorotic halos formation. Consequently, the transgenic tobacco plants described in the present study might be utilized for phytoremediation of residual xenobiotics in the environment and might represent a model for engineering plants that resist to pathogen attack.     

抗草甘膦抗虫植物表达载体的构建及其转基因烟草的分析

构建了含草甘膦抗性突变基因（aroAM12）和人工合成重组Bt抗虫基因（Bts1m）的植物表达载体pCM12_s1m。aroAM12基因的表达由CaMV35S启动子控制,Bts1m基因的表达由2E_CaMV35S启动子和Ω因子控制。通过农杆菌介导,将aroAM12和Bts1m基因转化到烟草中,转基因烟草通过在含草甘膦的MS培养基上筛选而获得。Southern blot分析表明所有经过草甘膦筛选出的转化植株都整合有aroAM12基因,约70%的转化植株同时整合有aroAM12和Bts1m基因。Northern blot、Immunodot blot分析进一步证明整合的两个基因在转录、翻译水平上均进行了表达,不同植株之间表达存在着差异。草甘膦抗性和虫试实验证明,获得的转基因烟草对草甘膦和烟青虫具有很强的抗性。     

Characterization of Glutathione S-Transferase Isoforms in Three Maize Inbred Lines Exhibiting Differential Sensitivity to Alachlor

Glutathione S-transferases (GSTs) are a family of isozymes that catalyze the conjugation of glutathione to several xenobiotics, including a number of important herbicides. Several GST isoforms have been identified in maize (Zea mays L.). In this study we focused on three isoforms, GST I, II, and IV, derived from homo-or heterodimerization of two subunits GST-29 and GST-27, which have been shown to be responsible for reactivity to alachlor. The expression of these isoforms was examined in three inbred lines of maize that showed tolerance, susceptibility, and intermediate resistance to alachlor (2-Cl-N-[2,6-diethylphenyl]-N-[methoxymethyl]acetamide) treatment. The different isoforms were separated by anion-exchange chromatography and subunits were quantified by western blot analysis. GST assays were performed against both 1-Cl-2,4-dinitrobenzene and alachlor. This analysis showed that the susceptible and intermediate lines exhibit impaired function in the GST-27 and GST-29 subunits, respectively. In addition, this study suggests that GST IV is the principal, detoxifying enzyme for alachlor, although GST I and II are required to achieve tolerance to high rates of the herbicide.     

The expression of a maize glutathione S-transferase gene in transgenic wheat confers herbicide tolerance,both in planta and in vitro

Maize (Zea mays), in common with a number of other important crop species, has several glutathione S-transferase (GST) isoforms that have been implicated in the detoxification of xenobiotics via glutathione conjugation. A cDNA encoding the maize GST subunit GST-27, under the control of a strong constitutive promoter, was introduced into explants of the wheat (Triticum aestivum L.) lines cv. Florida and L88-31 via particle bombardment, using the phosphinothricin acetyltransferase (pat) gene as a selectable marker. All six independent transgenic wheat lines recovered expressed the GST-27 gene. T₁ progeny of these wheat lines were germinated on solid medium containing the chloroacetanilide herbicide alachlor, and tolerance to this herbicide was correlated with GST-27 expression levels. In glasshouse sprays, homozygous T₂ plants were resistant not only to alachlor but also to the chloroacetanilide herbicide dimethenamid and the thiocarbamate herbicide EPTC. These additional GST-27 activities, demonstrated via over-expression in a heterologous host, have not been described previously. T₂ plants showed no enhanced tolerance to the herbicides atrazine (an s-triazine) or oxyfluorfen (a diphenyl ether). In further experiments, T₂ wheat plants were recovered from immature transgenic scutella cultured on medium containing 100 mg/l alachlor, a concentration which killed null segregant and wild-type scutella. These data indicate the potential of the maize GST-27 gene as a selectable marker in wheat transformation.     

Green fluorescent protein as a marker for expression of a second gene in transgenic plants.

The use of transgenic crops has generated concerns about transgene movement to unintended hosts and the associated ecological consequences. Moreover, the in-field monitoring of transgene expression is of practical concern (e.g., the underexpression of an herbicide tolerance gene in crop plants that are due to be sprayed with herbicide). A solution to these potential problems is to monitor the presence and expression of an agronomically important gene by linking it to a marker gene, such as GFP. Here we show that GFP fluorescence can indicate expression of the Bacillus thuringiensus cry1Ac gene when co-introduced into tobacco and oilseed rape, as demonstrated by insect bioassays and western blot analysis. Furthermore we conducted two seasons of field experiments to characterize the performance of three different GFP genes in transgenic tobacco. The best gene tested was mGFP5er, a mutagenized GFP gene that is targeted to the endoplasmic reticulum. We also demonstrated that host plants synthesizing GFP in the field suffered no fitness costs.     

Overexpression of glutathione S-transferase gene increases salt tolerance of arabidopsis

The Suaeda salsa glutathione S-transferase gene (GST) was introduced into arabidopsis under the control of the cauliflower mosaic virus 35S promoter. Transformants were selected for their ability to grow on medium containing kanamycin. Southern and northern blot analyses confirmed that GST was transferred into the arabidopsis genome, and the GST and GPX activities in transgenic plants (GT) were much higher than in wild-type plants (WT). There were no obvious morphological or developmental differences between transgenic and wild-type plants. One transgenic homozygous line (GT_6–8) and WT plants were evaluated for salt tolerance and gene expression. Seed germination and seedling salt tolerance were improved after overexpression of GST in arabidopsis; the photosynthesis rate and the fresh weight of the GT_6–8 line were distinctly higher than those of WT plants after NaCl treatment. Glutathione content increased substantially in salt-stressed arabidopsis plants of both genotypes, and the glutathione pool in GT_6–8 plants was more oxidized than in WT plants under both control and stressful conditions. The MDA content, an indicator of lipid peroxidation, increased in WT plants but was not affected distinctly in GT_6–8 seedlings after NaCl treatment. Results from different tests indicated that the expression of the GST gene promoted a higher level of salt tolerance in vivo in transgenic arabidopsis plants.     

Production of transgenic male sterile tobacco plants with the cDNA encoding a ribosome inactivating protein in Dianthus sinensis L.

The ribosome inactivating protein (RIP) gene from D. sinensis was used as a cytotoxin gene to induce male sterility in tobacco plants. The TA29 promoter, obtained by PCR amplification from tobacco, was fused to the RIP cDNA, and the chimaeric molecule was then introduced into tobacco plants by Agrobacterium-mediated transformation. Out of twenty-one independent transformants, twenty transgenic tobacco plants exhibited male sterility. Southern blot analysis revealed that four of the transgenic plants contained a single copy of the RIP gene, while the rest of the transgenic tobacco plants had two to four copies of the gene. The transgenic male sterile plants set seeds normally when pollinated with pollens from untransformed control plants, indicating that the RIP gene does not affect the pistil development. Furthermore, the seed yield of the transgenic plant was similar to that of the untransformed, self-pollinated control plant. A light microscopic observation of anther cross sections clearly showed that the tapetal tissue of the anther was selectively and completely destroyed causing male sterility. This study suggests that the RIP gene can be used as a cytotoxin gene for induction of male sterility in the plant.     

Cloning and expression of a cDNA encoding a maize glutathione-S-transferase in E. coli.

The isolation and characterization of a family of maize glutathione-S-transferases (GST's) has been described previously. These enzymes are designated GSTs I, II and III based on size, substrate specificity and responsiveness to safeners. GST III has been shown to act on the herbicide alachlor as well as the commonly used substrate 1-chloro-2,4-dinitrobenzene (CDNB). Clones were isolated from a maize cDNA library in lambda gt10. Three clones contained the entire coding region for GST III. The sequences of these clones were consistent with the known amino terminal GST III protein sequence. Moreover, expression of one of these clones in E. coli resulted in a GST activity as measured with both CDNB and alachlor, proving that at least one of the clones encodes an active GST III species. With the enzyme expressed in E. coli it will become possible to study enzyme structure-function relationships ex planta. While a number of different GST proteins are present in maize tissue the GST III gene is present in single or low copy in the genome.     

Characterization of Transgenic Tobacco Plants Containing Bacterial bphc Gene and Study of Their Phytoremediation Ability

Genetically modified plants can serve as an efficient tool for remediation of diverse dangerous pollutants of the environment such as pesticides, heavy metals, explosives and persistent organic compounds. Transgenic lines of Nicotiana tabacum containing bacterial bphC gene from the degradation pathway of polychlorinated biphenyls (PCBs) were tested. The product of the bphC gene – enzyme 2,3-dihydroxybiphenyl-1,2-dioxygenase is responsible for cleaving of the biphenyl ring. The presence of bphC gene in transgenic plants was detected on DNA, RNA and protein level. The expression of the bphC/His gene was verified after purification of the enzyme from plants by affinity chromatography followed by a Western blot and immunochemical assay. The enzyme activity of isolated protein was detected.Efficient transformation of 2,3-DHB by transgenic plants was achieved and the lines also exhibited high production of biomass. The transgenic plants were more tolerant to the commercial PCBs mixture Delor 103 than non-transgenic tobacco. And finally, the higher decrease of total PCB content and especially congener 28 in real contaminated soil from a dumpsite was determined after cultivation of transgenic plant in comparison with non-transgenic tobacco. The substrate specificity of transgenic plants was the same as substrate specificity of BphC enzyme.     

Overexpression of AtFRO6 in transgenic tobacco enhances ferric chelate reductase activity in leaves and increases tolerance to iron-deficiency chlorosis

The Arabidopsis gene FRO6(AtFRO6) encodes ferric chelate reductase and highly expressed in green tissues of plants. We have expressed the gene AtFRO6 under the control of a 35S promoter in transgenic tobacco plants. High-level expression of AtFRO6 in transgenic plants was confirmed by northern blot analysis. Ferric reductase activity in leaves of transgenic plants grown under iron-sufficient or iron-deficient conditions is 2.13 and 1.26 fold higher than in control plants respectively. The enhanced ferric reductase activity led to increased concentrations of ferrous iron and chlorophyll, and reduced the iron deficiency chlorosis in the transgenic plants, compared to the control plants. In roots, the concentration of ferrous iron and ferric reductase activity were not significantly different in the transgenic plants compared to the control plants. These results suggest that FRO6 functions as a ferric chelate reductase for iron uptake by leaf cells, and overexpression of AtFRO6 in transgenic plants can reduce iron deficiency chlorosis.     

Production of HIV-1 p24 protein in transgenic tobacco plants

The production of antigens for vaccines in plants has the potential as a safe and cost-effective alternative to traditional production systems. Toward the development of a plant-based expression system for the production of human immunodeficiency virus type I (HIV-1) p24 capsid protein, the p24 gene was introduced into the genome of tobacco plants using Agrobacterium tumefaciens-mediated gene transfer. Southern blot analyses confirmed the presence of the p24 coding sequence within the genome of transgenic lines. Western blot analysis of protein extracts from transgenic plants identified plant-expressed p24 protein that cross-reacted with a p24-specific monoclonal antibody, thus confirming the maintenance of antigenicity. Quantification of the p24 protein using enzyme-linked immunosorbent assay (ELISA) estimated yields of approx 3.5 mg per g of soluble leaf protein. Similar accumulation levels of p24 were also detected in T1 plants, confirming that the p24 gene is transmitted stably. Our results indicate that plant-based transgenic expression represents a viable means of producing p24 for the development of HIV vaccine and for use in HIV diagnostic procedures.     

Study on the biochemical characterization of herbicide detoxification enzyme, glutathione S-transferase

To gain further insight into herbicide detoxification, we studied the herbicide activity and specificity toward glutathione S-transferases from human and rice. In this study, the genes of the plant specific phi and tau class GST enzymes from Oryza sativa (OsGST) and human pi class GST enzyme (hGSTP1-1) were cloned and expressed in Escherichia coli with the pET and pKK vector systems, respectively. The gene products were purified to homogeneity by GSH Sepharose affinity column chromatography. The herbicide specificity of the enzymes was investigated by enzyme-catalyzed conjugation of GSH with chloroacetanilide, diphenylether and chloro-s-triazine herbicides. The hGSTP1-1 showed very high specific activity toward atrazine. On the other hand, the phi class OsGST enzymes showed high specific activity toward chloroacetanilide herbicides, acetochlor, alachlor and metolachlor. The tau class GST enzymes displayed remarkable activity toward the diphenylether herbicide, fluorodifen. From these results, we conclude that the phi and the tau class GST enzymes show herbicide specificities and also they play an important role in the detoxification reaction of plant toward herbicides.     

Inhibition of Ethylene Biosynthesis Enhances Vegetative Bud Formation without Affecting Growth and Development of Transgenic Tobacco Plants

The role of ethylene in vegetative bud formation was investigated using transgenic tobacco plants expressing an antisense tomato 1-aminocyclopropane-carboxylic acid synthase (ACS) gene. Northern blot hybridization showed that the accumulation of ACS mRNA was strongly reduced in the bud-forming leaf explants of the transgenic plants. Consequently, these transgenic tissues exhibited low ACS enzyme activity, 1-aminocyclopropane-carboxylic acid (ACC) content and ethylene production, and at the same time the tissue capacity to generate buds was greatly enhanced. However, it was also noted that the antisense ACS gene did not inhibit the endogenous ACS gene expression in intact transgenic tobacco plants. The growth and development of the transgenic tobacco was almost identical to control plants with respect to height, internode number, leaf morphology, and flowering time. Furthermore, mature leaves of transgenic tobacco had similar chlorophyll content, stomatal conductance, photosynthetic ability, and transpiration rates compared to control plants. These results demonstrated that ethylene plays an important role in bud formation in tobacco tissue culture.     

干旱耐逆基因（HS1）转化甘薯获得转基因植株

以甘薯（1pomoeabatatas（L.）Lam．）品种栗子香的胚性悬浮细胞为受体材料,用根癌农杆菌介导法,获得了表达除草剂抗性基因bar基因的转HSl基因甘薯植株。共计380个遗传转化的胚性细胞团,在添加2mg／L2．4-D、100mg／L Carb和10mg／L Glu（glufosinate）的固体Ms培养基上选择培养9周后,得到了12个Glu抗性愈伤组织。将这些抗性愈伤组织转移到添加1mg／L ABA、100mg／L羧苄青霉素和10mg／L Glu的固体MS培养基上,其中的3个抗性愈伤组织再生出拟转基因植株。PCR鉴定它们为转基因植株。Southern blot分析表明,HS1基因已整合到基因组中。转基因植株具有稳定的除草剂抗性。结薯观察实验结果表明,转基因植株结薯正常。     

Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.     

Transgenic Tobacco Plants Constitutively Overexpressing a Rice Thaumatin-like Protein (PR-5) Show Enhanced Resistance to Alternaria alternata

Overexpression of antifungal pathogenesis-related (PR) proteins in crop plants has the potential for enhancing resistance against fungal pathogens. Thaumatin-like proteins (TLPs) are one group (PR-5, permatins) of antifungal PR-proteins isolated from various plants. In the present study, a plasmid containing a cDNA of rice tlp (D34) under the control of the CaMV-35S promoter was introduced into tobacco plants through Agrobacterium-mediated transformation system. A considerable overproduction of TLP was observed in transformed tobacco plants by Western blot analysis. There was a large accumulation of tlp mRNA in transgenic plants as revealed by Northern blot analysis. Southern blot analysis of the DNA from transgenic tobacco plants confirmed the presence of the rice tlp gene in the genomic DNA of transgenic tobacco plants. Immunoblot analysis of intracellular and extracellular proteins of transgenic tobacco leaves using a Pinto bean TLP antibody demonstrated that the 23-kDa TLP was secreted into the extracellular matrix. T₂ progeny of regenerated plants transformed with TLP gene were tested for their disease reaction to Alternaria alternata, the brown spot pathogen. Transgenic tobacco plants expressing TLP at high levels showed enhanced tolerance to necrotization caused by the pathogen. This revised version was published online in July 2006 with corrections to the Cover Date.     

雪花莲凝集素基因（gna）的改造及其抗蚜性

用定点突变方法对编码雪花莲凝集素（Galanthus nivalis agglutinin,GNA)前体蛋白的DNA序列进行了改造和转基因烟草9Nicotana tabacum L.）抗蚜性的研究。结果表明,将GNA编码序列中含有的稀有密码子改造后,GNA的表达水平从占总可溶性蛋白的0.17％增加到0.25％,转基因烟草的抗蚜性也随之增强,从平均抑制桃蚜（Myzus per-sicae(Sulzer））虫口密度63.7％显地提高到71.0％。     

Manipulation of plant tolerance to herbicides through co-ordinated metabolic engineering of a detoxifying glutathione transferase and thiol cosubstrate

The diphenyl ether herbicide fomesafen can be used selectively in soybean (Glycine max) due to its rapid detoxification by tau class glutathione transferases (GmGSTUs) which preferentially utilize the endogenous thiol homoglutathione (hGSH) as cosubstrate. Soybean cDNAs encoding GmGSTU21, which is highly active in detoxifying fomesafen, and an hGSH synthetase (GmhGS) have been cloned and functionally identified in Escherichia coli. Tobacco plants, which have limited GST activities towards fomesafen and which accumulate glutathione (GSH), rather than hGSH, have been transformed with either GmhGS alone, or a dual construct of GmhGS-GmGSTU21, both under the control of constitutive promoters. Using either construct, the transgenic tobacco accumulated hGSH, with a concomitant increase in GSH content. Segregating T1 plants were analysed for thiol content and GST activity towards fomesafen with GSH and hGSH as cosubstrates, and then scored for photobleaching injury caused by applications of fomesafen. These studies showed that hGSH accumulation alone gave no significant protection against the herbicide and that tolerance was only seen in plants which contained appreciable concentrations of hGSH and GmGSTU21 activity. Tolerance in the dual transformants was associated with the metabolism of radiolabelled fomesafen to inactive hGSH-derived conjugates, while susceptible lines were unable to detoxify the herbicide. These studies confirm the combined importance of specific GSTs and their preferred thiol cosubstrates in conferring herbicide selectivity traits in planta.     

Expression of the glutathione <Emphasis Type="Italic">S</Emphasis>-transferase gene (NT107) in transgenic <Emphasis Type="Italic">Dianthus superbus</Emphasis>

Using an Agrobacterium-mediated transformation method based on wounding cultured immature seeds with carborundum (600 mesh) in liquid, auxin-regulated tobacco glutathione S -transferase (GST) (NT107) constructs were used to transform Dianthus superbusL. A 663 bp DNA band was found in the transgenic plant genome by PCR analysis using NT107-1 and NT107-2 primers, and a Southern blot analysis showed that the DIG-labelled GST gene was hybridized to the expected amplified genomic DNA fragment from transgenic D. superbus. An overexpression of NT107 led to a twofold increase in GST-specific activity compared to the non-transgenic control plants, and the GST overexpression plants showed an enhanced acclimatization in the soil. To investigate whether an increased expression of GST could affect the resistance of photosynthesis to environmental stress, these plants were subjected to drought and various light intensities from 100 to 3000 mol m^–2s^–1. Copper accumulation and the translocation rate were also analysed in the transgenic lines, and the GST overexpression plants were found to synthesize phytochelatin (PC), which functions by sequestering and detoxifying excess copper ions.These two authors contributed equally to this work