Distinguishing transgenic from non-transgenic Arabidopsis plants by 1H NMR-based metabolic fingerprinting

We have recently reported the construction of an nuclear magnetic resonance （NMR）-based metabonomics study platform, Automics. To examine the application of Automics in transgenic plants, we performed metabolic fingerprinting analysis, i.e., 1H NMR spectroscopy and multivariate analysis, on wild-type and transgenic Arabidopsis. We found that it was possible to distinguish wild-type from four transgenic plants by PLS-DA following application of orthogonal signal correction （OSC）. Scores plot following OSC clearly demonstrates significant variation between the transgenic and non-transgenic groups, suggesting that the metabolic changes among wild-type and transgenic lines are possibly associated with transgenic event, We also found that the major contributing metabolites were some specific amino acids （i.e., threonine and alanine）, which could correspond to the insertion of the selective marker BAR gene in the transgenic plants. Our data suggests that NMR-based metabonomics is an efficient method to distinguish fingerprinting difference between wild-type and transgenic plants, and can potentially be applied in the bio-safety assessment of transgenic plants.     

Profiling phenolic metabolites in transgenic alfalfa modified in lignin biosynthesis

Soluble phenolics, wall-bound phenolics and soluble and core lignin were analyzed in transgenic alfalfa with genetically down-regulated O-methyltransferase genes involved in lignin biosynthesis. High performance liquid chromatography and principal component analysis were used to distinguish metabolic phenotypes of different transgenic alfalfa genotypes growing under standard greenhouse conditions. Principal component analysis of HPLC chromatograms did not resolve differences in leaf metabolite profiles between wild-type and transgenic plants of the same genetic background, although stem phenolic profiles were clearly different between wild-type and transgenic plants. However, the analytical methods clearly differentiated two non-transgenic alfalfa cultivars based on either leaf or stem profiles. Metabolic profiling provides a useful approach to monitoring the broader biochemical phenotypes of transgenic plants with altered expression of lignin pathway enzymes.     

Metabolic profiles of genetically modified potatoes using a combination of metabolite fingerprinting and multivariate analysis

Comprehensive metabolite fingerprinting of transgenic potatoes that constitutively express human beta amyloid, curdlan synthase (CRDS), and glycogen synthase (glgA); and of wild-type potatoes was carried out using FT-IR and ¹H NMR spectroscopy in combination with multivariate analyses. Comparison of metabolic patterns between transgenic and wild-type potatoes revealed that there were neither quantitative nor qualitative differences in metabolites between transgenic potatoes expressing human beta amyloid, CRDS or glgA, and non-transformed control potatoes. However, there were metabolic differences between two control potato lines — one that was fresh and the other stored. After 1 week of storage, comprehensive metabolite patterns were significantly modified. Although the differences between CRDS and glgA transgenic and control potato lines were small, PCA analysis of FT-IR and ¹H NMR spectral data identified two distinct control lines. These results suggest that the comprehensive metabolite changes in control potato lines, which occurred after 1 week of storage, were greater than the differences between CRDS and glgA transgenic and wild-type potato lines. Thus, the combination of FT-IR and ¹H NMR spectral data and multivariate analysis was valuable for the detection of comprehensive differences in metabolic profiles between transgenic and non-transformed control plants, even though peak-signal overlap prevented assignment of pure compounds. The combination of FT-IR and ¹H NMR spectral data and multivariate analysis is a simple and rapid method for evaluation of the metabolic equivalence of GM crops.     

Transgenic plants expressing geminivirus movement proteins: abnormal phenotypes and delayed infection by Tomato mottle virus in transgenic tomatoes expressing the Bean dwarf mosaic virus BV1 or BC1 proteins

Transgenic tomato plants expressing wild-type or mutated BV1 or BC1 movement proteins from Bean dwarf mosaic virus (BDMV) were generated and examined for phenotypic effects and resistance to Tomato mottle virus (ToMoV). Fewer transgenic plants were recovered with the wild-type or mutated BC1 genes, compared with the wild-type or mutated BV1 genes. Transgenic tomato plants expressing the wild-type or mutated BV1 proteins appeared normal. Interestingly, although BDMV induces only a symptomless infection in tomato (i.e., BDMV is not well adapted to tomato), transgenic tomato plants expressing the BDMV BC1 protein showed a viral disease-like phenotype (i.e., stunted growth, and leaf mottling, curling, and distortion). This suggests that the symptomless phenotype of BDMV in tomato is not due to a host-specific defect in the BC1 protein. One transgenic line expressing the BC1 gene did not show the viral disease-like phenotype. This was associated with a deletion in the 3' region of the gene, which resulted in expression of a truncated BC1 protein. Several R0 plants, expressing either wild-type or mutated BV1 or BC1 proteins, showed a significant delay in ToMoV infection, compared with non-transformed plants. R1 progeny plants also showed a significant delay in ToMoV infection, but this delay was less than that in the R0 parents. These results also demonstrate that expression of viral movement proteins, in transgenic plants, can have deleterious effects on various aspects of plant development.     

Secretion and folding of human growth hormone in Escherichia coli

Abstract

Phytoremediation is the use of plants for the treatment of environmental pollution, including chlorinated organics. although conceptually very attractive, removal and biodegradation of chlorinated pollutants by plants is a rather slow and inefficient process resulting in incomplete treatment and potential release of toxic metabolites into the environment. In order to overcome inherent limitations of plant metabolic capabilities, plants have been genetically modified, following a strategy similar to the development of transgenic crops: genes from bacteria, fungi, and mammals involved in the metabolism of organic contaminants, such as cytochrome p-450 and glutathione substrate catabolic genes, natural or engineered, for the simultaneous remediation of a range of pollutants, such as usually found in contaminated sites, e.g., chlorinated solvent, metals, and nitroaromatics. In addition, biodegradation of many xenobiotics are catalyzed by similar, broad-substrate enzymes, such as cytochrome P-450 monoxygenases, glutathione S-transferases, and fungal peroxidases, that can potentially be used for the treatment of multiple pollutants. Moreover, the introduction of multiple transgenes involved in different phases of the metabolism of xenobiotics in plants, i.e., uptake by roots and the different phases of the green liver model, would allow enhancing both the removal and metabolism of several toxic compounds and could therefore help overcome a major limitation inherent to phytoremediation, i.e., the threat that accumulated toxic compounds would volatilize or otherwise contaminate the food chain. An important barrier to the application of transgenic plants for bioremediation in the field is associated with the true or perceived risk of horizontal gene transfer to related wild or cultivated plants. Therefore, it is likely that the next generation of transgenic plants will involve systems preventing such a transfer, for instance by the introduction of transgenes into chloroplastic DNA or the use of conditional lethality genes (Davison, 2005). Since bacteria naturally exchange plasmids via conjugation, endophytes that gain genes involved in pollutant degradation might not be considered ‘genetically modified’ and may be subject to fewer restrictions in usage.     

Metabonomics evaluation of urine from rats given acute and chronic doses of acetaminophen using NMR and UPLC/MS

Urinary metabolic perturbations associated with acute and chronic acetaminophen-induced hepatotoxicity were investigated using nuclear magnetic resonance (NMR) spectroscopy and ultra performance liquid chromatography/mass spectrometry (UPLC/MS) metabonomics approaches to determine biomarkers of hepatotoxicity. Acute and chronic doses of acetaminophen (APAP) were administered to male Sprague-Dawley rats. NMR and UPLC/MS were able to detect both drug metabolites and endogenous metabolites simultaneously. The principal component analysis (PCA) of NMR or UPLC/MS spectra showed that metabolic changes observed in both acute and chronic dosing of acetaminophen were similar. Histopathology and clinical chemistry studies were performed and correlated well with the PCA analysis and magnitude of metabolite changes. Depletion of antioxidants (e.g. ferulic acid), trigonelline, S-adenosyl-l-methionine, and energy-related metabolites indicated that oxidative stress was caused by acute and chronic acetaminophen administration. Similar patterns of metabolic changes in response to acute or chronic dosing suggest similar detoxification and recovery mechanisms following APAP administration.     

Effect of crop rotation and soil cover on alteration of the soil microflora generated by the culture of transgenic plants producing opines

The culture of transgenic Lotus corniculatus plants producing opines, which are bacterial growth substrates, leads to the selection of rhizospheric bacteria able to utilize these substrates. We have investigated the fate of the opine-utilizing community over time under different experimental conditions following elimination of selective pressure exerted by the transgenic plants. These plants were removed from the soil, which was either left unplanted or replanted with wild-type L. corniculatus or wheat plants. The density of opine-utilizing bacteria in the fallow soils remained essentially unchanged throughout the experiment, regardless of the soil of origin (soil planted with wild-type or transgenic plants). When wild-type Lotus plants were used to replace their transgenic counterparts, only the bacterial populations able to utilize the opines were affected. Long-term changes affecting the opine-utilizing bacterial community on Lotus roots was dependent upon the opine studied. The concentration of nopaline utilizers decreased, upon replacement of the transgenic plants, to a level similar to that of normal plants, while the concentration of mannopine utilizers decreased to levels intermediate between transgenic and normal plants. These data indicate that: (i) the opine-utilizing bacterial populations can be controlled in the rhizosphere via plant-exudate engineering; (ii) the interaction between the engineered plants and their root-associated micro-organisms is transgene specific; and (iii) alterations induced by the cultivation of transgenic plants may sometimes be persistent. Furthermore, opine-utilizing bacterial populations can be controlled by crop rotation. Therefore, favouring the growth of a rhizobacterium of agronomic interest via an opine-based strategy appears feasible.     

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.     

A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type （ecotype Col-o） were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly, the phosphinothricin acetyl transferase （PAT）, the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.     

Spectral editing and pattern recognition methods applied to high-resolution magic-angle spinning 1H nuclear magnetic resonance spectroscopy of liver tissues

Principal component analysis (PCA) has been applied to three nuclear magnetic resonance (NMR) spectral editing methods, namely, the Carr-Purcell-Meiboom-Gill spin-echo, diffusion editing, and skyline projection of a two-dimensional J-resolved spectrum, obtained from high-resolution magic-angle spinning NMR spectroscopy of liver tissues, to distinguish between control and hydrazine-treated rats. The effects of the toxin on rat liver biochemistry were directly observed and characterized by depleted levels of liver glycogen, choline, taurine, trimethylamine N-oxide, and glucose and by elevated levels of lipids and alanine. The highly unsaturated omega-3-type fatty acid was observed for the first time in hydrazine-treated rat liver. The contributions of the metabolites to the separation of control from dosed liver tissues varied depending on the type of spectral editing method used. We have shown that subtle changes in the metabolic profiles can be selectively amplified using a metabonomics approach based on the different NMR spectral editing techniques in conjunction with PCA.     

A comparative proteomics approach to detect unintended effects in transgenic Arabidopsis

Recently, as part of biosafety assessments, unintended effects have been given much attention. In this study, we applied a proteomics approach to elucidate the unintended effects of random T-DNA insertion in transgenic plants. Separated proteins extracted from 12 transgenic Arabidopsis thaliana with different T-DNA insertion sites and from wild-type (ecotype Col-o) were analyzed. In the transgenic plants, 102 significantly altered protein spots were detected, in which 59 were up-regulated and 43 down-regulated. MALDI-TOF MS analysis showed that most of these expression level-altered proteins were involved in energy transfer, oxidative respiration and photosynthesis. However, none of these proteins was a toxic protein or allergen. Using plants with or without cold treatment, a natural environmental stress, as controls, we found that the number of the altered proteins was even less in those transgenic plants than those triggered by the cold treatment, suggesting that the transgenic events had a weaker impact on the plants than the environmental stresses. Interestingly,the phosphinothricin acetyl transferase (PAT), the BAR-encoded protein, was detected in nine out of twelve different T-DNA insertion lines at five different insertion sites. These data suggest that the most significant impact of transgenic events on the host plants is from the transgene itself, i.e., from the predictable intended effects, rather than unintended effects. This study also suggests that the proteomics approach has the potential to detect the unintended effects in transgenic plants.     

A GC-based metabonomics investigation of type 2 diabetes by organic acids metabolic profile

"Metabonomics" method requires the development of rapid, advanced analytical tools and GC will play an important role for its special advantage. In this study we show the application of GC-based metabonomics to investigate the control and type 2 diabetes (DM2) patients by urinary organic acids metabolic profile. After peak matching, multivariate statistical analysis methods: principal components analysis (PCA) and partial least squares-discriminant analysis (PLS-DA) were used. The results showed that there was a relationship between organic acids metabolic profiles and DM2, and PLS-DA can distinguish the DM2 patients from the control. Five organic acids as potential biomarkers were identified.     

Quantitative approaches for analysing fluxes through plant metabolic networks using NMR and stable isotope labelling

The quantitative analysis of metabolic networks is a prerequisite for understanding the integration and regulation of plant metabolism and for devising rational approaches for manipulating resource allocation in plants. The analysis of steady state stable isotope labelling experiments using nuclear magnetic resonance (NMR) spectroscopy has developed into a powerful method for determining these fluxes in micro-organisms and its application to heterotrophic plant metabolism is increasing. After an introductory discussion of the well known role of stable isotopes in pathway delineation, the review considers their application to metabolic flux analysis in plants. These applications are divided into two groups – small scale analyses of fluxes through particular pathways and large scale analyses of multiple fluxes through metabolic networks – and the problems caused by the complexity of intermediary metabolism in plants are discussed. It is concluded that metabolic flux analysis provides a powerful method for defining the metabolic phenotype of wild type, mutant and transgenic plants and that its development should be pursued.     

The 2b silencing suppressor of a mild strain of Cucumber mosaic virus alone is sufficient for synergistic interaction with Tobacco mosaic virus and induction of severe leaf malformation in 2b-transgenic tobacco plants

Tobacco plants infected simultaneously by Tobacco mosaic virus (TMV) and Cucumber mosaic virus (CMV) are known to produce a specific synergistic disease in which the emerging leaves are filiformic. Similar developmental malformations are also caused to a lesser extent by the severe strains (e.g., Fny) of CMV alone, but mild strains (e.g., Kin) cause them only in mixed infection with TMV. We show here that transgenic tobacco plants expressing 2b protein of CMV-Kin produce filiformic symptoms when infected with TMV, indicating that only 2b protein is needed from CMV-Kin for this synergistic relationship. On the other hand, transgenic plants that express either the wild-type TMV genome or a modified TMV genome with its coat protein deleted or movement protein (MP) inactivated also develop filiformic or at least distinctly narrow leaves, while plants expressing the MP alone do not develop any malformations when infected with CMV-Kin. These results show that either TMV helicase/replicase protein or active TMV replication are required for this synergistic effect. The effect appears to be related to an efficient depletion of silencing machinery, caused jointly by both viral silencing suppressors, i.e., CMV 2b protein and the TMV 126-kDa replicase subunit.     

AtNHX1基因对草木樨状黄芪的转化和耐盐性表达研究

应用RT-PCR技术从100mmol/LNaCl胁迫处理的拟南芥幼中克隆得到编码液泡膜Na /H 逆向转运蛋白的AtNHX1基因cDNA 编码ORF.并在该ORF上游分别插入CaMV 35启动子和TMV RNA5'UTR的Ω片段,而在下游插入NOS polyA构建真核表达盒,进而将该表达盒插入双元植物表达栽体pNT质粒的T-DNA区构建了携带AtNHX1 基因的植物表达载体质粒pNT-AtNHX1.将pNT-AtNHX1 导入农杆菌LBA4404,用农杆菌介导法将AtNHX1 基因导入豆科牧草草木樨状黄芪中,共获得103株Kan抗性再生植株.通过对农杆菌茵液浓度、侵染时间和乙酰丁香酮浓度等影响转化效率的因素进行优化,初步建立了稳定的草木樨状黄芪农杆菌转化体系.经过PCR检测、Southern杂交和RT-PCR检测表明,AtNHX1 基因已被成功整合到草木樨状黄芪基因组中,并且能够正常转录.野生型和转基因株系诱发的愈伤组织进行耐盐生长实验,结果显示相同盐胁迫条件下,转基因愈伤组织的相对生长率显著高于野生型愈伤组织.施加梯度NaCl胁迫后,植株叶片K ,Na 含量和叶片相对电导率测定结果显示,转基因植物叶片比野生型积累更多的Na 和K ,维持较高的K /Na ;转基因株系叶片相对电导率显著低于野生型.上述结果表明,AtNHX1 基因的导入和表达在提高草木樨状黄芪耐盐性的同时减轻了盐胁迫对植物细胞膜的伤害.关键词: AtNHX1 草木樨状黄芪农杆菌遗传转化耐盐性.