植物体中的过氧化物酶体

过氧化物酶体参与了包括氧化氢反应、长链脂肪酸的β-氧化等几乎所有的必需代谢途径。植物过氧化物酶体在植物体抗病和抗衰老过程中发挥作用。介绍了植物过氧化物酶体与亚硫酸盐氧化酶以及植物过氧化物酶体抗衰老、生物发生和动力学等方面的研究进展。     

Transgenic plants as a source of polyhydroxyalkanoates

Polyhydroxyalkanoates (PHAs) make a large class of biodegradable biopolymers that are naturally synthesized by numerous microorganisms. These biopolymers could be an alternative to commonly used plastics based on petroleum. Production of PHAs in bioreactors using microorganisms is not widely applied due to its unprofitability. Using transgenic plants for this purpose may be cheaper and more environmental friendly because the biosynthesis of PHAs in plants is based only on water, mineral salts, CO₂ and light. Additionally, plants are not capable of degrading PHAs as bacteria do, and extraction of PHAs from plant tissues is not always necessary. The main objective of this work is a review of possibilities of PHA biosynthesis in transgenic plants and presentation of general information on properties and potential application possibilities of these biopolymers. The possibility of syntheses and accumulation of PHA in several transgenic plants has been studied for some years. Many experiments were performed on model plant Arabidopsis thaliana, however, the research has also revealed a great potential of transgenic crop plants such as camelina (Camelina sativa), tobacco (Nicotiana tabacum) or sugarcane (Saccharum officinarum) as a good sources of PHAs. The highest level of PHAs accumulation in plants was achieved in transgenic A. thaliana (up to 40% of the dry weight of the leaf), and among crop plants in C. sativa (up to 20% of the dry weight of the seed). Increasing knowledge on PHAs permits expansion of the possibilities of these biopolymers use even at a low level of their accumulation in plant tissues.     

Cytokinins and cytokinin-binding sites in transgenic potato plants

The introduction of the gene for cytokinin biosynthesis into the potato genome led to a manifold increase in the level of cytokinins (zeatin and zeatin riboside) in transgenic plants grown in vitro. The high amount of endogenous cytokinins in 20-day-old plants of clone 1339-3A correlated with high cytokinin-binding capacity of ribosomes that is presumably attribute to a cytokinin receptor.     

Quantitative GFP fluorescence as an indicator of recombinant protein synthesis in transgenic plants

The utility of green fluorescent protein (GFP) for biological research is evident. A fluorescence-based method was developed to quantify GFP levels in transgenic plants and protein extracts. Fluorescence intensity was linear with increasing levels of GFP over a range that encompasses transgene expression in plants by the cauliflower mosaic virus 35S promoter. Standard curves were used to estimate GFP concentration in planta and in protein extracts. These values were consistent with ELISA measurements of GFP in protein extracts from transgenic plants, indicating that the technique is a reliable measure of recombinant GFP expression. The levels of in planta GFP expression in both homozygous and hemizygous plants was then estimated. Homozygous transgenic plants expressed twice the amount of GFP than hemizygous plants, suggesting additive transgene expression. This methodology may be useful to simplify the characterization of transgene expression in plants.Abbreviations ELISA Enzyme-linked immunosorbent assay - HRP Horseradish peroxidase - GFP Green fluorescent protein Communicated by M.C. Jordan     

Analysis of the alternative pathways for the beta-oxidation of unsaturated fatty acids using transgenic plants synthesizing polyhydroxyalkanoates in peroxisomes

Degradation of fatty acids having cis-double bonds on even-numbered carbons requires the presence of auxiliary enzymes in addition to the enzymes of the core beta-oxidation cycle. Two alternative pathways have been described to degrade these fatty acids. One pathway involves the participation of the enzymes 2, 4-dienoyl-coenzyme A (CoA) reductase and Delta(3)-Delta(2)-enoyl-CoA isomerase, whereas the second involves the epimerization of R-3-hydroxyacyl-CoA via a 3-hydroxyacyl-CoA epimerase or the action of two stereo-specific enoyl-CoA hydratases. Although degradation of these fatty acids in bacteria and mammalian peroxisomes was shown to involve mainly the reductase-isomerase pathway, previous analysis of the relative activity of the enoyl-CoA hydratase II (also called R-3-hydroxyacyl-CoA hydro-lyase) and 2,4-dienoyl-CoA reductase in plants indicated that degradation occurred mainly through the epimerase pathway. We have examined the implication of both pathways in transgenic Arabidopsis expressing the polyhydroxyalkanoate synthase from Pseudomonas aeruginosa in peroxisomes and producing polyhydroxyalkanoate from the 3-hydroxyacyl-CoA intermediates of the beta-oxidation cycle. Analysis of the polyhydroxyalkanoate synthesized in plants grown in media containing cis-10-heptadecenoic or cis-10-pentadecenoic acids revealed a significant contribution of both the reductase-isomerase and epimerase pathways to the degradation of these fatty acids.     

Analysis of integration sites of T-DNA insertions in transgenic tobacco plants

DNA fragments containing T-DNA/plant DNA junctions isolated from 17 transgenic tobacco plants were amplified using inverse PCR. Analysis of the nucleotide sequences of 34 cloned DNA fragments revealed 100% homology with vector sequences outside T-DNA in 10 cases. Nine nucleotide sequences had homology with the repeats in the tobacco genome. The percentage of homology varied from 70 to 90%, with the identified repeats belonging to different types. In most clones no homology was revealed with the GENEBANK sequences. Alignment of the sequences truncated during the integration of the left and the right borders of the T-DNA insertions demonstrated significant clusterization (10 bp region) of truncation sites for the left border. Five sequences had identical truncation sites (+23 T) that showed the perferable use of this nucleotide. The AT content varied from 51 to 72% which was close to the total percentage of AT pairs in the tobacco genome.     

T-DNA structure in transgenic tobacco plants with multiple independent integration sites

Summary Transgenic tobacco plants were produced by inoculation of leaf disks withAgrobacterium tumefaciens harboring a disarmed binary vector containing soybean leghemoglobin Lbc3 and glycinin G2 genes. Physical and genetic characterization of these plants indicated that one to six copies of DNA from the vector were transferred and maintained in the plant genome. Approximately 30% of the copies transferred were found to be incomplete or rearranged and in some cases joined as inverted repeats. The transferred DNA was found at multiple genetic loci in five of the six cases examined. In one plant, kanamycin-resistance traits were at four independent chromosomal positions, although two were genetically linked at about 3 centimorgans. Thus,Agrobacterium-mediated DNA transfer to plants has some characteristics in common with “natural” systems in animals, such as retroviral or P-element derived systems, some characteristics in common with “artificial” systems, such as microinjection, electroporation, or calcium phosphate coprecipitation techniques, and some novel characteristics.     

Perspectives on the production of polyhydroxyalkanoates in plants

Abstract Poly-β-hydroxybutyrate (PBH) was recently shown to be produced in genetically engineered plants which expressed the genes from Alcaligenes eutrophus responsible for the formation of PHB from acetoacetyl-CoA. The transgenic plants accumulated PHB as granules which were similar in size and appearance to the bacterial PHB granules. These observations suggest that large scale production of PHB and other polyhydroxyalkanoates in genetically altered crop plants may be feasible.     

Polyhydroxyalkanoate synthesis in transgenic plants as a new tool to study carbon flow through beta-oxidation

Transgenic plants producing peroxisomal polyhydroxy- alkanoate (PHA) from intermediates of fatty acid degradation were used to study carbon flow through the beta-oxidation cycle. Growth of transgenic plants in media containing fatty acids conjugated to Tween detergents resulted in an increased accumulation of PHA and incorporation into the polyester of monomers derived from the beta-oxidation of these fatty acids. Tween-laurate was a stronger inducer of beta-oxidation, as measured by acyl-CoA oxidase activity, and a more potent modulator of PHA quantity and monomer composition than Tween-oleate. Plants co-expressing a peroxisomal PHA synthase with a capryl-acyl carrier protein thioesterase from Cuphea lanceolata produced eightfold more PHA compared to plants expressing only the PHA synthase. PHA produced in double transgenic plants contained mainly saturated monomers ranging from 6 to 10 carbons, indicating an enhanced flow of capric acid towards beta-oxidation. Together, these results support the hypothesis that plant cells have mechanisms which sense levels of free or esterified unusual fatty acids, resulting in changes in the activity of the beta-oxidation cycle as well as removal and degradation of these unusual fatty acids through beta-oxidation. Such enhanced flow of fatty acids through beta-oxidation can be utilized to modulate the amount and composition of PHA produced in transgenic plants. Furthermore, synthesis of PHAs in plants can be used as a new tool to study the quality and relative quantity of the carbon flow through beta-oxidation as well as to analyse the degradation pathway of unusual fatty acids.     

Bacterial synthesis of biodegradable polyhydroxyalkanoates

Various bacterial species accumulate intracellular polyhydroxyalkanoates (PHAs) granules as energy and carbon reserves inside their cells. PHAs are biodegradable, environmentally friendly and biocompatible thermoplastics. Varying in toughness and flexibility, depending on their formulation, they can be used in various ways similar to many nonbiodegradable petrochemical plastics currently in use. They can be used either in pure form or as additives to oil-derived plastics such as polyethylene. However, these bioplastics are currently far more expensive than petrochemically based plastics and are therefore used mostly in applications that conventional plastics cannot perform, such as medical applications. PHAs are immunologically inert and are only slowly degraded in human tissue, which means they can be used as devices inside the body. Recent research has focused on the use of alternative substrates, novel extraction methods, genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive.     

Vectors with rare-cutter restriction enzyme sites for expression of open reading frames in transgenic plants

Cloning of long open reading frames (ORFs) into plant gene expression vectors and transfer of the chimeric expression cassettes into binary vectors is often hampered by the presence of restriction enzyme cleavage sites internal to the open reading frame (ORF) to be expressed. We therefore modified the commonly used expression vector pRT100 [7] and several pGPTV binary vectors [2] by replacing 6 bp restriction sites with 8 bp sequences recognized by rare-cutter restriction enzymes.     

Eicosapentaenoic acid: biosynthetic routes and the potential for synthesis in transgenic plants

Long chain polyunsaturated fatty acids are now known to play important roles in human health. In particular, eicosapentaenoic acid (20:5Delta(5,8,11,14,17); n-3: EPA) is implicated as a protective agent in a range of pathologies such as cardiovascular disease and Metabolic Syndrome (Syndrome X). Eicosapentaenoic acid is currently sourced from fish oils, the presence of this fatty acid being due to the dietary piscine consumption of EPA-synthesising micro-algae. The biosynthetic pathway of EPA has been elucidated, and contains several alternative metabolic routes. Progress in using "reverse engineering" to transgenically mobilize the trait(s) for EPA are considered. In particular, the prospect of producing this important polyunsaturated fatty acid in transgenic oilseeds is highlighted, as is the urgent need for a sustainable replacement for diminishing fish stocks.     

Isoprene synthesis protects transgenic tobacco plants from oxidative stress

Isoprene emission represents a significant loss of carbon to those plant species that synthesize this highly volatile and reactive compound. As a tool for studying the role of isoprene in plant physiology and biochemistry, we developed transgenic tobacco plants capable of emitting isoprene in a similar manner to and at rates comparable to a naturally emitting species. Thermotolerance of photosynthesis against transient high-temperature episodes could only be observed in lines emitting high levels of isoprene; the effect was very mild and could only be identified over repetitive stress events. However, isoprene-emitting plants were highly resistant to ozone-induced oxidative damage compared with their non-emitting azygous controls. In ozone-treated plants, accumulation of toxic reactive oxygen species (ROS) was inhibited, and antioxidant levels were higher. Isoprene-emitting plants showed remarkably decreased foliar damage and higher rates of photosynthesis compared to non-emitting plants immediately following oxidative stress events. An inhibition of hydrogen peroxide accumulation in isoprene-emitting plants may stall the programmed cell death response which would otherwise lead to foliar necrosis. These results demonstrate that endogenously produced isoprene provides protection from oxidative damage.     

Isoprene synthesis in plants: lessons from a transgenic tobacco model

Isoprene is a highly reactive gas, and is emitted in such large quantities from the biosphere that it substantially affects the oxidizing potential of the atmosphere. Relatively little is known about the control of isoprene emission at the molecular level. Using transgenic tobacco lines harbouring a poplar isoprene synthase gene, we examined control of isoprene emission. Isoprene synthase required chloroplastic localization for catalytic activity, and isoprene was produced via the methyl erythritol (MEP) pathway from recently assimilated carbon. Emission patterns in transgenic tobacco plants were remarkably similar to naturally emitting plants under a wide variety of conditions. Emissions correlated with photosynthetic rates in developing and mature leaves, and with the amount of isoprene synthase protein in mature leaves. Isoprene synthase protein levels did not change under short-term increase in heat/light, despite an increase in emissions under these conditions. A robust circadian pattern could be observed in emissions from long-day plants. The data support the idea that substrate supply and changes in enzyme kinetics (rather than changes in isoprene synthase levels or post-translational regulation of activity) are the primary controls on isoprene emission in mature transgenic tobacco leaves.     

Strategies for antiviral resistance in transgenic plants

Genetic engineering offers a means of incorporating new virus resistance traits into existing desirable plant cultivars. The initial attempts to create transgenes conferring virus resistance were based on the pathogen-derived resistance concept. The expression of the viral coat protein gene in transgenic plants was shown to induce protective effects similar to classical cross protection, and was therefore distinguished as 'coat-protein-mediated' protection. Since then, a large variety of viral sequences encoding structural and non-structural proteins were shown to confer resistance. Subsequently, non-coding viral RNA was shown to be a potential trigger for virus resistance in transgenic plants, which led to the discovery of a novel innate resistance in plants, RNA silencing. Apart from the majority of pathogen-derived resistance strategies, alternative strategies involving virus-specific antibodies have been successfully applied. In a separate section, efforts to combat viroids in transgenic plants are highlighted. In a final summarizing section, the potential risks involved in the introduction of transgenic crops and the specifics of the approaches used will be discussed.     

Biodegradable polyhydroxyalkanoates as carriers for antitumor agents]

The possible use of biodegradable polyethers of microbial origin (polyhydroxyalkanoates) as matrices for deposition of daunorubicin (rubomycin), an antitumor anthracycline, was studied. The tablet dosage form of various rubomycin load (from 1 to 60% w/w) was prepared by cold compaction under pressure. The in vitro kinetics of the rubomycin release from the polymer matrix was investigated. It was shown that the rubomycin release to the medium resulted from the drug solution and diffusion within various periods, from tens hours to several weeks and months depending on the load. When the rubomycin load was under 20% w/w the drug release was prolonged and directly proportional to the observation time. When the rubomycin concentration was under 5%, the drug release kinetics corresponded to the type of the zero order reaction with prolonged release without sharp efflux at the initial stage of the observation. The findings showed that the polyhydroxyalkanoates were applicable as matrices for deposition of rubomycin and preparation of drugs with prolonged action.