Pathogen resistance of transgenic tobacco plants producing caffeine

Caffeine (1,3,7-trimethylxanthine) is a typical purine alkaloid, and produced by a variety of plants such as coffee and tea. Its physiological function, however, is not completely understood, but chemical defense against pathogens and herbivores, and allelopathic effects against competing plant species have been proposed. Previously, we constructed transgenic tobacco plants, which produced caffeine up to 5 microg per gram fresh weight of leaves, and showed them to repel caterpillars of tobacco cutworms (Spodoptera litura). In the present study, we found that these transgenic plants constitutively expressed defense-related genes encoding pathogenesis-related (PR)-1a and proteinase inhibitor II under non-stressed conditions. We also found that they were highly resistant against pathogens, tobacco mosaic virus and Pseudomonas syringae. Expression of PR-1a and PR-2 was higher in transgenic plants than in wild-type plants during infection. Exogenously applied caffeine to wild-type tobacco leaves exhibited the similar resistant activity. These results suggested that caffeine stimulated endogenous defense system of host plants through directly or indirectly activating gene expression. This assumption is essentially consistent with the idea of chemical defense, in which caffeine may act as one of signaling molecules to activate defense response. It is thus conceivable that the effect of caffeine is bifunctional; direct interference with pest metabolic pathways, and activation of host defense systems.     

利用转基因植物生产药用蛋白研究进展

简要评述了国内外利用转基因植物生产药用蛋白的研究现状、发展趋势,以及转基因植物生产药用蛋白的基本方法、应用研究等。尽管目前植物作为药用蛋白的生物反应器受到诸多因素限制,优点与问题并存,但利用转基因植物生产药用蛋白是植物基因工程研究领域的一个新的发展趋势。     

Generation of transgenic wheat plants producing high levels of the osmoprotectant proline

Plasmid DNA (pBI-P5CS), containing the selectable neomycin phosphotransferase-II `npt II' gene for kanamycin resistance and the reporter -glucuronidase `gus' gene as well as the Vigna aconitifolia ¹-pyrroline-5-carboxylate synthetase `P5CS' cDNA that encodes enzymes required for the biosynthesis of proline, was delivered into wheat plants using Agrobacterium-mediated gene transfer via indirect pollen system. Southern, northern and western blot analysis demonstrated that the foreign gene had been transferred, expressed and integrated into wheat chromosomal DNA. Salinity test indicated that proline acts as an osmoprotectant and its overproduction in transgenic wheat plants results in the increased tolerance to salt.     

Metabolite fingerprinting in transgenic lettuce

Metabolite fingerprinting has been achieved using direct atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) and linked gas chromatography (GC-APCI/EI-MS) for transgenic lettuce (Lactuca sativa L. cv. Evola) plants expressing an IPT gene under the control of the senescence-specific SAG12 promoter from Arabidopsis thaliana (P(SAG12)-IPT). Mature heads of transgenic lettuce and their azygous controls were maintained under defined conditions to assess their shelf life. Transgenic lettuce plants exhibited delayed senescence and significant increases (up to a maximum of threefold) in the concentrations of three volatile organic compounds (VOCs), corresponding to molecular masses of 45, 47 and 63, when compared with heads from azygous plants. These VOCs were identified as acetaldehyde (45), ethanol (47) and dimethyl sulphide (63). The increase in dimethyl sulphide was paralleled by an accumulation of reactive oxygen species (ROS) in the heads of transgenic plants. These results demonstrate the applicability of metabolic fingerprinting techniques to elucidate the underlying pleiotropic responses of plants to transgene expression.     

Features of lettuce transgenic plants with ifn-alpha2b gene regenerated after Agrobacterium rhizogenes-mediated transformation

"Hairy" roots of lettuce Lactuca sativa and regenerated plants with interferon-alpha2b gene had been obtained via Agrobacterium rhizogenes-mediated transformation. According to the results of PCR and rt-PCR analyses the studied plants had ifn-alpha2b gene. The regenerated plants differed from the plants of wild type by elongated internodes, early flower-bearing stem formation and purple coloration of leaves in artificial illumination conditions.     

A novel double T-DNA system for producing stack and marker-free transgenic plants

This study aimed to develop a new vector system to remove selection genes and to introduce two or more genes of interest into plants in order to express them in a coordinated manner. A multigene expression vector was established based on pCamBIA2300 using a selectable marker gene (SMG)-free system based on the combination of the isocaudamer technique and double T-DNA. The vector DT7 containing seven target genes was constructed and introduced into tobacco using Agrobacterium-mediated transformation. Twenty-one of 27 positive transgenic plants contained both T-DNA regions. The co-transformation frequency was 77.8 %. The frequency of unlinked integration of two intact T-DNAs was 22.22 % (6/27). The frequency of removal of SMG from transgenic T1 plants was 19.10 %. These results suggest that this vector system was functional and effective for multigene expression and SMG-free transgenic plant cultivation. At least seven target genes can be co-expressed using this system. Overall, these findings provide a new and highly effective platform for multigene and marker-free transgenic plant production.     

Improved drought tolerance without undesired side effects in transgenic plants producing trehalose

Most organisms naturally accumulating trehalose upon stress produce the sugar in a two-step process by the action of the enzymes trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP). Transgenic plants overexpressing TPS have shown enhanced drought tolerance in spite of minute accumulation of trehalose, amounts believed to be too small to provide a protective function. However, overproduction of TPS in plants has also been found combined with pleiotropic growth aberrations. This paper describes three successful strategies to circumvent such growth defects without loosing the improved stress tolerance. First, we introduced into tobacco a double construct carrying the genes TPS1 and TPS2 (encoding TPP) from Saccharomyces cerevisiae. Both genes are regulated by an Arabidopsis RuBisCO promoter from gene AtRbcS1A giving constitutive production of both enzymes. The second strategy involved stress-induced expression by fusing the coding region of ScTPS1 downstream of the drought-inducible Arabidopsis AtRAB18 promoter. In transgenic tobacco plants harbouring genetic constructs with either ScTPS1 alone, or with ScTPS1 and ScTPS2 combined, trehalose biosynthesis was turned on only when the plants experienced stress. The third strategy involved the use of AtRbcS1A promoter together with a transit peptide in front of the coding sequence of ScTPS1, which directed the enzyme to the chloroplasts. This paper confirms that the enhanced drought tolerance depends on unknown ameliorated water retention as the initial water status is the same in control and transgenic plants and demonstrates the influence of expression of heterologous trehalose biosynthesis genes on Arabidopsis root development.     

Iron accumulation and enhanced growth in transgenic lettuce plants expressing the iron- binding protein ferritin

We have produced transgenic lettuce plants accumulating the iron storage protein ferritin. The integration of the ferritin gene and expression levels in leaves were examined by Southern- and Western-blot analysis, respectively. It was shown that transgenic lettuce plants contained iron levels ranging from 1.2 to 1.7 times that of the control plants, however, the manganese content in transgenic lettuce plants was similar to that in the control. Enhanced growth of transgenic lettuces was observed at the early developmental stages, resulting in weights 27–42% greater than those of control plants. Transgenic lettuce had photosynthesis rates superior to those of the controls, and grew larger and faster compared with the controls during the period of 3 months from germination. These results demonstrate the possibility of producing lettuce plants with high yield, high iron content and rapid growth rate. Received: 21 March 1999 / Accepted: 14 September 1999     

Use of the gene of antimicrobial peptide cecropin P1 for producing marker-free transgenic plants

The marker-free transgenic tobacco plants carrying a synthetic gene encoding the antimicrobial peptide cecropin P1 (cecP1) under the control of the cauliflower mosaic virus 35S RNA promoter were produced. The binary vector pBM, free of any selective genes of resistance to antibiotics or herbicides intended for selecting transgenic plants, was used for transformation. The transformants were screened on a nonselective medium by detecting cecropin P1 in plant cells according to the antibacterial activity of plant extracts and enzyme immunoassay. According to the two used methods, 2% of the analyzed regenerants were transformants. The resulting marker-free plants displayed a considerably increased resistance to microbial phytopathogens—the bacterium Erwinia carotovora and fungus Sclerotinia sclerotiorum. Thus, the gene cecP1 can be concurrently used as a target gene and a screening marker. The utility of cecP1 as a selective gene for direct selection of transformed plants is discussed.     

Induced expression of a chimeric gene construct in transgenic lettuce plants using tobacco pathogenesis-related protein gene promoter region

The expression of a stress- and salicylic acidinducible protein gene from tobacco, PR1a protein gene, was determined after its Introduction to lettuce (Lactuca sativa L.) plants. The 5 flanking 2.4 Kb fragment from PR1a gene was joined to the bacterial -glucuronidase (GUS) gene (PR-GUS) and introduced into lettuce cotyledons by Agrobacterium-mediated gene transfer using a binary vector containing a kanamycin-resistance gene as a selectable marker. As a control with constitutive expression, the chimeric gene consisting of CaMV 35S RNA promoter and GUS gene (35S-GUS) was used. An improved method for shoot formation directly from lettuce cotyledons was used effectively for transformation, shortening the time for regeneration. In 70% or more of kanamycin-resistant regenerated lettuce plants, into which PR-GUS or 35S-GUS was introduced, high GUS activity and integration of the chimeric gene into the lettuce genome were detected. By treatment with salicylic acid, GUS activity increased 3- to 50-fold in PR-GUS transformants, however, no increase was detected in 35S-GUS plants. These results showed that the promoter of the stress-inducible tobacco PR1a protein gene was introduced into lettuce plants, and the introduced chimeric gene was expressed normally under the regulated control of the PRla promoter.Abbreviations BA N6-benzyladenine - GUS -glucuronidase - NAA -naphthaleneacetic acid - Km kanamycin - Km^s kanamycin resistant - Km⁰ kanamycin sensitive - NPT- II neomycin phosphotransferase II - PR pathogenesis-related - SA salicylic acid - MS Murashige and Skoog medium - NOS nopaline synthase     

Marker-free transgenic plants

Selectable marker genes are widely used for the efficient transformation of crop plants. In most cases, selection is based on antibiotic or herbicide resistance. Due mainly to consumer concerns, a suite of strategies (site-specific recombination, homologous recombination, transposition and co-transformation) have been developed to eliminate the marker gene from the nuclear or chloroplast genome after selection. Current efforts concentrate on systems where marker genes are eliminated efficiently soon after transformation. Alternatively, transgenic plants are produced by the use of marker genes that do not rely on antibiotic or herbicide resistance but instead promote regeneration after transformation. Here, the merits and shortcomings of different approaches and possible directions for their future development are discussed.     

Modification of non-protein thiols contents in transgenic tobacco plants producing bacterial enzymes of cysteine biosynthesis pathway

Conditions of achieving the maximal accumulation of sulfhydryl metabolites in the leaves of tobacco were explored. Simultaneous production of bacterial O-acetylserine (thiol)-lyase and serine acetyltransferase resulted in the increased thiols contents as compared to single transformants and controls. However, leaf discs feeding experiments differently affected thiols concentration in different plant groups and suggested that the most promising strategy to obtain plants with a high level of non-protein thiol-containing compounds might be sulfate feeding to plants overproducing serine acetyltransferase.     

Engineering production of antihypertensive peptides in plants

To date, a number of antihypertensive peptides (AHPs) have been identified. Most of these are derived from proteins present in common edible consumables, including milk, egg, and plant foods. Consumption of these foods serves as means of AHP delivery and thus contributing favorable health benefits. It is hypothesized that food crops, either over-expressing AHP precursor proteins or producing particular peptides as heterologous components, may serve as viable vehicles for production and delivery of functional foods as alternative hypertension therapies. In recent years, genetic engineering efforts have been undertaken to add value to functional foods. Pioneering approaches have been pursued in several crop plants, such as rice and soybean. In this review, a summary of current tools used for discovery of new AHPs, as well as strategies and perspectives of capitalizing on these AHPs in genetic engineering efforts will be presented and discussed. The implications of these efforts on the development of functional foods for preventing and treating hypertension are also presented.     

Insect-resistant transgenic brinjal plants

A synthetic cry1Ab gene coding for an insecticidal crystal protein (ICP) of Bacillus thuringiensis (Bt) was transferred to brinjal (eggplant) by cocultivating cotyledonary explants with Agrobacterium tumefaciens. Transformant plants resistant to kanamycin were regenerated. Hybridization experiments demonstrated gene integration and mRNA expression. Double-antibody sandwich ELISA analysis revealed Bt toxin protein expression in the transgenic plants. The expression resulted in a significant insecticidal activity of transgenic brinjal fruits against the larvae of fruit borer (Leucinodes orbonalis). The results also demonstrated that a synthetic gene based on monocot codon usage can be expressed in dicotyledonous plants for insect control.     

Colonization of adult lettuce plants by Bremia lactucae

Studies were made of the infection by Bremia lactucae of a range of lettuce cultivars at the adult plant stage, under conditions of artificial and natural epidemic. The pathogen invaded at least the ten oldest leaves of many cultivars, the leaf area affected declining from the oldest to the youngest leaves. Two assessment methods were compared: (1) the percentage area infection on individual leaves and (2) the percentage area infection on the inverted basal area as a whole. Results from the two methods agreed closely under conditions of artificial inoculation and in a natural epidemic. It is concluded that both methods should be used for detailed studies, but method 1 restricted to the oldest five leaves. At least two assessment dates are required since on individual cultivars there were differences in the leaf area infected at various assessment dates. This is probably influenced by the cycle of growth and sporulation of B. lactucae and by the effects of invasion by secondary pathogens such as Botrytis cinerea.     

Salt-induced inhibition of phosphorus transport in lettuce plants

Copper interferes with numerous physiological processes and can be toxic at low concentrations. We examined the effect of a 2-week exposure to various concentrations of copper on the structure of the nucleus in maize root meristem cells by transmission electron microscopy. Copper uptake and distribution in the plants, as well as DNA synthesis were also investigated. Treatment with Cu induced alterations in the structure of the nucleus, resulting mainly in a greater density of chromatin. Morphometric analysis showed that the part of the condensed chromatin in the nucleus might be increased by Cu, an observation that was correlated with decreased DNA synthesis as measured by incorporation of ³H-thymidine. Copper may therefore play a role in chromatin structure and one of the manifestations of Cu toxicity is an alteration in the structure of the nucleus, leading to decreased DNA synthesis.     

Regeneration of transgenic tamarillo plants

Media were developed to regenerate shoots from leaf pieces of tamarillo (Cyphomandra betacea (Cav.) Sendtner). Shoots were derived via organogenesis and could be easily rooted and transferred to the growth chamber. Transgenic tamarillo plants were produced using the binary vector pKIWI110 in the avirulent Agrobacterium strain LBA4404. All transgenic plants were kanamycin resistant and some plants expressed the D-glucuronidase (gusA) reporter gene and were chlorsulfuron resistant. Molecular evidence for transformation was obtained using PCR (polymerase chain reaction) and Southern hybridization. Inheritance of the transgenic phenotypes was demonstrated in seedling progeny.