Induced tyramine overproduction in transgenic rice plants expressing a rice tyrosine decarboxylase under the control of methanol-inducible rice tryptophan decarboxylase promoter

Tyramine, one of the various biogenic amines found in plants, is derived from the aromatic L-amino acid tyrosine through the catalytic reaction of tyrosine decarboxylase (TYDC). Tyramine overproduction by constitutive expression of TYDC in rice plants leads to stunted growth, but an increased number of tillers. To regulate tyramine production in rice plants, we expressed TYDC under the control of a methanol-inducible plant tryptophan decarboxylase (TDC) promoter and generated transgenic T(2) homozygous rice plants. The transgenic rice plants showed normal growth phenotypes with slightly increased levels of tyramine in seeds relative to wild type. Upon treatment with 1% methanol, the transgenic rice leaves produced large amounts of tyramine, whereas no increase in tyramine production was observed in wild-type plants. The methanol-induced accumulation of tyramine in the transgenic rice leaves was inversely correlated with the tyrosine level. These data indicate that tyramine production in rice plants can be artificially controlled using the methanol-inducible TDC promoter, suggesting that this promoter could be used to selectively induce the expression of other proteins or metabolites in rice plants.     

Enhanced resistance to citrus canker in transgenic mandarin expressing Xa21 from rice

Genetic engineering approaches offer an alternative method to the conventional breeding of Citrus sp. ‘W. Murcott’ mandarin (a hybrid of ‘Murcott’ and an unknown pollen parent) is one of the most commercially important cultivars grown in many regions around the world. Transformation of ‘W. Murcott’ mandarin was achieved by direct DNA uptake using a protoplast transformation system. DNA construct (pAO3), encoding Green Fluorescent Protein (GFP) and the cDNA of Xa21, a Xanthomonas resistance gene from rice, was used to transform protoplasts of ‘W. Murcott’ mandarin. Following citrus protoplast culture and regeneration, transformed micro calli were microscopically designated via GFP expression, physically isolated from non-transformed tissue, and cultured on somatic embryogenesis induction medium. More than 150 transgenic embryos were recovered and from them, ten transgenic lines were regenerated and cultured on rooting medium for shoot elongation. Transgenic shoots were micrografted and established in the greenhouse with 3–5 replicates per line. The insertion of Xa21 and GFP was confirmed by PCR and southern blot analysis. GFP expression was verified by fluorescence microscopy and western blot analysis revealed expression of Xa21 although it was variable among transgenic lines, as shown by RT-qPCR. Transgenic plants challenged with the citrus canker pathogen by syringe inoculation showed a reduction in lesion number and bacterial populations within lesions compared to non-transgenic control plants. Transgenic ‘W. Murcott’ mandarin lines with improved canker resistance via protoplast transformation from embryogenic callus with the Xa21 gene from rice are being evaluated under field conditions to validate the level of resistance.     

Enhanced drought tolerance of transgenic rice plants expressing a pea manganese superoxide dismutase

We investigated the role that manganese superoxide dismutase (MnSOD), an important antioxidant enzyme, may play in the drought tolerance of rice. MnSOD from pea (Pisum sativum) under the control of an oxidative stress-inducible SWPA2 promoter was introduced into chloroplasts of rice (Oryza sativa) by Agrobacterium-mediated transformation to develop drought-tolerant rice plants. Functional expression of the pea MnSOD in transgenic rice plants (T1) was revealed under drought stress induced by polyethylene glycol (PEG) 6000. After PEG treatment the transgenic leaf slices showed reduced electrolyte leakage compared to wild type (WT) leaf slices, whether they were exposed to methyl viologen (MV) or not, suggesting that transgenic plants were more resistant to MV- or PEG-induced oxidative stress. Transgenic plants also exhibited less injury, measured by net photosynthetic rate, when treated with PEG. Our data suggest that SOD is a critical component of the ROS scavenging system in plant chloroplasts and that the expression of MnSOD can improve drought tolerance in rice.     

Fitness of transgenic Anopheles stephensi mosquitoes expressing the SM1 peptide under the control of a vitellogenin promoter

Three transgenic Anopheles stephensi lines were established that strongly inhibit transmission of the mouse malaria parasite Plasmodium berghei. Fitness of the transgenic mosquitoes was assessed based on life table analysis and competition experiments between transgenic and wild-type mosquitoes. Life table analysis indicated low fitness load for the 2 single-insertion transgenic mosquito lines VD35 and VD26 and no load for the double-insertion transgenic mosquito line VD9. However, in cage experiments, where each of the 3 homozygous transgenic mosquitoes was mixed with nontransgenic mosquitoes, transgene frequency of all 3 lines decreased with time. Further experiments suggested that reduction of transgene frequency is a consequence of reduced mating success, reduced reproductive capacity, and/or insertional mutagenesis, rather than expression of the transgene itself. Thus, for transgenic mosquitoes released in the field to be effective in reducing malaria transmission, a driving mechanism will be required.     

Altered secondary myogenesis in transgenic animals expressing the neural cell adhesion molecule under the control of a skeletal muscle alpha-actin promoter

The majority of skeletal muscle fibers are generated through the process of secondary myogenesis. Cell adhesion molecules such as NCAM are thought to be intricately involved in the cell-cell interactions between developing secondary and primary myotubes. During secondary myogenesis, the expression of NCAM in skeletal muscle is under strict spatial and temporal control. To investigate the role of NCAM in the regulation of primary-secondary myotube interactions and muscle fusion in vivo, we have examined muscle development in transgenic mice expressing the 125-kD muscle-specific, glycosylphosphatidylinositol- anchored isoform of human NCAM, under the control of a human skeletal muscle alpha-actin promoter that is active from about embryonic day 15 onward. Analysis of developing muscle from transgenic animals revealed a significantly lower number of myofibers encased by basal lamina at postnatal day 1 compared with nontransgenic littermates, although the total number of developing myofibers was similar. An increase in muscle fiber size and decreased numbers of VCAM-1-positive secondary myoblasts at postnatal day 1 was also found, indicating enhanced secondary myoblast fusion in the transgenic animals. There was also a significant decrease in myofiber number but no increase in overall muscle size in adult transgenic animals; other measurements such as the number of nuclei per fiber and the size of individual muscle fibers were significantly increased, again suggesting increased secondary myoblast fusion. Thus the level of NCAM in the sarcolemma is a key regulator of cell-cell interactions occurring during secondary myogenesis in vivo and fulfills the prediction derived from transfection studies in vitro that the 125-kD NCAM isoform can enhance myoblast fusion.     

Phenotype characteristics of transgenic male mice expressing human aromatase under ubiquitin C promoter

To study the significance of the increased ratio of the estrogen/androgen concentration for the male reproductive functions, we have generated transgenic mice expressing human P450 aromatase under a promoter providing ubiquitous and permanent transgene expression (AROM+ mice). AROM+ male mice are characterized by elevated serum estradiol and prolactin (Prl) concentrations, combined with markedly reduced testosterone levels. The mice are present with a multitude of structural and functional alterations in the reproductive organs such as cryptorchidism, Leydig cell hyperplasia, disrupted spermatogenesis and infertility. Furthermore, the mice develop infravesical obstruction associated with the rhabdosphincter atrophy and rudimentary accessory sex glands. Interestingly, the mammary gland in AROM+ males undergo a ductal and alveolar development morphologically resembling terminally differentiated female mammary glands, and express several signaling proteins typical for female mammary glands. Some of the abnormalities seen in AROM+ mice are similar to those described in both mice and humans exposed to diethylstilbestrol (DES) in utero. The importance of the AROM+ model may lie in its predictability, i.e. the model suggests which abnormalities of the human reproductive functions may be associated with the increased ratio of estrogen/androgen concentrations in early life and at adult age as well.     

Activity of a maize ubiquitin promoter in transgenic rice

We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants.     

Enhanced resistance to stripe rust disease in transgenic wheat expressing the rice chitinase gene RC24

Stripe rust is a devastating fungal disease of wheat worldwide which is primarily caused by Puccinia striiformis f. sp tritici. Transgenic wheat (Triticum aestivum L.) expressing rice class chitinase gene RC24 were developed by particle bombardment of immature embryos and tested for resistance to Puccinia striiformis f.sp tritici. under greenhouse and field conditions. Putative transformants were selected on kanamycin-containing media. Polymease chain reaction indicated that RC24 was transferred into 17 transformants obtained from bombardment of 1,684 immature embryos. Integration of RC24 was confirmed by Southern blot with a RC24-labeled probe and expression of RC24 was verified by RT-PCR. Nine transgenic T₁ lines exhibited enhanced resistance to stripe rust infection with lines XN8 and BF4 showing the highest level of resistance. Southern blot hybridization confirmed the stable inheritance of RC24 in transgenic T₁ plants. Resistance to stripe rust in transgenic T₂ and T₃ XN8 and BF4 plants was confirmed over two consecutive years in the field. Increased yield (27–36 %) was recorded for transgenic T₂ and T₃ XN8 and BF4 plants compared to controls. These results suggest that rice class I chitinase RC24 can be used to engineer stripe rust resistance in wheat.     

Expression of a rice glutelin promoter in transgenic tobacco

A chimeric gene consisting of the 5 flanking sequences of a rice glutelin gene (Gt3) linked to the chloramphenicol acetyltransferase (CAT) coding segment was introduced into tobacco via Agrobacterium tumefaciens-mediated transformation. CAT enzyme activity could be detected in extracts from seeds as early as 8 days after flowering and obtained a maximum level at 16 days after flowering, the onset of overall protein accumulation. Significant expression of CAT activity in non-seed tissues occurred in some, but not all plants, suggesting possible chromosome position effects on non-seed tissue expression. A positive correlation was observed between expression levels in seeds and gene copy numbers.Author for correspondence     

Enhanced resistance to blast fungus and bacterial blight in transgenic rice constitutively expressing OsSBP, a rice homologue of mammalian selenium-binding proteins

The rice Oryza sativa selenium-binding protein homologue (OsSBP) gene encodes a homologue of mammalian selenium-binding proteins, and it has been isolated as one of the genes induced by treating a plant with a cerebroside elicitor from rice blast fungus. The possible role of OsSBP in plant defense was evaluated by using a transgenic approach. Plants overexpressing OsSBP showed enhanced resistance to a virulent strain of rice blast fungus as well as to rice bacterial blight. The expression of defense-related genes and the accumulation of phytoalexin after infection by rice blast fungus were accelerated in the OsSBP overexpressors. A higher level of H(2)O(2) accumulation and reduced activity of such scavenging enzymes as ascorbate peroxidase and catalase were seen when the OsSBP-overexpressing plants were treated with the protein phosphatase 1 inhibitor, calyculin A. These results suggest that the upregulation of OsSBP expression conferred enhanced tolerance to different pathogens, possibly by increasing plant sensitivity to endogenous defense responses. Additionally, the OsSBP protein might have a role in modulating the defense mechanism to biotic stress in rice.     

水稻谷蛋白启动子驱动下的ipt基因在转基因烟草中的表达

利用农杆菌系统介导 ,采用叶盘转化法 ,将在水稻谷蛋白启动子驱动下的外源ipt基因导入烟草植株中 ,经过抗生素筛选、PCR与测序分析检测出转基因植株。成熟的转基因烟草种子经过ELISA细胞分裂素试剂盒检测 ,发现iPAs含量为对照的 2 .43倍 ,此外 ,种子的重量也增加了 7.8%。     

Inducible transformation of cells from transgenic mice expressing SV40 under lac operon control.

If it were possible to clone in vitro cells of any type, at any stage of differentiation, from an extensively characterized animal such as the mouse, many areas of cell biology would benefit. Indeed, it would be even more helpful if these cells could subsequently be restored to their normal in vivo phenotype whenever required. Here, we describe a step on the pathway to such an idealized "clonable" mouse. In principle, it seeks to link a "universal" transforming agent to a regulatory system that is relatively simple, yet quite foreign to the mouse. A plasmid containing the bacterial lac operator/promoter region linked to the SV40 large T antigen and a vector containing the lac repressor that can be expressed in mammalian cells were coinjected into fertilized mouse oocytes utilizing the standard techniques for generating transgenic mice. Two progeny were obtained that express large T antigen in the presence, but not the absence, of the nonmetabolizable lac inducer, isopropyl-beta-thio-D-galactoside. This report characterizes fibroblast cell lines established from these transgenics that are readily transformed in vitro with isopropyl-beta-thio-D-galactoside. A significant proportion of the cells are restored to their "normal" (nontransformed phenotype) when isopropyl-beta-thio-D-galactoside is removed.     

Glucose intolerance and reduced islet blood flow in transgenic mice expressing the FRK tyrosine kinase under the control of the rat insulin promoter

The FRK tyrosine kinase has previously been shown to transduce beta-cell cytotoxic signals in response to cytokines and streptozotocin and to promote beta-cell proliferation and an increased beta-cell mass. We therefore aimed to further evaluate the effects of overexpression of FRK tyrosine kinase in beta-cells. A transgenic mouse expressing kinase-active FRK under control of the insulin promoter (RIP-FRK) was studied with regard to islet endocrine function and vascular morphology. Mild glucose intolerance develops in RIP-FRK male mice of at least 4 mo of age. This effect is accompanied by reduced glucose-stimulated insulin secretion in vivo and reduced second-phase insulin secretion in response to glucose and arginine upon pancreas perfusion. Islets isolated from the FRK transgenic mice display a glucose-induced insulin secretory response in vitro similar to that of control islets. However, islet blood flow per islet volume is decreased in the FRK transgenic mice. These mice also exhibit a reduced islet capillary lumen diameter as shown by electron microscopy. Total body weight and pancreas weight are not significantly affected, but the beta-cell mass is increased. The data suggest that long-term expression of active FRK in beta-cells causes an in vivo insulin-secretory defect, which may be the consequence of islet vascular abnormalities that yield a decreased islet blood flow.     

Enhanced stress-tolerance of transgenic tobacco plants expressing a human dehydroascorbate reductase gene

To analyze the physiological role of dehydroascorbate reductase (DHAR, EC 1.8.5.1) catalyzing the reduction of DHA to ascorbate in environmental stress adaptation, T1 transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants expressing a human DHAR gene in chloroplasts were biochemically characterized and tested for responses to various stresses. Fully expanded leaves of transgenic plants had about 2.29 times higher DHAR activity (units/g fresh wt) than non-transgenic (NT) plants. Interestingly, transgenic plants also showed a 1.43 times higher glutathione reductase activity than NT plants. As a result, the ratio of AsA/DHA was changed from 0.21 to 0.48, even though total ascorbate content was not significantly changed. When tobacco leaf discs were subjected to methyl viologen (MV) at 5 mumol/L and hydrogen peroxide (H2O2) at 200 mmol/L, transgenic plants showed about a 40% and 25% reduction in membrane damage relative to NT plants, respectively. Furthermore, transgenic seedlings showed enhanced tolerance to low temperature (15 degrees C) and NaCl (100 mmol/L) compared to NT plants. These results suggest that a human derived DHAR properly works for the protection against oxidative stress in plants.     

Production of marker-free transgenic rice expressing tissue-specific Bt gene

The hybrid Bacillus thuringiensis (Bt) δ-endotoxin gene Cry1Ab/Ac was used to develop a transgenic Bt rice (Oryza sativa L.) targeting lepidopteran insects of rice. Here, we show the production of a marker-free and tissue-specific expressing transgenic Bt rice line L24 using Agrobacterium-mediated transformation and a chemically regulated, Cre/loxP-mediated DNA recombination system. L24 carries a single copy of marker-free T-DNA that contains the Cry1Ab/Ac gene driven by a maize phosphoenolpyruvate carboxylase (PEPC) gene promoter. The marker-free T-DNA was integrated into the 3′ untranslated region of rice gene Os01g0154500 on the short arm of chromosome 1. Compared to the constitutive and non-specific expression of the P _Actin1 :Cry1Ab/Ac:T _Nos gene in the control Bt rice line T51-1, the P _Pepc :Cry1Ab/Ac:T _Nos gene was detected only in the leaf and stem tissues of L24. More importantly, compared to high levels of CRY1Ab/Ac proteins accumulated in T51-1 seeds, the CRY1Ab/Ac proteins were not detectable in L24 seeds by Western blot analysis. As demonstrated by insect bioassay, L24 provided similar level of resistance to rice leaffolder (Cnaphalocrocis medinalis) as T51-1. The marker-free transgenic line L24 can be used directly in rice breeding for insect resistance to lepidopteran insects where absence of Bt toxin protein in the seed is highly desirable.     

Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene

The cbnA gene encoding the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9 was introduced into rice plants. The cbnA gene was expressed in transgenic rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. Growth and morphology of the transgenic rice plants expressing the cbnA gene were not distinguished from those of control rice plants harboring only a Ti binary vector. It is thus possible to breed transgenic plants that degrade chloroaromatic compounds in soil and surface water.     

Enhanced fungal resistance in transgenic cotton expressing an endochitinase gene from Trichoderma virens

Mycoparasitic fungi are proving to be rich sources of antifungal genes that can be utilized to genetically engineer important crops for resistance against fungal pathogens. We have transformed cotton and tobacco plants with a cDNA clone encoding a 42 kDa endochitinase from the mycoparasitic fungus, Trichoderma virens. Plants from 82 independently transformed callus lines of cotton were regenerated and analysed for transgene expression. Several primary transformants were identified with endochitinase activities that were significantly higher than the control values. Transgene integration and expression was confirmed by Southern and Northern blot analyses, respectively. The transgenic endochitinase activities were examined in the leaves of transgenic tobacco as well as in the leaves, roots, hypocotyls and seeds of transgenic cotton. Transgenic plants with elevated endochitinase activities also showed the expected 42 kDa endochitinase band in fluorescence, gel-based assays performed with the leaf extracts in both species. Homozygous T2 plants of the high endochitinase-expressing cotton lines were tested for disease resistance against a soil-borne pathogen, Rhizoctonia solani and a foliar pathogen, Alternaria alternata. Transgenic cotton plants showed significant resistance to both pathogens.