Tissue-specific expression directed by an Arabidopsis thaliana pre-ferredoxin promoter in transgenic tobacco plants

We have isolated and analyzed a pre-ferredoxin gene from Arabidopsis thaliana. This gene encodes a 148 amino acid precursor protein including a chloroplast transit peptide of 52 residues. Southern analysis shows the presence of a single copy of this ferredoxin (Fd) gene in the A. thaliana genome. Its expression is tissue-specific and positively affected by light. Response times, both to dark and light conditions, are remarkably rapid.A chimeric gene consisting of a 1.2 kb Fd promoter fragment fused to the -glucuronidase reporter gene was transferred to tobacco. This fusion gene is expressed in a tissue-specific way; it shows high levels of expression in green leaves, as compared to root tissue.     

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     

Bacterial pathogen phytosensing in transgenic tobacco and Arabidopsis plants

Plants are subject to attack by a wide range of phytopathogens. Current pathogen detection methods and technologies are largely constrained to those occurring post‐symptomatically. Recent efforts were made to generate plant sentinels (phytosensors) that can be used for sensing and reporting pathogen contamination in crops. Engineered phytosensors indicating the presence of plant pathogens as early‐warning sentinels potentially have tremendous utility as wide‐area detectors. We previously showed that synthetic promoters containing pathogen and/or defence signalling inducible cis‐acting regulatory elements (RE) fused to a fluorescent protein (FP) reporter could detect phytopathogenic bacteria in a transient phytosensing system. Here, we further advanced this phytosensing system by developing stable transgenic tobacco and Arabidopsis plants containing candidate constructs. The inducibility of each synthetic promoter was examined in response to biotic (bacterial pathogens) or chemical (plant signal molecules salicylic acid, ethylene and methyl jasmonate) treatments using stably transgenic plants. The treated plants were visualized using epifluorescence microscopy and quantified using spectrofluorometry for FP synthesis upon induction. Time‐course analyses of FP synthesis showed that both transgenic tobacco and Arabidopsis plants were capable to respond in predictable ways to pathogen and chemical treatments. These results provide insights into the potential applications of transgenic plants as phytosensors and the implementation of emerging technologies for monitoring plant disease outbreaks in agricultural fields.     

Amyloid-like protein inclusions in tobacco transgenic plants

The formation of insoluble protein deposits in human tissues is linked to the onset of more than 40 different disorders, ranging from dementia to diabetes. In these diseases, the proteins usually self-assemble into ordered β-sheet enriched aggregates known as amyloid fibrils. Here we study the structure of the inclusions formed by maize transglutaminase (TGZ) in the chloroplasts of tobacco transplastomic plants and demonstrate that they have an amyloid-like nature. Together with the evidence of amyloid structures in bacteria and fungi our data argue that amyloid formation is likely a ubiquitous process occurring across the different kingdoms of life. The discovery of amyloid conformations inside inclusions of genetically modified plants might have implications regarding their use for human applications.     

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%。     

Molecular cloning of the complete 11S seed storage protein gene of Coffea arabica and promoter analysis in transgenic tobacco plants

In this paper, we present the complete nucleotide sequence of the csp1 gene from Coffea arabica coding for the 11S-globulin seed storage protein. To investigate the sequences responsible for the regulated expression of this seed-specific coffee storage protein gene, about 1 kb of the 5'-upstream region from the csp1 gene was isolated using inverse polymerase chain reaction (IPCR) and then sequenced. Several DNA boxes were found in this coffee sequence that had similarity to those previously identified as being essential for grain (endosperm) specific expression in other plants. To study the ability of this sequence to direct grain-specific expression, the whole fragment, as well as a series of 5' deletions, was fused to the reporter gene β-glucuronidase (uidA) and analysed in transgenic Nicotiana tabacum plants. GUS measurements showed that all the deletions of the csp1 promoter directed the expression of the reporter gene in tobacco grain but not in the other tissues examined. GUS activities also revealed that the csp1 promoter constructs function as very strong promoters by comparison to the strength of the cauliflower mosaic virus (CaMV) 35S promoter. Therefore, this 11S promoter could represent a useful tool to change the expression of targeted genes in the grain of transgenic coffee plants.     

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants

The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the Arabidopsis MinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.     

Isolation of the Chinese rose sHSP gene promoter and its differential regulation analysis in transgenic Arabidopsis plants

In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s response to high temperature.     

Developmental regulation and tissue-specific differences of heat shock gene expression in transgenic tobacco and Arabidopsis plants

The heat shock (hs) response during plant growth and development was analyzed in tobacco and Arabidopsis using chimaeric -glucuronidase reporter genes (hs-Gus) driven by a soybean hs promoter. Fluorimetric measurements and histochemical staining revealed high Gus activities in leaves, roots, and flowers exclusively after heat stress. The highest levels of heat-inducible expression were found in the vascular tissues. Without heat stress, a developmental induction of hs-Gus was indicated by the accumulation of high levels of Gus in transgenic tobacco seeds. There was no developmental induction of hs-Gus in Arabidopsis seeds. In situ hybridization to the RNA of the small heat shock protein gene Athsp17.6 in tissue sections revealed an expression in heat-shocked leaves but no expression in control leaves of Arabidopsis. However, a high level of constitutive expression of hs gene was detected in meristematic and provascular tissues of the Arabidopsis embryo. The developmental and tissue-specific regulation of the hs response is discussed.Abbreviations hs heat shock - Hsp heat shock protein(s) - hs Gus: heat-inducible Gus gene(s) - HSE heat shock element(s) - HSF heat shock factor - X-gluc 5-bromo-4-chloro-3-indolyl--D-glucuronide - Gus -glucuronidase - DAF days after flowering - SAR scaffold attachment region     

Variability of organ-specific gene expression in transgenic tobacco plants

Variability of expression of introduced marker genes was analysed in a large number of tobacco regenerants from anAgrobacterium-mediated transformation. In spite of standardization of sampling, considerable variation of GUS and NPTII expression was observed between individual transformants at different times of analysis and in different parts of the same plant. Organ-specificity of root versus leaf expression conferred by the par promoter from the haemoglobin gene ofParasponia andersonii in front of thegus gene showed a continuous spectrum. GUS expression in roots was found in 128 out of 140 plants; expression in leaves was found in 46 plants, and was always lower than in the corresponding roots. NPTII expression regulated by the nos promoter also showed a continuous spectrum. Expression levels were generally higher in roots than in leaves. Plants with high GUS expression in leaves showed high NPTII activity as well. A positive correlation between the level of NPTII expression and the numbers of integrated gene copies was noted. Chromosomal position effects and physiological determination are suggested as triggers for the variations. The transformed regenerated tobacco plants were largely comparable to clonal variants.