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991.
Marker-free transgenic white poplar (Populus alba L., cv ‘Villafranca’) plants, expressing the PsMT A1 gene from Pisum sativum for a metallothionein-like protein, were produced by Agrobacterium tumefaciens-mediated transformation. The 35SCaMV-PsMT A1 -NosT cassette was inserted into the ipt-type vector pMAT22. The occurrence of the abnormal ipt-shooty phenotype allowed the visual selection of transformants, while the yeast site-specific recombination R/RS system was responsible for the excision of the undesired vector sequences with the consequent recovery of normal marker-free transgenic plants. Molecular analyses confirmed the presence of the 35SCaMV-PsMT A1 -NosT cassette and transgene expression. Five selected lines were further characterized, revealing the ability to withstand heavy metal toxicity. They survived 0.1 mM CuCl2, a concentration which strongly affected the nontransgenic plants. Moreover, root development was only slightly affected by the ectopic expression of the transgene. Reactive oxygen species were accumulated to a lower extent in leaf tissues of multi-auto-transformation (MAT)-PsMTA1 plants exposed to copper and zinc, compared to control plants. Tolerance to photo-oxidative stress induced by paraquat was another distinctive feature of the MAT-PsMTA1 lines. Finally, low levels of DNA damage were detected by quantifying the amounts of 8-hydroxy-2′-deoxyguanosine in leaf tissues of the transgenic plants exposed to copper.  相似文献   
992.
The efficacy of the ipt-type Multi-Auto-Transformation (MAT) vector system to transform the extensively grown cassava cultivar “KU50” was evaluated. This system utilizes the isopentenyltransferase (ipt) gene as morphological marker for visual selection of transgenic lines. The extreme shooty phenotype (ESP) of transgenic lines is lost due to the removal of ipt gene mediated by the yeast Rint/RS system. As a result, phenotypically normal shoots, considered marker-free transgenic plants, could be obtained. When transforming KU50 cassava cultivar with two different ipt-type MAT vectors, transformation frequency at 19–21% was observed. Among the total number of ESP explants, 32–38% regained normal extended shoot phenotype and 88–96% of which were confirmed to represent the marker-free transgenic plants. This is the first demonstration of the efficacy of Rint/RS system in promoting excision of ipt marker gene in cassava specie, with the consequent rapid production of marker-free transgenic plants. The high efficiency of this system should facilitate pyramiding a number of transgenes by repeated transformation without having to undergo through laborious, expensive and time-consuming processes of sexual crossing and seed production. The generation of marker-free, thus environmentally safe, genetically modified cassava clones should also ease the public concerns regarding the use of transgenic cassava in both food and nonfood industries.  相似文献   
993.
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995.
The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.  相似文献   
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997.
The effect of salt stress on leaf morphology and functionality was studied in three Populus alba genotypes differing in tolerance to salinity: 6K3 (sensitive), 2AS11 (moderately tolerant), and 14P11 (tolerant). Plants were subjected to an intense and progressive salt stress from 50 to 250 mM NaCl by 50 mM steps at 10-day intervals. The micromorphological results highlighted phenotypic variation among the three genotypes already in control plants, with the genotype 14P11 having significantly smaller epidermal cells and higher stomatal density. Salt-treated plants modulated differently the expansion of stomata compared with epidermal cells. Regression analysis showed significant correlations between decrease of stomatal area and stomatal conductance (gs) in genotypes 14P11 and 6K3. So, the common reduction of stomatal area could be an early mechanism to save water in this species. However, only genotype 14P11 showed further significant decrease of this trait under the highest salinity level, combined with a significant reduction in leaf length. In addition, this genotype showed the lowest leaf abscission rate at the end of salt stress period. The genotype 6K3 was severely affected by leaf necrosis and showed the highest leaf abscission rate in salt stress conditions. In the moderately tolerant genotype 2AS11, an intermediate plastic behaviour in both leaf morphology and physiology was observed during the experiment. The phenotypic variation among the three genotypes in terms of micromorphology and stomatal conductance is discussed in relation to plant functionality in salt stress conditions. Overall results suggest that leaf morphological habit contributes to salt tolerance in P. alba.  相似文献   
998.
Enzymatic hydrolysis of hybrid poplar treated by ammonia recycle percolation (ARP) was studied applying cellulase enzyme supplemented with additional xylanase or pectinase. The effect of xylanase addition was much more significant than pectinase addition. Conversion of ARP‐treated hybrid poplar to ethanol was carried out by simultaneous saccharification and fermentation (SSF) and SS and cofermentation (SSCF). The maximum ethanol yield observed from the SSCF experiment was 78% of theoretical maximum based on the total carbohydrate (glucan + xylan). The same feedstock was also treated by soaking in aqueous ammonia (SAA), a batch pretreatment process with lower severity than ARP. The test results indicated that relatively high severity is required to attain acceptable level of digestibility of hybrid poplar. In order to lower the severity of the pretreatment, addition of H2O2 was attempted in the SAA. Addition of H2O2 significantly enhanced delignification of hybrid poplar due to its oxidative degradation of lignin. Several different H2O2 feeding schemes and different temperature profiles were attempted in operation of the SAA to investigate the effects of H2O2 on degradation of lignin and carbohydrates in hybrid poplar. More than 60% of lignin in hybrid poplar was removed with stepwise‐increase of temperature (60–120°C after 4h of reaction). Increase of carbohydrate degradation was also observed under this condition. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009  相似文献   
999.
Comparative data is presented on glucose and xylose release for enzymatic hydrolysis of solids produced by pretreatment of poplar wood by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, flowthrough (FT), lime, and sulfur dioxide (SO2) technologies. Sugar solubilization was measured for times of up to 72 h using cellulase supplemented with β‐glucosidase at an activity ratio of 1:2, respectively, at combined protein mass loadings of 5.8–116 mg/g of glucan in poplar wood prior to pretreatment. In addition, the enzyme cocktail was augmented with up to 11.0 g of xylanase protein per gram of cellulase protein at combined cellulase and β‐glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose to evaluate cellulase–xylanase interactions. All pretreated poplar solids required high protein loadings to realize good sugar yields via enzymatic hydrolysis, and performance tended to be better for low pH pretreatments by dilute sulfuric acid and sulfur dioxide, possibly due to higher xylose removal. Glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments, xylanase leverage on glucan removal decreased at high cellulase loadings. Washing the solids improved digestion for all pretreatments and was particularly beneficial for controlled pH pretreatment. Furthermore, incubation of pretreated solids with BSA, Tween 20, or PEG6000 prior to adding enzymes enhanced yields, but the effectiveness of these additives varied with the type of pretreatment. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009  相似文献   
1000.
Pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP) catalyzes the reversible interconversion of fructose-6-phosphate and fructose-1,6-bisphosphate, a key step in the regulation of the metabolic flux toward glycolysis or gluconeogenesis. To examine the role of PFP in plant growth, we have generated transgenic Arabidopsis plants that either overexpress or repress Arabidopsis PFP sub-unit genes. The overexpressing lines displayed increased PFP activity and slightly faster growth relative to wild type plants, although their photosynthetic activities and the levels of metabolites appeared not to have significantly changed. In contrast, the RNAi lines showed significantly retarded growth in parallel with the reduced PFP activity. Analysis of photosynthetic activity revealed that the growth retardation phenotype of the RNAi lines was accompanied by the reduced rates of CO2 assimilation. Microarray analysis of our transgenic plants further revealed that the altered expression of AtPFPβ affects the expression of several genes involved in diverse physiological processes. Our current data thus suggest that PFP is important in carbohydrate metabolism and other cellular processes. These authors contributed equally to this study.  相似文献   
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