In higher plants, DREB1/CBF-type transcription factors play an important role in tolerance to low temperatures, drought, and high-salt stress. These transcription factors bind to CRT/DRE elements in promoter regions of target genes, regulating their expression. In this study, we cloned and characterized a novel gene encoding a DREB1 transcription factor from dwarf apple, Malus baccata (GenBank accession number: EF582842). Expression of MbDREB1 was induced by cold, drought, and salt stress, and also in response to exogenous ABA. Subcellular localization analyses revealed that MbDREB1 localizes in the nucleus. A yeast activity assay demonstrated that the MbDREB1 gene encodes a transcription activator, which specifically binds to DRE/CRT elements. Compared with wild-type plants, transgenic Arabidopsis overexpressing MbDREB1 showed increased tolerance to low temperature, drought, and salt stresses. Analysis of the MbDREB1 promoter revealed an ABA-responsive element (ABRE), an inducer of CBF expression 1 (ICE1)-like binding site, two MYB recognition sites, and three stress-inducible GT-1 boxes. GUS activities driven by the MbDREB1 promoter in transgenic Arabidopsis increased in response to ABA, cold temperature, drought, and salt treatments. Interestingly, the expression of both ABA-independent and ABA-dependent stress-induced genes (COR15a and rd29B, respectively) was activated under normal growth conditions in Arabidopsis overexpressing MbDREB1. These results suggest that MbDREB1 functions as a transcription factor and increases plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways.

Dwarf apple MbDREB1 enhances plant tolerance to low temperature, drought, and salt stress via both ABA-dependent and ABA-independent pathways.

ion between fibre lignin and laccase in qualitative difference in temporal phases than laccase does alone. Resulting distinguished lignin modifications may have consequences for subsequent fibre behaviour (Barsberg 2002). Lund & Felby (2001) applied a range of mediators in laccase-modification of spruce and pine fibres and found ABTS and PPT (phenothiazine-10-propionic acid) to be effective in enhancing wet tensile strength of paper handsheets made from the pulp. Similar results were obtained with ABTS by Elegir et al. (2005, 2007). Treatment of fibres with laccase and gallic acid were also reported to change wet tensile strength (Chandra et al. 2004b), indicating that laccase-mediator system can influence properties of the fibre surfaces. Various mediators are therefore currently tested in MDF production with laccase in order to achieve higher reactivity between laccase and lignin and better board properties (Müller et al. 2006). The unfortunately limited availability of cheap and non-toxic mediators is of course a restricting factor in such approaches (Rochefort et al. 2004, Morozova et al. 2007). So far, in laccase-mediated bleaching of lignocellulosic fibres 1-hydroxybenzotriazole and the lignin-derived phenols acetosyringone and syringaldehyde acid have been positively tested as mediators whereas negligible effects were seen with p-coumaric acid (Ibarra et al. 2006, Camamero et al. 2007, Gutiérrez et al. 2007). Oppositional ABTS-mediated depolymerisation and polymerisation effects on kraft lignin samples (González Arzola et al. 2006, Hernández Fernaud et al. 2006) imply that the doses and times of mediators and enzymes applied have to be expected to play an important role in activating of fibre material. In line with this, dosages of 4-hydroxybenzoic acid were most influential in laccase-mediated modification of paper pulp (Chandra et al. 2004a). Further to applications of mediators to improve mechanical properties by laccase action, recent studies suggest that a successive xylanase and laccase treatment with or without mediators might also be useful in efficiently modifying fibre surfaces at least in paper production. As the gallic acid mediator treatment, successive xylanase-laccase treatment has been reported to increase the wet tensile strength of paper (Chandra & Raugaskas 2005, Kapoor et al. 2007). 2-component adhesives based on lignin and enzymes In the wood composite industry, addition of lignosulphonates (spent sulphite liquor) or kraft lignins from sulphate pulping, to conventional PF resins is targeted at replacing parts of the phenol component made from petrochemicals (e.g. see Roffael & Rauch 1971, Shen 1974, Nimz 1983, Ayla & Nimz 1984, Danielson & Simonson 1998a,b, Tejado et al. 2007; further reading in Chapter 16 of this book). Formaldehyde from the PF resin may act in such mixtures as a cross-linker between the reactive sites of the technical lignins and the synthetic phenol binder (Kuo et al. 1991, Vázquez et al. 1999, El Mansouri & Salvadó 2006, 2007). As first

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Wood production, wood technology, and biotechnological impacts

Objectives: The aim of this study was to assess whether Escherichia coli phylogenetic groups were associated with the site of infection and the level of antibiotic resistance in community-acquired bacteraemia (CAB). Methods: The population-based cohort study included 1533 unique isolates of E. coli from Danish patients with CAB during a 10 year period. Triplex PCR was used to classify the phylogenetic groups, and susceptibility testing was performed by disc diffusion. Data were analysed using contingency tables and logistic regression. Results: Overall, 65.9% of the 1533 E. coli isolates belonged to phylogroup B2, 16.6% to D, 13.1% to A and 4.4% to B1. B2 was the most prevalent group for all sites of infection, ranging from 69.9% in cases with a urinary tract site of infection to 54.8% in cases with a hepatobiliary tract site of infection. Antibiotic resistance to one and more than three antibiotics, respectively, was most frequent in group D (11.4%/33.9%), followed by A (5.5%/26.9%), B1 (5.9%/19.1%) and B2 (6.7%/7.5%). Regression analysis, with group B2 as reference, confirmed that groups A and B1 were associated with a site of infection other than the urinary tract and that groups A and D were associated with resistance to antibiotics including ampicillin, sulphonamide, trimethoprim, gentamicin and quinolones. Conclusions: Phylogenetic group B2 was predominant in E. coli CAB. This was the least resistant of the four groups. Phylogroups A and B1 were associated with sites of infection other than the urinary tract, and resistance to multiple antibiotics was most prevalent for groups A and D.

Escherichia coli phylogenetic groups are associated with site of infection and level of antibiotic resistance in community-acquired bacteraemia: a 10 year population-based study in Denmark

CRISPR-Cas9 assisted gene disruption in the higher fungus Ganoderma species

Production of Useful Terpenoids by Higher-Fungus Cell Factory and Synthetic Biology Approaches

A highly efficient transformation system mediated by polyethylene glycol was developed for the cultivated mushroom Pleurotus ostreatus. Eighty to 180 integrative and stable-resistant colonies appeared per mug of DNA per 10(7) viable protoplasts in a transformation experiment with the hygromycin B phosphotransferase gene (hph), which is about 40-1800 times higher than that previously reported in P. ostreatus. One hundred to 150 transformants emitting green fluorescence were observed per mug of DNA per 10(7) viable protoplasts in a transformation with the green fluorescent protein gene, but green fluorescence disappeared 30 h after transformation, suggesting that the green fluorescent protein gene was only transiently expressed in P. ostreatus. Plasmid pAN7-1 was also transferred into two important cultivated mushrooms, Ganoderma lucidum and Lentinus edodes, and 120-150 and 85-100 transformants per mug of DNA per 10(7) viable protoplasts were obtained, respectively, which is seven to 38 times and 24-28 times greater than previously reported. These data indicate that this new polyethylene glycol-mediated transformation procedure is highly efficient for mushrooms, and could be a useful tool in mushroom improvement by gene engineering.

A highly efficient polyethylene glycol‐mediated transformation method for mushrooms

Simple methods for the preparation of polymerase chain reaction (PCR) templates from Escherichia coli, Saccharomyces cerevisiae and Oryza sativa using a microwave and by boiling are described without the need for any special chemical reagent such as a detergent. The microwave and boiling methods were robust for the preparation of both plasmid and genomic templates of E. coli, as was the boiling approach for S. cerevisiae DNA. The microwave procedure was only suitable for dry yeast colonies with fair efficiency as absence of water (H2O) was critical for a successful PCR. Oryza sativa genomic DNA was amplified with both approaches with satisfactory efficiency. Escherichia coli plasmids can be prepared using a microwave with a yield equivalent to 15–20% of an alkaline lysis mini-prep. PCR templates were also efficiently generated from Pichia pastoris and Ganoderma lucidum by using the boiling method.

Rapid and efficient generation of pcr templates from escherichia coli, saccharomyces cerevisiae and oryza sativa using a microwave and by boiling

Most DNA shuffling methods currently used require PCR process. A novel method of DNA shuffling without PCR process is described, taking advantage of the feature of some restriction enzymes whose recognition sites differ from their cleavage sites, thus giving rise to different cohesive ends. These cohesive ends can be rejoined at their native sites from different parental sequences, generating new sequences with various combinations of mutations.

A novel method of DNA shuffling without PCR process

A method for the rapid recovery of DNA from agarose gel slices for polymerase chain reaction (PCR) applications was described. DNA bands were excised from agarose gel and diluted with distilled water. One-minute microwave treatment at full power was performed, immediately followed by 1-min centrifugation. The supernatant thus recovered can be directly used in PCR amplifications with minimal delay. This protocol represents an ultrafast and inexpensive alternative to commercial kits.

Qiang Wang

Papers

A highly efficient polyethylene glycol‐mediated transformation method for mushrooms

Rapid and efficient generation of pcr templates from escherichia coli, saccharomyces cerevisiae and oryza sativa using a microwave and by boiling

A novel method of DNA shuffling without PCR process

Rapid recovery of dna from agarose gel slice using a microwave

Simultaneous detection of seven mutations with seven forward primers and one common reverse primer in a single PCR step.