scholarly journals Use of Multiplex Reverse Transcription-PCR To Study the Expression of a Laccase Gene Family in a Basidiomycetous Fungus

2003 ◽  
Vol 69 (12) ◽  
pp. 7083-7090 ◽  
Author(s):  
Tania González ◽  
María C. Terrón ◽  
Ernesto J. Zapico ◽  
Alejandro Téllez ◽  
Susana Yagüe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Reverse Transcription ◽  
White Rot Fungi ◽  
Gene Families ◽  
Veratryl Alcohol ◽  
White Rot ◽  
Laccase Gene ◽  
Reverse Transcription Pcr ◽  
Basidiomycetous Fungus ◽  
Laccase Genes

ABSTRACT Laccases produced by white rot fungi are involved in the degradation of lignin and a broad diversity of other natural and synthetic molecules, having a great potential for biotechnological applications. They are frequently encoded by gene families, as in the basidiomycete Trametes sp. strain I-62, from which the lcc1, lcc2, and lcc3 laccase genes have been cloned and sequenced. A multiplex reverse transcription-PCR method to simultaneously study the expression of these genes was developed in this study. The assay proved to be quick, simple, highly sensitive, and reproducible and is particularly valuable when numerous samples are to be analyzed and/or if the amount of initial mRNA is limited. It was used to analyze the effect of 3,4-dimethoxybenzyl alcohol (veratryl alcohol) and two of its isomers (2,5-dimethoxybenzyl alcohol and 3,5-dimethoxybenzyl alcohol) on differential laccase gene expression in Trametes sp. strain I-62. These aromatic compounds produced different induction patterns despite their chemical similarity. We found 2,5-dimethoxybenzyl alcohol to be the best inducer of laccase activity while also producing the highest increase in gene expression; 3,5-dimethoxybenzyl alcohol was the next best inducer. Transcript amounts of each gene fluctuated dramatically in the presence of these three inducers, while the total amounts of laccase mRNAs seemed to be modulated by a coordinated regulation of the different genes.

Transformation of Industrial Dyes by Manganese Peroxidases from Bjerkandera adusta and Pleurotus eryngii in a Manganese-Independent Reaction

1998 ◽  
Vol 64 (8) ◽  
pp. 2788-2793 ◽  
Author(s):  
A. Heinfling ◽  
M. J. Martínez ◽  
A. T. Martínez ◽  
M. Bergbauer ◽  
U. Szewzyk
Keyword(s):  
White Rot Fungi ◽  
Veratryl Alcohol ◽  
Pleurotus Eryngii ◽  
White Rot ◽  
Reactive Black 5 ◽  
Bjerkandera Adusta ◽  
Independent Manner ◽  
Phthalocyanine Dyes ◽  
Specific Activities ◽  
Dye Oxidation

ABSTRACT We investigated the transformation of six industrial azo and phthalocyanine dyes by ligninolytic peroxidases from Bjerkandera adusta and other white rot fungi. The dyes were not oxidized or were oxidized very little by Phanerochaete chrysosporiummanganese peroxidase (MnP) or by a chemically generated Mn3+-lactate complex. Lignin peroxidase (LiP) from B. adusta also showed low activity with most of the dyes, but the specific activities increased 8- to 100-fold when veratryl alcohol was included in the reaction mixture, reaching levels of 3.9 to 9.6 U/mg. The B. adusta and Pleurotus eryngii MnP isoenzymes are unusual because of their ability to oxidize aromatic compounds like 2,6-dimethoxyphenol and veratryl alcohol in the absence of Mn2+. These MnP isoenzymes also decolorized the azo dyes and the phthalocyanine complexes in an Mn2+-independent manner. The reactions with the dyes were characterized by apparentKm values ranging from 4 to 16 μM and specific activities ranging from 3.2 to 10.9 U/mg. Dye oxidation by these peroxidases was not increased by adding veratryl alcohol as it was in LiP reactions. Moreover, the reaction was inhibited by the presence of Mn2+, which in the case of Reactive Black 5, an azo dye which is not oxidized by the Mn3+-lactate complex, was found to act as a noncompetitive inhibitor of dye oxidation byB. adusta MnP1.

scholarly journals Enhanced Production of Ligninolytic Enzymes by a Mushroom Stereum ostrea

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
K. Y. Usha ◽  
K. Praveen ◽  
B. Rajasekhar Reddy
Keyword(s):  
Solid State ◽  
Copper Sulphate ◽  
White Rot Fungi ◽  
Ligninolytic Enzymes ◽  
Tween 80 ◽  
Veratryl Alcohol ◽  
White Rot ◽  
Tween 20 ◽  
Laccase Production ◽  
Enhanced Production

The white rot fungi Stereum ostrea displayed a wide diversity in their response to supplemented inducers, surfactants, and copper sulphate in solid state fermentation. Among the inducers tested, 0.02% veratryl alcohol increased the ligninolytic enzyme production to a significant extent. The addition of copper sulphate at 300 μM concentration has a positive effect on laccase production increasing its activity by 2 times compared to control. Among the surfactants, Tween 20, Tween 80, and Triton X 100, tested in the studies, Tween 80 stimulated the production of ligninolytic enzymes. Biosorption of dyes was carried out by using two lignocellulosic wastes, rice bran and wheat bran, in 50 ppm of remazol brilliant blue and remazol brilliant violet 5R dyes. These dye adsorbed lignocelluloses were then utilized for the production of ligninolytic enzymes in solid state mode. The two dye adsorbed lignocelluloses enhanced the production of laccase and manganese peroxidase but not lignin peroxidase.

Alternative splicing in plants

2008 ◽  
Vol 36 (3) ◽  
pp. 508-510 ◽  
Author(s):  
Craig G. Simpson ◽  
Dominika Lewandowska ◽  
John Fuller ◽  
Monika Maronova ◽  
Maria Kalyna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Alternative Splicing ◽  
Reverse Transcription ◽  
Protein Function ◽  
Mrna Levels ◽  
Reverse Transcription Pcr ◽  
Multiple Genes ◽  
The Impact

The impact of AS (alternative splicing) is well-recognized in animal systems as a key regulator of gene expression and proteome complexity. In plants, AS is of growing importance as more genes are found to undergo AS, but relatively little is known about the factors regulating AS or the consequences of AS on mRNA levels and protein function. We have established an accurate and reproducible RT (reverse transcription)–PCR system to analyse AS in multiple genes. Initial studies have identified new AS events confirming that current values for the frequency of AS in plants are likely to be underestimates.

scholarly journals Expression of the Laccase Gene from a White Rot Fungus in Pichia pastoris Can Enhance the Resistance of This Yeast to H2O2-Mediated Oxidative Stress by Stimulating the Glutathione-Based Antioxidative System

2012 ◽  
Vol 78 (16) ◽  
pp. 5845-5854 ◽  
Author(s):  
Yang Yang ◽  
Fangfang Fan ◽  
Rui Zhuo ◽  
Fuying Ma ◽  
Yangmin Gong ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Gene Expression ◽  
Pichia Pastoris ◽  
Oxidative Damage ◽  
White Rot ◽  
Antioxidative System ◽  
White Rot Fungus ◽  
Laccase Gene ◽  
Content Type ◽  
Glutamylcysteine Synthetase

ABSTRACTLaccase is a copper-containing polyphenol oxidase that has great potential in industrial and biotechnological applications. Previous research has suggested that fungal laccase may be involved in the defense against oxidative stress, but there is little direct evidence supporting this hypothesis, and the mechanism by which laccase protects cells from oxidative stress also remains unclear. Here, we report that the expression of the laccase gene from white rot fungus inPichia pastoriscan significantly enhance the resistance of yeast to H2O2-mediated oxidative stress. The expression of laccase in yeast was found to confer a strong ability to scavenge intracellular H2O2and to protect cells from lipid oxidative damage. The mechanism by which laccase gene expression increases resistance to oxidative stress was then investigated further. We found that laccase gene expression inPichia pastoriscould increase the level of glutathione-based antioxidative activity, including the intracellular glutathione levels and the enzymatic activity of glutathione peroxidase, glutathione reductase, and γ-glutamylcysteine synthetase. The transcription of the laccase gene inPichia pastoriswas found to be enhanced by the oxidative stress caused by exogenous H2O2. The stimulation of laccase gene expression in response to exogenous H2O2stress further contributed to the transcriptional induction of the genes involved in the glutathione-dependent antioxidative system, includingPpYAP1,PpGPX1,PpPMP20,PpGLR1, andPpGSH1. Taken together, these results suggest that the expression of the laccase gene inPichia pastoriscan enhance the resistance of yeast to H2O2-mediated oxidative stress by stimulating the glutathione-based antioxidative system to protect the cell from oxidative damage.

Melanoidin-containing wastewaters induce selective laccase gene expression in the white-rot fungus Trametes sp. I-62

2008 ◽  
Vol 159 (2) ◽  
pp. 103-109 ◽  
Author(s):  
Tania González ◽  
María Carmen Terrón ◽  
Susana Yagüe ◽  
Howard Junca ◽  
José María Carbajo ◽  
...  
Keyword(s):  
Gene Expression ◽  
White Rot ◽  
White Rot Fungus ◽  
Laccase Gene

scholarly journals Transcriptional and Enzymatic Profiling of Pleurotus ostreatus Laccase Genes in Submerged and Solid-State Fermentation Cultures

2012 ◽  
Vol 78 (11) ◽  
pp. 4037-4045 ◽  
Author(s):  
Raúl Castanera ◽  
Gúmer Pérez ◽  
Alejandra Omarini ◽  
Manuel Alfaro ◽  
Antonio G. Pisabarro ◽  
...  
Keyword(s):  
Gene Family ◽  
Wheat Straw ◽  
Pleurotus Ostreatus ◽  
Submerged Fermentation ◽  
Expression Profiles ◽  
Water Soluble ◽  
White Rot ◽  
Accurate Estimation ◽  
Laccase Gene ◽  
Laccase Genes

ABSTRACTThe genome of the white rot basidiomycetePleurotus ostreatusincludes 12 phenol oxidase (laccase) genes. In this study, we examined their expression profiles in different fungal strains under different culture conditions (submerged and solid cultures) and in the presence of a wheat straw extract, which was used as an inducer of the laccase gene family. We used a reverse transcription-quantitative PCR (RT-qPCR)-based approach and focused on determining the reaction parameters (in particular, the reference gene set for the normalization and reaction efficiency determinations) used to achieve an accurate estimation of the relative gene expression values. The results suggested that (i) laccase gene transcription is upregulated in the induced submerged fermentation (iSmF) cultures but downregulated in the solid fermentation (SSF) cultures, (ii) theLacc2andLacc10genes are the main sources of laccase activity in the iSmF cultures upon induction with water-soluble wheat straw extracts, and (iii) an additional, as-yet-uncharacterized activity (Unk1) is specifically induced in SSF cultures that complements the activity of Lacc2 and Lacc10. Moreover, both the enzymatic laccase activities and theLaccgene family transcription profiles greatly differ between closely related strains. These differences can be targeted for biotechnological breeding programs for enzyme production in submerged fermentation reactors.

scholarly journals Gene Transcription in Lactarius quietus-Quercus petraea Ectomycorrhizas from a Forest Soil

2008 ◽  
Vol 74 (21) ◽  
pp. 6598-6605 ◽  
Author(s):  
P. E. Courty ◽  
M. Poletto ◽  
F. Duchaussoy ◽  
M. Bu�e ◽  
J. Garbaye ◽  
...  
Keyword(s):  
Gene Expression ◽  
Forest Soil ◽  
Reverse Transcription ◽  
Quercus Petraea ◽  
Primary Metabolism ◽  
Extraradical Mycelium ◽  
Root Tips ◽  
Reverse Transcription Pcr ◽  
Fungal Genes

ABSTRACT Extracting fungal mRNA from ectomycorrhizas (ECMs) and forest soil samples for monitoring in situ metabolic activities is a significant challenge when studying the role of ECMs in biogeochemical cycles. A robust, simple, rapid, and effective method was developed for extracting RNA from rhizospheric soil and ECMs by adapting previous grinding and lysis methods. The quality and yield of the extracted RNA were sufficient to be used for reverse transcription. RNA extracted from ECMs of Lactarius quietus in a 100-year-old oak stand was used to construct a cDNA library and sequence expressed sequence tags. The transcripts of many genes involved in primary metabolism and in the degradation of organic matter were found. The transcription levels of four targeted fungal genes (glutamine synthase, a general amino acid transporter, a tyrosinase, and N-acetylhexosaminidase) were measured by quantitative reverse transcription-PCR in ECMs and in the ectomycorrhizospheric soil (the soil surrounding the ECMs containing the extraradical mycelium) in forest samples. On average, levels of gene expression for the L. quietus ECM root tips were similar to those for the extraradical mycelium, although gene expression varied up to 10-fold among the samples. This study demonstrates that gene expression from ECMs and soil can be analyzed. These results provide new perspectives for investigating the role of ectomycorrhizal fungi in the functioning of forest ecosystems.

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