ISSN 10227954, Russian Journal of Genetics, 2013, Vol. 49, No. 10, pp. 999–1003. © Pleiades Publishing, Inc., 2013.
Original Russian Text © D.V. Vasina, D.S. Loginov, O.N. Mustafaev, I.V. GoldenkovaPavlova, O.V. Koroleva, 2013, published in Genetika, 2013, Vol. 49, No. 10, pp. 1149–1154.
Basidiomycetes, an infectious agent that causes
white rot, has a unique enzyme complex that can effi
ciently degrade and mineralize lignin . Currently, it
is thought that laccase, which belongs to the family of
coppercontaining oxidases, is one of the key enzymes
of this complex (EC 18.104.22.168). The enzyme catalyzes
the reduction of molecular oxygen to water and the
concurrent oxidation of an electron donor without an
intermediate step of the peroxid formation .
Although laccases have been studied for more than
100 years, their biosynthetic mechanism is still of con
siderable interest. There are many papers on how
nitrogen and carbon sources, as well as different
inducers, affect the laccase biosynthesis [3–5]. A vari
ety of organic substances, mainly of phenolic and aro
matic origin, as well as inorganic compounds, e.g.,
copper sulfate, are used as inducers. However, in most
studies on laccase synthesis, the principle aim was to
increase enzyme production instead of investigating
the biosynthetic process.
Currently, the main tools for studying biosynthesis
come from proteomics. These include 2D electro
phoresis, mass spectrometry, and others. This
approach allows one to identify individual proteins,
which makes it possible to suggest their involvement in
a variety of biochemical processes [6, 7]. Nevertheless,
in the case of basidiomycota, the proteomic methods
are difficult to apply as far as there are no annotated
genomes available, which makes it difficulty to iden
tify proteins. One alternative may be an approach
based on the comparative analysis of transcripts that
originated from fungi grown with or without the
inducer, which helps in the identification of differen
tially expressed genes [8, 9]. The induction of the lac
case biosynthesis with copper sulfate is a convenient
model for this purpose.
The aim of this work was to find suggested genecan
didates involved in the laccase biosynthesis in the basid
using the comparative analy
sis of transcriptomes and current proteomic methods.
MATERIALS AND METHODS
(Wulfen) Pilat, 1939), from the col
lection of the Komarov Botanical Institute, Russian
Academy of Sciences (LEBIN), was kindly presented
by Dr. N.V. Psurtseva.
The laccase producer
was grown on GLP medium as described previously
. The medium included 10.0 g/L glucose, 3.0 g/L
peptone, 0.6 g/L KH
, 0.001 g/L ZnSO
0.4 g/L K
, 0.0005 g/L FeSO
O, 0.05 g/L
, and 0.5 g/L MgSO
O. For induction,
the cultivation medium was supplied with 0.25 g/L
. The fungal mycelium was collected after
3days of growth.
Measurement of laccase activity.
The enzymatic activ
ity was measured spectrophotometrically using the syrin
galdazine as a substrate (Sigma,
= 65000 M
solution buffered with 0.1 M Naacetate (pH 4.5)
. The amount of enzyme needed to oxidize 1
of substrate in 1 mL of reaction mixture for 1 min was
assumed to be the unit of the activity.
Isolation of total RNA.
Total RNA was purified from
the mycelium samples with the use of the QIAamp
RNA kit (QIAGEN, United States).
Range of Gene Candidates Involved in Biosynthesis of Laccase
D. V. Vasina
, D. S. Loginov
, O. N. Mustafaev
, I. V. GoldenkovaPavlova
, and O. V. Koroleva
Bach Institute of Biochemistry, Russian Academy of Sciences, Moscow, 119071 Russia
Timiryazev Institute of Plant Physiology, Russian Academy of Sciences, Moscow, 127276 Russia
Received December 28, 2012
—A comparative analysis of transcripts from the basidiomycota
grown with and without
an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive par
allel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high
level of expression and belong to different gene ontology categories were identified. Also, a large number of
transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may rep
resent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is
induced are selected as genecandidates involved in the laccase biosynthetic pathway.