ISSN 1021-4437, Russian Journal of Plant Physiology, 2006, Vol. 53, No. 2, pp. 223–230. © MAIK “Nauka /Interperiodica” (Russia), 2006.
Published in Russian in Fiziologiya Rastenii, 2006, Vol. 53, No. 2, pp. 250–258.
orchids, native to tropical Asia, come
in all colors, except blue and true red. Flavonoids are
the major ﬂower pigments and play an important role in
UV protection, plant–microbe interactions, and male
fertility [1–3]. Chalcone synthase (CHS) is the key
enzyme in ﬂavonoid biosynthesis pathway, which cata-
lyzes the condensation of three acetate residues from
malonyl-CoA with 4-coumaroyl-CoA to produce nar-
ingenin chalcone .
CHS has been extensively studied in parsley, petu-
nia, soybean, and other plants [4–7] and used to manip-
ulate ﬂower color [8, 9]. Both sense and antisense con-
structs encoding CHS have been obtained to reduce the
content of CHS mRNA and CHS activity to produce
fainter colored or even white ﬂowers. What is more
inspiring is that the introduction of heterogeneous dihy-
droﬂavonol 4-reductase gene, another key gene for
anthocyanin synthesis, into petunia resulted in the
appearance of brick-red ﬂower color due to
synthesis of a pigment absent from this plant .
Though CHS have been studied extensively, no
information is available about
tiple ﬂower colors of
prompt us to inves-
tigate the pigment biosynthetic pathway in its ﬂowers.
This study reports the isolation of CHS-encoding gene
ﬂowers by PCR ampliﬁca-
tion. A putatively functional
gene was deter-
mined. Southern blot was carried out to describe its
genomic organization in
tion-PCR was used to investigate the spatial and tempo-
ral expression of
Results indicated that
was moderately transcribed in petals and lips at the
stages when the anthocyanin was accumulated at the
MATERIALS AND METHODS
, cv. Formosa rose, plants
with ﬂowers in mauve color were grown as usually in a
greenhouse of the Institute of Horticulture, Agricultural
Science College of Shanghai.
DNA extraction, PCR ampliﬁcation, DNA clon-
ing and sequencing.
Genomic DNA was extracted
from leaves by a modiﬁed method of Dellaporta .
The tissue was ﬁrst grounded to ﬁne powder with liquid
nitrogen. 10 ml of preheated to 65
C extraction buffer
(1.5% (w/v) SDS, 0.5 M NaCl, 0.01 M Tris–HCl, pH 8,
0.02 M EDTA, pH 8, and 2% (v/v) 2-mercaptoethanol)
was added to 2-g sample. The mixture was incubated at
Cloning and Characterization of a Novel Chalcone Synthase Gene
Y. Y. Han, F. Ming, J. W. Wang, J. G. Wen, M. M. Ye, and D. L. Shen
Institute of Genetics, State Key Laboratory of Genetic Engineering, Research Center of Gene Diversity
and Designed Agriculture, Fudan University, Handan Road 220, Shanghai, 200433, China;
Received March 14, 2005
—Chalcone synthase (CHS, EC 220.127.116.11) is the key enzyme involved in ﬂavonoid and anthocyanin
biosynthesis. A complete DNA sequence of chalcone synthase gene designated
was isolated by means
of usual and then inverse polymerase chain reactions from genomic DNA of an orchid,
cv. Formosa rose. Nucleotide sequence analysis based on alignment with published
contained an intact open reading frame of 1173-bp with one 109-bp intron at the conserved
site. The deduced polypeptide (PCHS1) from
comprised 390 amino acids with a predicted mol wt of
42.5 kD. PCHS1 showed 61–65% identities with CHS from other plants and retained most of the conserved
residues. Some putative
-regulatory elements were present at the 5' and 3' ﬂanking regions of
ern blot analysis predicted at least four
-like genes, thus indicating the presence of a small multigene
Relative quantitative RT-PCR showed that
is expressed in petals at early ﬂower
development as well as in lip tissue when the ﬂower has just opened.
Key words: Phalaenopsis hybrida - orchid - chalcone synthase - gene expression - ﬂanking region - anthocyanins
: CHS—chalcone synthase; IRE—inverted repeat ele-
ment; RT-PCR—reverse transcription polymerase chain reaction.
The text was submitted by the authors in English.