Isolation and Characterization of Chalcone Synthase Gene Isolated from Dendrobium Sonia Earsakul
To isolate and characterize chalcone synthase gene in anthocyanin biosynthetic pathway during flower development of Dendrobium Sonia Earsakul. The gene was isolated from floral tissues of the orchid by reverse transcriptase polymerase chain reaction. Characterization of the gene considered to its relatedness to chalcone synthase gene in other orchid plants elucidated by construction of a neighbor-joining phylogenetic tree. Gene expression pattern related to flower development and pigmentation was investigated by relative quantification real time polymerase chain reaction. A complete coding sequence was obtained and sequence analysis revealed that the gene of Dendrobium Sonia Earsakul consisted of 1,188 bp. Blast analysis and multiple alignments showed that the chalcone synthase gene of Dendrobium Sonia Earsakul shares high homology to chalcone synthase gene of Dendrobium genus particularly Dendrobium hybrid Uniwai prince. Phylogenetic tree revealed that chalcone synthase of Dendrobium genus are highly conserved. The chalcone synthase gene of Dendrobium Sonia Earsakul was highly expressed in young flower bud with no pigmentation and the expression was sharply decreased when young flower bud started accumulation of pigments. Expression of chalcone synthase gene was then maintained at the same level until young bud developed into fully opened flowers.
Received: June 24, 2010;
Accepted: August 16, 2010;
Published: September 24, 2010
Anthocyanin is a type of flavonoid that is a major factor in flower pigmentation.
It contributes a range of colors from red to margenta, orange to red and purple
(Tanaka et al., 2005). Biosynthesis of these
pigments is controlled by a series of enzymes. Chalcone synthase gene (chs)
encodes chalcone synthase (CHS) which is an important enzyme in anthocyanin
biosynthetic pathway. CHS supplies chalcone, the precursor of anthocyanin molecules,
via processes of decarboxylation, condensation, cyclization and aromatization
reaction of p-coumaroyl and three molecules of malonyl-CoA (Ferrer
et al., 1999).
Chalcone synthase was investigated in many plant species. Their flower
colors were successfully manipulated through modification this structural gene
such as Torenia Petunia and Gentian (Fukusaki
et al., 2004; Hanumappa et al., 2007;
Nakatsuka et al., 2008; Suzuki
et al., 2000; Tanaka et al., 1998).
In orchids, there are few data reported in this anthocyanin biosynthetic gene
such as Bromheadia (Liew et al., 1998)
Phalaenopsis (Han et al., 2006), while
only one publication was reported in Dendrobium hybrids (Mudalige-Jayawickrama
et al., 2005).
Dendrobium Sonia Earsakul is widely cultivated for commercial production
as cut flower orchid in Thailand. Its flower color is purple. To gained insight
in color production of this orchid plants. We isolated cDNA clone of chs
gene. BLAST analysis and phylogenetic tree was performed to see their relationship
to those of orchids. Expression profile of gene in floral tissue at five developmental
stages was investigated by real time PCR analysis.
MATERIALS AND METHODS
RNA extraction: Flower tissues of D. Sonia Earsakul grown at
five stages as shown in Fig. 1 were pooled and utilized for
total RNA extraction by modified CTAB method described by Sambrook
et al. (1989). For relative quantification real time PCR, total RNA
of flower tissues grown in different developmental stages (Fig.
1) were separately extracted as described above.
Cloning the chs gene: The first strand cDNA was synthesized from total RNA using MMLV reverse transcriptase (Fermentas, Canada). OligodT including M13 reverse sequences, 5′CAG GAA ACA GCT ATG ACC ATG TTT TTT TTT TTT TTT TT 3′, was used as a primer. Degenerate primers, 3′endF 5′TAT CCG GAY TAC TAC TTC AGR ATT ACC A 3′ and 5′endR 5′GGC GTT GTT CTC GGC GAG GTC TTT GGC3′, were designed from a conserved region of chs genes from many plant species and employed to amplify partial chs gene. Degenerate and specific primers, 5′endF 5′ GAA TAG GGA GGG AGT TAA TTA ATG GC 3′ and 5′endR were used to amplify 5′end of the chs gene while a pair of primers 3′endF and M13R 5′CAG GAA ACA GCT ATG ACC ATG 3′ were used to amplify the 3′end of the gene. The amplified fragments were ligated to pDrive cloning vector and used for transformation of E.coli DH5α. All positive clones containing the inserts were subjected to sequencing analysis.
Sequence analysis: Nucleotide and amino acid sequence were analyzed
using BLAST SEARCH (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
and clustalW (http://www.ebi.ac.uk/Tools/clustalw2/).
Protein analysis system (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi)
was used to predict the simple physical properties of the sequence and functional
domains of the protein.
Phylogenetic tree analysis: Multiple alignment of amino acid sequences were compared among chs from various orchids and other plant species using clustalW program. The phylogenetic tree was constructed using neighbor joining method.
Relative quantification real time PCR analysis: The expression of chs
gene at different growth stages of flower buds was investigated by relative
real time PCR. The 18S rRNA was used as the reference control. Relative changed
in chs gene expression was calculated by using the 2-ΔΔCt
method (Livak and Schmittgen, 2001). Flower tissues of
D. Sonia Earsakul were separated into five growth stages (Fig.
1). The total RNA of each stage was used as the RNA templates. The reaction
of 15 μL consisted of 7.5 μL RNA direct MASTER with SYBR I dye chemistry,
0.5 μM of each primer (a primer set of forward primer 5'AAT CGA ATC ATG
CTT TAC CAA CAA GGC TGC 3'and reverse primer 5'TGA ACA AAC GAC GAG AAC TCG 3'
were used for partial chs gene amplification and a primer set of forward
primer 5'GCT ACT CGG ATA ACC GTA GT 3'and reverse primer 5'ACC AGA CTT GCC CTC
CAA TG 3' were employed to amplification of 18S rRNA gene) and 150 ng of RNA
template. RT-PCR was performed by denaturation at 95°C for 1 min and followed
by reverse transcription at 61°C for 20 min. PCR was immediately done after
reverse transcription was completed by denaturation the DNA at 95°C for
1 min followed by 45 cycles of 95°C for 20 s, 58°C for 20 sec and 72°C
for 45 sec. DNA melting was performed after amplification had completed to allow
fluorescence measurements of non specific products and analyze of expected DNA
product by melting curve analysis.
||Dendrobium Sonia Earsakul flower was categorised into
five growth stages. Stage 1 is the early stage of flower budding. Stage
2 is the closed bud without pigmentation. Stage 3 is the near-open bud with
slight pigmentation. Stage 4 is the opened bud. Stage 5 is the fully opened
This research was carried out at Center for Agricultural Biotechnology, Kasetsart University Nakhon Pathom, Thailand. Dendrobium Sonia Earsakul was cultivated in nursery at Plant Unit, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand.
Isolation of chs gene from D. Sonia Earsakul: To isolate
a chs gene, first strand cDNA of D. Sonia Earsakul was synthesized
by reverse transcription using oligodtM13R primer. Subsequently, a conserved
region of chs gene sequence was amplified using primers named 3'endF
and 5'endR (Fig. 2) and a fragment of about 440 bp was obtained.
Sequence analysis revealed that the gene fragment showed high homology to chs
genes of orchid plants. To obtain a full-length coding sequence, the 3' and
5' ends of the chs gene were PCR-amplified using primers as detailed
in Fig. 2. Fragments of 663 and 552 bp were obtained after
amplification of the 3' and 5' ends. Gene assembly of the three fragments using
SeqMan software (DNA Star, Inc.) showed that a complete coding sequence of chs
gene was obtained. The gene comprised of 1,188 bp and encodes 395 amino
acids with the calculated molecular mass of 43.04 kDa and isolectric point (pI)
of 6.22. The complete cDNA and deduced amino acid sequences were deposited in
GenBank with accession no. AM490639 and CAM32716, respectively.
Characterization of chs gene: Protein-protein Blast analysis of deduced amino acid sequence revealed that CHS of D. Sonia Earsakul has high homology to those of Dendrobium genus. The CHS contains putative conserved domains which are the characteristics of chalcone synthase. Multiple alignment of orchid CHS using ClustalW confirmed that the CHS of D. Sonia Earsakul shares high degree of identity to those of CHS isolated from Dendrobium orchids like D. hybrid Uniwai prince (99%) and D. nobile (97%) (Table 1). The CHS of D. Sonia Earsakul also showed high similarity to those of Bromheadia genus (93%). However, the CHS of D. Sonia Earsakul shares less similarity to those isolated from Oncidium and Phalaenopsis orchids, except for one cultivar of Phalaenopsis (AAY83389).
Phylogenetic analysis of chalcone synthase: A phylogenetic tree of plant
CHS proteins was constructed based on amino acid sequences (Fig.
3). The tree showed two clades of CHS proteins. The CHS of D. Sonia
Earsakul was grouped with those of Dendrobium, Bromheadia,
a Phalaenopsis plant and those of other monocots and dicots whereas
the CHS of Oncidium and Phalaenopsis orchids fell into the other
||The schematic representation of chs gene fragments
of D. Sonia Earsakul isolated by RT-PCR. AAAAAAM13R was used as a
primer for first strand cDNA was synthesized. Primers and 5'endR were used
to amplify 440 bp. partial chs gene. Primers 5'endF and 5'endR were
used to amplify 552 bp. 5'end of the chs gene. Primers 3'endF and
M13R were used to amplify 663 bp the 3'end of the gene
|| Percentage of similarity at nucleotides and amino acid sequences
of chs cDNA of D. Sonia Earsakul comparison with other orchid species
||The phylogenetic tree of plant CHS. The tree of CHS proteins
was generated using clustalW and MEGA4. Numerals next to the branch represent
percentage of bootstap values from 1,000 replications. The bar indicates
an evolutionary distance of 0.02%. Accession number in the database of each
chs protein was placed in front
||Relative quantification of the chs gene expression
from flower buds of D. Sonia earsakul
Expression of chs gene in flower of D. Sonia Earsakul: Relative quantification real time PCR analysis revealed that the chs gene was highly expressed in young flower buds (stage 1, Fig. 1) with no pigmentation (Fig. 4). The expression was sharply decreased when young flower buds started to develop and accumulation of pigments was seen. From this developmental stage, the level of expression was maintained at nearly the same level throughout to the end of flower development.
A complete cDNA of chs gene of D. Sonia Earsakul was obtained. The gene comprised of 1188 bp and encodes 395 amino acids. The CHS of D. Sonia Earsakul shared high degree of homology to those of Dendrobium species and to a lesser extend to those of Bromheadia orchid. Alignment of CHS of D. Sonia Earsakul to those of Phalaenopsis and Oncidium plants revealed that they shared less similarity. Phylogenetic tree of CHS proteins revealed that the CHS of D. Sonia Earsakul was categorized into the same clade as Dendrobium CHS whereas the CHS proteins of Phalaenopsis and Oncidium orchids were grouped in the other clade.
Sequence analysis showed that the deduce amino acid sequence contains feature
domains of which are characteristics of chalcone synthase as shown in Fig.
3. This suggested that the CHS could be function in the processes of decarboxylation,
condensation, cyclization and aromatization reaction of p-coumaroyl and
three molecules of malonyl-CoA (Ferrer et al., 1999).
Gene expression pattern of chs gene in D. Sonia Earsakul is similar
to that in other plant such as Oncidium (Chiou and
Yeh, 2008) Phalaenopsis (Han et al., 2005)
and Gentian (Nakatsuka et al., 2005).
The expression pattern of chs gene showed that the gene was expressed
in all flower development stages. This finding is consistent with the fact that
chs gene is a common enzyme necessary for flavone and anthocyanin biosynthesis
(Martin et al., 1991; Nakatsuka
et al., 2005). The expression of chs gene is highest at the
early stage of flower formation. This might reflect the fact that chs gene
is one of early biosynthetic genes of flavone and anthocyanin pathways (Davies,
2004; Martin and Gerats, 1993; Nakatsuka
et al., 2005; Pelletier et al., 1999).
Its product is subsequently required for production of structural genes and
enzymes downstream in all flavone and anthrocyanin biosynthesis (Nakagawa
et al., 2008).
The most important basic for manipulating orchid flower color is to gain information of the key enzymes in anthocyanin synthetic pathway. In this study, the data of chs gene from D. Sonia Earsakul through gene isolation, characterization and gene expression during flower development could be a tool for flower color manipulation in this plant.
This research is supported by the Center for Agricultural Biotechnology, Postgraduate Education and Research Development Office, Commission on Higher Education, Ministry of Education. And it was partially supported by thesis and dissertation support fund, Graduate school, Kasetsart University.
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