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Structural
Analysis of a Gene Cluster Encoding DFR-like Proteins from Rice Chromosome 4
LEI
Hai-Yan1,2#, ZHOU Bo1#￥,
ZHANG Yu1, HONG Guo-Fan1,2*, HAN Bin1*
(
1 National Center for Gene Research, Shanghai Institutes for
Biological Sciences, the Chinese Academy of Sciences,
Shanghai
200233, China;
2
Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese
Academy of Sciences,
Shanghai
200031, China )
Abstract Sequencing analysis of the 323 kb contig of rice
chromosome 4 identified a gene cluster encoding 7 dihydroflavonol-4-reductase
(DFR)-like proteins within a 56 kb region. The 7 DFR-like genes were found to
be arranged in a tandem array, and all of them comprised 6 exons and 5 introns.
Analysis of the predicted amino acid sequences demonstrated that these 7
proteins shared strong similarities with DFR and other enzymes of the
phenylpropanoid biosynthesis pathway. RT-PCR revealed the expression pattern of
the 7 genes was different in various rice tissues. The structural and
functional features of these 7 DFR-like genes and their evolutionary
implications are discussed.
Key
words gene cluster; rice; Arabidopsis;
dihydroflavonol-4-reductase (DFR); Oryza sativa enzyme of flavonoid
biosynthesis (OsEFS)
Flavonoids
are secondary metabolites widespread among plants and involved in many plant
functions such as UV protection, defense against pathogen attack, legume
nodulation and pollen viability[1,2]. Dihydroflavonol-4-reductase
(DFR) catalyses the first common step in the flavonoid biosynthetic pathway
leading to anthocyanins and proanthocyanidins. The latter compounds are also
known as anthocyanogens and condensed tannins. Some of the flavonoids, the
anthocyanidins, anthocyanins and tanins are responsible for the red, purple and
brown pigmentation of flowers, fruits, seeds and other plant tissues and organs[3].
Since these products are not essential for the viability of the plants,
flavonoid biosynthesis represents an excellent model system in which to study
the regulation of a complex biosynthetic pathway. Thus the genetic control of
flavonoid biosynthesis has been studied in several model plants including maize、snapdragon、petunia
and Arabidopsis[4,5]. Most of the genes encoding DFR have
been cloned from many plants, such as Z. mays, moring glories, P.hybrida,
grape[6–9]
and Arabidopsis[5] etc., and their sequences are well
conserved among plant species.
In
this study, the 323 kb contig of rice chromosome 4 was completely sequenced and
analyzed. A large gene cluster consisting of seven predicted DFR-like protein
genes in a tandem array was found to be located in the 56 kb region of the
contig. The deduced protein sequences of these seven genes all shared
significant sequence similarities with DFRs and BANYULS[10] in Arabidopsis.
The BANYULS gene encodes a DFR-like protein and is a marker of early
seed coat development. Mutations in the BANYULS gene lead to precocius
accumulation of anthocyanins in immature seed coat in Arabidopsis.
Because DFR and BANYULS both involved in the flavonoid synthesis
in plant, we deduced that these seven genes may have similar functions in rice
and designated them as OsEFS (Oryza sativa enzyme of flavonoid
biosynthesis). Each gene of this cluster was named according to their order in
the 56 kb fragment. RT-PCR was performed to elucidate the expression pattern of
each gene in this cluster. The structural and functional features of these
seven DFR-like genes and their evolutionary implications are discussed.
1 Materials and Methods
1.1
Plant materials and growth conditions
Seeds
of rice (Oryza sativa indica Guangluai 4) were germinated
at 37 ℃
and the seedlings were grown in the light at 30 ℃
for 3 days for RNA extraction of root and bud. Leaves were collected after 10
days growing. Flowers and young panicles were prepared from 3 month old plants[11].
1.2
Cloning and sequencing of BAC clones
A
BAC (bacterial artificial chromosome) contig, which was anchored on the region
from 110.0 cM (centimorgan) to 111.5 cM of chromosome 4, was constructed by
using colony hybridization and chromosome walking. The contig consisted of 20
BAC clones which came from two BAC libraries of Oryza sativa Guangluai
4, and the genetic markers used as probes were provided by rice genome program
(RGP) in Japan[12,13]. Six tiled BAC clones (BAC H0410G08, H0315F07,
H0613A10, B0808H03, H0105C05, H0323C08) with minimum overlaps were sequenced
and analyzed. The BAC DNA was purified by cesium chloride gradient
centrifugation, and subcloned into pBluescript II plasmid vector (Stratagene)
after sonication. Subclones were sequenced at both ends using the DYEnamicTM
ET dye terminator kit (Amersham Pharmacia) and analyzed on Megabase100
(Amersham Pharmacia). The sequence data were assembled using PHRED/PHRAP
software. Homology searches were performed using the Blast program[14].
GENSCAN program was used to predict possible genes in this contig[15].
The
nucleotide sequences of the BACs H0410G08 and B0808H03 have been submitted into
the EMBL database under the accession numbers AL512546 and AL512545.
1.3
Oligonucleotides
All
oligonucleotides used in this study were synthesized by Sangon company, China;
except for Oligo dT-adaptor primer which was provided by RNA RCR Kit. The
sequences of all oligunucleotides were shown in Table 1.
1.4
RT-PCR
Total
RNA of roots，buds，leaves，flowers
and young panicles of rice were extracted following the manufacture’s
instruction (Qiagen RNeasy Plant mini kit). For RT-PCR, 1 μg
of DNase-treated total RNA was reverse-transcribed with gene-specific antisense
primers and AMV reverse transcriptase using an RNA PCR kit (TaKaRa, Japan), and
the entire reaction mixture was used as a template in the subsequent PCR. Each
PCR cycle consisted of 94 ℃
denaturation for 30 s, 60 ℃
annealing for 30 s, 72 ℃
extension for 2 min, for 30 cycles.
2 Results
2.1
OsEFS gene cluster was revealed in the 56 kb fragment
Six
overlapping BACs representing the 323 kb region of rice chromosome 4 were
sequenced and analyzed by using the gene identification software GENSCAN to
predict the location of the genes in this contig. The prediction result showed
that seven DFR-like genes were clustered in a 56 kb fragment, and the predicted
protein sequences of these 7 genes all had high similarities to DFR of
rice and BANYULS of Arabidopsis. The similarities between 7 OsEFS
proteins and BANYULS of Arabidopsis are 55.52%–60.18%,
and the similarities to DFR in rice are 48.19%–55.56%.
One mutator-like transposon was found to be located between OsEFS3 and OsEFS4.
These 7 genes all had the same transcription direction and contained 6 exons
and 5 introns. Though the length of each gene in genomic level is different
from each other, the lengths of their coding regions are almost same. The
average G+C content of each gene’s coding and noncoding region is similar to
other plant genes (Table 2). The predicted seven OsEFS protein sequences had
high similarities with each other (Table 3). The relevant features of each
deduced protein were shown in Table 4. It is interesting to find that other 6
OsEFS proteins are all acidic proteins except that the isoelectric point of
OsEFS4 is 7.27. Though the amino acid compositions of 7 proteins are similar,
the basic amins acid Arg is higher in OsEFS4 than in other six proteins. The
number of basic amino acid Arg in OsEFS1-3, 5-7 protein is 17, 11, 12, 17, 13,
12, respectively, however the number in OsEFS4 is 25.
2.2
Transcript levels of OsEFS gene cluster
he
expression of the OsEFS gene cluster in different tissues of rice was
examined by RT-PCR. Total RNAs were extracted from roots, buds, leaves, flowers
and young panicles. These RNA were all treated with DNase to remove any
contaminated genomic DNA. As a control, amplification by RT-PCR was performed
using two primers (actin-P1/actin-P2) specific for the rice actin 1 gene (ACT1).
The gene-specific primers (OsEFSs 5′/
OsEFSs 3′,
see Table 1) were designed according to the GENSCAN prediction result. Each
gene’s corresponding PCR fragment was sequenced and confirmed that they all
came from the transcript of OsEFS. The amplification fragments of seven
genes were transferred to membranes and hybridized with corresponding genomic
DNA.
Results
of RT-PCR and Southern blot revealed that seven OsEFSs transcribed at
different levels in five rice tissues [Fig.1 (A), (B)]. We found that seven
genes all had strong expressions in flowers and young panicles and they had
different transcription in other three tissues. The expression pattern of seven
genes may be due to that these genes involved in the biosynthesis of
anthocyanins.
Fig.1 RT-PCR analysis of OsEFS
transcripts in various tissues
(A) RT-PCR products detected by ethidium
bromide staining. (B) RT-PCR products detected by Southern blot hybridization.
From left to right is root, immature shoot, mature leaf, panical and immature seed,
respectively. The products of the transcripts of 7 genes were confirmed by
sequencing.
3 Discussion
We
found a large gene cluster encoding seven OsEFS genes in the 56 kb
fragment of chromosome 4 of Oryza sativa ssp. indica cv. Guangluai
4. Protein sequence analysis demonstrated that OsEFS belongs to a superfamily
of NADP(H) binding oxido-reductases as defined by Baker et al.[16].
Comparison with various databases revealed similarities to known genes encoding
the dihydroflavonol reductase (DFR) of rice and many other plant species and to
BANYULS of Arabidopsis. A phylogenetic tree was constructed using the
CLUSTALX program and is presented in Fig.2. The tree summarizes the theoretical
evolutionary distances among the different NADPH-dependent oxido-reductase
superfamily members and BANYULS and OsEFS. The DFR proteins from several plant
species represent a separate cluster from which OsEFSs and BANYULS are
excluded. The motif of 13 amino acid residues common to DFRs and thought to
define their substrate specificity are not found in BANYULS and seven OsEFS[10].
However, these 13 conserved amino acids were found in rice DFR[17].
DFR genes have been cloned from rice[17] 、sorghum[18]
, barley[19] , maize[6], Arabidopsis[5]
etc.. Though the copy numbers of DFR gene in each species are different, they
mostly locate in a single locus. BANYULS[10] has been cloned
from Arabidopsis recently and it encodes a DFR-like portein. The BANYULS
locates in the chromosome 4 of Arabidopsis and the DFR gene of Arabidopsis
locates in the chromosome 1. Devic et al.[10] thought that BANYULS is not another copy
of DFR of Arabidopsis, and it may encode the leucoanthocyanidin
synthesis. OsEFSs have a closer evolution relation with BANYULS than that of
rice DFR. From all these thoughts, we can conclude that OsEFS gene
cluster were not a second DFR gene in rice and they may have a closer
function with BANYULS.
Fig.2 Phylogenetic tree of the members of the
superfamily of NADPH-dependent oxido-reductase including 7 OsEFS and BANYULS
The tree was constructed by the program CLUSTALX
1.0.
The
predicted protein sequences of seven OsEFS genes showed high
similarities to each other. From the phylogenetic tree analysis, we found that
seven OsEFS genes formed four evolution branchs and the branchs were
consistent with their localization order in the gene cluster. Members of gene
families were often found to be located in the gene cluster in genome. Five
perioxicase genes were found in rice chromosome 4[20]. Three nit
genes were located in the 13.8 kb region in Arabidopsis genome[21].
Gene duplication and subsequent divergence are thought to play important roles
in evolution of genes[22]. The putative evolution mechanism of the
seven OsEFS genes by duplication was shown in Fig.3. At least, three
gene duplication processes must be involved in generating seven tandem copies
from a single copy, and a transposon insertion might occur prior to the second
duplication. The insertion of this transposon led to the phenomenon that
previous three genes had higher similarities than other four genes. Transposons
were often found in the intergenic regions of gene clusters in the genome[23].
Fig.3 Schematic reprensentation of the
putative evolution mechanism of the 7 OsEFS genes by duplication in rice
In
summary, we found a large gene cluster encoding seven DFR-like protein in the
56 kb fragment of rice chromosome 4. RT-PCR analysis revealed the expression
pattern of these seven genes. We discussed the structural and functional
features of these seven genes. The putative evolutionary mechanism of this gene
cluster was also discussed in this text.
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Received:
March 27, 2002
Accepted: May 21, 2002
This
work was supported by grants from the Ministry of Science and Technology, the
Chinese Academy of Sciences and the Shanghai Municipal Commission of Sciences
and Technology
#
These authours contribute equally
￥Present
address: Department of Plant Pathology, Ohio State University, Columbus, Ohio,
45210, USA
*Corresponding
author: HAN Bin: Tel, 86-21-64825260; Fax, 86-21-64825775; e-mail,
[email protected]. HONG Guo-Fan: Tel, 86-21-64822885; Fax,
86-21-64825775; e-mail, [email protected].