全 文 ：Received 19 May 2003 Accepted 28 Aug. 2003
Supported by the National Natural Science Foundation of China (30170491).
* Author for correspondence. Tel(Fax): +86 (0)10 64889381; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (5): 588-594
Cloning and Expression of Two Chalcone Synthase and a Flavonoid 35-
Hydroxylase 3-end cDNAs from Developing Seeds of Blue-grained
Wheat Involved in Anthocyanin Biosynthetic Pathway
YANG Guo-Hua1, 2, LI Bin1, GAO Jian-Wei1, LIU Jian-Zhong1, ZHAO Xue-Qiang1,
ZHENG Qi1, TONG Yi-Ping3, LI Zhen-Sheng1*
(1. State Key Laboratory of Plant Cell and Chromosome Engineering, Institute of Genetics and Developmental Biology,
The Chinese Academy of Sciences, Beijing 100101, China;
2. College of Chemistry and Life Sciences, Tianjin Normal University, Tianjin 300074, China;
3. Research Center for Eco-environmental Sciences, The Chinese Academy of Sciences, Beijing 100085, China)
Abstract: Using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of
cDNA ends (RACE) strategies, two chalcone synthase (CHS) cDNAs were cloned from developing seeds of
blue-grained wheat, both of the deduced peptides contain 394 amino acids, and share 98.9% of amino acid
sequence identity and the nucleot ide sequences have the identity of 96.0%, and one flavono id 35-
hydroxylase (F3 5 H) 3-end cDNA was isolated. Four CHS genomic DNAs were cloned from Thinopyrum
ponticum (Podp.) Z. W. Liu et R. R.-C. Wang (ThpCHS.tg), blue-grained wheat (TaCHS.bg), white-grained
offspring of light blue-grained wheat (TaCHS.wg) and Chinese Spring (2n=42)(TaCHS.csg), respectively.
Although these four genomic DNAs were isolated from different materials, they are very high homologous
and each has one intron. The difference of the four CHS genomic DNAs mainly exists in intron. Through
DNA alignment we found that one CHS cDNA (TaCHS.t1) came from one of the parents, Th. ponticum, the
other one (TaCHS.w1) had the identity of 100% with white grain parent. This indicated that CHS genes from
two parents expressed at the same developing stage in blue-grained wheat. Southern blotting analysis
showed that they have at least four copies in wheat, the copy numbers in different color grains are not
significantly different, but they are different from that of Th. ponticum. CHS in blue-grained wheat belongs
to a CHS multifamily. Reverse Northern analysis indicated that the CHS expressed strongly in the
developing blue-grained seeds at early stage (15 d after flowering, DAF), but F3 5 H and dihydroflavonol 4-
reductase (DFR) transcripts accumulated less than that of CHS at early stage. However, at the later
developing stage (21 DAF), F3 5H and DFR t ranscripts accumulated more than that of CHS, the
transcripts of CHS could hardly be detected. The expression order of the three genes is the same as the
order of the biosynthetic steps in anthocyanin biosynthesis. At the same time, CHS genes cloned from
seeds have not been detected in leaves of blue-grained wheat, but F3 5 H and DFR expressed strongly in
leaves. This showed that the expression of CHS genes cloned by us had tissue specificity. RT-PCR
indicated that the transcripts of F3 5H accumulated a lot in the developing seeds of blue- and white-
grained wheats at 21 DAF, but the transcripts of CHS and DFR accumulated in the blue-grained wheat more
than those of white-grained wheat and Chinese Spring at the same deve loping stage. Therefore, we
proposed that anthocyanin biosynthetic pathway existed in blue-grained wheat and the expression of the
secondary structure genes in anthocyanin biosynthetic pathway was coordinately regulated by regulatory
gene(s) during the period of blue pigment formation.
Key words: blue-grained wheat; anthocyanin biosynthetic pathway; chalcone synthase (CHS);
flavonoid 35-hydroxylase (F3 5 H); reverse Northern analysis
Blue-grained wheat was derived from the hybrid of Triti-
cum aestivum (2n=42)× Thinopyrum ponticum (2n=70).
The F1 hybrid was backcrossed to various wheat cultivars
and a blue-grained substitution line with 42 chromosomes
was derived (Li et al., 1982; 1983). In the selfing progeny of
this substitution line, a plant with 41 chromosomes and its
grains with blue pigment were iso lated . Th is p lant is a
monos omic subst itu tion o f one chromos ome from Th.
ponticum for 4D of common wheat. The blue pigmentation
gene or gene(s) controlling the formation o f blue pigment
is located on the Th. ponticum chromosome(s) and espe-
cially express ed in the aleurone layer of endosperm and
has distinct dosage effect. Four kinds of grains, which were
dark blue, medium blue, light blue and white, were observed
YANG Guo-Hua et al.: Cloning and Expression of Two Chalcone Synthase and a Flavonoid 35-Hydroxylase 3-end cDNAs
from Developing Seeds of Blue-grained Wheat Involved in Anthocyanin Biosynthetic Pathway 589
in a single spike of the selfed blue monosomic plant (Li et
al., 1983; 1986). The chromosome fragment(s) from Th.
ponticum detected by GISH was co-s egregated with the
blue-grained character (Yang et al., 2002). We predicted
that the gene(s) controlling the formation of blue pigment
in aleurone layer was o riginated from the genome of Th.
ponticum. Prev ious biochemical analys is results showed
that the blue-grained aleurone layer consis ted of at least
eight different pigments (Gao et al., 2000; 2001). It s ug-
gested that the gene(s) from Th. ponticum is clos ely re-
lated with the production of flavonoids. To iden tify the
identity of the blue-grained gene(s), we planned to clone
all the genes involved in the anthocyanin biosynthetic path-
way in b lue-grained wheat and tried to establish the rela-
tionsh ip between the express ion patterns of these genes
and the blue-pigmentation character and ultimately found
the gene(s) directly responsible for the blue-pigmentation.
Although the genetics of the blue-grained wheat has been
well characterized, the molecular mechanism of the biosyn-
thet ic pathway of blue pigments in the blue grain is s till
unclear yet.
Chalcone synthase (CHS) catalyzes the first step in the
biosyn thes is o f flavonoids (Holton and Corn ish, 1995;
Shirley, 2002), which are important for the pigmentation of
flowers and other parts of plan ts. Genes encoding CHS
constitu te a multigene family in which the copy number
varies among plant species and functional divergence ap-
pears to have occurred repeatedly (Koes et al., 1990; Shirley,
2001; Shirley, 2002; Yang et al., 2002). CHS gene-specific
expression in soybean seed coats shows that multiple CHS
genes are expressed in seed coats (Todd and Vodkin, 1996).
The expression of CHS is an important control step in the
biosynthesis of flavonoids. CHS transcription is regulated
by endogenous programs and in response to environmen-
tal signals (Thain et al., 2002). Aida et al. (2000) reported
the modificat ion of flower co lo r in to ren ia (Torenia
fournieri) by re-introduction of the dihydroflavonol-4-re-
ductase (DFR) gene o r the CHS gene. Chalcone and s til-
bene synthases (CHS and STS) catalyze condensation re-
actions of p-coumaroyl-CoA and three C (2)-unit from ma-
lonyl-CoA, but catalyze different cyclization reactions to
produce naringenin chalcone and resveratrol, respectively
(Suh et al., 2000).
Flavonoids are a diverse group of pheno lic secondary
metabolites. Many of the compounds belonging to this
group are potent antioxidants in vitro and epidemiological
studies suggest a direct correlation between high flavonoid
intake and decreased risk of cardiovascular disease, can-
cer and other age-related diseases. Enhancing flavonoid
biosynthesis in chosen crops may provide new raw materi-
als that have the potential to be used in foods designed for
specific benefits to human health (Verhoeyen et al., 2002;
Bartel and Matsuda, 2003). Analysis of the flavonoids ac-
cumulated in tissues from mutant lines implies that (genetic)
control of flavonoid biosynthesis is highly tissue-specific
(Koes et al., 1990). The production of anthocyanins in fruit
tissues is h ighly cont rolled at the developmental level
(Jaakola et al., 2002). The ubiquitous plant enzymes phe-
nylalanine ammonia-lyase (PAL) and CHS are key biosyn-
thet ic catalys ts in pheny lpropano id and flavonoid
assembly, respectively (Moore et al., 2002). The maize Lc
and C1 genes expressed in petunia differentially activate
the promoters of the chalcone synthase genes CHSA and
CHSJ in the same way as the homologous petunia genes
do (Quattrocchio et al., 1993).
1 Materials and Methods
1.1 Plant materials
All materials were kept in the laboratory of 705 Research
Group, State Key Laboratory of Plant Cell and Chromo-
some Engineering, Institute of Genetics and Developmen-
tal Biology, The Chinese Academy of Sciences (CAS).
Young spikes of dark blue-grained wheat were harvested at
15 and 21 DAF, respectively . White-grained wheat from
the progeny of blue-grained monosomic line and the back-
cross parent, Chinese Spring, were planted in the field. The
spikes were cut and frozen in liquid nitrogen and stored in
a –80 ℃ refrigerator for total RNA extraction.
1.2 RNA extraction and RT-PCR
Total RNAs were isolated from developing seeds of dark
blue-grained at different developing stages and its young
seedlings . We applied an economical, modified SDS/phe-
nol method to extract RNA. The total RNA samples were
digested with DNaseⅠ before use.
Four microgrammes of mixed total RNA of blue-grained
wheat seeds at different developmental stages were reverse
t rans cribed in a volume of 20 µL as des cribed as
InvitrogenTM. Two microlitres of the first-strand cDNA so-
lution dilu ted five times was used for PCR, which had a
total volume of 50 µL including 0.2 µmol/L degenerate oli-
gonucleotide of each primer and 2.5 unit of TaKaRa LA Taq
DNA polymerase with GC bufferⅠ. The 20-b degenerate
fo rward primer has the sequence 5-(C/T)T(A/T/G/C)
ATGATGTA(C/T)CA(A/G)GG(A/T/G/C)TG-3 and the re-
verse primer has the s equence 5- (A/T/G/C)CC(A/G)AA
(A/T/G/C)CC(A/G)AA(A/T/G/C)A(A/G)(A/T/G/C)AC(A/
T/G/C)CCCCA-3, which were used to amplify CHS frag-
ment from blue-grained wheat. PCR was conducted in a
Acta Botanica Sinica 植物学报 Vol.46 No.5 2004590
DNA Thermal Cycler (Perkin-Elmer Gene Amp PCR system
9600). The amplification product was fractionated in 0.8%
agarose gel and recovered by DNA Fragment Quick Purifi-
cation/Recover Kit (Dingguo, Beijing).
Accord ing to the sequence o f DFR in GenBank
(accession number: AY209183), the full length primers were
us ed : DFR1F 5-ATGGACGGGAATAAAGGGCCGG-3;
DFR1R 5-CGCAGCAGCGCTGGCTTATTATGT-3. F35H
fo rwa rd degene rate p rimer was d es igned a s 5-
AAAGAATTCTT(T/C)ACIGC(A/T/G/C)GGIAC(A/T/G/C)
GA (T /C)ACI- 3 an d th e r ev er s e p r imer as 5 -
AAATCTAGAICC(A/T/G/C)AT(A/T/G/C)AT(A/T/G/C)
GCCCA(T/G/C)AT(G/A)TTIAC-3.
1.3 5-RACE and 3-RACE
CHS gene-specific primers (GSP1-2) were deduced from
the sequence of CHS-p. The 5-RACE was carried out es-
sentially according to the manufactu re’s instructions us-
ing the kit from Invit rogenTM Life Technologies (www.
invitrogen.com). Total RNA mixture from different develop-
ing stages of blue-grained seeds was reversibly transcribed
using 5- (A/T/G/C)CC(A/G)AA(A/T/G/C)CC(A/G)AA(A/
T/G/C)A(A/G)(A/T/G/C)AC(A/T/G/C)CCCCA-3 as the
primer. After purification and terminal transferase reaction,
5 µL of the resulting TdT product was used as a template in
the first-round PCR in the p resence of the Abridged An-
c h o r p r ime r ( I n v i t r o g e n TM) a n d GS P 1 ( 5 -
CATGGCGGTGATCTCCGAGCAG-3). For the second
round PCR, 5 µL of the diluted first-round PCR product was
re-amplified with AUAP (Inv it rogenTM) and GSP2 (5-
TTGTTCTCGGCGATGTCTTTGG-3).
For 3-RACE, two CHS gene-specific primers (GSP3-4),
were designed according to the sequence of CHS fragment.
In the first round PCR, the abridged universal amplification
p r i m e r ( A U A P ) a n d C H S - G S P 3 ( 5 -
CAGGATGGCCAAGCAACCACTG-3) were used and to-
tal RNA mixture was reversibly trans cribed us ing AP
(InvitrogenTM) as the primer. The product was used as tem-
plate for the first round PCR. For the second round PCR, 2
µL of the diluted first round PCR product was re-amplified
wit h AUAP ( Inv i t rog en TM) an d CHS -GSP4 (5 -
CACTGGAGAGGGTAAGGAGTGG-3). At the same time,
two F35H gene-specific primers (GSP1-2) were synthe-
sized according to the sequence of F35H-p. F35H-GSP1:
5-TCGACGCCACTCTCCCTTCCTCG-3, F35H-GSP2: 5-
AGGACTGCGAGGTGGACGGCTAC-3. The method of
PCR amplification of F35H 3-end is the same as that of
CHS.
A final PCR was performed to create a full-length ver-
sion of the CHS gene that completely lacked endogenous
5-untranslated region. The following PCR primers were
us ed: CHS1F 5-ATGGCGGCGACGATGACGGTGGA-3;
CHS1R 5-CTAGGCTGTGACTGGGACGCTAT-3. These
primers were used to clone the CHS genes from genomic
DNAs of Th. ponticum, blue-grained wheat, white-grained
wheat and Chinese Spring by PCR.
1.4 Southern and reverse Northern blotting analyses
Genomic DNAs were digested by endonucleases and
then fractionated in 0.8% agarose gel, blotted onto Hybond-
N+ membranes by capillary act ion with 0.4 mol/L NaOH
overnight and cross-linked to the Hybond-N+ membranes
via baking at 80 ℃ for 2 h.
Pre-hybridization, hybridization and washing of the fil-
ter were performed as described (Sambrook et al., 1989)
with minor modifications, we used an economical and ef-
fective hybridization s olution which con tains 0.3 mol/L
sodium phosphate (pH 7.2), 2% BSA, 1 mmol/L EDTA and
7% SDS.
Reverse Northern probe was labeled as the following
procedure: adding 4 µg total RNA, 1.5 µL Oligo-dT, and
RNase-free water up to a total volume of 12.5 µL to a 1.5 mL
eppendorf tube, 65 ℃ water bath for 15 min, then put the
tube immediately on ice for at least 2 min. Add the second
mixture which contains 1 µL mixture (dATP, dGTP, dTTP 10
mmol/L each), 5 µL 5×buffer, 1 µL RNasin (40 U/µL), 1 µL
M-MLV (200 U/µL) or reverse transcriptase (SuperScript
ⅡTM), 2.5 µL [α-32P]dCTP (10 Ci/µL) and RNase-free wa-
ter to a total volume of 12.5 µL to the first mixture, mixed
thoroughly. The tube was incubated at 42 ℃ fo r 50 min,
then added the same volume (25 µL) of 0.4 mol/L NaOH to
the tube to denature the labeled probe at room temperature
for 10 min. The procedures of hybridization, washing and
auto-radiography were the same as that of Southern blot-
ting hybridization.
1.5 DNA sequencing and analysis
The amplified CHS and F35H fragments, and the CHS
and DFR full-length genes were subsequently purified and
cloned into pGEM-T Easy Vector (Promega, USA). The se-
quences of the clone were determined by automatic se-
quencing using the ABI PRISMTM Big DyeTM Terminator
Cycle (Applied Biosystem/Perkin-Elmer, San Jose, CA), then
the nucleo tide s equences were compared with the s e-
quences in NCBI using BLAST program. The positive frag-
ments or genes were defined as CHS-p, DFR and F35H-p.
2 Results
2.1 Isolation of two CHS cDNAs, one DFR cDNA and a
F35H (belongs to P450) cDNA 3-end
Through DNA sequencing, we obtained two d ifferent


YANG Guo-Hua et al.: Cloning and Expression of Two Chalcone Synthase and a Flavonoid 35-Hydroxylase 3-end cDNAs
from Developing Seeds of Blue-grained Wheat Involved in Anthocyanin Biosynthetic Pathway 593
through sequence alignment. This indicates that genes on
the pathway at the primary steps controlling the secondary
metabolites of anthocyanin biosynthetic pathway are very
conservative during the plant evolution. Our results sug-
ges ted that CHS expressed in wheat seeds with t iss ue
specificity, but F35H and DFR expressed not only in seeds
but also in seedlings, which accumulated in young leaves
may have other function(s). In the blue-grained wheat, CHSs
iso lated were derived from common wheat and Th.
ponticum. This suggests that there must have some genes
from Th. pont icum express ed normally in blue-grained
wheat, and there may have s ome key genes to cont rol or
contribute to the blue pigmentation.
The reverse Northern blotting analysis suggested that
CHS expressed prior to F35 H, and DFR expressed later
than F35H did. This order is iden tical to the expression
pattern of genes in the anthocyanin biosynthetic pathway
in other plant species (Holton and Cornish, 1995; Shirley,
2002). Therefore, there may exist an anthocyanin biosyn-
thetic pathway in the formation of blue pigment. RT-PCR
res ults s howed that there may have some regulatory
gene(s) to regulate the expression of thes e genes on the
pathway in the developing seeds of white-grained and blue-
grained wheats, but the regulatory pattern in blue-grained
seeds may not be the same as that in white-grained wheat
derived from blue-grained monosomic line and Chinese
Spring.
Acknowledgements: We thank Dr. ZHANG Xue-Yong
for his critical review of this manuscript.
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