全 文 ：ISSN　1007-7626
CN　11-3870 Q
中国生物化学与分子生物学报
Chinese Journal of Biochemistry and Molecular Biology
2004年 8 月
20(4):428 ～ 433
Cloning and Sequence Analysis of the Full-length cDNAs Encoding rpeA
and rpeB of R-Phycoerythrin from Corallina officinalis
WANG Sheng , ZHONG Fu-di1), WU Zu-jian , LIN Qi-ying , XIE Lian-hui＊
(Institute of Plant Virology , Fujian Agriculture and Forestry University , Fuzhou 350002 , China)
Abstract　The full-length cDNAs encoding rpeA and rpeB from Corallina officinalis L.were cloned with the
primers derived from the DNA sequence (AF510986)using RACE method.The sequences of these clones
were analyzed , and there were two open reading frames (ORF)that were co-transcribed into a 2257 bp
polycistronic transcript (AF542554).The structure of this cDNA is arranged as 5′-UTR-rpeB-intergenic
region-rpeA-3′-UTR with 493 bp , 534 bp , 101 bp , 495 bp and 634 bp , respectively.Shine-Dalgarno (SD)
sequences are found both at 13 bp upstream to the initiator of rpeA and rpeB , which are TAAGGAGA for rpeB
and TAAGGAAA for rpeA , respectively.According to the ORF finder prediction , another potential ORF was
identified at 110 bp downstream to the stop codon of rpeA with the SD-like motif at 9 bp upstream to the
predicted initiation codon.To the best of our knowledge , this is the first report of the cDNA sequence analysis
for rpeA and rpeB of phycoerythrin from the marine red algae.
Key words　Corallina officinalis L., phycoerythrin , rapid amplification of cDNA ends(RACE)
珊瑚藻 R-藻红蛋白 rpeA 和 rpeB 基因全长 cDNA克隆与序列分析
王　盛 , 钟伏弟1) , 吴祖建 , 林奇英 , 谢联辉＊
(福建农林大学植物病毒研究所 ,福州　350002)
摘要　依据珊瑚藻(Corallina officinalis L.)藻红蛋白 rpeA和 rpeB 的 DNA序列(AF510986)设计引物 ,
通过 PCR-RACE方法扩增得到 rpeA和 rpeB 的 cDNA序列.序列分析表明 ,该序列采用多顺反子转录
策略 ,全长 2257 bp(AF542554),排布顺序为 5′UTR-rpeB-间隔区-rpeA-3′UTR.5′非编码区 493 bp ,
rpeB 基因 534 bp ,基因间隔区 101 bp , rpeA基因495 bp ,3′非编码区 634 bp.在 rpeA 和 rpeB 的基因起
始密码子上游均存在类似原核核糖体结合的 Shine-Dalgarno (SD)序列.在 rpeA 基因终止密码子下
游110 bp处还存在着一个可能的开放阅读框架.经检索 GenBank发现 ,真核红藻藻红蛋白中尚无有
关 cDNA序列的报道.
关键词　珊瑚藻 ,藻红蛋白 ,快速分离 cDNA末端(RACE)
中图分类号　Q943.2
Received:October 17 ,2003;Accepted:December 16 , 2003
Supported by the Key Project of Fujian Province(No.2000H004 and No.2001Z127)
＊Corresponding author.Tel:86-591-3769704 ,Fxa:86-591-3769704 ,E-mail:xielh@fjau.edu.cn
1)Who contributed equally to this article
收稿日期:2003-10-17 ,接受日期:2003-12-16
福建省科技厅重大项目(No.2000H004和No.2001Z127)
1)该作者与王盛对本文有同等贡献
王盛 ,男 , 1972年 7月生 ,博士
　　Phycobiliproteins function as a major light harvesting
protein-pigment complex in the cyanobacteria and the
eukaryotic algae
[ 1] .Among these proteins , phycoerythrins
(PE)are characterized with strong absorption bands in
the green region of the spectrum.These red-colored
proteins fall into three spectroscopically distinct classes:
DOI :10.13865/j.cnki.cjbmb.2004.04.002
C-phycoerythrins(λmax ～ 565 nm ), B-phycoerythrins
(λmax ～ 540-560 nm , shoulder ～ 495 nm), and R-
phycoerythrins(λmax ～ 565 nm , 545 , and 495 nm)[ 2] .
Phycoerythrin allows high efficiency of light transfer to the
photosynthetic centers in the thylakoid membrane through
the associated pigments
[ 3] .Only a single type of
phycoerythrin is present in each organism
[ 4] .
Most of the genes encoding the components of
phycobilisome have been cloned and characterized in
cyanobacteria
[ 5 ～ 8] .In red algae , the genes encoding
phycobilisome components have been located
[ 9 , 10] , but so
far only a few of them have been sequenced
[ 11] .As the
main components of phycoerythrin , the genes encoding
alpha and beta subunits have been cloned only from a few
kinds of red algae , such as Rhodella reticulate , R .
violacea , Porphyra purpurea , P.tenera , P .yecoensis ,
Polysiphonia boldii , Griffithsia monilis , Aglaothamnion
neglectum and Gracilaria lemanneiformis.On other
aspects , such as gene structure , transcription and
translation regulation , there is much less information
available.Apt and Grossman[ 12] presumed that the
sequence of cpeB and cpeA were co-transcribed in the red
algae Aglaothamnion neglectum .At the same time , Roell
and Morse
[ 13]
also reached the same conclusion in the
macrophytic red alga Polysiphonia boldii.Kim and
Fujita
[ 14]
confirmed the co-transcription of cpeB and cpeA
genes in Porphyra yezoensis using the nested RT-PCR
analysis.But there were no any direct evidence about the
co-transcription of beta and alpha subunit genes.
Moreover , the untranslated region (UTR)plays a very
important role on translational regulation , and
characterization of these sequences may be useful in
understanding the mechanism of phycoerythrin s synthesis
and algal photosynthesis.
In this study , we cloned the cDNA encoding R-
phycoerythrin alpha and beta subunits from C.officinalis
using RACE method , and analyzed these cDNA
sequences.This is the first report of the full-length cDNA
sequence coding for the alpha and beta subunits of
phycoerythrin from red algae.
1　Materials and Methods
1.1　Algae materials
Samples of Corallina officinalis were collected from
Putian Bay , Fujian , China.After collection , the
materials were washed with distilled water , frozen in
liquid nitrogen , and stored at -50℃until use.
1.2　RNA isolation and first-strand cDNA synthesis
The total RNA was extracted from Corallina
officinalis using UNIQ-10 Spin Column Total RNA
Isolation Kit (Shanghai Sangon Biological Engineering
Technology and Service CO., LTD., China).The
cDNAs were synthesized according to the manufacturer s
protocol , using primers , 3′-CDS , 5′-CDS and SMARTⅡA Oligo provided by the SMARTTM RACE cDNA
Amplification Kit (BD Bioscience Clontech Company ,
USA).
1.3　cDNA amplification
According to the corresponding of DNA sequence in
the GenBank database (AF510986), the sequence-specific
primers , from Gsp1 to Gsp8 , were designed (Table1).
The cDNA sequences of rpeA were amplified by nested
PCR.The 3′RACE was first amplified with Gsp1 and
UPM(Universal Primer Mix), and then with Gsp2 and
NUP (Nested Universal Primer).For the 5′RACE , the
first amplification usedGsp3 and UPM , and then Gsp4 and
NUP.The cDNA sequence of rpeB was amplified by the
same method as described above , using Gsp5 and Gsp6 for
3′RACE , Gsp7 and Gsp8 for 5′RACE.The primers ,
UPM-long primer , UPM-short primer and NUP , were
provided by the SMART
TM
RACE cDNA amplification Kit
(BD Bioscience Clontech Company , USA).
Table 1　Primer sequence for RACE
Name of primer Sequence of primer
Gsp1 5′-GCTGATGCAGCAGGACGTT-3
Gsp2 5′-GCGGCAAGATTAGAAGCAGC-3
Gsp3 5′-GCTTCTAATCTTGCCGCAGCTC-3
Gsp4 5′-CGTCCTGCTGCATCAGCAGCGC-3
Gsp5 5′-ATGGCTGCTTGTCTACGTGACG-3
Gsp6 5′-GCTTAGCTGCAGAAGTTGCTGC-3
Gsp7 5′-CACCGTCACGTAGACAAGCAG-3
Gsp8 5′-GTGCTTGAAGGTCACTACCACC-3
1.4　PCR production linking to the vector , cloning
and sequencing
The PCR products were gel-purified using
UltraClean
TM
15 (MO BIO laboratories , Inc.USA)
according to the manufacturer s protocol.The purified
PCR products were cloned into pMD18T-Vector (TakaRa
Biotechnology Co., Ltd., Dalian , China), and screened
with blue-white selection[ 15] .The clones were confirmed
by restriction analysis , and then sent to BioAsia Company(Shanghai , China)for sequence analysis.
2　Results
2.1　Analysis of full-length cDNA
The full-length cDNA of rpeBA was obtained using
RACE method , and confirmed by 3′-end and 5′-end
sequence analysis of the cDNA clones.The 3′-RACE cDNA
clone of rpeB(clone C)contains all sequences of rpeA ,
while the 5′-RACE cDNA clone of rpeA (clone B)contains
the sequences of rpeB (Fig.1).Thus , we concluded that
the gene of rpeB and rpeA were co-transcribed in the red
algae Corallina officinalis.The full-length cDNA was a
2 257 bp-polycistronic transcript.The structure of this cDNA
is arranged as 5′-UTR-rpeB-intergenic region-rpeA-3′-UTR
with 493 bp , 534 bp , 101 bp , 495 bp and 634 bp ,
respectively (Fig.2).After searching the GenBank
database , there were no any other cDNA sequences for
rpeBA of phycoerythrin from the eukaryotic red algae.
429No.4 WANG Sheng et al:Cloning and Sequence Analysis of Full-length cDNAs Encoding rpeA and rpeB of R-Phycoerythrins 　
Fig.1　Schematic diagram of the cloning for the full-length cDNA of rpeBA
IR shows the intergenic region.The clone A and B show the 3′and 5′RACE cDNAs of rpeA , respectively.The clone C and D show the 3′and 5′RACE
cDNAs of rpeB , respectively
Fig.2　Nucleotide sequences of cDNA and deduced amino acid sequences for rpeA and rpeB
The oligonucleotides in f rame are SD core mot if s.The asterisk indicates stop codon.The intergenic region are underline.Another potential ORF predicted by
ORF finder are in the shadow.This cDNA sequence was submitted to GenBank database with accession number AF542554
430 Chinese Journal of Biochemistry and Molecular Biology Vol.20
Fig.3　Amino acid alignments of the PEαand β subunits of Corallina officinalis
Stars indicate identical amino acid residues in the different sequences
　　The predicted molecular mass of rpeA and rpeB was
17.6 kD and 18.6 kD , respectively , which were
significantly lower than the observed value.The predicted
molecular mass would be increased to 19 kD and 21 kD ,
respectively , if the two or three tetrapyrrole chromophores
associated with the polypeptides are included in the
431No.4 WANG Sheng et al:Cloning and Sequence Analysis of Full-length cDNAs Encoding rpeA and rpeB of R-Phycoerythrins 　
calculation.The predicted pIs of the rpeB and rpeA
polypeptides were 5.29 and 5.34 , respectively.The AT
content at the 3′site of the triplet was 84.2% for rpeA
and 80.9% for rpeB , showing a tendency of AT
enrichment at the third nucleotide of the genetic codes ,
the characteristics of chloroplast genes
[ 16] .
2.2　Comparison with known red algae sequences
The deduced amino acid sequence was compared
with corresponding sequence of other eukaryotic red algae
Rhodella reticulata (AF114823), Rhodella violacea(L02188), Porphyra purpurea (U38804), Porphyra
tenera (D89877), Porphyra yezoensis (D89878),
Polysiphonia boldii (Z14094), Griffithsia monilis
(Z98528), Aglaothamnion neglectum (Z11907),
Gracilaria lemaneiformis (AF275685) by alignment
procedure(Fig.3).The shaded amino acids in Fig.3
were the binding sites for the associated pigments.We
identified these bilin attachment sites by comparing our
sequences with the sequence of Gastroconium coulteri
[ 4] .
The rpeB and rpeA genes in the ten species of eukaryotic
red algae were highly conservative , especially in the
neighboring sites for bilin attachment.The similarities
between those red algae and Corallina officinalis are from
68% to 81% at the nucleotide level and from 71% to
89% at amino acid level for rpeB .For rpeA , they share
the similarities from 76% to 81% at the nucleotide level
and 82% to 90% at amino acid level(Table 2).
Table 2　Amino acid sequences identity(%)between the PE subunits aligned in the Fig.3
AF510986 AF114823 L02188 U38804 D89877 D89878 Z14094 Z98528 Z11907 AF275685
AF510986 - 71 84 89 89 89 84 80 80 85
AF114823 n - 73 72 72 72 71 70 68 72
L02188 88 n - 87 86 86 80 79 78 84
U38804 87 87 n - 99 99 88 84 83 91
D89877 87 89 98 n - 100 88 84 84 92
D89878 86 88 97 99 n - 88 84 84 92
Z14094 86 81 84 84 84 n - 82 81 87
Z98528 88 82 85 84 84 84 n - 84 83
Z11907 82 76 80 80 79 79 82 n - 81
AF275685 90 85 84 84 84 83 84 79 n -
　　Similarities between β subunits are shown in the
upper right half , and those between αsubunits in the
lower left.Abbreviations are as in the Fig.3.There are
notαsubunit sequences of red algae Rhodella reticulata(AF114823)in GenBank , so “n” indicates that there is
not a comparison.
2.3　Prediction of the secondary structure of amino
acid sequence
The secondary structure of rpeB may contain helix
66.67%, coil 21.47%, extension 6.21% and turn
5.65%;the rpeA may contain helix 72.56%, coil
20.12%, extension 3.05% and turn 4.27% using the
online predictive software-SOPMA.
2.4　Analysis of untranslated region(UTR)
The cDNA has long untranslated region (UTR),
including 5′UTR of 493 bp , an intergenic region of 101
bp , and 3′UTR of 634 bp.In the prokaryotes , the
Shine-Dalgarno(SD)sequences act as ribosome binding
sites (RBS).However , most SD sequences vary in
slightly the core sequence of GGAGG.The core SD motifs
are GGAG , GAGG , and AGGA in bacteria[ 17] .In
Corallina officinalis , such sequences are found both at 13
bp upstream to the initiation codon of rpeA and rpeB ,
TAAGGAGA for rpeB and TAAGGAAA for rpeA.After
analysis by ORF finder , another potential ORF was
identified at 110 bp downstream to the stop codon of rpeA
with the typical SD-like motif at 9 bp upstream to the
predicted initiation codon.It uses ATG as initiation codon
and TAA as stop codon.The predicted ORF is 210 bp
encoding 69 amino acid residues.But the conserved
sequences , motifs and domains were not found at both
amino acid and nucleic acid level by the online predictive
softwares provided by the NPSA and NCBI.
The secondary structure of the untranslated region
was predicted by the RNAdraw software.The complex
stem-loop was found at both 5′ and 3′UTR.The
initiation codon and SD-like sequence of rpeB and rpeA
were both buried in the stem-loops.As we know , the
translation initiation region (TIR)is important for the
translation regulation.Those stem-loops may affect the
binding of ribosome initiation complex , and consequently
may affect the efficiency of translation.
3　Discussion
　　Phycobilisomes are photosynthetic antenna apparatus
in red algae and prokaryotic cyanobacteria , and consist of
phycobiliproteins and linker polypeptides
[ 11] . It is
organized into two distinct structural domains , the core
and rods.The core is composed of allophycocyanin
(APC) and is in direct contact with thylakoid
membranes. The rods are composed of stacked
phycobiliprotein hexamers that radiate from the core.
432 Chinese Journal of Biochemistry and Molecular Biology Vol.20
Phycocyanin(PC)is located at the end proximal to the
core on the rods , whereas phycoerythrin (PE)is at the
distal end
[ 18] . Light energy absorbed by PE is
sequentially transferred to PC , then to APC and finally to
chlorophyll
[ 19] .It is interesting that phycobilisomes of C.
officinalis do not contain phycocyanin and
allophycocyanin
[ 20]
and thus differ from phycobilisomes of
other red algae.We cloned and sequenced the rpeB and
rpeA that are the main components of PE.
The obtained sequence is a sequence for
polycistronic transcript and has SD-like motifs that act as
ribosome binding sites in the prokaryotes.At the same
time , it possesses a poly(A)tail that is the representative
characteristic of eukaryotes.It has both prokaryotic and
eukaryotic characteristics.Moreover , the AT content at
the third nucleotide of the triplet shows a tendency of AT
rich , characteristic of chloroplast genes[ 16] .So , the rpeA
and rpeB must be located on the plasmid genome of C.
officinalis.
PE has three distinct subunits:α, β , and γ
polypeptides.Theαand β subunit are present in a ratio of
1∶1 in PE.The co-transcript of rpeA and rpeB may be
useful to keep this ratio.After analyzing the UTR
sequences , we found both of the two subunits have SD-
like sequences 13 bp upstream to their initiation codon.
Studies on the expression of polycistronic transcripts of
chloroplast suggest that , similar to prokaryotes , the
chloroplast translation initiation complex binds internally
to the chloroplast transcript but not to its 5′-terminus.
The binding of initiation complex is mediated by the SD-
like sequence in which the SD-like sequence is located in
close proximity to the initiation codon
[ 21] .In prokaryotes ,
the direct RBS binding and internal initiation of
translation is usually independent of upstream sequence
and allows the simultaneous translation of several ORFs in
a polycistronic transcript
[ 22] .The sequences in close
proximity to the initiation codon of two subunits do not
share any homology at all. If ribosomes bind
independently to each initiation site by making some
initiation sites more attractive to ribosomes than others ,
the balance between the two subunits of PE may be
broken.In order to keep the balance , the two subunits
may be expressed one by one.
A number of important questions remain unanswered
regarding theαand β subunits from red algae.We still do
not understand the way by which they interact with γPE
subunits and the mechanism of regulation between plastid
encoded αβ PE subunits and nuclear-encoded γPE
subunits.The answers will require more sequence data
and more detailed biochemical researches.
Acknowledgments　We thank Dr.Duan Yongping and Prof.Hu
Fangping for a thoughtful review of the manuscript.
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433No.4 WANG Sheng et al:Cloning and Sequence Analysis of Full-length cDNAs Encoding rpeA and rpeB of R-Phycoerythrins