全 文 ：Comparison of Phycobiliproteins from Gracilaria lemaneiformis (Rhodophyceae)
and Its Pigment Mutants in Spectral and Molecular Respects
SUI Zheng_Hong , ZHANG Xue_Cheng＊ , CHENG Xiao_Jie
(College of Marine Life Sciences , Ocean University of Qingdao , Qingdao 266003 , China)
Abstract:　Comparative studies of absorption spectra of phycobiliproteins of Gracilaria lemaneiformis Greville
and its pigmental mutants were conducted in this study.The results showed that the absorption spectra of phy-
coerythrins(PE)from different material changed significantly , while those of phycocyanins(PC)and allophy-
cocyanins(APC)were basically similar.In order to disclose the essence of the difference , partial sequences of
the subunit genes of PE of Qingdao strain of G.lemaneiformis (qd)and its pigmental mutants were deter-
mined.The amino acid sequences were deduced and used to explain spectral shifts of PE from the pigmental
mutants.The amino acid sequences of PE resembled each other , and several residues changed among qd and
its pigmental mutants.Residue substitutions were found in a region consisting of amino acids which determined
the secondary structure and subunits interactions , thus might influence the confirmation and interaction of sub-
units , and further caused spectral deviation.
Key words:　Gracilaria lemaneiformis;hydrophobicity;pigment mutant;phycoerythrin gene;secondary
structure
　　Phycobilisomes exist in all Rhodophyta , Cyanobac-
teria , and some of Dinoflagellate and Cryptophyta , play-
ing the key role in harvesting light for photosynthesis.As-
sembled phycobilisome contains linker proteins , chro-
mophores , and phycobiliproteins , consisting of phycoery-
thrin (PE), phycocyanin (PC) and allophycocyanin(APC).The phycobilisomes are located on the surface of
the thylakoid membrane as polymers.Light energy is har-
vested by PE , transferred to PC , APC and chlorophyll se-
quentially and used to drive photosynthesis[ 1] .
PE has characteristic absorption spectra due to the
composition of chromophores , including phycourobilin(PUB)(Amax 498 nm)and phycoerythrobilin (PEB)(Amax 540 nm and 565 nm).In some species of red al-
gae , PE shows three_peak spectrum.In other species ,
however , it confers a specific two_peak spectrum.It is
believed that PE with the two_peak type is less progressive
than that with the three_peak type , which has been pro-
posed as an evolutionary mark
[ 2 ,3] .
PE has three dissimilar subunits , the α, β , and γ
ploypeptides , presenting in a ratio of 6∶6∶1[ 4] , or 6∶3∶
1[ 5] .The αand β subunits in the red algae have been
characterized[ 6 , 7] .They are encoded on the plastid
genome , usually forming an operon with the arrangement
from β to α.
In this study , we compare the absorption spectra of
phycobiliproteins of Gracilaria lemaneiformis (GL), in-
cluding the wild type (qd)and its pigment mutants ,
clone their PE genes , and investigate the relationship
among the materials in the spectral and molecular re-
spects.The results showed that the absorption spectra of
PE from different materials change significantly.The
putative explanation is the residue substitutions in a region
consisting of amino acids which determine the secondary
structure and subunits interactions , thus may influence
the confirmation and interaction of subunits , and further
cause spectral deviation.
1　Materials and Methods
1.1　Materials
Gracilaria lemaneiformis Greville of Qingdao strain(qd)was collected from Zhanshan Bay , Qingdao.Cul-
tures were grown in SWM medium[ 8] and exposed to light
intensity of 40μmol·m-2·s-1 with a light_dark rhythm of
12∶12 and temperature at 21 ℃ to 23 ℃.
MNNG(N_Methyl_N′_Nitro_N_Nitrosoguandine)was
used to treat young gametophytes of qd with a concentra-
tion of 40 mg/L for 30 min.After 30 days cultivating ,
mutants with different colors from that of qd were selected
under the anatomy microscope when they had grown to be
visible and then transferred to be individually cultivated.
Two strains were cultivated to grow up normally with a dif-
ferent color(yellow=ye and green=gr)from that of the
wild type(qd)which was red brown.
1.2　Methods
1.2.1　Separation of PE and spectral analysis　The
algal tissue was suspended in 0.65 mol/L K_Na phos-
phate buffer(pH 7.0)at 0.2 g of wet weight/mL and
homogenized at room temperature using a mortar and pes-
tle.The resulting suspension was passed through a French
pressure cell(ASLM Instruments)at 10 000 psi , and the
lysed cells were incubated in the presence of 1%Triton
X_100 for 30 min at room temperature.The suspension
was centrifuged at 10 000g for 15 min , and the
Received:2001-08-08　Accepted:2001-12-29
Supported by the National Natural Science Foundation of China(39670405 and 300000126).
＊Author for correspondence.E_mail:.
植　物　学　报　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　　
Acta Botanica Sinica　2002 , 44(5):557-561
supernatant was layered on a 0.25-2.0mol/L linear su-
crose gradient in 0.65 mol/L K_Na phosphate(pH 7.0).
Ultracentrifugation was in a SCP_55H rotor at 48 000
r/min for 1 h at 20 ℃.Purified PE was dialyzed against
1 mmol/L K_Na phosphate buffer overnight at 4 ℃, and
total PE was isolated by hydroxyapatite chromatogra-
phy[ 9] .The absorption spectrum of PE was detected with-
in the wavelength ranging from 400 to 700 nm by a spec-
trometer UV_240(Hitachi Co.).
1.2.2 　DNA isolation[ 10] 　The fresh samples were
ground in a 1.5 mL eppendorf tube using a specially de-
signed pestle , and immediately mixed with 25 to 30 μL
buffer (100 mmol/L Tris_HCl , pH 8.0 , 20 mmol/L
EDTA , 1.4 mol/L NaCl , 3% cetyl trimethyl ammonium
bromide , 0.2%(V/V)β_mercaptoethanol)and then in-
cubated at 60 ℃ for 30 min with occasional gentle stir-
ring.The samples were extracted with an equal volume of
chloroform_isoamylalcohol , and the aqueous phase was re-
covered and precipitated with isopropanol , pelleted by
centrifugation at 15 000 r/min , washed in 70%ethyl al-
cohol , vacuum_dried and redissolved in TE (10 mmol/L
Tris_HCl , pH 8.0 , 1 mmol/L EDTA).The samples were
incubated at 37 ℃for 10 to 15min , and then centrifuged
at 15 000 r/min for 10min.The solution , containing nu-
cleic acids and some low molecular weight polysaccha-
rides , was removed from the tube by pipette , discarding a
pellet composed largely of polysaccharides with some
trapped nucleic acids.DNA concentrations were deter-
mined by Pharmacia Gene Quant RNA/DNA calculator.
1.2.3　PCR　Primers were synthesized by Sangon Co.(CA), with sense sequence of ACCGTCGTATGGC(AT)
GCTT , and anti_sense sequence of GCACC(TCA)GC(GA)ATACCCCATT.PCR amplification was performed
on 9600 PCR cycler (product of PE Co.of USA).The
PCR circle parameters were 94 ℃80 s , 52 ℃2 min , 72℃80 s ,35 cycles.
1.2.4　Gene isolation　After PCR amplification , frag-
ments of interests were recovered by low melting point
agarose.Then it was constructed into dT vector
(pGEM5zf(+)/EcoR Ⅴ)[ 11] .Standard methods were
used for screening by complement selection[ 12] and PCR
amplifying using the conditions described above.
1.2.5　Sequencing and analysis 　 Sequencing was
performed on a PE_ABI377 DNA sequencer (PE Co.of
USA).The softwares of DNASIS(ver.5.0)and PROSIS(ver.5.0)were employed to analyze the result.The
softwares were products of Hitachi Software Engineering
Co.Ltd.DNASIS is aimed to align the sequences and to
give open reading frame , while PROSIS analyses amino
acids according to their attribution to the secondary struc-
ture of proteins.
2　Results
2.1 　Characterization of the absorption spectra of
G.lemaneiformis and its pigment mutants
The absorption spectra of PE of the mutants and wild
type are shown in Figs.1 to 3.The absorption spectrum of
Fig.1.　Absorption spectra of phycoerythrin(PE)of the wild type
of Gracilaria lemaneiformis of Qingdao strain (qd)and those of its
pigment mutants.
qd refers to the wild type.Ye refers to the yellow mutant and gr the
green one.
Fig.2.　Absorption spectra of phycocyanin(PC)of the wild type
of Gracilaria lemaneiformis of Qingdao strain (qd)and those of its
pigment mutants.
qd refers to the wild type.Ye refers to the yellow mutant and gr the
green one.
Fig.3.　Absorption spectra of allphycoerythrin (APC)of the wild
type of Gracilaria lemaneiformis of Qingdao strain(qd)and those of
its pigment mutants.
qd refers to the wild type.Ye refers to the yellow mutant and gr the
green one.
558　 植物学报　Acta Botanica Sinica　Vol.44　No.5　2002
PE of the wild type and the yellow mutant were similar ,
with three_absorption peaks at 496 nm , 562 nm and 540
nm respectively (Fig.1).However , the green one was
characterized by two_peak absorption at 496 nm and 562
nm respectively.The 540 nm peak in qd and ye was re-
placed by an even shoulder in gr.
On the contrary , the PC and APC displayed little
difference between the wild type and pigment mutants at
614 nm and 650 nm respectively (Figs.2 ,3).
2.2　Cloning and alignment of the PE genes of the
wild type and pigment mutants
To inquire into the molecular shift of absorption
spectrum variety of PE , PE genes of the wild type of qd
and those of its pigment mutants were partially se-
quenced.The sequences were situated near the C_termi-
nus of the β subunit and near the N_terminus of theαsub-
unit compared to that of other red algae[ 6] .The sequence
consisted of 309 nucleotides of the β subunit , 255(in qd
and ye)or 81(in gr)nucleotides of theαsubunit and 55
nucleotides of the spacer.The sequence might code for
102 amino acids of the β subunit and 85(in qd and ye)
or 27(in gr)amino acids of theαsubunit.The deduced
amino acids sequences were aligned(Fig.4).
The high rate of similarity could be seen among
them.The similarity of nucleotide acids was 99.2% be-
tween qd and ye , and 96% between qd and gr , respec-
tively.There was only one residue replacement between
qd and ye , and nine between qd and gr with the similarity
of amino acids 99.5%(between qd and ye)and 92.
27%(between qd and gr), respectively.
2.3　The secondary structure of protein of PE
Deduced amino acids of PE were examined for the
structural prediction of proteins(Fig.5).It was similar to
the secondary structure of PE between qd and ye except
the region from amino acid 158 to 164.The coil and β
sheet structure in the wild type was replaced by helix in
ye.The hydrophobicity of this region was correspondingly
decreased compared with that of the wild type (Fig.6).
There were three regions with different secondary
structures of the PE between the wild type and gr , which
were amino acids from 29 to 35 , from 77 to 89 and from
126 to 129(Fig.5).The only hydrophobicity variety be-
tween the wild type and gr was produced from the second
region , i.e.the amino acid from 77 to 89.
3　Discussion
It has been proved that 498 nm peak was produced
by PUB , and 540 nm and 562 nm peaks were produced
by PEB[ 13] .The disappearance of 540 nm peak in gr may
be caused by the change of PEB.However , there are
many aspects that relate to the polymer formation of PE
and its photoactivity , which will influence the spectral
character , such as the attachment of the chromophores to
the apoprotein and the components involved in this pro-
cess
[ 14-16] .On phycobiliprotein , gr mutant has a more
profound change than that of ye mutant.There are highly
conserved amino acids in phycobiliproteins where αand β
subunits conjugate with each other to form phycobilisome ,
and amino acids combine with chromophores and interact
with the attached chrompohores
[ 17] .These residues are
found to be constant in all kinds of phycobiliproteins or in
certain kinds.In the sequence we have obtained , none of
these amino acids are changed in the mutants , such as
Cys(7)of gr which is responsible for chromophore at-
tachment and both Arg (9)and Asp (10)which will in-
teract with the chromophores.So the conserved residues
may not cause the spectral change of gr.The solely differ-
ent secondary structure of PE of ye from the wild type and
one of gr s (i.e.the section from amino acid 126 to
129), even with the hydrophobicity deviation of protein in
ye , cannot impose on the PE.It has been proven by the
stability of the spectrum trait of ye , compared with that of
the wild type.
But the other varying secondary structure of gr from
the wild type , i.e.the fragment from amino acid 29 to 35
and from 77 to 89 , does affect the protein , because there
are conserved amino acids in it.There are three amino
acids with important functions that are related to linker in-
teractions in the fragment from 29 to 35.Flanking the
area are a few of amino acids fundamental to the exact for-
mation of the secondary structure of PE.The hydropho-
bicity change takes place in the region from amino acid 77
to 89 when compared with that of the wild type , in addi-
tion to the functional amino acids enclosing which is vital
for trimer_trimer interaction of the subunits of PE in the
region.It is speculated that the protein secondary struc-
tural change within these regions may alter the spatial
transformation of PE polymer , and the protein interactions
of gr.Gr has a two_peak absorption style of PE , which is
Fig.4.　Alignment of deduced amino acid sequence of phycoerythrin(PE)of the wild type of Qingdao strain of Gracilaria lemaneiformis(qd)
and its pigment mutants.＊, designates different residues between qd and its green pigment mutant(gr). , designates those between qd and its yellow pigment mutant
(ye).The subunits are interrupted by short bars.
SUI Zheng_Hong et al:Phycobiliproteins from Gracilaria lemaneiformis and Its Pigment Mutants in Spectral and Molecular Respects 559　
Fig.5.　The secondary structure of the phycoerythrin gene of qd and its pigment mutants.
Residues marked by arrowheads are highly conserved amino acids in both theαand β subunits.Single star designates highly conserved residues
in either theαsubunit orβ subunit.+ and - indicate residues with the net or negative charge in all phycobiliprotein types.Bold residues are
100% conserved in all phycobiliprotein types , including TEA(Terminal Energy Acceptor , which are mostly linker proteins)proteins.Under-
lined residues construct regions of gr with different secondary structures from the wild type , and that different structures between the wild type
and ye are shown in the boxed region.Denominators H , C and T stand for the helix , the β sheet and the coil , and the turn structure repective-
ly.The amino acid sequence belongs to the wild type.The preceding part belongs to the beta subunit , while the later isα.They are interrupt-
ed by the short bar.
Fig.6.　Hydrophobic curve of the wild type of Gracilaria lemaneiformis(qd)and those of its pigment mutants.
The curve of qd , ye and gr are shown in images A , B and C , respectively.
560　 植物学报　Acta Botanica Sinica　Vol.44　No.5　2002
different from the wild type and ye , and may be produced
by this protein structural transformation.It is suggested
that the secondary structure of photosynthesis protein may
influence the photosynthesis process.To disclose the pho-
tosynthetic mechanism , more details are needed , such as
the biochemical and molecular diversities of chromophore
and other enzymes related to the formation of phyco-
biliprotein polymers.
The mutants are useful material in studying life pro-
cess , especially pigment mutants of plants.The mutants
that we have obtained (gr and ye)have maintained their
biochemical characters for six years with vegetative
growth.The mutagen might affect other genes because the
mutants show different traits from the wild type in phyco-
biliprotein constitution and high temperature tolerance(unpublished data).
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龙须菜及其色素突变体藻胆蛋白的光谱及分子特征的比较
隋正红　张学成＊　程晓杰
(青岛海洋大学海洋生命学院 , 青岛 266003)
摘要:　对龙须菜(Gracilaria lemaneiformis Greville)及其色素突变体藻胆蛋白吸收光谱进行了比较研究 ,结果显示不
同藻株藻红蛋白的吸收光谱有显著的变化 ,而藻蓝蛋白和别藻蓝蛋白的基本相同。我们克隆了龙须菜及其色素突
变体的藻红蛋白亚基的部分基因序列 ,用该基因序列推导出的氨基酸序列进行分析以揭示这一变化的分子机理 ,
结果显示除几个氨基酸残基的替换外 ,几株藻间的藻红蛋白的氨基酸序列十分相似 , 一些氨基酸的替换发生在决
定藻红蛋白二级结构及亚基间相互作用的区域 , 可能会影响藻胆蛋白的构型及相互作用 ,导致光谱性质的变化。
关键词:　龙须菜;疏水性;色素突变体;藻红蛋白基因;二级结构
中图分类号:Q945.11　　　文献标识码:A　　　文章编号:0577-7496(2002)05-0557-05
收稿日期:2001-08-08　接收日期:2001-12-29
基金项目:国家自然科学基金(39670405和 300000126)。
＊通讯作者。E_mai l:.
(责任编辑:贺　萍)
SUI Zheng_Hong et al:Phycobiliproteins from Gracilaria lemaneiformis and Its Pigment Mutants in Spectral and Molecular Respects 561