全 文 ：广 西 植 物 Guihaia 27(2)：224— 230 2007年 3月
Phylogenetic analysis and cloning of psaB
gene of Dunaliella salina
LU Zhao-Ming，LIU Hong-Tao，ZANG Wei-Dong，XUE Le-Xun*
(Laboratory for Cell Biology，Zhengzhou University，Zhengzhou 450052．China)
Abstract：One pair of degenerate primers was designed according to conserved motifs of the psaB (A2 subunit of
photosystem I)of Chlamydomonas reinhardtii，Chlamydomonas moewusii，Chlorella wulgaHs，and a total RNA of
Dunaliella salina was extracted with TRIzol reagent
． A eDNA fragment，about 1．8 kb in length，from green algal D
．
salina was obtained through RT-PCR method．The resulting PCR product was cloned into T-vector and screened to
determine its sequence．Homologous analysis of the deduced amino acid sequence was performed by BLAST and sub—
sequently compared to GenBank data． The obtained eDNA sequence was 1815 bp long，which encoded 605 amino
acids(OenBank accession number：AY820754)．The sequence shared high homologue with the following psaB：92
for Chlamydomonas reinhardtii，91 for Chlamydomonas moewusii，86 for Chlorella vulgarls，85 for Mes0sti 一
撇 viride，85 for Physcomitrella pat~s subsp．Patens and 84 for Nephroselmis olivacea
． It can be concluded
that the cloned sequence is psaB eDNA fragment from D
． salina．In addition，codon bias analysis shows that the con—
tent of A and T (35．7 and 39．17 ，respectively)is used significantly more frequently in the third position compo—
sition than that of G and C (7．27 and 17．85 ，respectively)，that is to say，the codons of psaB of D
． salina are
mainly composed of NNA and NNT．At the same time，through phylogenetic analysis of the Chlorophyta with psaB
gene，it is shown that D．salina is the nearest to the most species of Haematococcaceae in the phylogenetic relation—
ship，which is contrary to previous opinion that D．salina is the nearest to the most species of Chlamydomonadaceae in
the phylogenetic relationship．These studies wil help clarify the genetic background of D
． salina．
Key words：Dunaliella salina；A2 subunit；psaB；cDNA；degenerate primer
CLC number：Q943 Docum ent code：A Article ID：1000—3142(2007)02—0224—07
Genetic engineering of microalgae has been greatly
developed．At present，various kinds of microalgae，in—
cluding prokaryotic microalgae cyanobacterium，and eu—
karyotic microalgae Chlamydomonas reinhardtii，chlo—
rella，etc，have been used to pruduce a large number of
bioactive substances． Dunaliella salina，an unicelular
green alga，which was one of the most halotolerant eu—
karyotes，originally described by Dunal in 1938(Avron et
a1．，1992)，has a thin cellular membrane without a rigd
cel wall，and a single，large cup-shaped chloroplast with
its photosynthetic thylakoid membranes，pyreniod，and
starch，and abundanceS-carotene globules．Studies on the
genetics of Dunaliella have been conducted for decades
at a few laboratories(Ge ng et a1．，2003；Li et a1．，2003；
Chai et a1．，2004；Wang et a1．，2005)．
Photosystem I(PSI)complex is an iron-sulfur type
of reaction center(RC)which consists of 1 1～ 13 sub—
units and catalyzes the photoinduced oxidation of plasto—
cyanin(Pc)／cytochrome(cyt)c6 and reduction of ferre—
doxin／￡ avodoxin(Golbeck et a1．，1991；Bretel，1997)．
The PSI reaction center contains two major core proteins
psaA and psaB (Fish et a1．，1985)，The following cofac—
tors are bound to the psaA／psaB heterodimeric polypep—
tides(82～ 83 kDa)：P700 (a pair of chlorophyll mole—
Received date：2005’05_17 Accepted date：2006-01-30
基金项目：国家自然科学基金(30600006)；河南省医学科技创新人才工程项目(2001006)[supported by the National Natural Science
Foundation of China(30600006)；Henan Innovation Scholar Project for Medical Science and Technology(2001006)3
作者简介：鲁照明(1966一)，男 ，河南原阳人，博士，从事分子生物学研究，(E-mail)lzm310@zzu．edu．cn o
。通讯作者(Author for correspondence，E-mail：xuelx@371．net)
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2期 鲁照明等：杜氏盐藻psaB基因eDNA克隆及系统进化分析 225
cules)，A0(monomerie chlorophyl1)，A1(phylloquinone)
and FX (4Fe-4S center)．The terminal acceptors FA and
FB (4Fe-4S centers) are located in the psaC subunit
(8．9 kDa)．In addition，psaA，psaB，psbD，r C2，and
rbcL are good sources of phylogenetie information at a
deep level(Nishiyama et a1．，1999)． The plastid genes
psaA，psaB and rpsl4，encoding the photosystem I reae—
tion center chlorophyl proteins and ribosomal protein
CS14，are organized into an operon on the circular plastid
genome(Wu et a1．，1999)．
In this study，one pair of degenerate primer was de—
signed according to conserved motifs of the psaB (A2
subunit of photosystem I) of Chlamydomonas rein—
hardtii，Chlamydomonas moeumsii，Chlorella vulgaris
and Mesostigma viride，and a total RNA of Dunaliella
salina(D．salina)was extracted with TRIzol reagent．A
eDNA fragment from green alga D．salina was obtained
through RT-PCR method．Cloning of the psaB eDNA
can further easily obtain the upstream strong promoter
which provide theoretic basis for the construction of
transgene D．salina bioreactor and promote the studies of
the phylogenetie relationships among D．salina，other al—
gae and high plants．These studies are expected to pro—
vide a new insight into molecular mechanisms of electron
transfer in PSI．
1 Materials and Methods
1．1 Algal and bacterial strains
Dunaliella salina (UTEX 1 644 Teod) was pur-
chased from the University of Texas，USA，E．coli JM109
was kept in our laboratory．
1．2 Culture of Duna liella sa lina
Dunaliella ⅡZ was grown in batch cultures in
iquid PKS medium at 27℃ under continuous irradiance
of 4 500 Lux，cycle ratio of light and dark is 12 h：12 h．
1．3 Primers
According to conserved motifs of the psaB (A2 sub—
unit of photosystem I)of Chlamydomonas reinhardtii，
Chlarnydomonas moewusii，and Chlorella vulgaris，two
pieces of highly conservative regions(AWQGNFE and
RGYWQE)were found．A pair of degenerate primers
was designed as folows： Pl： 5一GGNTGGCARG—
GNAAYTTYGAR一3； P2： 5_YTCYTGCCARTAN(：C—
NCX一3．W here N was random base pairs，Y R and X
stand for C／T，G／A and G／T，respectively．
1．4 Total RNA extraction
After eultured for 4 days，cells of D．salina about 1
×1 ／mL were harvested by centrifugation under 4 000
g fOr RNA extraction，the total I A was isolated wi th
TI zol(Invitrogen) and measured by eleetrophoresis
and UV analysis．
1．5 RT-PCR amplication of psaB eDNA
Two microgram of total RNA was reverse-tran—
scribed into eDNA with Reverse Transeriptase (AMV
First strand eDNA synthesis Kit，Shanghai Sangon Co．
Ltd)folowi ng the protocol recommended by ma nufae—
turer．The product was then subjected to PCR-amplifi—
cation wi th the degenerate primers above-mentioned．
PCR amplification was performed using Biometra Ther—
mal Cycler．PCR reactions contained 10×PCR Buffer 5
L，P1 1 L(50／~mol／?L)，P2 ltzL(50 mol／ L)，Taq
polymerase 0．5 L(5 U／uL)，dNTP 4 L(10 mol／／~L)，
template DNA 1 L and O 37．5 L，with a final vol—
ume of 50 uL．PCR program was described as folows．
95℃ Dredenatrue lmin，then 94 ℃ 30 S，5O℃ 30S，72
℃ 90 S，3O cycles，72℃ lOmin．The RT-P(二R produets
were examined on eleetrophoresis in the 1％ agarose gels
with ethidium bromi de stain．
1．6 Purification and cloning of PCR product
The PCR products were separated by 1 agarose
gel ecectrophoresis and then the band of interest was pu—
rifled according to ma nufaetruer’s instruetion of the
DNA gel extraction Kit(Hangzhou Vitagene Bioehemi—
cal Technique Co．Ltd，China )，and inserted into the
pMD18T-veetor．Competent cells of E coli JM109 were
transformed with the ligation product，then grown on
LB-agar plates containng 100 p．g／mL ampicillin，8O ug／
mL X-gal and 80,ug／mL IPTG．White colonies were
eultured in a 3ml LB liquid medium containing 100 ug／
mL ampicillin．Plasmi d DNA mini-preparation，purifiea—
tion and enzyme digestion were carried out according to
Molecular Cloning：A Laboratory Manual(Sambrook et
a1．，1989)．
1．7 Sequencing and homolog analysis of the psaB gene
The positive recombinant plasmids DNAs were se—
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226 广 西 植 物 27卷
quenced by the Shanghai Sangon Co． Ltd．The amino
acid sequences were deduced from the cDNA data with
DNAMAN software．Sequence analysis was performed
using blast of NCBI(http || ．ncbi． nih．gov／ 2000b。
BLAST／)．
Table 1 Results of ultraviolet absorbency of total RNA
1．8 Phylogenetic analysis of the pasB gene
In all ana lyses taxa belonging to families of the
psaB gene of Chlorophyta were given in Table 4．The
nucleotide sequences alignments from psaB gene were
constructed using CLUSTAL software．All phylogenetic
analyses were carried out using TreeView software．
2 Results
2．1 Identification of RNA quality
Formaldehyde denatured electrophoresis on agarose
gel showed the total RNA quality met the need of RT‘
PCR (Fig．1)．Two clear bands of 28S rRNA and 18S
rRNA isolated wi th TRIzol reagent were shown in Fig．
1．The brightness of 28S rRNA was nearly 2 times that
of 18S rRNA。showi ng that total RNA isolated is con—
sistent with ultraviolet ana lysis of the yield and purity of
tota1 RNA．
Fig．1 Electrophoresis of the D．salina total RNA
2．2 RT-PCR and cloning of the psaB
The agarose gel electrophoresis results of the RT-
PCR from cpDNAs of D．salina were shown in Fig．2．A
fragment of interest was around 1800bp long，which was
purified and subcloned into pMD18一T vector．The re—
combinant plasmids(pMD18一T-psaB)were identified
with EcoRI and SalI，which were included in the multiple
cloning sites(MCS)of pMD18一T vector，and a correct
fragm ent was obtained(Fig．3)．
Fig．2 Agarose gel electrophoresis analysis
of the PCR products
1：Molecular size marker：Sangon GeneRuler DNA
Ladder mix；2：1．8 kb of PCR product．
2．3 The deduced amino acids sequences and its homolog
of the psaB
The multiple alignments of the deduced 605 ami no
acid polypeptides of D． “Z 舢 psaB cDNA showed that
the cloned psaB cDNA shared more high identities wi th
other species in ami no acid(Fig．4)：92 for Chlamydo—
DqOI1／JS reinhardtii，9 1 9／6 for Chlarnydomonas moewusii，
86 for Chlorella vulgaris，85 for Mesostigma
viride，85 9／6 for Physcornitrella patens subsp．Patens and
84 fOr Nephroselmis olivacea．
3000bp
2000bp
Fig．3 Identification of the recombinant plasmid
containing the psaB fragment
1：Molecular size marker：Sangon GeneRulerTMDNA
Ladder mix：2：pMD1 8-T—psaB／EcoR I+Sal I
2．4 Analysis of codon bias
For each ami no acid，the table lists the number of
times a given codon occurs in the D．salina psaB gene．
The codon usage in the D．salina psaB gene is ap—
parently biased(GC 39．4，AT 9／6 60．6)(Table 2)．It
can be seen that A and T (35．7 and 39．17 ，respective—
ly)are used significantly more frequently in the third posi—
tion composition than G and C(7．27％ and 17．85 ，re—
spectively)，that is to say，the codons of psaB of D．salina
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2期 鲁照明等：杜氏盐藻psaB基因 cDNA克隆及系统进化分析 227
Fig．4 Alignment of 1he deduced amino acid sequences of the psaB
D．s：Dunaliella salina；C．r：Chlam3,domonas reinhardtii；C．m：Chlamydomonasmoewusii；C．v：Chlorella vulgaris；M
． v：Mesostigma viride
蕊豳
S r m V V S r m V V S r 啪 V V S r m V V S r m V V S r m V V S r m V V S r m V V S r n V V ㈠
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228 广 西 植 物 27卷
Table 2 Codon usage in the Dunaliella
salina psaB gene
are mainly composed Of NNA and NNT．
2．5 Phylogenetic analysis of the pasB gene
All the spedes betonging tO Chlorophyta were given
in 1、able 3．In order tO clarify the phylogenetic relation-
sl1ips between D salina and other spedes of the Chloro—
phyta(Fig．5)，alignments of the amino add encoded by
psaB gene were constructed using CLUSTAL software；
phylogenetic trees were constructed using TreeView soft—
ware,As shown in Fig．5，D．salina is the nearest tO the
most species of Haema tococcaceae in the phylogenetic re_
lationship．
3 Discussions
Unicellular green alga，Dunaliella salina，is one of
the most halotolerant eukaryotes．To date，there iS a lit—
tle information about genetic background of D．salina．
Degenerate primer is a compound constituting of a great
deal of oligonucleotide，of these compounds，one or more
differences can be found．PCR with degenerate primer
help detect new genes(Fietto eta1．，2002)．In the pres—
ent study，degenerate primers were designed according tO
conserved amino acid motifs of known species，and con—
generic genes can be screened by PCR，which is a simple
and feasible method(Jiang et a1．，2003)．
According tO conserved motifs of the psaB (A2
subunit of photosystem I)of Chlamydomonas rein—
hardtii，Chlamydomonas moewusii，Chlorella vulgaris
and Mesostigma viride，two pieces of highly conservative
Table 3 All the species belonging to Chlorophyta
regions(AWQGNFE and RGYWQE)were found．A
pair of degenerate primers was designed．A cDNA frag—
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2期 鲁照明等 ：杜氏盐藻 psaB基因 cDNA克隆及系统进化分析 229
_J r_[
厂
L
广
L
广L
— __J L
L-厂 L
r
L
厂
L[
广
U_-
L厂
I
I r
’
L[：
厂‘
厂
Fig．5 Phylogenetic chart of psaB in the Chlorophyta
ment from green algal D．salina was obtained through
RT-PCR method．Homologous analysis of the deduced
amino acid sequences was performed by BLAST and
subsequently compared tO GenBank data．The obtained
cDNA sequence was 1815 bp in length，which encoded
605 amino acids (GenBank accession number．
AY820754)．The sequences shared high homologue with
the followi ng psaB：92 for Chlamydomonas rein—
hardti，9 1 for Chlamydomonas moewusi，86％ for
Chlorela vulgaris，85％ for Mesostigma viride，85％ for
Physcomitrella patens subsp．Patens and 84％ fOr —
phroselmis olivacea． The findings indicate that the
cloned sequence is a psaB cDNA fragment from D．sali—
na ．
In higher plants，the studies of the psaB gene main—
ly focus on special operon of psaA-psaB-rpsl4(Wu et
a1．，1999)．The plastid genes psaA，psaB and rpsl4，en—
coding the photosystem I reaction center chlorophyl
proteins and ribosomal protein CS14，respectively，are or—
ganized into an operon on the circular plastid genome．In
the upstream，the activity of promoter is very strong．So
the author can obtain the full psaB，psaA and upstream
promoter sequences by 5’一RACE and genome walking
(data not shown)．These studies are expected to provide
a new strategy into bioreactor of transgene in D．salina．
The chloroplast genome is an interesting system for
studies of codon bias．Several complete chloroplast ge—
nome sequences are available from plants as well as from
red，brown ，and green algae． The chloroplast genome
codes for a limited set of proteins involved in protein
synthesis and photosyn thesis(Ohyama et a1．，1986；Shi—
nozaki et a1．，1986；Hiratsuka et a1．，1989)．Chloroplast
genes of D．salina have a codon usage that reflects the
genome compositional bias of a high A+ T content with
the translated psaB gene encoding the photosystem I A2
protein(Tab．2)．The codon usage of algal psaB corre—
sponds more closely to the lim ted tRNA population of
the chloroplast and is very sim lar tO the codon use ob—
served in the chloroplast genes of the green alga
Chlamydomonas reinhardtii(Morton BR，1996)．
Opinions on the basal relationship of Chlorophyta
vary considerably and no phylogenetic tree wi th signifi—
cant statistical support has been obtained．Here，we re—
port phylogenetic analyses using psaB gene sequences of
50 representative algal species of Chlorophyta．Analyses
at the nucleotide level could not resolve the basal rela—
tionship with statistical cordidence(Nishiyama et a1．，
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23O 广 西 植 物 27卷
2004)． Furthermore，Bryophyte monophyly inferred u—
sing amino acid sequences has a good statistical founda—
tion and is not rejected statistically by other data sets
(Nishiyama eta1．，2004)．In the study，the analyses，u—
sing translated ami no add sequences，indicate that D．5a—
lina is the nearest to the most species of Haematococ—
caceae in the phylogenetic relationship，which is contrary
to previous opinion that D．salina is the nearest to the
most species of Chlamydomonadaceae in the phylogenetic
relationship(Hou eta1．，2004；Hou eta1．，2006)．So we
can further know the genetic background of D salina
through various species of Haematococcaceae and find
various new functional genes in D．salina under the con—
dition of the research backgrounds．
杜氏盐藻 psaB基因cDNA克隆及系统进化分析
鲁照明，刘红涛，臧卫东，薛乐勋
(郑州大学 细胞生物学研究室，郑州 450052)
摘 要：根据真核生物莱茵衣藻(Chlamydomonas reinhardtii)、Chlamydomonas moewusii及 Chlorella vul—
garis等光系统I反应中心蛋白psaB基因的氨基酸高度保守序列，设计一对简并引物，利用 TRhol试剂提取
杜氏盐藻(Dunaliella salina)细胞的总 RNA，通过 RT—PCR，得 到的一 段长为 1．8 kb左右 的 eDNA片段。
PCR产物经 T_A克隆并测序以及测序结果推导成氨基酸序列进行同源性比较，表明所克隆的 1815bp序列为
杜氏盐藻光系统 I反应中心 psaB基因的 eDNA片段，OenBank收录号为 AY820754。根据已经得到的 psaB
的核苷酸序列推导成氨基酸序列与一些已知物种的psaB氨基酸序列相比较，同源性分别为 c^ m d0 0”ns
reinhardtii 92 ，C^ Znm d0m0 ns moewusii 91 ，Chlorella vulgaris 86 ，Mesostigma viride 85％，Phy—
s∞mitrella patens subsp．Patens 85％，Nephroselmis olivacea 84 。此外 ，psaB密码子偏爱性分析表明 ：杜氏
盐藻 psaB基因第三位密码子 A和 T的组成分别为35．7 和39．17 ，而 G和 c分别为 7．27 和 17．85 ，
即杜氏盐藻 psaB基因密码子的组成大多为 NNA和NNT。根据 psaB基因的特征，作者对绿藻门的 5O个物
种的psaB基因作了进化分析，结果表明：杜氏盐藻与 Haematococcaceae中的大多数种类进化地位最为接近，
这为更进一步弄清杜氏盐藻的遗传背景提供了理论依据。
关键词：杜氏盐藻；A2亚基 ；psaB；eDNA；简并引物
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