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
2007年 4 月
23(4):262 ～ 270
Received:November 11 ,2006;Accepted:January 16 , 2007
＊Corresponding author Tel:023-68250086;E-mail:limy@swu.edu.cn
收稿日期:2006-11-11 , 接受日期:2007-01-16
＊联系人 Tel:023-68250086;E-mai l:limy@swu.edu.cn
Molecular Characterization and Functional Prediction of a Novel Leaf
SAG Encoding a CBS-domain-containing Protein from Coleus blumei
ZHU Qin-Long , LI Ming-Yang＊ , LIU Guang-De ,LI Yan-Dong , SUI Shun-Zhao , GUO Tie-Ying
(College of Horticulture and Landscape , Institute of Ornamental Plants , Southwest University , Chongqing　400716, China)
Abstract　Leaf senescence is considered as one of important factors to decrease ornamental values of foliage
plants.In the attempt to study and understand the molecular mechanism of leaf senescence , a senescent leaf
cDNA library of Coleus blumei was constructed and a small EST library was obtained.According to the
sequence of an EST fragment with a cystathionine beta synthase (CBS)domain , a novel leaf senescence-
associated gene (SAG)full-length cDNA encoding a CBS-domain-containing protein , denoted Cbcbs , was
rapidly cloned using a strategy of RACE combined with cDNA library.The full length of the Cbcbs gene was
859 bp long(accession No.EF076754)and contained a 609 bp open reading frame (ORF)encoding a 202
amino acid protein.One stop codon (TAA)was found in 5′UTR and one possible polyadenylation signal ,
AATAAA , and a pentanucleotide motif , ATTTA , were found in 3′UTR.The CbCBS contained a predicted
mitochondrial targeting peptide in the N-terminal region , two conserved and intact CBS domains , four casein
kinase Ⅱ(CK Ⅱ)phosphorylation sites , three protein kinase c (PKC)phosphorylation sites and one tyrosine
sulfation (TS) site.Sequence comparisons and phylogenetic analysis showed that CbCBS was a novel
senescence or stress-associated protein.The prediction analysis of secondary structure and three dimensional
structure of CbCBS suggested that the chief function of the protein was decided by the CBS domain pair.The
expression pattern of Cbcbs in leaves was analyzed by RT-PCR.It was demonstrated that Cbcbs gene was a
senescence-associated gene(SAG)and expressed in all leaf stages , young stage (Y)being the lowest and
terminal senescence stage (S3)being the highest , and was upregulated along with the leaf senescence.
Function analysis showed that the mature CbCBS maybe acts as a sensor of cellular energy status and directly or
indirectly regulates cellular energy levels to increase ATP content in mitochondria during periods of metabolic
stress of senescent leaves.
Key words　Coleus blumei;CBS domains;Cbcbs gene;senescence-associated genes;leaf senescence
彩叶草叶片衰老相关新基因 Cbcbs 的分子特征和功能分析
祝钦泷 , 　李名扬＊ , 　刘光德 , 　李艳冬 , 　眭顺照 , 　郭铁英
(西南大学园艺园林学院 , 西南大学花卉研究所 , 重庆 400716)
摘要　叶片衰老是观叶植物观赏性降低的重要因素之一.为研究彩叶草叶片衰老变化的分子机理 ,
在构建彩叶草衰老叶片 cDNA 文库及小型 EST 库的基础上 , 以 1 条新的具有胱硫醚-β-合酶
(cystathionine beta synthase , CBS)结构域的EST 序列为探针 ,通过 RACE与文库结合的方法 ,克隆了 1
个具有 1对完整 CBS 结构域的全长 cDNA , Cbcbs.Cbcbs cDNA 全长 859 bp ,包含 1个 609 bp的 ORF
框 ,编码 202个氨基酸.其 5′UTR区含有 1个终止子 TAA , 3′UTR区含有推测的加尾信号 AATAAA
和ATTTA元件.CbCBS N端含有线粒体转运肽 ,具有 2个保守的 CBS 结构域 , 4个酪蛋白激酶 Ⅱ
(casein kinase Ⅱ ,CKⅡ)磷酸化位点 ,3个蛋白激酶 C(protein kinase c ,PKC)磷酸化位点和 1个酪氨
酸硫化(tyrosine sulfation ,TS)位点.序列比较和进化分析表明 ,CbCBS是与衰老或应急相关的蛋白.
二级结构和三级结构预测表明 ,CbCBS的功能主要由 CBS 结构域决定.RT-PCR分析表明 ,该基因在
DOI :10.13865/j.cnki.cjbmb.2007.04.005
叶的各个时期均有表达 ,但随叶片衰老进程的加快而表达增加 ,是一个叶衰老相关基因(SAG),推
测在线粒体中成熟的 CbCBS 可能作为细胞能量传感器 ,在叶衰老引起的能量应急中参与细胞能量
水平的调节.
关键词　彩叶草;胱硫醚-β-合酶结构域;Cbcbs基因;衰老相关基因;叶片衰老
中图分类号　Q786
　　Leaf senescence , the final phase of the leaf
developmental process that ultimately leads to organ
death , is a genetically programmed process[ 1] , during
which a major transition is from autotrophic to
heterotrophic metabolism involved in chlorophyll (Chl)
degradation , chloroplast breakdown , proteolysis , nucleic
acid hydrolysis , and the redistribution of nitrogen and
phosphorus from the degraded products to developing
organs
[ 2 , 3] .The senescence process involves dramatic
differential gene expression and is believed to be regulated
by specific genes
[ 4 ～ 6] .During leaf senescence , expression
of many genes , involved in photosynthesis and other
anabolic processes , are downregulated , while others ,
designated as senescence-associated genes (SAGs), are
upregulated.SAG expression is required for senescence
because both transcription and translation inhibitors can
effectively delay the leaf senescence
[ 4 , 7] .In the past
decade , hundreds of SAGs have been cloned from
different plant species , including Arabidopsis , barley ,
Brassica , maize , cucumber , rice , tobacco , radish ,
asparagus and soybean
[ 4 , 8 , 9] .Especially , in recent years ,
thousands of potential SAGs have been identified by using
genomics approaches
[ 4 , 8 , 10 ～ 13] , which encode
transcription factors , kinases , proteinases , lipases ,
RNases and others proteins with unknown functions.In
these unknown-function SAGs , there is a cystathionine-β-
synthase (CBS) domain-containing unknown protein
which is found in senescing leaves of Arabidopsis using
SSH technology and encoded by AT5g10860
[ 8] .
The CBS domain , named after Cystathionine Beta
Synthase by Bateman in the study of the genome of the
archaebacterium Methanococcus jannaschii , is an
evolutionarily conserved protein domain that is widespread
and found in the proteome of archaebacteria , prokaryotes ,
and eukaryotes
[ 14] .Up to now , there are 124 proteins
with CBS domains in green plants including 51in
Arabidopsis and 43 in Rice
[ 15] .Crystal-lographic studies
show that this domain consists of a conserved(β1)-α1-β2-
β3-α2 pattern , in which the first β-strand (β1)does not
always show up as a clear β-sheet in some of the crystal
structures , and usually comes in tandem repeats that form
a CBS pair or so-called Bateman domain , a symmetrical
intramolecular dimeric structure that forms a cleft between
the two CBS domains which could form a ligand binding
site
[ 16] .The CBS pair occurs in several different unrelated
proteins (inosine 5′-monophosphate dehydrogenase(IMPDH), cystathionine-beta-synthase , AMP kinase ,
and chloride channels) performing different functions(metabolic enzymes , kinases , and channels), in all
kingdoms of life.Mutations in conserved residues within
CBS domains of , respectively , cystathionine-β-synthase ,
IMPDH , AMP kinase , and chloride channels can cause
several human hereditary diseases , including
homocystinuria , retinitis pigmentosa , hypertrophic
cardiomyopathy , myotonia congenital , etc[ 17 ～ 19] .
Depending on the CBS-domain-containing proteins , CBS
domains have been proposed to affect multimerization and
sorting of proteins , channel gating , and ligand
binding
[ 17] .In some cases , CBS domains may act as
sensors of intracellular metabolites by being activated by
AMP and inhibited by ATP
[ 20] .Although CBS-domain-
containing proteins are widely distributed and perform
multi-functions , the research of the domain mainly focus
on humans hereditary diseases and there is hardly a report
about the CBS proteins in leaf senescence.
Coleus blumei (also known as Solenostemon
scutellarioides.), one of perennial herbs of the genus
Coleus of the family Lamiaceae , is a famous foliage plant
with multicolor on leaves and a medical plant rich in
Rosmarinic Acid(RA)[ 21 , 22] .Up to now , Coleus blumei
has been studied on photosynthesis , hormone regulation ,
RA synthesis , and tissue culture , but absence of leaf
senescence research , especially in the field of molecular
biology.Furthermore , leaf senescence reduces the value
of foliage plants.Therefore , to more study and understand
the molecular mechanism of leaf senescence , a senescent
leaf cDNA library of Coleus blumei was constructed and a
small EST library was obtained.According to the
sequence of an EST fragment , a novel leaf SAG full-
length cDNA encoding a CBS-domain-containing protein ,
denoted Cbcbs , was rapidly cloned using a strategy of
RACE combined with cDNA library
[ 23 , 24] , and its
molecular characterization , protein structure , function ,
and expression were analyzed , which laid the groundwork
to further research the effects of a CBS-domain-containing
protein in leaf senescence.
1　Materials and Methods
1.1　Plant materials and growth conditions
A C.blumei red variety was obtained from College
of Horticulture and Landscape , Southwest University , and
was grown at 25 ℃ and 16 hours light 8 hours dark
photoperiod in an HP1000GS artificial weather box(Wuhan Ruihua Instrument & Equipment Co., Ltd ,
263No.4 ZHU Qin-Long et al:Molecular Characterization and Functional Prediction of a Novel Leaf SAG 　
China).
1.2 　Isolation of RNA , Construction of a cDNA
library and EST sequencing
Total RNA of C.blumei senescent leaves was
extracted using W6711 Total RNA Extractin Kit(Watson ,
China)according to the manufacturer s instructions.The
cDNA library was constructed using SMART
TM (Switch
mechanism of the 5′end of the RNA transcript)cDNA
Library Construction Kit according to the manufacturer s
instructions (Clotech Ltd., USA).The primary library
had a titer of 4.2×105 pfu ml in which 94% clones
were recombinants and the insert cDNAs were from 0.4 to
2 kb in length.A total of 25 well-isolated single plaques
were selected randomly and converted to pTriplEx2
according to the kit manufacture s instruction.Each
clone was sequenced from the 3′to 5′end with the primer
provided by the kit.
1.3　Cloning of a CBS protein gene using RACE
method combined with cDNA library
In SMART
TM
cDNA library , the cDNAs were
oriented cloning into the TriplEx2 vector sites specifically
at Sfi Ⅰ A and Sfi Ⅰ B.There are two specifical
primers , 5′P (5′TriplEx sequencing primer:5′-
TCCGAGATCTGGACGAGC-3′) and 5′NP (5′TriplEx
LD-Insert Screening primer: 5′-CTCGG
GAAGCGCGCCATTGTGTTGGT-3′) in the upstream of
inserted sites of the vector , and one specifical primer 3′P(3′ TriplEx LD-Insert Screening prime: 5′-
TAATCGACTCACTATAGGGC GAATTGG-3′) is in the
downstream of inserted sites.According to the sequence
of the EST for Cbcbs , two gene specific primers (GSP),
5′GSP1 (5′-TCCCACCTTCATTCACCACC-3′) and 5′
GSP2 (5′-CGGAATG TGCCTGATGCGGTTA-3′), were
designed to amplify the cDNA 5′end with 5′P and 5′
NP. The specific primer CbcbsF (5′-
GAGCCTTCACTTTACTCTTACTGGC-3′), according the
5′end sequence designed , and the 3′P were used to
amplify the full-lengh cDNA.
1.3.1　5′RACE of Cbcbs
In the 5′RACE amplification , 1 μl 100× diluted
primary cDNA library as template and 1.5 units of Taq
DNA Polymerase (TaKaRa , Dalian , China)were used in
a 50 μl PCR reaction for primary amplication with primers
5′GSP1 and 5′P.Then 0.5 μl PCR product was used
for the nested PCR amplification in a 50μl PCR reaction
with primers 5′GPS2 and 5′NP.The 2 reactions were
carried out on an Eppendorf 5331 Mastercycler
(Eppendorf , Germany)under the following conditions:
predenaturation at 94℃ for 5 min , 20 cycles at 94℃for
1 min , 65℃(each subsequent cycle was run at 0.5℃
less than the preceding cycle)for 1min and 72℃ for 1
min , and then 15 cycles at 94℃ for 1 min , 55℃ for 1
min and 72℃ for 1 min , followed by the final extension
at 72℃for 10min.The 5′PCR product was purified and
cloned into pMD18-T vector followed by sequencing.
1.3.2　Generation of the full-length cDNA of Cbcbs
In the full-length cDNA amplification , 1 μl 100×
diluted primary cDNA library as template and 1.5 units of
Pfu DNA polymerase (TaKaRa , Dalian , China)were
used in a 50 μl PCR reaction with primers CbcbsF and 3′
P , using the following conditions:predenaturation at
94℃ for 5 min , 35 cycles at 94℃ for 1 min , 62℃ for
1min and 72℃ for 1min , followed by the final extension
at 72℃ for 10 min.Immediately after the reaction , the
tube was added with 1.5 units of Taq DNA polymerase
and further incubated at 72 ℃ for 20min for dA-tailing of
the PCR product.The PCR product was purified and
cloned into pMD18-T vector followed by sequencing.
1.4　Sequence analysis , protein structure and function
prediction of Cbcbs
Sequence alignments and molecular mass calculation
of the predicted protein were carried out using programs
from Clustal W (http: www.ebi.ac.uk clustalw )and
DNAstar program package , respectively.ORF finding ,
domain and BLAST were done using NCBI (http: www.
ncbi.nlm.nih.gov).Protein structure predictions , the
subcellular localization and function prediction were
carried out on websites (http: www.expasy.org ,
http: www.softberry .com and http: www.cbs.dtu.dk
services )and Gene3D program packages (http: www.
biochem.ucl.ac.uk bsm cath Gene3D ).The tertiary
structure models were performed by SWISS-MODEL(http: swissmodel.expasy.org SWISS-MODEL.html)
using compatative modeling method and showed with
PyMol software(http: www.pymol.org).Protein signal
peptide and cleavage sites predictions , transit peptide
predictions , and phosphorylation site predictions were
carried out on SignalP(http: www.cbs.dtu.dk services
SignalP ), TargetP 1.1 (http: www.cbs.dtu.dk
services TargetP ), and Prosite (http: www.expasy..
ch tools scanprosite ), respectively.Gene functional
prediction was performed by GoFigure program (http:
udgenome.ags.udel.edu gofigure ).
1.5　RT-PCR expression analysis
Semi-quantitative reverse transcription-PCR (RT-
PCR)was performed to detect the expression of Cbcbs in
different developmental and senescent stages of leaves
which were classified into five , young (Y), mature(M),
early senescence (S1), middle senescence (S2)and
terminal senescence(S3), according to the chlorophyll
levels
[ 8] .2 μg of each total RNA sample was used for
Oligo(dT)18 directed reverse transcription (RevertAidTM
M-MuLV Reverse Transcriptase , MBI , Lithuania).1 μl
total first strand cDNA from each sample was used as
template in a 50 μl standard Taq PCR reaction using
primer pair CbcbsF and CbcbsR ( 5′-
GCTCCACAACAGCAAGTGAAATC-3′).Annealed at 56℃, amplifications were taken for 24 and 35 cycles.For
internal control , primers Actin1 (5′-CTCGGC
AGTTGTGGTAAACATG-3′) and Actin2 ( 5′-
264 Chinese Journal of Biochemistry and Molecular Biology Vol.23
CCGTGTCGCTCCTGAAGAGC-3′) were synthesized to
simultaneously amplify the actin gene fragment according
to DQ423374(a Coleus blumei actin fragment , cloned by
our lab.)in the 24-cycle amplification.
2　Results
2.1　Sequence analysis of the EST of Cbcbs
A small EST library of 25 clones from a cDNA
library derived from the red senescent leaves of C.coleus
were sequenced and analyzed for the purpose of providing
some information of the leaf senescence in this plant.
Among them , we identified a 459 bp EST (Fig.2)that
encoded an 93 amino acid fragment which has higher
identity (91%)to the unknown protein with CBS domains
of Arabidopsis , Atcbs(Accession No.At5g10860), 89%
identity to the CBS1 protein of Hyacinthus orientalis ,
Hocbs(Accession No.AAT08729), and 86% identity to
the CBS domain containing protein of Oryza sativa , Oscbs(Accession No.ABF98755), respectively.
2.2　Cloning of the full-length cDNA of Cbcbs
A total of 10 μl aliquots of the PCR products were
separated on 1.2% agarose gel after 5′RACE and full-
length amplification all using 1 μl 100×diluted primary
cDNA library as template , respectively.The 5′RACE
product was about 500 bp and the full-length cDNA was
about 950 bp obtained using the RACE combined with
cDNA library method(Fig.1).The precise length of the
Cbcbs gene was 859 bp long (accession no.EF076754)
by sequencing and contained a 609 bp open reading frame(ORF)encoding a 202 amino acid protein , similar to
other plant CBS genes.There was a 5′untranslated region(UTR)of 73 bp upstream from the start codon (ATG ,
74—76 bp)that conformed to the predicted translation
start site for eukaryotic genes (A GXXATGG)except the
last C
[ 25] , and one stop codon(62—64 bp)found in the
5′UTR confirmed that the cDNA contained the entire
ORF.The 3′UTR was 177 bp long downstream from the
stop codon including the poly (A)and rich in A T(about
67% A +T).One possible polyadenylation signal
AATAAA was found at 127 bp position downstream from
the stop codon.A pentanucleotide motif , ATTTA , which
was considered to confer instability to the mRNA
[ 26] , was
found in the 3′UTR(797—801 bp)(Fig.2), and did
not always function
[ 27] .The analysis on Cbcbs the G C
content of the revealed that the G C content in the coding
region of the gene was 49%, similar to other plant CBS
genes , such as Atcbs(45%)and Oscbs(49%).
2.3　Conservation , structural features and functional
analysis of the deduced CbCBS Protein
By using the software of EditSeq of DNAstar program
package , the calculated isoelectric point (pI) and
molecular weight of the deduced CbCBS protein were
predicted to be about 9.23 and 22.25 kD respectively.
NCBI blastp indicated that CbCBS showed wide
similarities to CBSs from other plants and low homologies
Fig.1 　The amplication of 5′RACE(A) and the full-
length(B)of Cbcbs by a RACE combined with cDNA
library strategy
M:DL2000 DNA marker;1:5′RACE product;
2:full-length cDNA PCR product
to other CBSs from Bacteria.When pairwise aligned on
the whole molecule scale , it showed 84% identities and
92% positives to HoCBS , 80% identities and 88%
positives to AtCBS and 43% identities and 66%positives
to CBS from Burkholderia cepacia AMMD(ABI89676).
NCBI Conserved Domain search detected 2 CBS domains(Fig.3), S67-I117 and K137-V184 , in tandem
arrangements , which was similar to other CBS
proteins
[ 17] .
SignalP 3.0 prediction did not find any secretory
pathway signal peptide , but TargetP 1.1 predicted that
CbCBS had a probability of 0.667 to have a mitochondrial
targeting peptide (mTP)with a most likely cleavage site
between H35 and E36 (Fig.2), furthermore , the same
predicted result was obtained by using MITOPROT(Prediction of mitochondrial targeting sequences , http:
ihg.gsf.de ihg mitoprot.html).The predicted mTP
character of CbCBS was identical with the mTP of
AtCBS
[ 28] .The analysis above suggested that CbCBS was
a mitochondrial precursor protein encoded by nuclear
gene.NetNGlyc 1.0 prediction did not find any repeat
structure or N-glycosylation site , but Prosite predicted 3
significant protein kinase c phosphorylation sites(PKCps), 4 significant casein kinase II phosphorylation
sites(CKIIps)and 1 tyrosine sulfation site (TSs)(Fig.
4), which suggested that phosphorylation and sulfation
may be a posttranslational modification of CbCBS to
activate functions.
The multi-alignment by Clustal W program showed that
the deduced CbCBS had high similarity with other CBS
sequences from plants (Fig.4).In these plant CBS
sequences , AAL67493 was a senescence-associated putative
protein from Narcissus pseudonarcissus
[ 29] , AAU04402 was
encoded by a stress resistance gene by PCR-Select cDNA
Subtraction in Citrus
[ 30]
and At5g10860 was isolated from a
SSH library of senescing leaves of Arabidopsis
[ 8] .
265No.4 ZHU Qin-Long et al:Molecular Characterization and Functional Prediction of a Novel Leaf SAG 　
Fig.2　cDNA sequence and the deduced amino acid sequence of CbCBS
The ATG initiation codon and TAG termination codon are in bold and italics.A termination codon TAA before the initiation codon is bold-underlined.
The putative polyadenylation signals are boxed.The “ATTTA” element is underlined twice.5′GSP1 and5′GSP2 indicate gene specific primers for 5′
RACE.CbcbsF is a forward primer for ful l-length PCR and paired with CbcbsR(reverse primer)for RT-PCR.
indicates a cleavage site of deduced mitochondrial targeting peptide.The two CBS domains are marked with gray background.The EST sequence
is underlined.The numbers for the nucleotides on the right are based on the cDNA(accession no.EF076754)and the numbers on the left are based
on the deduced amino acide sequence
Fig.3　The conversed cystathionine-β-synthase(CBS)domains predicted by NCBI conserved domain search
　　Phylogenetic analysis showed that CbCBS , AtCBS ,
AAU04402 , AAL67493 and HoCBS , formed a closely
related subgroup , which further grouped with OsCBS and
ZmCBS (AAU93534) and belonged in one plant CBS
group(Fig.5).The other CBS group consisted of bacteria
CBS proteins(Fig.5).The results of multi-alignment and
phylogenetic analysis suggested that the CbCBS may be
related to senescence or stress resistance in plants.
SOPMA predicted that the secondary structure of
CbCBS was mainly composed ofα-helices(42.08%)and
random coils (26.73%), while extended strands(20.30%)and β-strands(10.89%)contributed a little.
On the level of secondary structure , each CBS domain was
composed of a standard β1-α1-β2-β3-α2 unit , and the β1-
strand in the two domains was clear.Oneα-helix , K127-
E134 , was between two CBS domains(Fig.4).
In order to better characterize the CbCBS protein , a
comparative modeling of three dimensional structure of
CbCBS was performed using SWISSMODEL. The
theoretical CBS pair structure for CbCBS was portrayed
against the template of the CBS1 and CBS2 domains of
Streptococcus pyogenes inosine-5′-monophosphate
dehydrogenase (SpIMPDH)(Protein Data Bank code
1ZFJ)by Zhang et al.[ 16] using crystallography (Fig.6).
In predicted tertiary structure of CbCBS (Fig.6A), the
CBS pair was a symmetrical structure that was composed
of two(β1)-α1-β2-β3-α2 structures(in both domains , β1 is
not clearly resolved as a β-sheet) in an antiparallel
arrangement.The interface of the 2 CBS domains was
formed by a β-plate that consisted of the antiparallel β2-β3
sheets of each CBS domain , and a cleft , which could
form a ligand binding site , was formed by the interface
266 Chinese Journal of Biochemistry and Molecular Biology Vol.23
　　　　　　　　
Fig.4　The multiple sequence alignment of CbCBS with homologous proteins
CKIIps in box indicates the casein kinase Ⅱ phosphorylation sites;PKCps in box indicates the protein kinase c phosphorylation sites;TSs , tyrosine
sulfation si te , is in bold-underlined.Shades in black represent identical amino acids among the homologous proteins.The secondary structure of
cystathionine-β-synthase(CBS)domain predicated by SOPMA.The β1-α1-β2-β 3-α2 unit i s conserved among different CBS domainswith the exception
of the first β-st rand(β1)that does not always show up as a clear β-sheet in some of the crystal st ructures.The α-helices are depicted as dark gray
rectangles , whereas the light gray arrows represent the β-strands.AAU04402(Citrus limon);AAL67493(Narcissus pseudonarcissus);
At:Arabidopsis thaliana(accession no.At5g10860);Cb:Coleus blumei(accession no.EF076754);Ho:Hyacinthus orientalis (accession no.
AAT08729);Os:Oryza sativa (japonica cultivar-group)(accession no.ABF98755)
Fig.5　Phylogenetic tree analysis of Coleus blumei CBS protein by the neighbor joining method in Clustal W
The arrow indicates CbCBS position in the phylogenetic tree.As:Azoarcus sp.EbN1(accession no.YP -161064);Bc:Burkholderia cepacia
AMMD(accession no.YP-776010);Bd:Burkholderia dolosa AUO158 (accession no.ZP -00984293);Bv:Burkholderia vietnamiensis G4
(accession no.ZP-00426232);Bs:Burkholderia sp.383(accession no.YP -371784);Bx:Burkholderia xenovorans LB400(accession no.YP -
553640);Bt:Burkholderia thailandensis E264(accession no.YP -439711);Mf:Methylobaci llus flagellatus KT(accession no.YP-546468);Pn:
Polaromonas naphthalenivorans CJ2(accession no.ZP-01020351);Pp:Pseudomonas putida KT2440(accession no.NP -742371)
between the two CBS domains.In the CBS pair , the four
α-helices were positioned at one end(top), whereas the
loops connecting the β2-β3 sheets were at the other end of
the structure(bottom).As could be seen from Fig.6 , the
267No.4 ZHU Qin-Long et al:Molecular Characterization and Functional Prediction of a Novel Leaf SAG 　
predicted structure of CBS pair of CbCBS was most similar
to the CBS domains structures of SpIMPDH.This result
indicated that the CbCBS shared the same conserved CBS
domains with SpIMPDH , implying that CbCBS , based on
the CBS domains , might have partial similar functions
with IMPDH , a bacterial protein whose function and
crystal structure had been verified previously.
Because of lacking detailed function information
about known CBS-domain-containing protein genes from
plant in the Genbank , the GoFigure Annotator , a program
of gene function prediction , was used to analyse Cbcbs.
The result displayed that CbCBS was localized into
mitochondrion (score:5739) having partial IMPDH
activity (low score:3938), and was concerned with
carbohydrate metabolism (score:5975) and GMP
biosynthesis(score:6177).CbCBS was closely related to
cellular energy metabolism and participated in regulation
of cellular energy levels.
Fig.6　The three-dimensional models of the deduced CBS domains of CbCBS(A)and a CBS pair of SpIMPDH from
Protein Data Bank code 1ZFJ(B)
The models were predictedwith SWISS-MODEL and showedwith PyMol.α-helices are shown in red , β-strands in yellow and loops in green lines.The
two CBS domains , CBS1(left)and CBS2(right), have a(β1)-α1-β 2-β3-α2 structure (in both domains , β 1 is not clearly resolved as a β-sheet).
The interface between the two CBS domains forms a cleft , which could form a ligand binding site
2.4　Expression analysis of Cbcbs in leaves
Based on RT-PCR results (Fig.7), Cbcbs was
expressed in all tested different stages of leaves with
different levels , the terminal senescence stage (S3)being
the highest , followed by middle senescence stage (S2),
early senescence stage (S1)and mature stage (M), the
young stage (Y)being the lowest.In the 24-cycle
amplification of RT-PCR , Cbcbs expression in young leaf
was not observed until 35-cycle amplification.During leaf
senescence , the Cbcbs expression was enhanced and
senescence upregulated , therefore , the gene was a
senescence-associated gene.
3　Discussion
For the purpose of studying on foliage plants
senescence-associated genes , a senescent leaves cDNA
library of Coleus blumei has been constructed using
SMART techniques.Based on a small EST library , a
novel EST fragment encoding a CBS domain was
obtained.For bypassing the time-consuming and tedious
cDNA screening method , a RACE combined with cDNA
library strategy
[ 23 , 24]
was employed to clone the full-length
of Cbcbs.The result showed that the way was a rapid and
effective method for cloning full-length gene from a
library.
The CbCBS had 203 amino acids with two conserved
CBS domains and multi-phosphorylation sites.NCBI
Blastp showed that CbCBS had high homology to plant
Fig.7　The RT-PCR expression analysis of Cbcbs in leaves
of different stages
Y:young stage;M:mature stage;S1:early senescence stage;
S2:middle senescence stage;S3:terminal senescence stage
CBSs with the same structure and following by other CBSs
from bacteria.Five sequences with high scores of identity
in Blastp were selected to align with CbCBS , and the
result(Fig.4)indicated that their secondary structures of
the CBS pair were similar besides sequence homolog ,
implying their functional similarity. Moreover ,
phylogenetic analysis showed that CbCBS belonged to a
closely related subgroup of senescence or stress consisting
of AtCBS , AAU04402 , AAL67493(Fig.5).The results
of multi-alignment and phylogenetic analysis were in
268 Chinese Journal of Biochemistry and Molecular Biology Vol.23
conformity with the RT-PCR that Cbcbs expression was
senescence upregulated , which suggested that CbCBS was
encoded by a senescence-associated gene in senescent
leaves.
Because protein domains are evolutionarily conserved
units with shared structural (sequence and folding
pattern)and functional properties , usually , each protein
domain performs a specific function.However , the CBS
domain is widespread in all species and occurs in proteins
with completely different functions (e.g., channels ,
kinases , metabolic enzyme)[ 17] .The fact indicates that
the CBS domain may be a basic functional structural unit
and play an important role in different functional proteins.
In known CBS proteins , there are no functional
information on plant CBS proteins , therefore ,
bioinformatics methods were used to analyze CbCBS.
TargetP 1.1 andMITOPROT are special programs to
predicate mitochondrial targeting peptide and GoFigure
was an integrative gene function annotator.By using the
three programs respectively , the same result was obtained
that the CbCBS was a mitochondrial precursor protein with
a 35 amino acids mTP and encoded by nuclear gene.To
test the reliability of these programs , the AtCBS , a
mitochondrial precursor protein with a 39 amino acids
mTP and cleavage site between S39 and E40
[ 28] , was
analyzed and the predicated result accorded with the
experimental result , which indicated that the analysis of
CbCBS using these programs was credible.
The CbCBS had two CBS domains only;hence the
function of the protein was closely related to the function
of the CBS pair.As small intracellular modules , CBS
domains tandem pair together to form a stable globular
domain
[ 16] , which has been shown to bind ligands with an
adenosyl group such as AMP , ATP and S-AdoMet[ 20] .In
cystathionine-beta synthase , the CBS domains are
involved in regulation by S-AdoMet[ 32] , and in AMPK ,
CBS domain pairs bind AMP or ATP
[ 20] .The CBS
domains from IMPDH and the chloride channel CLC2 bind
ATP
[ 20] .The phenomenon that CBS domains occur in
several different proteins indicates that the pair of CBS
may regulate the activity of attached enzymatic or other
domains , besides acting as binding domains for adenosine
derivatives.In some cases , CBS domains may act as
sensors of cellular energy status by being activated by
AMP and inhibited by ATP
[ 20] .
IMPDH is the key enzyme in the de novo guanosine
nucleotide biosynthesis , catalysing the first step unique to
GMP synthesis.The enzyme is composed of two parts , a
CBS pair and a GMP reductase active region.Though the
CBS pair is inserted between alpha helix two and strand
three of IMPDH , without the CBS domains the catalytic
activity of the enzyme is found to be normal
[ 33] .
Therefore , the chief function of CbCBS may be not related
to GMP synthesis but associated with CBS domains ,
which is coincident with Go Figure prediction to the low
score of IMPDH activity and contrary to GMP synthesis.
The prediction of GMP synthesis is based upon IMPDH
activity.
AMPK (AMP-activated protein kinase) is a
heterotrimer composed of an alpha , beta and gamma
subunit
[ 34] .The gamma subunit is composed of two CBS
pairs.Because the quantitative difference of CBS pairs
between AMPK and CbCBS , the result of the CBS
domains comparative modeling shows similarity between
CbCBS and IMPDH.One of two CBS pairs in AMPK has
similar structure with the CBS pair in CbCBS predicated
by SWISSMODEL (data not shown).Mutations in
conserved residues within CBS domains of AMPK cause a
human hereditary disease , familial hypertrophic
cardiomyopathy
[ 35 , 36]
which indicates that the CBS pairs
belong to the constituent parts of function.As a sensor of
cellular energy content , AMPK plays an important role in
restoring the cellular ATP balance and regulating the
whole body energy storage and expenditure during periods
of metabolic stress
[ 37] .When the cellular energy content
drops (low ATP , high AMP), through a CBS pair
binding AMP and phosphorylation by an AMPK kinase ,
AMPK is activated
[ 38] .Once activated , AMPK switches
on catabolic pathways(ATP production)and switches off
anabolic pathways(ATP consumption)raising the cellular
ATP level
[ 39] .CbCBS has not only a CBS pair but also
multi-phosphorylation sites , which suggests that CbCBS
has similar function with CBS domains of AMPK.
The chief function of the mitochondria is to create
energy (ATP) for cellular activity by the process of
aerobic respiration.As a source of reactive oxygen
species , mitochondria participate in senescence and
programmed cell death
[ 40] .In senescent leaves , when
chlorophyll levels have dropped by 50%, mitochondrial
respiration provide the energy for nutrient remobilization ,
and the population of mitochondria increase
significantly
[ 41] .In mitochondria , the mature CbCBS
maybe acts as a sensor of cellular energy status and
directly or indirectly regulates cellular energy levels to
increase ATP content by the CBS pair combined with
AMP and CBS domains phosphorylation , or as a subunit
of a kinase , respectively , during periods of metabolic
stress of senescent leaves.
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