全 文 ：水稻中一个谷胱甘肽转移酶基因的克隆、
表达和酶活性分析 ?
胡廷章1 ,2 , 黄小云2 , 肖国生2 , 屈霄霄1
(1 重庆大学生物工程学院 , 重庆 400044 ; 2 重庆三峡学院生物系 , 重庆 404000)
摘要 : OsGSTL1 是位于水稻 3 号染色体上的一个λ类谷胱甘肽转移酶基因 , 由 8 个内含子和 9 个外显子组
成 , 编码一个由 243 个氨基酸组成的多肽链。将 OsGSTL1 克隆到酵母表达载体 pYTV 上 , 转化大肠杆菌 ,
然后再转化酵母菌 PEP4。Western印迹分析表明外源 OsGSTL1 基因在转基因酵母中表达 , 分析半乳糖诱导
表达的酵母粗提液的谷胱甘肽转移酶活性表明 : 转基因酵母的谷胱甘肽转移酶较非转基因酵母和未诱导的
酵母高 , 说明 OsGSTL1 在转基因酵母中受半乳糖的诱导表达 , 具有谷胱甘肽转移酶活性。
关键词 : 谷胱甘肽转移酶 ; 水稻 ; Western印迹分析 ; GST 活性
中图分类号 : Q 78 文献标识码 : A 文章编号 : 0253 - 2700 (2008) 06 - 688 - 05
Molecular Cloning , Expression and Activity Analysis
of a Glutathione S-transferase in Rice *
HU Ting-Zhang1 ,2 , HUANG Xiao-Yun2 , XIAO Guo-Sheng2 , QV Xiao-Xiao1
(1 Bioengineering Collegeof Chongqing University, Chongqing 400044 , China; 2 Department of Biology,
Chongqing ThreeGorges University, Chongqing 404000 , China)
Abstract: OsGSTL1 is a glutathione S-transferase (GSTs, EC 2.5 .1 .18 ) , lambda class gene in chromosome 3 of rice
( Oryza sativa L .) . It includes 8 introns and 9 exons, and encodes a protein of 243 amino acid residues . The OsGSTL1
gene was cloned into pYTV vector and was transformed intoyeast strain PEP4 . Western blot analysis showedthe exogenous
OsGSTL1 was expressed in transformed yeast . GST activity of crude extracts of yeast showed the OsGSTL1 transgenic yeast
had higher levels of GST activitiesthancontrol yeasts, whichdemonstratethe OsGSTL1 has glutathione S-transferaseactivity .
Key words: Glutathione S-transferase; Oryza sativa L .; Western blot analysis; GST activity
GlutathioneS-transferases (GSTs, EC 2 .5.1 .18)
are scavenging enzymes that detoxify cellular xenobio-
tics and toxins by catalyzing the conjugation of these
substrates with a tripeptide glutathione . In animals,
the first discovered GST was a result of its importance
in the metabolism and detoxification of drugs in the
1960s . In plant, the first recognized GST was from
maize as its GST activity to be responsible for conjuga-
ting the chloro-S-triazine atrazine with GSH in 1970 .
GST activities, thecorresponding enzymes andgenese-
quences have been identified in animals, plants and
fungi ( Wilce and Parker, 1994; Sheehan et al. ,
2001) .
The plant GST gene families are large and highly
diverse . Thereare 48 members in Arabidopsis, over 25
in soybean ( Glysinemax L .) , 42 inmaize ( Zea mays
L .) , and 59 in rice ( Oryza sativa L .) (McGonigle et
al. , 2000; Dixon et al. , 2002b; Wagner et al. ,
云 南 植 物 研 究 2008 , 30 (6) : 688～692
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2008.08038
? ?Foundation item: 国家自然科学基金 ( 30771464 ) , 重庆市教育委员会科学技术研究项目 ( KJ071112 ) , 重 庆三峡学院资助项目
(2007-Sxxyyb-04 )
Received date: 2008 - 03 - 15 , Accepted date: 2008 - 05 - 20
作者简介 : 胡廷章 (1965 - ) 男 , 教授 , 博士 , 主要从事植物分子生物学研究。E-email : tzhu2002@ yahoo. com. cn
2002; Soranzo et al. , 2004) . Basedon proteinhomol-
ogyandgeneorganization, plants GSTshavebeen cate-
gorized into fiveclasses: phi (F ) , zeta (Z) , tau (U) ,
theta ( T) and lambda (L ) (Dixon et al. , 2002b) .
The GST proteins have evolved by gene duplica-
tion to perform a range of functional roles using the
tripeptide glutathione ( GSH) as a cosubstrate or co-
enzyme . Classically, GSTs catalyze the transfer of the
tripeptide glutathione ( γ-glutamyl-cysteinyl-glycine;
GSH) to a cosubstrate ( R-X ) containing a reactive
electrophilic center to form a polar S-glutathionylated
reaction product ( R-SG ) ( Dixon et al. , 2002b) .
Some GSTs have been shown to have glutathione pero-
xidase activity, with the GSTs using glutathione to re-
duce organic hydroperoxides of fatty acids and nucleic
acids to the corresponding monohydroxyalcohols ( Cum-
mins et al. , 1999; Dixon et al. , 2002a) . And GSTs
catalyze the isomerization of maleylacetoacetate to fu-
marylacetoacetate (Dixon et al. , 2000 ) . Some GSTs
also have ligand functions (Edwards et al. , 2000; Le-
derer and Boger, 2005) . Somestudies havealso impli-
cated GSTs as stress signalingproteins of some cell sig-
naling pathways (Loyall et al. , 2000) . GSTs also have
function in cellular redox homeostasis or regulate apop-
tosis ( Loyall et al. , 2000; Kampranis et al. , 2000;
Dixon et al. , 2002a; Kunieda et al. , 2005 ) .
In this report, OsGSTL1 cDNA (AF 237487) was
isolated from Oryza sativa cv . Zhonghua 11 . Expres-
sion and activity of OsGSTL in yeast strain PEP4 was
studied . The results demonstrate OsGSTL1 is a gluta-
thione S-transferase (GSTs, EC 2 .5 .1 .18) .
Materials and methods
Culture of Oryza sativa cv . Zhonghua 11
The seeds of Oryza sativa cv . Zhonghua11 were sterilized,
soaked in water at 28℃ for 2 d, and then grownat 28℃ under a
16?8 h light?dark photoperiodat an intensityof approximately 250
μE m- 2 s - 1 . Seven-day-old seedlingswere collected and frozen
in liquid nitrogen and stored at - 80℃ .
Cloning and analysis of OsGSTL1 gene
Total RNA from the leaves and roots of the seedlings were
isolated with Trizol reagent ( Gibco-BRL ) ( Vidmar et al. ,
2000) . Reverse transcriptionswere performed with Olig (dT) 18
as 3′primer and1μg RNA as templates .
The OsGSTL1 sequence was amplified from cDNA by re-
versetranscriptase-PCR usingTaqDNA polymeraseand30 cycles
of 94℃ for 30 s, 58℃ for 45 s, and 72℃ for 90 s, with the
combinations of specific primers OsGSTL1 f ( 5′-CACCACAAA
ATGGCCGCAGCTGCAGCA-3′) and OsGSTL1 r ( 5′-GGCAAC-
CTTAAGATGCGT-3′) . A CACCACAAA sequence was intro-
duced to the 5′-end of forward primer OsGSTL1 f . This PCR
product was cloned into pENTR?D-TOPO vector ( Invitrogen,
USA) and sequenced . The nucleotide sequence and the putative
amino acid sequence were analyzed with the DNAMAN and
blastP ( http:?www. ncbi. nlm.nih.gov?) , respectively .
Expression of OsGSTL1 gene in yeast strain PEP4
The OsGSTL1 gene that had been cloned into pENTR?D-
TOPO vector was cloned intopYTV vector ( Invitrogen, USA) by
LR reaction, transformed into E. coli . DH5α, and then the cor-
rect expression vector was transformed into yeast strain PEP4 .
The OsGSTL1 protein was expressed in PEP4 cell after 19 h incu-
bation with SC-Uramediumincluding2% glucoseand 4 h induc-
tion with 2% galactose .
Western blot analysis
The crude protein extracts were prepared from the yeast .
Theyeast was homogenized in the Lysis buffer ( 50 mmol?L Tris
pH 7 .5 , 1 mmol?L EDTA , 100 mmol?L NaCl , 0 . 1% TritonX-
100 , 1 mmol?L DTT , 1 mmol?L PMSF) and centrifuged at 4℃ .
The supernatant was extracted with trichloroacetic acid and cen-
trifuged at 4℃ , andthen precipitatewaswashed twicewith etha-
nol . The crudeprotein extractswas used to Western blot analysis
and enzyme assay . Protein concentration in crude extracts was
determined with the BioRad Protein Assay Kit ( Bradford,
1976) .
Proteins (15μg) were separated in SDS-PAGE and trans-
ferred onto Immobilon membrane (Millipore, USA) with a semi-
dry transfer cell (Millipore, USA) . The recombinant OsGSTL1
polypeptides expressed in yeast strain PEP4 was detectedwith an
antibody raised against His-tag (Novagen, USA) .
Activity assay of OsGSTL1
Protein concentration in crude extracts was adjusted to 0 .5
mg?ml . GST activitywasmeasured spectrophotometrically (Habig
et al. , 1974; Takesawa et al. , 2002) . One unit of activitywas
defined as the amount of enzyme required forming 1μM product
per minute at 30℃ .
Results
Isolation and bioinformative analysis of OsGSTL1
OsGSTL1 gene was isolated from Oryza sativa cv .
Zhonghua 11 by RT-PCR . It includes8 introns and 9 ex-
ons, and encodes aprotein of 243 amino acid residues .
9866 期 HU Ting-Zhang et al . : Molecular Cloning, Expression and Activity Analysis of a Glutathione . . .
Fig . 1 Alignment of the peptide sequences of the OsGSTL1 with Lambda GSTs from Arabidopsis
( AtGSTL1 , AtGSTL2 ) , maize ( In2-1 ) and wheat (Cla30) .
The position of theputative active site cysteinyl residues are shaded gray . Residues that are identical
and conserved between sequences are marked with asterisk and colon, respectively .
The analysisof protein function domain suggested OsG-
STL1 has certain characteristics of glutathione-S-trans-
ferases . Alignment of the peptide sequences showed
OsGSTL1 are a putative Oryza Sativa glutathione S-
transferase, lambda class (OsGSTL) accordingly Dixon
et al. (2002a) . Lambda class members have a cysteine
redidue in active center which distinguishes them from
other class members which have a serine residue in
same site ( Fig. 1) .
Expression of OsGSTL1 transgene in yeast
To investigatethe functionof OsGSTL1 , the OsG-
STL1 gene was cloned into pYTV vector ( Invitrogen,
USA ) andwas transformed into yeast strain PEP4 . The
results of Western blot analysis showed the exogenous
OsGSTL1 was expressed in transformed yeast, whereas
no exogenous OsGSTL1 was detected in nontransformed
yeast (Fig . 2) .
Fig . 2 Western-blot analysis of crude protein
extract fromOsGSTL1 transgenic yeast
M, protein marker; 1 , OsGSTL1 transgenic yeast induced by galatose;
2 , untreated OsGSTL1 transgenic yeast; 3 , non-transgenic yeast
induced by galatose; 4 , untreated non-transgenic yeast
096 云 南 植 物 研 究 30 卷
GST activity analysis of OsGSTL1
GST activity of crude extracts of yeast was mea-
sured with CDNB as thesubstrate . The findings showed
the OsGSTL1 transgenic yeast that induced with 2%
galactose contains higher levels of GST activities than
control yeasts . In yeast, transgenic strains with OsG-
STL1 have seven more times higher GSH conjugating
activity than non-transgenic strain (Table 1) .
Table 1 Specific activities of GST in protein
extracts from the transgenic yeast
Yeast strain
GST activity in CDNB
(μmol?min per mg protein)
Non-treatment Galactose treatment
Non-transformant 72 .. 2±9 ?. 6 63 .4±13 }. 5
OsGSTL1 ?transgenic yeast 67 ?. 6±11 .3 531 .4±64 . 4
* The data are means of three independent experiments±SD
Discussion
GSTL1 proteins which have a cysteine redidue in
active center are classified as lambda class GSTs, are
presented in all eukaryotes from fungi to mammals
(Dixon et al. , 2002a) . From an evolutional perspec-
tive, it has been postulated that GSTL1 proteins were
one of the most ancient genes . The structures of the
OsGSTL1 gene is very complex compared to those of
other genes because their coding sequences include
nine exons .
Some reports have shown lambda GSTs have no
GSH conjugating . The maize In2-1 and soybean
Gmin2-1 previously considered being not GSTs because
of lack GSH conjugating activity towards CDNB
(McGonigle et al. , 2000) . Arabidopsis lambda GSTs
(AtGSTL1 and 2 ) have no GSH conjugatingwith stan-
dard substrates, but have activity of thiol transferases
instead (Dixon et al. , 2002a) . Fromstructure analys-
is, the serine residue in active center of other four
classes ( phi , zeta, tau, and theta) in plants which all
have GSH conjugating activities is replaced by cysteine
residue in lamda class and DHAR in plants . This sub-
stitution was considered to change the catalyzed proper-
ty of lamda class and DHAR GSTs . However, yeast
strains which transformed with OsGSTL1 have elevated
GSH conjugating activity, transgenic strains with OsG-
STL1 have seven more times higher GSH conjugating
activity than non-transgenic strain; it is difficult to be-
lieve that OsGSTL1 does not have GSH conjugating ac-
tivity . We also noticed our measured values of GSH
conjugating activity towards CDNB for OsGSTL1 were
evidently higher than that of OsGSTF5 ( Cho et al. ,
2007) , AtGSTU26 and AtGSTF9 ( Nutricati et al. ,
2006) . But the values of GSH conjugating activity for
these GSTsweregot by prokaryotic expression proteins,
which were different from our results which were from
eukaryotic system . The result of Arabidopsis lambda
GSTs and maize in 2-1 which have been reported to
have no GSH conjugating activity also was from that of
prokaryotic expressionproteins . TheOsGSTL1 was also
expressed in prokaryotic expression system, but the
GSH conjugating activity was not fond ( data not
shown) . So, there are some differentials between
lambda class GSTs which got from prokaryotic expres-
sion systemand that fromeukaryotic system . We spec-
ulate that the activities of GSTs may be affected by
posttranslational modification such as phosphorylation
which is lacked in prokaryotic system . Certainly, more
evidence needs to be found to prove whether lambda
class GSTs has GSH conjugating . However, our results
providevaluable information to the understandingof the
plant GST family especially lambda class GSTs .
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