全 文 ：稻属中一个高度保守和组成型表达酪氨酸?丝氨酸-酸酸
双特性蛋白激酶基因的克隆与分析?
杨明挚1 , 黄兴奇2 , 张汉波1 , 陈善娜1 , 杨红玉3 , 程在全2
??
( 1 云南大学生命科学学院 , 云南 昆明 650091; 2 云南省农业科学院种质资源与生物技术研究所 ,
云南 昆明 650223; 3 昆明学院生命科学与生物技术系 , 云南 昆明 650118)
摘要 : 从水稻中克隆了一个在稻属植物中高度保守和组成型表达的丝氨酸?苏氨酸蛋白激酶基因 (OsSTK )。
该基因包含两个外显子和一个 114 bp的小内含子序列 , 预测编码一个 419 个氨基酸的蛋白质。该基因推导
的氨基酸序列与其它已知序列的一致性均低于 52 %。利用从不同种和类型的野生稻克隆的部分该基因序
列构建的系统树与野生稻的分类和进化关系相一致。OSPK N-端拥有一段富含丝氨酸、碱性氨基酸和带电
荷氨基酸的特异性导肽序列 , 其中包含“GDGDGDGDG”短重复序列。由于该基因蛋白激酶结构域中的
VIb, VIII 和 XI 亚结构域中同时具有酪氨酸蛋白激酶和丝氨酸?苏氨酸蛋白激酶的特性 , 推测该基因可能同
时具有催化酪氨酸和丝氨酸、苏氨酸磷酸化的双重功能。
关键词 : 稻属 ; 丝氨酸?苏氨酸 蛋白激酶基因 ; 保守性分析 ; 转录分析
中图分类号 : Q 943.2 文献标识码 : A 文章编号 : 0253 - 2700 (2009) 05 - 433 - 06
Cloning and Analyses of a Dual Specific Serine?Thronine Protein
Kinase Gene with High Conservative and Constitutive
Expression in Oryza (OsSTK )
YANG Ming-Zhi
1
, HUANG Xing-Qi
2
, ZHANG Han-Bo
1
, CHEN Shan-Na
1
,
YANG Hong-Yu3 , CHENG Zai-Quan2 * *
( 1 Life Science School , Yunnan University, Kunming 650091 , China; 2 Biotechnology— Genetic
Germplasm Institute, Yunnan Academy of Agricultural Sciences, Kunming 650223 , China;
3 Biologydepartment, Kunming Institutey, Kunming 650118 , China)
Abstract: A cytoplasmic serine?thronine protein kinase gene (OsSTK ) , had been cloned from Oryza genus . It was found
high conservative and constitutive expression in Oryza . OsSTK gene had two exons, separated by 114 bp short intron . The
open readingframe of OsSTK gene that predicted encoded a419 amino acids protein . The amino acid sequence of OsSTK
had low identities ( less than 53% ) with any other known protein kinase . The phylogenetic tree based on the partial DNA
sequences of OsSTK from different species and types of wild rice and cultivated rice, was close to the taxation system of
rice . Interestingly, OsSTK hada serine, including basic amino acids and chargedamino acids abundant polypeptide with a
“GDGDGDGDG”sequenceat N-terminal that had not been found in anyother genes . OsSTK may play dual specificity that
phosphorylates both serine?thronine and tyrosone, because the amino acids module of VIb, VIII and XI catalytic domain
have both the serine?thronine and tyrosine kinase characters .
云 南 植 物 研 究 2009 , 31 (5) : 433～438
Acta Botanica Yunnanica DOI : 10 .3724?SP. J . 1143 .2009.09096
?
?? ?Author for correspondence; E-mail : czquan-99@163 . com
Received date: 2009 - 05 - 15 , Accepted date: 2009 - 06 - 19
作者简介 : 杨明挚 (1970 - ) 男 , 博士 , 副教授 , 从事植物生理与分子生物学研究。 ?
Foun ?dation items: National Natural ScienceFoundation of China ( 300660090) , theYunnanNatural ScienceFoundation of China ( 200600007Q and
2008C004) and the department of science and technology of Yunnan province, China ( 2007PY01-24 )
Key words: Oryza; Serine?thronine protein kinase gene (OsSTK ) ; Conservation analysis; Transcription analysis
Protein kinaseplays a central roleinmultiplebio-
logical processes, includinggrowth, development, me-
tabolismof cell , stress response and signal transduction
(Natalia et al. , 1998 ) . Protein kinase catalyze the re-
versible transfer of theγ-phosphate fromATP to amino
acid side chains of proteins . Phosphorylation status of a
protein has profound effects on its activity and interac-
tionwithother proteins . It is estimated that 1% to 3%
of functional eukaryotic genes encode protein kinases .
Enzymes belongingto theprotein kinasesuperfamily are
related by homologous protein kinase catalytic domains .
Typically, eukaryotic protein kinases have been subdi-
vided into those that phosphorylate Serine and?or Thro-
nine, those that phosphorylate Tyrosine and those that
phosphorylate Histidine ( Julie et al. , 1995 ) . There
are 12 conserved regions referred to as subdomains,
someof which contain invariant or nearly invariant resi-
dues (Hanks et al. , 1991 ) . Crystal structure determi-
nations of protein kinasesdemonstratethe importanceof
these conserved residues in catalysis and conservation
of overall three-dimensional structure ( Julie et al. ,
1995) . Since the importanceof protein kinase in plant
biological process, the morewe know about protein ki-
nase, the more we will understand about the net work
of cell metabolism and signal transduction . During the
cloning of STK ( serine?thronine protein kinase) type
resistance gene analogs from different species and types
of wild rice, a highly conserved serine?thronineprotein
kinase gene in Oryza has been identified . Here, we
describe the cloningand characters of thisnovel protein
kinase gene .
1 Materials and methods
1 .1 Rice materials
Three wild rice species were collected from wild fields in
Yunnan province, including Oryza rufipogon Griff, O. officinalis
Wall ex watt and O. meyerana Baill . O. longistaminata was col-
lected from Africa . O. minuta was from south Asia . Another
type of O. rufipogonwas fromJiangxi province, China . Four dif-
ferent types of Oryzarufipogon used inour researchwere: Yuan-
jiang type ( collected from Yuanjiang in Yunnan province) , red
awn type (fromJinghong in Yunnan province) , erect type ( from
Jinghong in Yunnan province) , and Dongxiang type (from Jian-
gxi province) . All of the collected cultivated rice varieties and
wild rice speciesweregrown in green house in Kunming of Yun-
nan province for further use . The young green leaves were used
for genomic DNA and RNA extraction .
1 . 2 DNA isolation, finding and cloning of OsSTK gene
Plant genomic DNA was isolated by CTAB method from
young plant leaves, according to the manufacturer′s protocol
(Doyle and Doyle, 1987) . For theamplification of STK typere-
sistancegene analogues, a pair of degenerated primers (KF and
KR , table1) was synthesized . 50 pmol L - 1 of each primer (KF ,
KR) and 50 ng of genomic DNA were addedto50μl of PCR re-
action mixture with 5μl of 10× reaction buffer , 2. 5 mmol L - 1
MgCl2 , 0 . 2 mmol L - 1 of each dNTP, and 4 units of Pfu DNA
polymerase . Thirty cycles of PCR with denaturation at 95℃ for
40 s, reanealing at 55℃ for 40 s, and extension at 72℃ for 1
min, were performed ina DNA thermalcycler (PTC-200 , MJ re-
search) . PCR productswerepurifiedand ligatedto pGEM-T vec-
tor (Promega) and transformed into E. coli (DH5α) , according
to the manufacturer′s protocol . Random selected plasmids con-
taining inserts of the PCR products were sequenced by Sangon
Ltd . China . Multiple DNA sequences analysis and alignment
weremade by the software DNASIS . Amongthe cloned STK type
disease resistanceanalogues, we found one of them from O. ru-
fipogon, O. officinalis and O. meyerian, respectively, shared
highly identities . These DNA fragments may come from one ho-
mologous gene . By usingone of these sequences as bait, search-
ing in DNA database, we found acDNA cloneanda BAC clone,
both has more than 99% identities with the bait sequence . Ac-
cording to thecDNA and BAC clone, we identified OsSTK gene .
1 . 3 Conservation analysis of OsSTK gene in Oryza
After the comparison to the OsSTK gene partial sequences
from O. rufipogon, O. officinalis and O. meyeriana, a pair of
specific primers ( OTK1: 5′-GGCGGTGAAGCAGCTGGAC-3′,
OTK2 : 5′-GGGTGGAGACGTGGGTCTTGTC-3′) was synthesized
Table 1 Degenerated primers for STK type disease resistance gene analogs
Primer name Type Conserved amino acid sequences Primer sequences
KF ( upstream) STK FG (K?V?I?S) VY ( K?R) G TTYGGITCIGTITAYMRIGG
KR ( downstream) STK D ( V?I ) YS ( F?Y ) G ( V?I?M) AYICCRWARCTRTAIAYRTC
Y = C?T ; R = A?G; W = A?T; M = A?C ; I = deoxyyinosine
434 云 南 植 物 研 究 31 卷
accordingto the most conservational region in OsSTK gene . For
the amplification of OsSTK gene partial DNA sequences in differ-
ent rice materials, the PCR conditions of 50μl reaction mixture
weremodified and the same PCR procedure for all materials were
used described as above in 1.2 except for the reanealing tempera-
ture is 60℃ .All thePCR productsfromdifferentwildrice species
were purified and directly sequenced by Sangon ltd . . Multiple
DNA sequences analysis and alignment weremade by the software
DNASIS . Phylogenetic tree based on the OsSTK gene partial DNA
sequenceswas constructed by the software CLASTALw1.83 .
1 .4 Transcription level analysis of OsSTK gene
The young plant leaves of cultivated rice ( Hexi 35 ) ,
O. rufipogon, O. officinalis, O. meyeriana, O. longistaminata
and O. minuta were used for the extractionof total RNA by a to-
tal RNA extraction kit (Gene Company) according to themanu-
facturer′s protocol . The extracted total RNA was treated with
RNase free DNase for 30 min at 37℃ to remove the genomic
DNA . After 5 min denaturation at 70℃ , the SuperScript first-
strand synthesis system for RT-PCR kit ( Clontech) was used to
synthesize cDNA according to kit instructions . About 1μg total
RNA and Oligo dT that used to prime the cDNA were added to
the synthesis reaction . One-tenth volume of the final cDNA (2
μl) was added to 20μl PCR reaction mixture to amplify the Os-
STK genefragment as described above in1 .3 . The PCR products
were detected by 1 .0% agrose gel electrophoresis .
2 Results
2 .1 Bait sequences from three species of wild rice
Many of the disease resistance analogues belong-
ing to serine?thronine protein kinase (STK ) type have
been cloned from different species of wild rice . Among
these DNA sequences, 3 of themfromwild rice O. ru-
fipogon, O. officinalis, O. meyeriana, respectively,
shared more than 91% identities of the deduced amino
acid sequences ( Fig. 1 ) . These analogues may origin
from one same gene or gene family (OsSTK ) . There
were 3 bp in O. rufipogon, and 18 bp in O. meyeriana
deletion in these DNA sequences, respectively, com-
pared to that of O. officinalis . But the deletion did not
affect the predicted reading frame .
2 . 2 OsSTK gene in O. sativa (Japonica)
Using the cloned OsSTK DNA fragment from
O. rufipogon as bait sequence, searching in the NCBI
gene database with BLAST program, we found one
cDNA clone ( AK070226) without annotation and one
BAC clone (gi : 53793517 ) fromNo . 9 chromosomeof
rice . All these clones contained DNA sequences that
havemore than 99% identities with the bait sequence,
Fig . 1 Deduced amino acid sequences of three highly conserved STK typeprotein kinase analogues from three different species of wild rice
“ * ” indicates the amino acid identity and“ .” shows the amino acid similarity . The underlined sequences are the positions of the degenerated primers
5345 期 YANG Ming-Zhi et al. : Cloning and Analyses of a Dual Specific Serine?thronine Protein Kinase . . .
respectively . So, by analysis of these two clones we
found the OsSTK gene consists of two exons, and the
open reading frame separated by a short intron of 114
bp . OsSTK gene was predicted encoding a 419 amino
acids cytoplasmic serine?threonine protein kinase, with
a predicted isoelectric point at5 .52 .Likeother protein
kinase, the deduced OsSTK protein sequence contains
all the invariant and highly conserved amino acids
which characterize the 12 sub-domains (Fig. 2 ) . The
subdomain VIII of OsSTK was completely consensus to
the classical serine?threonine protein kinase . But the
subdomain VIb and XI does not closely resemble either
the consensus of typical serine?threonine nor tyrosine
protein kinase . The XI catalytic domain of OsSTK was
much consensus to the tyrosine protein kinase ( Table
2) . The catalytic domain of OsSTK was flanked by N-
and C-terminal regions of 114 and 30 residues, respec-
tively . The N-terminal residues were abundant with
serine and positively charged amino acids ( lysine and
arginine) . Searching in protein databases using the
program BLAST, we revealed no close relatives to the
N-andC-terminal regionsof OsSTK , butmorethan 150
characterized protein kinase sequences sharing identi-
ties greater than 50% with OsSTK catalytic domain
were detected . Most of these protein kinases were not
annotated yet . Among the annotated or partially anno-
tated protein kinase, themost similar onewas a auxin-
regulated dual specificity cytosolic kinase from tomato
(Lycopersicon esculentum) and a putative Avr9?Cf-9
rapidly elicited protein [ Oryza sativa ( japonica culti-
var-group) XP— 482765 .1 ] , analyzed by the software
of Clustalw1 .83 .
2 .3 Conservation analysis of OsSTK homologue
gene in different species of wild rice
By using the specific primers ( OSK1?OSK2 ) ,
about 400 bp specific DNA sequences were amplified
fromall the Oryzaematerials, including O. sativa, dif-
ferent types of O. rufipogon, O. officinalis, O. long-
istaminata, O. minuta and O. meyeriana . The OsSTK
partial DNA sequences and corresponding deduced amino
Fig . 2 Putative amino acid sequenceof OsSTK gene in O. sativa
The framed N-terminal sequence showed theN-myristoylation motif; Roman numbers indicate the 12 corresponding catalytic sub-domains
of theprotein kinase . The serine, charged amino acids abundant N-terminal was under- lined . The GenBank accession number
for the nucleotide sequence and the deduced amino acid sequence is DQ457011
Table 2 Comparison of the sub-domain Ⅵb, Ⅷ and Ⅺ of OSSTK , ADK1 with the corresponding sequences of typical
serine?thronine and Tyrosine types of protein kinases
Protein Kinase VIb VIII XI
Serine?threonine D L K P E N G ( T?S) XX ( Y?F) X AP E XX ( X ) 6 ?RXXX
OSSTK D F K A S N G T Y G Y C A P E CL ( X ) 6 ?RPAI
ADK1 ?* D I K P D N G T A R Y A SV N CR ( X ) 6 ?KPDY
AhSTKY D L K S D N G T Y R W MAPE CW ( X ) 6 )RPPI
ATN1 ?D L K P E N G T Y R W MAPE CW ( X ) 6 )RPNF
Tyrosine DL ( R?A ) A ( R?A ) N XP ( I?V ) ( K?R) W (T?M ) APE CW ( X ) 6 RPXF
634 云 南 植 物 研 究 31 卷
acid sequences shares identities up to 91% and 90% , res-
pectively . Phylogenetic tree based on the homologous of
DNA sequences was constructed and displayed in Fig.3 .
2 . 4 Transcription of OsSTK
About 400 bp specific DNA fragments canbe ampli-
fied fromall the Oryzae plants that we available by RT-
PCR . For all the extracted tRNA were treated by RNase
free DNase, the results proved the constitutively expres-
sion of OsSTK gene in all these plant leaves (Fig.4) .
3 Discussion
PCR based technology together with bioinformatics
was used to clone OsSTK coding for a novel protein ki-
nase which may displays both serine?thronine and ty-
rosine kinase characteristics . The properties of high
conservation and constitutive expression of OsSTK gene
in Oryzae plants imply the functional importanceof Os-
STK . The structure of OsSTK catalytic domain is simi-
lar to the first cloned disease resistant gene ( R gene)
PTO, which conferred to resistant Pseudomonas syrin-
gaepv . from tomato (Martin et al. , 1993) . Pto gene
encodes a 321 amino acids cytoplasmic serine?thronine
protein kinase and is proved to have the function of
self-phosphorelation .
Dual specificity protein kinasewhich can phospho-
rylate serine, threonine and tyrosine was found in
ADK1, ANK1 from Arabidopsis (Ali et al. , 1994;
Tregear et al. , 1996 ) , and in AhSTKY from peanut
(Rudrabhatla and Rajasekharan, 2002) .Thesub-doma-
ins VIb, VIII and XI of OsSTK were closely resemble
theADK1, AhSTKY and ANK1 (Table 2) . Sub-domain
VIII of OsSTK contains 3 tyrosine residues . In animal
tyrosine kinases, tyrosine residues between subdomains
VII and VIII are thought to serve as sites for autophos-
phorylation ( Hanks et al. , 1995) .
Phylogenetic treebasedon OsSTK genepartial DAN
sequences was close to the taxation systemof Oryzae pla-
nts (Fig. 3) . O. sativa, O. rufipogon, O. longistaminata
Fig . 3 Phylogenetic tree baseon the partial sequences of OsSTK
gene from different species and types of wild rice
( Neighbor-Joint tree, constructed by the sofltware of CLASTAL
w1 .83 , the numbers at every nodewere the support
rate in 100 times of test)
Fig . 4 RT-PCR results of OsSTK gene fragments from different species and types of wild rice and cultivated rice
m . indicates the DL2000 Markers; 1 , 2 . indicate the negative control and“1”is thenegative control with no DNA plate,“2”is thenegative con-
trol with RNase free DNase treated genomic DNA from O. sativa ( hexi No . 35) ; 3 . O. saytiva ( hexi No . 35 ) ; 4 . Dongxiang type of O. ru-
fipogon; 5 . Oryza longistaminata Chev er Roehr; 6 . Oryza minuta J . s . presl ; 7 . Oryza officinalis Wall ; 8 . Oryza meyeriana Ball ; 9 . Oryza
officinalis Wall ; 10 . Yuanjiang typeof O. rufipogon Griff; 11 . Erect type of O. rufipogon Griff; 12 . Red awn type of O. rufipogon Griff
7345 期 YANG Ming-Zhi et al. : Cloning and Analyses of a Dual Specific Serine?thronine Protein Kinase . . .
belongingto the same AA genotype ( Vaughan et al. ,
2003) , werelocated in thenearest position in thetree .
O. meyeriana belonging to GG genotype that located in
relatively farther position from other species of Oryzae
plants . O. officinalis and O. munita have partially
samegenotype, and located in a relative close position
in the tree . Becauses they belong to the CC and BBCC
genetypes, respectively .
Besides a specific peptide at the N-terminal ,
moreover, OsSTK contains an MGIFCCF motif which
matches the (MGXX [S?T?A?G?C?N] X) consensus for
N-myristoylation (Towler and Gordon, 1988) . This co-
translational modification is catalized by a myristoy-
CoA: protein N-myristoyl transferase (NMT) , for which
an activity was found in wheat ( Heuckeroth et al. ,
1988) . The previously studied animal , yeast and wheat
enzymes were found to myristoylate the amino group of
theglycineat position + 2, the sequence contextgreatly
affect the efficiency of reaction . It was found inparticu-
lar that the presence of either a serine, a cysteine or a
second glycine at position + 6 , the absence of apraline
or a charged amino acid at position + 3 and the absence
of praline at position + 7 were important ( Casey,
1995) . And these characters were found in OsSTK . N-
myristoylationoften has an important functional role, ei-
ther for protein-protein interaction or for protein-mem-
brane attachment ( Ishitani et al. , 2000) . N-myritoyla-
tion motif was recently found in calcineurin B-like pro-
teins (CBLs) which acted inCBL?CIPK signalingsystem
to mediate avarietyof responses to environmental stimu-
li in plants (Gao et al. , 2008) . It is therefore possible
that this co-translational modificationmight permit mem-
brane association in the caseof OsSTK .Thespecific N-
terminal sequences, abundant with serine, basic and
charged amino acids, may have specific role in cell po-
sitional and protein recorgnizition . No T [ D?E ] Y
modulewas found between sub-domain VII and VIII in
OsSTK, which is necessary for activation of MAP ki-
nase, an evidence that powerfully supported their reli-
ability as themembers of the rice MAPK family (Liu et
al. , 2007 ) . Therefore, OsSTK doesn′t belong to the
MAPK family . The real function of OsSTK gene is de-
served to be further confirmed .
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