全 文 ：热带亚热带植物学报 2003，l I(2)：127-131
l口T of Tropical and Subtroplcd Botany
水稻重复序列 RRD3缺失体介导gusA在
水稻愈伤组织中的表达
陈中健 刘 兵 王宏斌 王金发 ’ 刘 良式
(中山大学生命科学学院基因工程教育部重点实验室，广东 广州 510275)
摘要：将水稻中等重复序列 RRD3及其系列缺失体克隆到植物启动子检测载体中，通过根癌土壤杆菌介导转化水稻
愈伤组织，利用GUS组织化学方法检测其在水稻愈伤组织中的启动子活性。结果显示：全长 RRD3、410 bp及 150 bp
缺失体具有强的启动子活性，而 700bp、120bl,缺失体仅有弱的启动子活性。通过与RRD3系列缺失体在哺乳动物
CHO细胞中的启动子活性比较后推测：在RRD3中存在两个真核生物启动子的调控元件，一个对动物细胞的启动子起
正调控，但对植物细胞中的启动子起负调控作用；另一个调控元件仅对动物细胞的启动子起负调控，而对植物细胞启动
子无影响。此外在 RRD3序列中至少存在一个与 TATA盒相关的真核启动子核心元件，但在动物和植物细胞中的调
控方式不同。
关t词：水稻；植物启动子；瞬时表达；GUS组织化学检测
中圈分类号：Q943．2 文献标识码 ：A 文章编号：1005-3395(2003)02-0127-o5
Expression of gush,in Rice Calli Mediated by Deletants
of Ri ce Repetitive DNA Sequence RRD3
CHEN Zhong—j Jan LIU Bing WANG Hong—bin WANG J in—fa‘ LIU Liang—shi
(7he Key Laboratory of Gene e咖 ofMiniawy of Education,School of￡ Sciences，~ongshan Univers GlmI 510275，China)
Abstract：Rice repetitive DNA sequence RRD3 andits six serial delcCamts were im煳 d into the promoter-capture
vector ofplants． Thus， A gene c[m be used to assess the promoter activity ofinserted DNA． The promoter
activities of serial deletants were detected after co-culture ofrice calli with Agrobacterium LBA4404 O0n缸lining
diferent recombinant DNA bytransient GUS Coeta-glucuronidase)assay．The results indicatedthat total length of
RRD3，410bpand 150bpdeletantshad strongerpromoteractivities， whilethepromoteractivitiesin700bp and
120 bp deletants werereduced．There are somepositive／negative~ ntrol elements inR 3 sequence．
Key words：Rice；Plantpromoter；Transientexpression；GUShistochemical assay
The kinetics of reassociation reaction indicated
that over 50％ of the plant genome conta／necl many
repetitive sequences presenting in diferent copies[”．
Recent studies indicated that many eukaryotic gene
expressions were controlled by the repetitive tandom
s4~qucnces in 5’untranslated regions or by the
lt~ dved：2002—)6- 5 Aax~ted：2002-12—O6
Feeadaflen item：This researchwas supportedbyFoeadationof
National‘863’HighTechnologyProgram(BH-01-02-02-03)；Natural
ScienceFoundationofGuangdong]~fovilce (021656)；Foundation of
National‘PlantTransgenicProgram’(J00-A-009)．
’Correspondingauthor
repetitive SCq~lcnces 西 introns 功 e re~ titive
sequences played an important role’m regu~ 0n 0f
gene expressionrZ-~．Ott and Hansen~ have identified
so me enhanoerfragments from repetitive sequences’m
Arabidopsis thalianawith GUS assay．
RRD3 Was a moderately repetitve DNA
sequence isolated from rice gellon~ by reassociation
kinetics and S1 enzyme digestion．After the fragment
Was inserted into the promoterless vector PKK175-6．
the promoter activity of RRD3 was determined in E
colic71．Tl1etotallength0f E旺)3is 820bp containing4
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l28 热带 亚热 带植物 学报 第 11卷
A boxes一 曲 e promoter characteristic sequence．
Six serial deletants of RRD3 based on the TATA
boxes deletion in turn were cloned into C
(chloramphenicol acetyltransferase)vector and used to
transfect the CHO(Chinese hamster ovary)cel line．
ByCAT an alysis in transfected CHO cell line。 these
deletants appeared to have very diferent promoter
activitiests1．
W e have replaced the 35S promoter 0f Ti
vector pBI121 with RRD3． then transformed the
reconstructed plasmid into tobacco an d rice calli by
the Agrobacterium tumefaciens mediated method．
The promoter activity 0f RE D3 in plan ts was
determined by the expression of gusA gene in the
tran sform ed tobacco and rice callitg1．For study ofthe
regulatory pa~ern 0f RRD3． in this paper serial
deletantsofRRD3 from 5’ endwere insertedintothe
multiple cloning site of plan t promoter checking
vector PCAM BIA1391Z． Atter transformation
mediated by LBA44 ， the promoter activities 0f
serial deletants in rico calli were assayed．
1 Materials and methods
Plant material Oryza sativa L． subsp．
Japonic(Taipei 309)．
Strain and vectors E coli DH5 Q：
Agrobacterium tumefaciens LBA4404；
CAM BIA1391Z／1381Z；CAM BIA1301Z．
Construction of the prom oter checking
plasmids in plants The RRD 3 fragment was
isolated from pKK175-6 (Pharmacia) by EcoR I&
饿 II (Takara) digestion． then inserted into
pCAM BIA1381Z in forward／direction
． Th e serial
deletants of RRD3 were i~ated from the animal
promoter checking vector pCAT-Basic(Promega)and
inserted into pCAM BIA1391Z．pCAM BIA1381Z and
pCAMBIA1391Z (gift from Dr．Yin Zhongchao)were
designedforpromoterchecldn,ginplants，therewasno
promoter element upstream to gum gene．
Extraction, purification, digestion, ligation
and transformation of th e plasmids Th e
methods described in Molecular Cloning 2nd ed【 q
were used．
Transformation of A．tumefaciens LBA4404
by frozen-thawed method A．tumefaciens
LBA44O4was transform ed accordingtothemethodof
Dityatkintll1．
Development of rice cam Themature rice
seeds being stripped of seed coats were sterilized for
30 minutes in 20％ fV厂V) NaClO solution， then
washed by sterilized water man y times． cultured on
MSD medium (2，4—D 40 n g nai l)to develop calus
(26—29oC。 about 2 weeks in dark)． Select the
embryogenic calli．
Transient GUS histochemical assay in rice
cam Rice calli were co-cultivated wi th A．
tumefaciens LBA44O4 for 3 days．AfI何being washed
5-10 times wi th sterilized water， the calli were
immersed into the GUS chromogenic solution
(Kye(CN)6 5 mmol／L，K,Fe(CN)65 mmol／L，Nal-I~34
100 mmol／L，X-Glue(Amresco)0．3(、7 )，pH 7．o)，
incubated at 37℃ for 2--4 hours． Atter chromogenic
reaction rice calliwere rinsedwith 70％ ethano1． then
observed by microscopy an d taken photographs． In
chromogenic reaction the strict control was needed to
check the background signals．
2 Results
2．1 Constru ction, transform ation and identifi-
ca tion of th e recombinant PCANⅡI
containing RRD3 and its serial deletants
RRD3 fragm entan d serial deletants wereiso lated
by appropriate restriction digestion an d inserted into
pCAM BIA1381Z or pCAM BIA1391Z． All the target
fragm ents located in the 5’region upstream to gum
gene． Then the recombinant plasmids were used to
transform E coli DH5 Q an d the transfo rm an ts were
identified by Amp resistance．The plasmid DNA were
extracted an d iden tified by electrophoresis after RE
digestion． The clones inserted in forward direction
were en titled pCRRD 3， pC700， pC410， pC1 50 an d
pC120accordingto thelen gth．
2．2 Transformation of A． tumefac／ens LBA4404
with PCRRD3 and its se rial deletants
A．tumefaciens LBA44O4 was transformed with
recombinan t plasmids containing RRD3 an d serial
曩 嵩
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第 2期 陈中健等：水稻重复序列 RRD3缺失体介导 gusA在水稻愈伤组织中的表达 129
deletants by frozen-thawed procedure． Promoterless
pCAMBIA1391Z was used as the negative control in
GUS histoehemical assay and pCAM BIA1301Z
(containing CaMv 35S) was used as the positive
contro1．BothweretransformedintoA． tumefaciens
LBA4404．
The transform ed LBA4404 strains were
identified、 m ‘s-PCR． Because there Was no inner
gusA gene in A．tumefaciens．the transformants in
which gus fragment Can be amplified must contain the
foreign DNA．So we design ed a pair ofPCR primer in
the 400-420 an d l579-1 599 region ofgusA gen e，
respectively． Intransform antsthe 1．2kbproductwas
generated by gus-PCR while no PCR product was
generated in untransformed U3A4404(Figure 1)．
M 1 2 3 4 5 6 7
2O00
1O∞
750
5oo
250
100
Fig．1 Productofgus-PCR withLBA4404 aStemplate
M ：DIr2000marker；,1-6：LBA4404transformedwi th pC1301．
pcm~o3，pC70O，pC410，pCl50 and pC120，t~ ively)aS
template；7：untraasformcd LBA4404aS template
3 Transient GUS histochem ical assay in rice cam
Rice calli were co-cultivated for 3 days with A．
tumcfaciens LBA4404 containing RRD3 and serial
deletants， then washed by sterilized water for 5-10
times an d ilnmersed into the GUS chromogenic
solution． Calli were selected randomly for GUS
histochemical assay． The chromogenic activity Was
diferentin rice calico-cultivated with A tumefaciens
LBA4404 containing diferent plasmids(GUS activty
Was assessed by number an d intensity ofthe blue foei
on surface ofeali)：Chromogenic reactions were not
detec ted in untran sform ed cali or rice calli
CO-cultivated m LBA4404 (pCAMBIA1391Z)．
Strong GUS positive reac tions were observed in calli
CO-cultivated with LBA4404 (pCAMBIA1 30 1，
pCRRD3， pC410 and pC150) while weak GUS
positve reactions with pC700 and pC120 (Figure 2)．
These results indicated that there existed not only the
COre element of plant promoter but alsd
positive／negative regulatory elem ents for promoter in
I 3 sequence．
3 Discussion
3．1 Comparison of promoter activity of RRD3
and ~rial deletants in CHO cells and in
rice cam
Above results an d Our form er report~indicated
A B C D
_●
E F G H
Fig．2 TransientexpressionofGUSinTaipei309 calliaflereo-culfivationwi th A
． nm~efac／ens~ A4404
Rice callieo-cultivatedwithA：LBA4404(pC1301)；B：LBA4404(pCRRD3)；C：LBA4404(peT00)；
D：LBA4404(pC410)；E：LBA4404(pCl50)；F：LBA4404(pC120)；G：LBA4404(pC1391 ；
H：Untram form~ rice callus
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130 热 带亚热带植 物学报 第 ll卷
that there were promoter elements for plants and
promoter elemen ts for animals in RRD3 fragmen t，but
the regulatory pattern ofthe promoter was diferent in
animal cells compared with that in plant cells{Table
Table 1 Cl帅p_rt蚰n 0f promoter a~Ivlfles of RRD3 and
叠圈—■I deletants dIak啪 t host础 s
Total RRD3 (820 bp) had strong promoter
activity in CHO cells an d in rice calli， but promoter
activities of serial deletants of RRD3 fl骶 very
diferent in CH[O cells an d in rice calli． After 100 bp
fragment from 5’en d was deleted, the promoter
activity ofdeletant in CHO cells was weakened a little
while promoter activity in rice calli was weakened
significan tly． This result indicated that there was a
positive regulatory elem en t in the 100 bp moon, but
its regulatory eficiency in animals difered from that
in plants． Interestingly, after 410 bp fragm ent
(containingthe second TATA box)was deleted,the
promoter activity in CHO cells was depressed,but the
activity in rice calli was retrieved． 1【1le conclusion
showed that there existed an negative regulatory
elem en t for plan t promoter in the 100--410 bp region
from 5’end,deletion of this region caused retrieval
ofthe activityofplantpromoter；butin CHO cellsthis
region had positve regulatory eficiency to its
downstream elemen t an d the downstream elements
activity was lost after deletion ofthis region．After the
670 bp region from 5’end(containing l，2，3 TATA
boxes)was deleted,the promoter activityofdeletant
was still strong in rice calli and the promoter activity
in CHO cells was retrieved ． 111is indicated that the
forth TATA bo x was related to the promoter activity
in animal cells an d plant cells while an nega tively
regulatory elem ent existed in 41l一670 bp region
depressedthe activity ofanimal promoter．
Two pl血 lry conclusions can be made．First,two
regulatory elements of eukaryotic promoter presented
in RRD3 sequence．The elem ent in 100-4l0 bp region
was po sitive to animal promoter but was negative to
plant promoter；anotherelem ent in 411-670 bp region
was negative only to animal promoter．Second,there
was at least one promoter related to TATA box(the
forth TATA box) in RRD3 sequence， but the
regulatory pattern of the promoter in animal cells
seemedto be diferent from that in plant ce ls．Further
serial de letion ofRRD3 from 3’en d will help analyze
the relationship between promoter activity and other
7 bo xes
3．2 GenBank analysts of RRD3 sequence
Result from GenBank Blast search indicated that
about 554-820bpregionofRRD3washomologousto
conjectured intron of some sequences published in
GenBan k．
Table 2 cI唧舯 I．_岫 of -Oh betwem 蛐 and
∞q啦嘲 h Q田呲
‘Al homologous sequences wele dedved from rice(0哆阳 sa~a)
1【1le promoter activity of eukaryotic intron w
firstly obtained in animal cellsn 1【1le corresponding
paper about plant inmm w鹤 pub!i~hed only recently：
1【1le DNA ~,onstruot containing only first intron and
first exon ofmaize 幻 gene could promote the
expression of ‘sA gene in scutela of transgenic
wheat．After it was co mbined wi th promoter ofmaize
Ubiquitin gen e， this co nstmct could enhance the
expresion ofgusA gene signiflcantly~堋．IIl our results，
this DNA fragm en t derived from rice moderately
repetitive sequence also has promoter activity in rice
calli． Perhaps this sequence can function as promoter
when it exists asintron in rice genome．Ifthe inference
is right, the unknown regu~ted gene and regulatory
mechanism wi 1 be an interestingtopic for study．
rapid de velopment of plant molecular
．纛 幽
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第 2期 陈中健等：水稻重复序列 RRD3缺失体介导 A在水稻愈伤组织中的表达 131
biology, more and more genes were determined by
gene cloningtechnology such asmap clone， manyof
which wefe related to agronomic traits． Current
research works are mainly to recombine these genes
with proper vector and transfer them into plants for
the improvemen t of agronomic waits． Among all
above problems。 how to choose an appropriate
transformation vector wi th high eficient or tissue-
specific promoter is a key procedure． Therefore
isolation and identification of promoter elements for
plants wi ll play a significant role in transgenic
research．
In this paper the promoter activities of rice
repetitive sequen ce RRD3 an d its serial deletants are
assayed qualitatively．Further quantitative analysis and
site．directedmutationwiII contributeto determination
ofregulatory elements in RRD3．
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