全 文 ：Received 5 Mar. 2003 Accepted 3 Sept. 2003
Supported by the National Natural Science Foundation of China (39928006) and the State Key Basic Research and Development Plan of China
(G2000016205).
* Author for correspondence. Tel: +86 (0)10 62554247; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (3): 332-336
Construct Hairpin RNA to Fight Against Rice Dwarf Virus
MA Zhong-Liang, YANG Huai-Yi, WANG Rong, TIEN Po *
(Department of Molecular Virology and Bioengineering, Institute of microbiology, The Chinese Academy of Sciences,
Beijing 100080, China)
Abstract: Hairpin RNA (hpRNA) can induce post-transcriptional gene silencing, and was constructed with
128-754 bp of segment 8 of rice dwarf virus then placed under the control of the CaMV35S promoter and
used to transform rice cultivar “Zhonghua 11” via Agrobacterium tumefaciens. A total of 12 independent
lines containing hpRNA were obtained as demonstrated by Southern blotting analysis. Challenge inocula-
tion with rice dwarf virus (RDV) viruliferous leafhoppers (Nephotettix cincticeps) showed that T1 plants
containing the hpRNA transgene displayed high resistance or delayed and attenuated viral symptoms. In
contrast, transgenic lines expressing sense RNA showed severe symptoms similar to control plants trans-
formed with the vector alone. These results suggest that hpRNA confers high resistance to RDV in
transgenic rice.
Key words: post-transcriptional gene silencing; rice dwarf virus; hairpin RNA; transgenic rice
Injection of double-stranded RNA (dsRNA) into
Caenorhabditis elegans can trigger specific RNA
degradation, in a process known as RNA interference (RNAi)
( Fire et al., 1998). This process facilitates targeted post-
transcriptional gene silencing (PTGS) and has been applied
in studying the function of new genes (Fraser et al., 2000;
Tuschl, 2002). Transgenes designed to express double-
stranded or single-stranded self-complementary RNA
(hairpin RNA, hpRNA) have a similar post-transcriptional
gene silencing effect on plants (Waterhouse et al., 1998;
Wang and Waterhouse, 2000; Liu et al., 2002). Almost total
plants transformed with an intron-containing hairpin RNA
construct showed silencing (Smith et al., 2000).
In higher plants PTGS functions as a natural antiviral
defense because plant viruses are both initiators and tar-
gets of PTGS and encode proteins that suppress PTGS,
such as 2b protein of cucumber mosaic cucumovirus and
tomato aspermy cucumovirus, p25 of potato virus X and
HC-Pro (potyviral helper component proteinase) of potato
virus Y (Dougherty and Parks, 1995; Anandalakshmi et al.,
1998; Baulcombe, 1999; Ding, 2000; Marathe et al., 2000;
Carrington et al., 2001; Voinnet, 2001). In addition, PTGS
plays an important role in development and transposon
restriction (Anandalakshmi et al., 2000; Fagard et al., 2000;
Smardon et al., 2000; Grishok et al., 2001).
Rice dwarf virus (RDV) is a member of the Reoviridae,
Phytoreovirus possessing a dsRNA genome of 12 seg-
ments designated S1 to S12 according to their mobility in
polyacrylamide gel (Mao et al., 1998). It is transmitted by
leafhoppers (Nephotettix cincticeps) and systemically in-
fects rice and other graminae causing chlorotic flecks at the
sites of infection and stunting of the plant bodies. RDV is
one of the most economically-damaging viruses of rice in
Asia.
Our previous study found that the transgenic plants
harboring ribozyme were lost of their resistance to RDV
due to gene silencing (Han et al., 2000). In this study, we
tried to obtain transgenic rice carrying hpRNA to give high
resistance to RDV infection.
1 Materials and Methods
1.1 Materials
Rice (Oryza sativa L. ssp. japonica) cultivar “Zhonghua 11”
was used. Agrobacterium tumefaciens strain LBA 4404,
Escherichia coli JM 109, plasmid pBluescript KS and plant
expression vector pROK-2 were from our laboratory. All
endonucleases were the products of TaKaRa (Dalian,China)
Co.
1.2 Methods
1.2.1 Construction of hpRNA Chimeric gene SI (sense
and intron) was obtained by the method of gene splicing
by overlap extension PCR (Horton et al., 1989) using the
primers for 128-754 bp of S8 ORF of RDV: sense primers
were P1: 5-GGTCTAGATCACACAGCTCTCAGTTTCG-3;
P2 5 -GCTGACAACGAATTTAGTAGCAGCGTCTGC-3
and the primers for cotton intron were: P3: 5 -
GCTGACAACGAATGTAGTAGCAGCGTCTGC-3; P4:
5-GCGGATCCCTGTTTAATTTTGGAGTTAGC-3. After
MA Zhong-Liang et al.: Construct Hairpin RNA to Fight Against Rice Dwarf Virus 333
digesting with XbaⅠ/BamHⅠ, the PCR product was
cloned into pBluescript KS digested with XbaⅠ/BamHⅠ
to construct recombinant pBSI. The antisense (AS) gene
was amplified by PCR using antisense primers: P5:5-
CTGGATCCTCACACAGCTCTCTCAGTTTCG-3; P6:5-
TCGGTACCAATGTAGTAGCAGCGTCTG-3. PCR product
digested by BamHⅠ/KpnⅠ was cloned into pBSI cut by
the same enzymes to form recombinant pBSIAS.
1.2.2 Rice transformation The SI and the chimeric gene
of sense strand-intron-antisense strand (SIAS) were excised
from pBSI and pBSIAS and subcloned separately into
pROK-2, a plant expression vector containing a 35S pro-
moter from CaMV and terminator from the nopaline syn-
thase in addition to a selectable marker, neomycin
phosphotransferase, conferring G418 resistance.
Recominant vectors pROK-2-SI and pROK-2-SIAS (hpRNA,
Fig.1) were transferred from Escherichia coli JM109 into
Agrobacterium tumefaciens LBA 4404 by electroporation
using a BioRad Gene Pulser and were used to transform
rice. As a control, the vector pROK-2 alone was used to
transform rice.
The rice cultivar “Zhonghua 11” was used for
transformation. The transformation was performed essen-
tially as described by Li et al. (1993). A. tumefaciens LBA
4404 (pROK-2-SI), LBA 4404 (pROK-2-SIAS), LBA 4404
(pROK-2) were grown for 2 d at 28 ℃ and 250 r/min on LB
medium supplemented with 50 mg/L Kanamycin and 10
mg/L Rifampin. The bacteria were collected and suspended
at a density of 3×109-5×109 in AAM medium. Mature
seeds were dehusked and sterilized with 70% ethanol for 2
min and then washed with 1.5% NaClO for 30 min. They
were rinsed thoroughly with sterile water and allowed to
geminate on 2N6 medium at 25 ℃ in the darkness. After
three weeks, calli derived from scutella were subcultured
on fresh 2N6 medium for 4 d. Then, actively growing pieces
of calli (2-3 cm in diameter) were immersed in the bacterial
suspension for 30 min, transferred on to 2N6 AS medium
and incubated at 25 ℃ in darkness for 3 d. After co-
cultivation, the materials were rinsed thoroughly with 500
mg/L carbenicillin in sterile water, then placed on selection
medium 2N6 supplemented with 50 mg/L G418 and 500
mg/L carbenicillin, and cultured for three weeks. Colonies
of cells that had proliferated on the selection medium were
excised and cultured on a regeneration medium, N6S3
supplemented with 50 mg/L G418 and 500 mg/L carbenicillin,
at 28 ℃ under 1 500-2 000 lx illumination. Regenerated
plants were eventually transferred to soil in pots and grown
to maturity in a greenhouse.
1.2.3 Genomic DNA PCR and Southern blotting analysis
Plant genomic DNA was isolated according to the method
of Chaudhry et al. (1999). PCR analysis for detection of
transgenes was carried out using the same primers as those
used for the construction of hpRNA. PCR products were
analysed by electrophoresis on 1% agarose gel.
Procedures for restriction enzyme digestion, electro-
phoresis and Southern blotting analysis were according to
Sambrook and Russell (2001) or the manufacturer’s
recommendations. Ten to twenty micrograms of genomic
DNA was digested with SacⅠ, which liberated fragments
of various molecular weights depending on the insertion
site of the modified gene in the genome. This was fraction-
ated on a 1% agarose gel, denatured and transferred on to
a Hybond-N+ membrane (Amersham). A PCR-amplified, 32p-
labeled the same 634 bp fragment was used as a probe for
hybridization. Pre-hybridization and hybridization were car-
ried out at 65℃ in the 6× SSPE solution (including
Denhardt’s solution and 100 mg/mL salmon sperm DNA).
Filters were washed twice with 2×SSPE, 0.1% SDS for 15
min at room temperature and once with 0.1×SSPE, 0.1%
SDS for 15 min at 65 ℃. Autoradiography was carried out
using Kodak X-ray film.
1.2.4 Bioassays of transgenic plants Seeds from selfed
T0 transgenic plants (Southern blot positive for hpRNA
and sense strand) and control plants (containing vector
alone) were sown in individual pots. The T2 progeny plants
were generated from T1 parents exhibiting high resistance
to RDV and lower level of RDV accumulation in vivo. The
plants of T1 and T2 generations were challenged with the
Fujian isolate of RDV by inoculating with viruliferous leaf-
hoppers (Nephotettix cincticeps). The inoculation was
twice with leafhoppers at two insects per plant, and the
interval was about 5 d. After inoculation for 2 d, the insects
were removed from the plants and the viral symptoms were
observed during the entire period of plant growth at 25-32
℃ in greenhouse.
ELISA tests were performed as described by Crowther
(1995). Leaf samples of one and two days post-inoculation
were grown in modified carbonate buffer (Clark and Adams,
1997). Rabbit antiserum was used at dilution of 1:3 000
(V/V) and goat anti-rabbit peroxidase conjugates (Sigma)
were used at a dilution of 1:500 (V/V) in PEP buffer
(phosphate buffer saline containing 2% PVP and 0.2% BSA).
Fig.1. A scheme of hairpin RNA (hpRNA). 35S, 35S promoter
of CaMV; Nos, terminator of nopaline synthase.
Acta Botanica Sinica 植物学报 Vol.46 No.3 2004334
The optical density was measured at 470 nm using
Microplate Autoreader from Biorad. The sample from non-
inoculated rice plant leaf was used as blank control.
2 Results
2.1 Genomic DNA PCR and Southern blotting analysis of
T0 transgenic rice plants
The calli derived from scutella of the rice cultivar
“Zhonghua 11” were transformed with SI and SIAS via A.
tumerfaciens. A total of 12 independent transgenic plants
expressing SIAS (hpRNA) and 8 transgenic plants express-
ing SI was generated. The PCR of transgentic plant ge-
nomic DNA was performed (Fig.2). Southern blot experi-
ments were carried out after digestion of the genomic DNA
with SacⅠ. There was a single SacⅠ site in pROK-2 and
transgenes. Thus each band on DNA blot represented a
different integration event. As shown in Fig.3, hybridiza-
tion pattern in transgenic plants means that the plants origi-
nated from different transgenic lines. Southern analysis
demonstrated the integration of SI and SIAS into the ge-
nome of rice plants.
2.2 Resistance detection of T1 transgenic plants
The seeds from each line of T0 transgenic plants con-
taining SI or SIAS genes with one Southern blot band and
the seed from control plants were sown. Initially, the T1
and control plants were inoculated with the RDV-carrying
leafhoppers at three-leaf stage. The control plant devel-
oped typical white stripe and spot symptoms in the leaf
after about 10 d post-inoculation, followed by severe stunt-
ing and sterility after one to three month post-inoculation.
About 90% of T1 progenies generated from T0 plants car-
rying hpRNA exhibited high resistance to RDV and showed
no symptoms associated with RDV infection and were fully
fertile.
By comparison of symptom development and severity
of disease incidence in T1 progenies after three months
day post-inoculation, it is apparent that the relative dis-
ease percentage of T1 progenies generated from T0 plant
transformed by SI was 90%-100% while those generated
from T0 plants transformed by SIAS was 10%-12%. Thus
it could be suggested that hpRNA plays an important role
in providing resistance to RDV infection.
2.3 Virus titer in T1 and T2
A total of 62 T2 progeny plants generating from 5 T1
transgenic lines exhibited high resistance to RDV. Among
these plants, 42 were symptomless and 8 exhibited delayed
symptom development. The rest was susceptible to RDV
infection, with the rational resistance to susceptible plants
being about 4.2:1. ELISA results indicated that virus con-
centration was highly correlated with the disease symptom.
In T1 plants exhibiting no symptom and high resistance to
RDV infection, the OD470 value was less than 0.065, and in
the T2 plants this value was less than 0.040, which means
that no virus can be detected.
3 Discussion
We obtained transgenic rice containing hpRNA
construct. Ninety per cent of these plants showed high
resistance to RDV infection. In this study, the arms of
hpRNA were 634 bp, of which more than 200 bp length
DNA as arms can induce gene silencing in high efficiency
and stability (Jan et al., 2000). Although it has been shown
that 21-25 nucleotides (nt) dsRNA can induce RNA inter-
ference (RNAi) in C. elegan (Zamore et al., 2000; Elbashir
et al., 2001) and PTGS in plants (Hamilton and Baulcombe,
1999; Waterhouse et al., 2001), respectively, about 600 bp
genes as arms can be easily to operate and obtain more
effective plants to resistance RDV.
Recently hpRNA has been used in suppressing the ex-
pression of genes in cultured mammalian cells using 19-21
nt RNA, thus it avoids the non-specific antiviral response
if dsRNAs are longer than 30 nt (Williams, 1997). So it can
be possible to shut down whole family genes using hpRNA
construct by choosing conserved regions.
The constructs encoding hpRNA should efficiently trig-
ger PTGS or RNAi for wide range of genes and become a
useful tool for uncovering gene function and antivirus.
Fig.2. Transgenic rice detection by PCR. 1, DNA marker (DL
2000); 2, positive control; 3, line 1；4, line 2; 5, line 3；6, line
4.
Fig.3. Southern blotting analysis of transgenic rice. 1-4, 6,
hairpin RNA (hpRNA) strand-intron-antisense strand (SIAS); 5,
positive control; 7, 8, SI.
MA Zhong-Liang et al.: Construct Hairpin RNA to Fight Against Rice Dwarf Virus 335
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