Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a Better Gene-Silencing Strategy to Study Gene Functions in Rice
et al. (2011) Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a
Better Gene-Silencing Strategy to Study Gene Functions in Rice. PLoS ONE 6(3): e17444. doi:10.1371/journal.pone.0017444
Expression of RNA-Interference/Antisense Transgenes by the Cognate Promoters of Target Genes Is a Better Gene- Silencing Strategy to Study Gene Functions in Rice
Jing Li 0
Dagang Jiang 0
Hai Zhou 0
Feng Li 0
Jiawei Yang 0
Laifa Hong 0
Xiao Fu 0
Zhibin Li 0
Zhenlan Liu 0
Jianming Li 0
Chuxiong Zhuang 0
Roland Roberts, King's College London, United Kingdom
0 1 Key Laboratory of Plant Functional Genomics and Biotechnology, Education Department of Guangdong Province, South China Agricultural University , Guangzhou , People's Republic of China, 2 Department of Molecular, Cellular, and Developmental Biology, University of Michigan , Ann Arbor, Michigan , United States of America
Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/ organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/ or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/ antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.
Funding: This work was supported by the National Basic Research Program of China (2005CB120802, http://www.973.gov.cn/English/Index.aspx), the National
High Technology Research and Development Program of China (2007AA10A144, http://www.most.gov.cn/eng/programmes1/200610/t20061009_36225.htm), the
National Natural Science Foundation of China (30470983, http://www.973.gov.cn/English/Index.aspx) and Genetically Modified Organisms breeding Major
Projects (2008ZX08001-004, http://www.most.gov.cn/index.htm). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Plant genomic research has made remarkable progress in recent
years. The genome sequence of a plant provides the foundation for
detailed functional characterization of plant genes . Rice was
the first crop plant to have its complete genome sequenced .
Although 56,797 genes have been annotated from sequencing of
the rice genome [3,4], the functions of .60% of these predicted
genes are unknown. Therefore, one of the most challenging goals
of the rice functional genomics is to characterize the functions of
these unknown rice genes.
Reverse genetics is a powerful tool for assessing gene function
, and several reverse genetics approaches have been developed
in recent years for functional genomic studies. Transfer DNA
(TDNA) insertional mutagenesis that creates loss of function
mutations  is a very effective reverse genetics approach in
studying gene functions. Although T-DNA insertional mutagen
has been widely used, it has several disadvantages. One common
drawback is complex organizations of many T-DNA inserts,
resulting in an overall 40% to 50% failure rate in identifying the
exact T-DNA insertional site . Besides, T-DNA exhibits certain
integration preference and may therefore not saturate the entire
rice genome . As a result, only 27,551 rice genes were found to
be mutated by T-DNA insertions from collections of .400,000
independent rice T-DNA lines . In addition, T-DNA insertion
may lead to lethal phenotypes, preventing genetic studies of gene
functions, or cause no observable phenotype due to functional
redundancy of homologous genes.
Several alternative reverse genetic approaches to study gene
function, such as RNA interference (RNAi) and antisense RNA
technology could circumvent the limitations of T-DNA insertional
mutagenesis. In RNAi technology, the introduction of
doublestranded RNAs (dsRNAs) into cells inhibits the expression of the
corresponding endogenous gene at transcriptional and
posttranscriptional levels . RNAi could silence the expression of an
endogenous target gene without altering its gene structure or
producing the permanent loss of gene function. The partial gene
silencing-effect of the RNAi and antisense strategies could avoid
potential lethality of a T-DNA insertional mutation. In addition,
RNAi/antisense-initiated gene silencing could simultaneously
inhibit the expression of several homologous genes, thus overcoming
potential gene redundancy problems. These advantages have made
the RNAi and antisense RNA strategies the method of choice for
studying gene functions in plants in recent years.
The choice of promoter is a very important factor in RNAi and
antisense RNA strategies. The most commonly used promoters in
RNAi and antisense strategies are constitutive promoters, such as
the 35S promoter from cauliflower mosaic virus (pCaMV35S) 
and the promoter from the maize Ubiquitin-1 gene (pUbi1) .
Without species restriction, constitutive promoters drive high
expression in virtually all tissues/organs of transgenic plants
independently of tissue/organ-specific regulators or
developmental/environmental signals. However, the constitutive
promoterdriven expression of an RNAi/antisense-transgene could cause
pleiotropic phenotypes or embryo lethality by silencing the
expression of the target gene and its homologs, thus making it
extremely difficult to study the functions of the target gene or to
define a causal relationship between a silenced gene and the
observed phenotypic alterations. On the other hand, recent studies
revealed that constitutive promoters are not active in all cell types,
especially in cereal crops [12,13]. Therefore, gene functions
cannot be fully defined, as the expression pattern of an RNAi/
antisense transgene might not completely overlap with that of its
Regulated promoters such as organ/tissue- or developmental
stage-specific promoters [14,15] and
physically/chemically-inducible promoters [16,17,18,19,20] have been used in the past to
better control the expression of an RNAi/antisense transgene
avoiding the adverse effects of constitutive promoters. However,
these promoters have their own limitations as an
RNAi/antisensetransgene driven by a regulated promoter will only be expressed in
certain tissues/organs, at specific developmental stages, or in
response to a unique chemical/physical signal but has no effect on
the target gene in other relevant tissues/organs at certain
important developmental stages .
By contrast, a cognate promoter of a target gene should drive
the expression of a gene-knockdown RNAi/antisense-transgene in
the native expression domains of the targeted endogenous gene,
which could overcome many of the known limitations of
constitutive/regulated promoters in driving the expression of
gene-silencing transgenes to define the biological functions of their
corresponding endogenous genes.
In this study, we investigated the effectiveness of constitutive/
cognate promoter-driven RNAi/antisense-transgene in causing
growth/developmental phenotype in transgenic rice plants. Four
rice genes, Pyruvate Dehydrogenase Kinase 1 and 2 (OsPDK1 and
OsPDK2), Silencing Information Regulator 2 (OsSRT1), and
Metallothionein2b (OsMT2b), were selected for our studies. The physiological
functions of these four genes were previously studied by gene
silencing using constitutive promoter-driven RNAi/antisense
transgenes [22,23,24], however, our studies using the cognate
promoter-driven RNAi/antisense transgenes revealed additional
functions of these genes in regulating rice growth/development.
Our investigation with the two OsPDK genes also showed that the
cognate promoter approach could specifically reduce the
transcript level of one member gene without affecting the expression of
other members of a gene family.
The cognate promoter-driven expression of an
RNAitransgene revealed novel physiological functions of
Metallothioneins (MTs) are a family of low-molecular weight,
cysteine rich intracellular proteins that are thought to play
important roles in metal tolerance, detoxification, and homeostasis
in plants via binding heavy metals [22,25,26]. The rice genome
encodes 15 MT proteins that could be classified into four types
. OsMT2b, a type-2 MT, scavenges reactive oxygen species
[22,27]. Earlier studies using transgenic rice plants in which
OsMT2b was silenced by an OsMT2b-RNAi transgene driven by
the maize Ubi promoter showed that OsMT2b participates in
epidermal cell death  and is involved in root development and
seed embryo germination by modulating the endogenous cytokinin
To better understand the physiological functions of OsMT2b,
we generated an OsMT2b RNAi transgene driven by the cognate
promoter of the endogenous OsMT2b gene (Figure 1A) and
transformed the resulting pOsMT2b::OsMT2b-RNAi transgene into
wild-type rice plants. Ten independent transgenic lines were
obtained and carefully analyzed, among which 6 transgenic lines
exhibited phenotypic variations in the T0 generation. RNA blot
analyses found that the expression of the endogenous OsMT2b
gene was significantly reduced in two independent pOsMT2b::
OsMT2b-RNAi transgenic lines exhibiting the growth defects
(Figure 2A), while segregation analysis of T1 progeny of several T0
lines carrying single-copy transgene revealed a 3:1 ratio for normal
individuals vs. abnormal individuals. Analyses of the
morphological/developmental defects of the 6 independent T0 transgenic
lines and their offspring not only confirmed previously reported
phenotypic alterations, including smaller mature embryos and
reduced thickness of scutellum of embryos (Figure 2B), but also
discovered novel growth phenotypes such as smaller spikelets,
lower percentage of seed setting, and smaller seeds at the bottom
of spikes (Figure 2C). Our study thus revealed a functional role of
OsMT2b in spikelet/seed development, suggesting that the
cognate promoter-driven gene silencing is a better strategy than
the constitutive promoter-driven gene silencing to study gene
functions in rice.
Silencing of the rice OsSRT1 gene by cognate
promoterdriven OsSRT1-RNAi or OsSRT1-antisense transgenes
To further confirm our discovery, we generated a cognate
promoter-driven RNAi transgene for another rice gene, which
encodes a protein homologous to the SILENT INFORMATION
REGULATOR2 (SIR2), a highly conserved NAD+-dependent
protein deacetylase [29,30]. The rice genome encodes two
SIR2related proteins, named OsSRT1 and OsSRT2 [23,31]. An earlier
study showed that transgenic rice plants in which OsSIRT1 was
silenced by an OsSRT1-RNAi transgene driven by the CaMV35S
promoter exhibited brown dots on leaves, which became larger at
later stages, leading to premature leaf senescence .
Despite numerous attempts, we were unable to generate a single
pOsSRT1::OsSRT1-RNAi (Figure 1B) transgenic rice line from the
OsSRT1-RNAi transgene-transformed calli. We suspected that the
RNAi-mediated silencing of OsSRT1 in its native expression
domains prevented transformed calli to regenerate. To test our
hypothesis, we performed a Southern blot analysis with genomic
DNAs isolated from antibiotic-resistant calli and found that these
hygromycin-resistant calli carried the
hygromycin-B-phosphotransferase gene, the antibiotic marker gene of the pOsSRT1::
OsSRT1-RNAi plasmid and originated from different
transformation events (data not shown). We also performed RNA blot
analysis using total RNAs isolated from hygromycin-resistant and
control calli and found that the OsSRT1 transcript level was
significantly reduced in hygromycin-resistant calli (Figure 3A).
Given the successful generation of multiple transgenic lines when
an OsSRT1-RNAi transgene was driven by the CaMV35S
promoter , our use of a cognate promoter-driven
RNAitransgene revealed a novel role of OsSRT1 in tissue regeneration.
Figure 1. Schematic presentation of the constructed RNAi/antisense transgenes. (A, B) Positions and orientations of independently
amplified genomic/cDNA fragments for generating pOsMT2b::OsMT2b and pOsSRT1::OsSRT1 RNAi transgenes. (CE) Schematic presentation of
antisense transgenes of OsSRT1 (C), OsPDK1 (D) and OsPDK2 (E) driven by their cognate promoters. (F, G) Schematic presentation of the pUbi::OsPDK1
(F) and pUbi::OsPDK2 (G) antisense transgenes. Purple arrows represent promoters, blue right arrows indicate sense fragments, blue left arrows mean
antisense fragments, and blue bars denote introns.
Because no transgenic plants were obtained with the pOsSRT1::
OsSTR1-RNAi transgene, we created a pOsSRT1::OsSRT1
antisense transgene carrying the cognate promoter of the endogenous
OsSRT1 gene (Figure 1C), as an antisense transgene is less effective
in triggering gene silencing. A total of 12 independent transgenic
lines were produced but none of them exhibited any observable
growth alteration. However, at least 5 T0 lines segregated out T1
individuals displaying developmental defects with a 3:1 ratio of
normal plants vs. defective individuals (data not shown). Further
genetic studies suggested that the defective T1 plants are likely
homozygous for the pOsSRT1::OsSRT1-antisense transgene as they
failed to segregate out normal plants in 4 subsequent generations.
Two homozygous pOsSRT1::OsSRT1-antisense lines were selected
to determine the gene silencing effect of the
cognate-promoterdriven antisense transgene.
Although RT-PCR analysis detected no significant changes in
the OsSRT1 transcript level (Figure 3B-a), our immunoblot
experiment showed that the OsSRT1 protein abundance in the
two pOsSRT1::OsSIRT1-antisense transgenic lines was significantly
reduced (Figure 3B-d). Consistent with the known function of the
yeast/mammalian SIR2 proteins in deacetylating the acetylated
lysine-9 residue on histone 3 (H3K9) , an immunoblot assay
using antibodies raised against the acetylated H3K9 revealed the
increased H3K9 acetylation in the two selected transgenic lines
(Figure 3B-e) , further supporting a reduction of OsSRT1
abundance in the two selected transgenic lines. These homozygous
pOsSRT1::OsSRT1-antisense transgenic rice plants not only
displayed brown spots on the leaves and early senescence symptom
(Figure 3C), which are similar to what were previously observed on
pCaMV35S::OsSRT1-RNAi transgenic plants , but also
exhibited additional growth/developmental abnormalities, such as
decreased tillering capacity and lower seed setting (Figure 3C
and data not shown). Our studies using pOsSRT1::OsSRT1-RNAi/
antisense transgenes therefore further supported our conclusion
that expression of RNAi/antisense transgene using a cognate
promoter of the target gene is a better silencing strategy in
revealing its physiological functions in rice.
Direct comparison of the phenotypic differences of
constitutive and cognate promoters in driving the
expression of antisense transgenes in rice
To directly compare the differential effects of constitutive and
cognate promoters on silencing rice genes, we created two
antisense transgenes each for two highly-homologous rice genes
encoding pyruvate dehydrogenase kinase 1 and 2 (OsPDK1 and
OsPDK2), one using the maize Ubi promoter and the other with
the cognate promoters of the OsPDK genes (Figure 1D1G). An
earlier study showed that silencing the OsPDK1 gene by a
CaMV35S promoter-driven OsPDK1-RNAi transgene resulted in
a weak dwarf phenotype in transgenic rice plants .
Transformation of pOsPDK1::OsPDK1 and pOsPDK2::OsPDK2
antisense transgenes resulted in generation of 16 and 13
independent transgenic lines of Zhonghua 11, respectively. Both
transgenes caused two types of growth alterations.The first one is
slightly-reduced plant height (,10% reduction compared to the
control), resembling that of the previously-reported pUbi::
OsPDK1-RNAi transgenic lines . The other type of growth
defects included severe dwarfism (,90% shorter than the
control), single tillering, and semi-sterility (Figure 4A and 4B),
which were not observed in p35S::OsPDK1-RNAi transgenic rice
plants. RT-PCR analysis revealed a slight reduction of the OsPDK
transcript abundance in weakly dwarfed transgenic plants but
detected no OsPDK transcripts in severely dwarfed lines
(Figure 4C and 4D). Interestingly, despite high sequence
similarity between the two OsPDK genes, the antisense-triggered
gene silencing was quite specific as the transcript levels of OsPDK1
and OsPDK2 were not obviously changed in OsPDK2-antisense
and OsPDK1-antisense transgenic plants, respectively (Figure
4E). Consistently, the severely-dwarfed pOsPDK1::OsPDK1 and
pOsPDK2::OsPDK2-antisense transgenic lines also exhibited
unique phenotypes. The pOsPDK1::OsPDK1-antisense lines had
longer life cycle than the control plants with pale yellow leaves,
whereas pOsPDK2::OsPDK2-antisense dwarfs had shorter life cycle
than the control with darker green leaves (Figure 4A and 4B),
revealing different physiological functions for two highly
homologous rice proteins.
By contrast, expression of either OsPDK-antisense transgene
driven by the constitutively-active Ubi promoter failed to cause
extreme dwarfism but only resulted in the semidwarf phenotype
(,30% shorter than control plants) (Figure 5), which is slightly
stronger than that caused by the expression of
pUbi::OsPDK1RNAi transgene . Consistently, RT-PCR analysis revealed a
slight reduction of OsPDK1 or OsPDK2 transcript level in the
pUbi::OsPDK-antisense transgenic lines (Figure 4C and 4D). As
expected from the failure of the pUbi::OsPDK-antisense transgenes
to cause strong dwarfism, no obvious phenotypic difference was
observed between pUbi:OsPDK1- and pUbi:OsPDK2-antisense
transgenic plants. Taken together, our direct comparison study
clearly demonstrated the superiority of the cognate
promoterdriven transgenes in silencing the corresponding endogenous rice
genes to reveal their physiological functions.
In this study, we investigated the differential effects of constitutive
promoter-driven and cognate promoter-driven RNAi/antisense
transgenes on gene silencing and causing growth/developmental
defects in transgenic rice plants. By comparing the growth/
developmental phenotypes of our transgenic plants with those of
previously reported transgenic lines, we found that the expression of
the cognate promoter-driven RNAi/antisense transgenes often gave
rise to growth/developmental defects that were not observed on
transgenic lines expressing constitutive promoter-driven RNAi/
antisense transgenes of the same target genes. For example, some
pOsPDK1::OsPDK1-antisense transgenic lines were severe dwarfs
with yellow leaves, which were not observed in previously reported
pUbi/p35S::OsPDK1- antisense transgenic rice plants . On the
other hand, the use of a cognate promoter-driven RNAi/antisense
transgene could avoid potential lethal phenotype caused by
expression of a constitutive promoter-driven RNAi/antisense
transgene. For example, an earlier study reported that strong
silencing of the OsSRT1 gene caused a postembryonic lethal
phenotype in p35S::OsSRT1-RNAi transgenic plants , whereas
no such a phenotype was observed in our
pOsSRT1::OsSRT1antisense transgenic lines. Our results demonstrated that gene
silencing using cognate promoter-driven RNAi/antisense
transgenes was a more effective and physiologically relevant approach
than that driven by constitutive promoters to define the gene
functions in rice. We have so far adopted this approach to
investigate the physiological functions of more than 20 rice genes
Antisense RNA, with the formation of antisense/target dsRNA, is
a gene silencing mechanism resulting in mRNA degradation or
affecting mRNA translation [32,33]. It has been reported that the
binding position of the antisense RNA may determine
genesilencing mechanisms [34,35]. Antisense RNAs binding to the 39
untranslated region (39-UTR) represses translation , whereas
antisense RNAs pairing with the 59 UTR of the target gene could
cause mRNA degradation. The full-length of OsSRT1 (NM_
001058878) cDNA is 1891 bp, and the predicted antisense
transcript of the OsSRT1-antisense transgene would hybridize to
the region near the 39-end, between nucleotides 1206 and 1770, of
the endogenous OsSRT1 transcript. In pOsSRT1::OsSRT1-antisense
transgenic plants, the transcript level of the endogenous OsSRT1
gene was not obviously changed but the OsSRT1 protein level and
its predicted histone deacetylase activity were significantly reduced.
The same antisense-transgene construction strategy was used for 8
additional rice genes, and similar effects on the abundance of the
endogenous target genes and their protein products were observed
(data not shown). The full-length of OsPDK1 (NM_001056731.1)
and OsPDK2 (NM_001066936.1) cDNAs are 1535 bp and1480 bp,
Figure 5. Phenotypic comparison between pOsPDK::OsPDK-antisense and pUbi::OsPDK-antisense transgenic plants. Shown here from
left to right are six-week old soil-grown rice plants of the wild-type control (Ctrl) and a representative transgenic line carrying an antisense transgene
of pUbi::OsPDK1, pUbi::OsPDK2, pOsPDK1::OsPDK1 and pOsPDK2::OsPDK2.
respectively. The cDNA fragments used for making antisense
transgenes of OsPDK1 and OsPDK2 were derived from the regions
spanning 434845 bp and 153594 bp near the 59 ends of OsPDK1
and OsPDK2 cDNAs, respectively. In pOsPDK1::OsPDK1 and
pOsPDK2::OsPDK2-antisense transgenic progeny, the mRNA levels
of the corresponding target genes decreased significantly. Our
studies thus further supported an earlier hypothesis that antisense
RNA directed against the 59 UTR often results in degradation of the
target mRNA whereas antisense RNA targeted near the 39 UTR
causes translational inhibition.
Consistent with earlier discoveries that the gene-silencing
efficiency of antisense transgene is lower than that of
RNAitransgene, growth/developmental defects were only observed in the
homozygous progeny of OsSRT1/OsPDK1/OsPDK2-antisense
transgenic lines. However, such a lower gene-silencing efficiency could
be useful to avoid lethal phenotypes of RNAi-induced strong gene
silencing. For example, despite numerous attempts and successful
generation of antibiotic-resistant calli with an
pOsSRT1::OsSRT1RNAi transgene, no single pOsSRT1::OsSRT1-RNAi transgenic
plants was regenerated; however, we were quite successful in
generating pOsSRT1::OsSRT1-antisense transgenic lines with
reduced transcript level of the endogenous OsSRT1 gene. We suggest
that the antisense-mediated gene-silencing technology might be
more appropriate than the RNAi technology to study rice genes that
play roles in early stage of plant growth/development.
Gene redundancy is a major obstacle in functional genomic
studies. 53% and 68% of the non-transposable element-related
genes in rice and Arabidopsis are grouped into paralogous gene
families, respectively . Although family members show high
sequence homology at the nucleic acid level, they often have
different expression patterns and biological functions.
Genesilencing using a constitutive promoter-drive RNAi/antisense
transgene could simultaneously knockdown the intended target
gene and its potential homologs , making is extremely difficult
in assigning a given biological function to a member of that gene
family. For example, a recent report showed that four members of
OsAGO1 gene family, OsAGO1a, OsAGO1b, OsAGO1c, and
OsAGO1d, are highly similar in sequence with each other ,
and their transcript levels were all significantly reduced by the
expression of a constitutive promoter-driven OsAGO1-RNAi
transgene. In this work, we studied two members of the OsPDK
gene family, OsPDK1 and OsPDK2, which share 82% similarity at
the nucleotide level. OsPDK1 is expressed in mature leaves, while
OsPDK2 is mainly expressed in actively-growing tissues. As
expected, no phenotypic difference was observed in pUbi::
OsPDK1/pUbi::OsPDK2-antisense transgenic lines, making it
difficult to define the physiological function for each OsPDK gene.
However, transgenic plants in which the
OsPDK1/OsPDK2antisense transgene was driven by the corresponding cognate
promoter displayed different phenotypes. The
pOsPDK1::OsPDK1antisense transgene caused yellowish leaf color and longer life
cycle, whereas the expression of the pOsPDK2::OsPDK2-antisense
transgene resulted in darker green leaf color and a shortened life
cycle with precocious flowering. Our results thus suggested that
the expression of an antisense transgene by the cognate promoter
of its target gene might be a better strategy to study the
physiological functions of gene families.
Materials and Methods
Plant and Other Experimental Materials
Rice (Oryza sativa L. ssp. Japonica) cv. Zhonghua 11 was used
for all experiments. Transgenic plants were grown in a
greenhouse with normal daylight illumination. Escherichia coli
DH10B and Agrobacterium tumefaciens strain EHA105 were used
for cloning and transformation experiments. pCAMBIA1380
was used as the binary vector for Agrobacterium-mediated
Two RNAi transgenes (OsSRT1 and OsMT2b) and three
antisense transgenes (OsSRT1, OsPDK1 and OsPDK2) were
constructed (Text S1). These 5 transgenes were driven by the
cognate promoters of the corresponding target genes. To directly
investigate the differential effect of cognate promoters and
constitutive promoters on gene silencing, OsPDK1 and OsPDK2
antisense transgenes driven by the maize pUbi promoter were also
constructed. Primers were designed based on published cDNA
sequences of OsSRT1, OsMT2b, OsPDK1 and OsPDK2 (Table 1)
and were used to amplify gene-specific cDNA fragments from total
RNAs isolated from Zhonghua 11. The published genome
sequences were also used to locate the 2.0-kb genomic fragment
immediately upstream of the annotated ATG start codon for each
gene (Table 2), which were amplified by PCR using the primer
pairs listed in Table 1 and used as cognate promoters for RNAi/
antisense transgene construction. The intron fragments of RNAi
transgenes were directly amplified the genomic DNA of Zhonghua
11 (Figure 1A and 1B). Each of the constructed transgenes was
fully sequenced to ensure no PCR error before being transformed
into Agrobacterial cells.
To investigate the effectiveness of generated RNAi/antisense
transgenes in silencing their target genes, these transgenes was
then transformed into the A. tumefaciens strain EHA105, which
were used to transform rice calli generated from mature dry seeds
of Zhonghua11 following a previously described protocol .
Tranformed calli were allowed to generate T0 plants. After further
analyses, they were transferred into soil to produce T1 seeds for the
generation of T1 transgenic lines.
Total RNAs were extracted using the Trizol method
(Invitrogen) according to the manufacturers protocols. Briefly, 0.1 g plant
tissues from leaves and spikelets of different developmental stages
of control/transgenic rice plants were ground in liquid N2 to fine
powder, dissolved in the Trizol reagent, incubated at 1530uC for
5 min, mixed with chloroform (0.2 mL/1 mL Trizol reagent), and
centrifuged 12,0006 g at 28uC for 15 min. The resulting
supernatants were mixed with isopropanol (0.5 mL/1 mL Trizol
reagent), incubated at 1530uC for 10 min, and centrifuged at
12,0006 g for 10 min at 28uC to collect RNA pellets. After twice
washing with 75% ethanol, the resulting RNA pellets were dried
and resuspended in water or an appropriate buffer.
Reverse transcriptase-PCR analysis
First strand cDNAs were synthesized at 42uC for 1 h in a 20 mL
reaction that contains 2.0 mg of total RNAs, 4.0 mL of 56 reaction
buffer, 1.0 mL of oligo d(T)15 (50 mmol/L), 2.0 mL dNTP mix
(10 mM each), 1.0 mL Ribonuclease Inhibitor (40 U, TAKARA,
Japan), 1 mL AMV reverse transcriptase (5 U, TAKARA, Japan).
0.5 mL of the reaction product was used for subsequent PCR
amplification of gene-specific cDNA fragments in a 50 mL
reaction containing 40 mL of RNase-free H2O, 5 mL of 106
PCR buffer, 1 mL dNTP mix (10 mM each), 1 mL of forward
primer (10 mmol/L), 1 mL of reverse primer (10 mmol/L), and
0.4 mL of DNA polymerase (2.5 U/mL). The gene-specific primer
pairs used for the RT-PCR reactions were: gaagaagaagatgtcttgctg
and acagtagcagcatccatacg for OsMT2b; gtgcttgtgtgtcattctaccc and
ggacatggtggttcagttgaaccc for OsSRT1; tgggtctccatatatgttcac and
ggactcattccgcgacttac for OsPDK1; gccaggctctgggtcag and
cgggtcgcgccccacg for OsPDK2.
Locations in the BAC
Locations in the rice genome
Note: + means upstream of ATG and 2 means downstream of ATG.
RNA blot analysis
For RNA blot hybridization, equal amounts (,2030 mg) of
total RNAs were separated on 1.2% denaturing agarose gels
containing 12.5% formaldehyde and transferred on to a
HybondN nylon membrane (Amersham Biosciences). The hybridization
probes were amplified by gene-specific primers used for RT-PCR
analysis and were labelled using an [a-32P]-dCTP random
primelabelling system. Hybridization was performed at 42uC following a
previously described procedure . After hybridization, the
membrane was washed twice with 26 SSC containing 0.1% SDS
(w/v) and twice with 0.16 SSC containing 0.1% SDS (w/v) at
50uC, and the hybridization signals were visualized by Molecular
Imager PharosFX Plus System (Bio-Rad).
Tissues were collected from the transgenic and wild type plants,
and total proteins were extracted as described . The protein
extracts (100 mg per lane) were separated by SDS-polyacrylamide
gel electrophoresis and transferred to Pure Nitrocellulose Blotting
Membrane (Pall Corporation) using the wet transfer apparatus.
The membranes were incubated in blocking buffer (5% [w/v]
skimmed milk powder, 0.05% [v/v] Tween 20, 20 mM Tris-HCl,
and 500 mM NaCl, pH 7.5) for 1 h, washed 5 times (5 min each)
with TBST (0.05% [v/v] Tween 20, 20 mM Tris-HCl, and
Construction of RNAi/antisense transgenes.
Conceived and designed the experiments: CZ. Performed the experiments:
Jing Li DJ HZ FL JY. Analyzed the data: DJ JY LH. Contributed
reagents/materials/analysis tools: Z. Li HZ XF. Wrote the paper: Jing Li
DJ CZ Jianming Li Z. Liu.
1. The Arabidopsis Genome Initiative ( 2000 ) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana . Nature 408 : 796 - 815 .
2. Frantz J , Pinnock D , Klassen S , Bugbee B ( 2004 ) Rice: Characterizing the environmental response of a gibberellic acid-deficient rice for use as a model crop . Agron J 96 : 1172 - 1181 .
3. Rice Genome Annotation [http://rice.plantbiology.msu.edu/riceInfo/info. shtml#Genes].
4. International Rice Genome Sequencing Project ( 2005 ) The map-based sequence of the rice genome . Nature 436 : 793 - 800 .
5. Pastuglia M , Azimzadeh J , Camilleri C , Bouchez D ( 2003 ) Forward and reverse genetics in Arabidopsis: isolation of cytoskeletal mutants . Cell Biol Intl 27 : 249 - 250 .
6. Bouchez D , Hofte H ( 1998 ) Functional genomics in plants . Plant Physiol 118 : 725 - 732 .
7. Upadhyaya NM ( 2007 ) Rice Functional Genomics-Challenges, Progress and Prospects . New York : Springer. 500 p.
8. An G ( 2008 ) T-DNA Tagging Lines . In: Hirono H, Sano Y , Hirai A , Sasaki T. Biotechnology in Agriculture and Forestry: Rice Biology in the Genomics Era . Vol. 62 . New York : Springer. pp 95 - 106 .
9. Bernstein E , Caudy A , Hammond S , Hannon G ( 2001 ) Role for a bidentate ribonuclease in the initiation step of RNA interference . Nature 409 : 363 - 366 .
10. Odell JT , Nagy F , Chua N-H ( 1985 ) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter . Nature 313 : 810 - 812 .
11. Christensen A , Sharrock R , Quail P ( 1992 ) Maize polyubiquitin genes: structure, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation . Plant Mol Biol 18 : 675 - 689 .
12. Suwannaketchanatit C , Chaisuk P , Piluek J , Peyachoknakul S ( 2006 ) Evaluation of constitutive promoters for gene expression in Dendrobium protocorms and flowers . Kasetsart J (Nat Sci) 40 : 934 - 943 .
13. Rooke L , Byrne D , Salgueiro S ( 2000 ) Marker gene expression driven by the maize ubiquitin promoter in transgenic wheat . Ann Appl Biol 136 : 167 - 172 .
500 mM NaCl, pH 7. 5 ), and incubated with the primary antiserum (1:500 dilution) for 2 h at room temperature . After 5 rinses (5 min each) with TBST, the membranes were incubated with the secondary antibody (alkaline phosphatase-conjugated goat anti-rabbit IgG [ALP], 1 : 10000 dilution; Kirkegaard and Perry Laboratories) for 1.5 h at room temperature, washed 5 times (5 min each) with TBST, and subsequently incubated in the substrate buffer (0.33 mg/mL nitroblue tetrazolium [SigmaAldrich], 0 . 165 mg/mL BCIP [Bio-Basic], 0 . 1 M Tris, 0 . 1 M NaCl, and 5 mM MgCl2, pH 9.5) for several minutes in the dark, and the chemiluminescent signals were subsequently detected by autoradiography film .
14. Nakatsuka T , Pitaksutheepong C , Yamamura S , Nishihara M ( 2007 ) Induction of differential flower pigmentation patterns by RNAi using promoters with distinct tissue-specific activity . Plant Biotechnol Rep 1 : 251 - 257 .
15. Hirsche J , Engelke T , Voller D , Gotz M , Roitsch T ( 2009 ) Interspecies compatibility of the anther specific cell wall invertase promoters from Arabidopsis and tobacco for generating male sterile plants . Theor Appl Genet 118 : 235 - 345 .
16. Masclaux FG , Charpenteau M , Takahashi T , Pont-Lezica R , Galaud J-P ( 2004 ) Gene silencing using a heat-inducible RNAi system in Arabidopsis . Biochem Biophys Res Commun 321 : 364 - 369 .
17. Gatz C , Frohberg C , Wendenburg R ( 1992 ) Stringent repression and homogeneous derepression by tetracycline of a modified CaMV 35S promoter in intact transgenic tobacco plants . Plant J 2 : 397 - 404 .
18. Mett VL , Lochhead LP , Reynolds PH ( 1993 ) Copper-controllable gene expression system for whole plants . Proc Natl Acad Sci U S A 90 : 4567 - 4571 .
19. Ait-Ali T , Rands C , Harberd NP ( 2003 ) Flexible control of plant architecture and yield via switchable expression of Arabidopsis gai . Plant Biotechnol J 1 : 337 - 343 .
20. Zuo J , Chua NH ( 2000 ) Chemical-inducible systems for regulated expression of plant genes . Curr Opin Biotechnol 11 : 146 - 151 .
21. Vasil IK ( 1999 ) Molecular Improvement of Cereal Crops . Dordrecht: Kluwer Academic Publishers. 500 p.
22. Yuan J , Chen D , Ren Y , Zhang X , Zhao J ( 2007 ) Characteristic and expression analysis of a metallothionein gene, OsMT2b, down-regulated by cytokinin suggests functions in root development and seed embryo germination of rice . Plant Physiol 146 : 1637 - 1650 .
23. Huang L , Sun Q , Qin F , Li C , Zhao Y , et al. ( 2007 ) Down-regulation of a SILENT INFORMATION REGULATOR2-related histone deacetylase gene, OsSRT1, induces DNA fragmentation and cell death in rice . Plant Physiol 144 : 1508 - 1519 .
24. Jan A , Nakamura H , Handa H , Ichikawa H ( 2006 ) Gibberellin regulates mitochondrial pyruvate dehydrogenase activity in rice . Plant Cell Physiol 47 : 244 - 253 .
25. Cherian MG , Jayasuryab A , Bay B-H ( 2003 ) Metallothioneins in human tumors and potential roles in carcinogenesis . Mutat Res/Fundam Mol Mech Mutage 533 : 201 - 209 .
26. Leszczyszyn OI , Schmid R , Blindauer CA ( 2007 ) Toward a property/function relationship for metallothioneins: Histidine coordination and unusual cluster composition in a zinc-metallothionein from plants . Proteins 68 : 922 - 935 .
27. Wong HL , Sakamoto T , Kawasaki T , Umemura K , Shimamoto K ( 2004 ) Down-regulation of metallothionein, a reactive oxygen scavenger, by the small GTPase OsRac1 in rice . Plant Physiol 135 : 1447 - 1456 .
28. Steffens B , Sauter M ( 2009 ) Epidermal cell death in rice is confined to cells with a distinct molecular identity and is mediated by ethylene and H2O2 through an autoamplified signal pathway . Plant Cell 21 : 184 - 196 .
29. Smith JS , Brachmann CB , Celic I , Kenna MA , Muhammadi S , et al. ( 2000 ) A phylogenetically conserved NAD+-dependent protein deacetylase activity in the Sir2 protein family . Proc Natl Acad Sci U S A 97 : 6658 - 6663 .
30. Brachmann CB , Sherman JM , Devine SE , Cameron EE , Pillus L , et al. ( 1995 ) The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability . Genes Dev 9 : 2888 - 2902 .
31. Fu W , Wu K , Duan J ( 2007 ) Sequence and expression analysis of histone deacetylases in rice . Biochem Biophys Res Commun 356 : 843 - 850 .
32. Good L ( 2003 ) Translation repression by antisense sequences . Cell Mol Life Sci 60 : 854 - 861 .
33. Praveena S , Mishra AK , Dasgupta A ( 2005 ) Antisense suppression of replicase gene expression recovers tomato plants from leaf curl virus infection . Plant Sci 168 : 1011 - 1014 .
34. Coopera SR , Taylora JK , Miragliaa LJ , Dean NM ( 1999 ) Pharmacology of antisense oligonucleotide inhibitors of orotein expression . Pharmacol Ther 82 : 427 - 435 .
35. van der Krol AR , Mur LA , de Lange P , Mol JNM , Stuitje AR ( 1990 ) Inhibition of flower pigmentation by antisense CHS genes: promoter and minimal sequence requirements for the antisense effect . Plant Mol Biol 14 : 457 - 466 .
36. Lin H , Ouyang S , Egan A , Nobuta K , Haas BJ , et al. ( 2008 ) Characterization of paralogous protein families in rice . BMC Plant Biol 8 : 18 .
37. Elomaa P , Helariutta Y , Kotilainen M , Teeri TH ( 1996 ) Transformation of antisense constructs of the chalcone synthase gene superfamily into Gerbera hybrida: differential effect on the expression of family members . Molecular Breed 2 : 41 - 50 .
38. Wu L , Zhang Q , Zhou H , Ni F , Wu X , et al. ( 2009 ) Rice microRNA effector complexes and targets . Plant Cell 21 : 3421 - 3435 .
39. Hiei Y , Ohta S , Komari T , Kumashiro T ( 1994 ) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA . Plant J 6: 271 - 282 .
40. Yadetie F , Sandvik AK , Bergum H , Norsett K , Laegreid1 A ( 2004 ) Miniaturized fluorescent RNA dot blot method for rapid quantitation of gene expression . BMC Biotechnol 4 : 12 .
41. Yu S-M , Kuo Y-H , Sheu G , Sheu Y-J , Liu L-F ( 1991 ) Metabolic derepression of alpha-amylase gene expression in suspension-cultured cells of rice . J Biol Chem 266 : 21131 - 21137 .