Rice Science, 2007, 14(3): 235-238 Copyright 2007, China National Rice Research Institute. Published by Elsevier BV. All rights reserved
Identification of a Herbicide Safener AD-67 Inducible cDNA in Rice
YIN De-suo1, 2, SUN Xiao-qiong3, LI Ke1, 2, WANG Shi-quan1, 2, DENG Qi-ming1, 2, LI Ping1, 2(1 Rice Research Institute, Sichuan Agricultural University, Wenjiang611130, China; 2 Key Laboratory of Southwest Crop Genetic Resource and Improvement, Sichuan Agricultural University, Ministry of Education, Ya’an 625014, China; 3 Sichuan Agricultural University High-Tech CO., LTD, Wenjiang 611130, China)Abstract: A herbicide safener AD-67 inducible cDNA was identified in an indica rice variety 9311 by mRNA differential display. The
transcript was increased 6 h after sprayed with the safener solution, and 4 days later, the expression still could be detected. The
fragment was recycled from the poly-gel and sequenced, and homologous analysis revealed the cDNA was 100% identical to some
ESTs and cDNAs in rice database, and the amino acid sequence was 60-84% homologous to those of the Yippee genes in several
eukaryotes. The fragment was extended to the whole long cDNA, and thus a primer pair was designed. RT-PCR analysis for the
designed primer supported the induction result.
Key words: rice; chemical induction; cDNA; mRNA differential display; herbicide safener; inducible gene
Chemical inducible promoter, as a tool of controlling gene
specific gene expression in plant without affecting growth and
expression, is important for plant research in laboratory and
development maybe still an effective way for discovering new
field application. The chemical inducible system can control
transgene expression both temporally and spatially, which has
AD-67 is a safener widely used in maize herbicide EPTC
an advantage to the old systems including the constitutive
(S-ethyl dipropylcarbamothioate). However, few reports
promoter and tissue specific promoter systems. This made the
revealed its mechanism and inducible gene. In this study, the
system become a new and powerful strategy for gene safener was used as the inducer to detect inducible gene.
analysis [1-2]. After Gatz and Quail first reported the tetracycline
induction system in 1988 , several induction systems were
MATERIALS AND METHODS
established and developed, such as copper, ethanol,
glucocorticoid, estrogen, ecdysone agonist, benzothiadiazole
Growth of rice seedlings
(BTH) and safener inducible systems [3-10]. These systems have
some applications in laboratory, but not yet in field due to
Seeds of rice variety 9311 were sterilized in 0.1% HgCl2
for 5 min and rinsed with sterile water, and then planted into
Herbicide safeners selectively protect crop plant from
growth chamber with cool-white fluorescent lamps under a
herbicide damage without reducing activity in target weed
photoperiod of 16 h/8 h (light/dark), 28℃ and a humidity of
species. The working mechanism of safener is not very clear,
85%. The seedlings were cultured in MS liquid medium and
several hypotheses were established to explain how the
grown over three weeks when the third leaves fully expanded.
safeners prevent the detrimental effects of herbicide, but many
At this stage, the plants are more sensitive to exogenous
studies produced inconsistent results. Generally, safener could
increase crop tolerance to herbicide . The activities of some
genes were increased when plant was treated with safener, such
Treatment with AD-67 and extraction of total RNA
as the cytochrome P450 and glutathione S-transferases (GST)
Rice seedlings were treated by foliar spray with solution
genes, but these genes were constitutively or temporally
of AD-67 (3 g/L) in acetone, with acetone as the control. All of
expressed with development . In 1991, Hershey’s group
the leaves were collected at 6 h, 12 h, 1 d, 2 d, 3 d, 4 d and 5 d
isolated two genes induced by benzenesulfonamide herbicide
after the treatments and stored at -80 ℃. Total RNA was
safener in maize, and the safener was used as a promoter to
extracted using Trizol agent (TaKaRa) according to the
develop a new inducible gene expression system for plant .
manufacturer instruction. After digestion by DNase, the RNA
This suggests that searching of compounds capable of inducing
was resuspended in DEPC water and diluted to 1 µg/µL, then
Received: 5 March 2007; Accepted: 6 July 2007 Corresponding author: LI Ping ([email protected]) Synthesis of the first strand cDNA
This paper was translated from its Chinese version in Chinese Journal of Rice Science, Vol. 21, No. 4, 2007, Pages 439-442.
Synthesis procedure was according to Promega M-MLV
manufacturer instructions. The 25 µL reaction system contained
Sequencing and RT-PCR analysis
2 µg RNA, 40 pmol anchor primer, 5 µL 5×RT buffer, 10
Nucleic acid and protein homology searches were
mol/L dNTPs of 5 µL, 25 U RNasin, 200 U M-MLV. The total
performed using the BLAST program at National Center of
RNA and primer were incubated at 70℃ for 5 min, followed
Biotechnology Information. Some data scanning were
by adding other components. The mixture was incubated at
conducted with DNAMAN6.0 bioinformatics software. Primers
42℃ for 1 h to complete the reaction, then at 95℃ for 10 min
responding to the cDNA were designed by Primer5.0, and
to end the reaction. Finally, the mixture was diluted to 100 µL
RT-PCR was carried out to check the expression of cDNA. Two
primers designed for RT-PCR were: Yipa, 5’CAGCAAAC
CCATCAATC 3’; Yips, 5’TGGTCCTCGCCA GATA 3’.
The first strand cDNA products were used for PCR
amplification with three anchor primers and twelve arbitrary
primers. The anchor primers were: XTH1, 5’TGCCGAAGC TTTTTTTTTTTA3’; XTH
Differential display and sequencing
G3’; XTH3, 5’TGCCGAAGCTTTTTTTTTTTC3’. The arbitrary
The fragments with significant differences between the
primes were: DD10, 5’TGCCGAAGCTTTGGTAGC3’; DD12,
control and treatment were selected, and one cDNA designed as
5’TGCCGAAGCTTGGAGAGT3’; DD18, 5’TGCCGAAGCT
ADT3-8 (Fig. 1) was used to sequence. After the sequence
TTGGTCAC3’; DD20, 5’TGCCGAAGCTTTGGTCAT3’; DD31,
being edited manually to remove vector and ambiguous
5’TGCCGAAGCTTTGGTCTG3’; DD33, 5’TGCCGAAGCTT
sequence, a fragment with final length of 256 bp was obtained
GGAGGAA3’; DD34, 5’TGCCGAAGCTTTGGTGAC3’; DD35,
5’TGCCGAAGCTTTGGTGAG3’; DD36, 5’TGCCGAAGCT
TGGAGGAT3’; DD38, 5’TGCCGAAGCTTGAT TGGC3’;
Homologous analysis of nucleotide and protein sequence
DD54, 5’TGCCGAAGCTTTGGTTCC3’; DD60, 5’TGCCGAA
Nucleotide sequence search by BLASTN revealed that
PCR was carried out in a 20 µL reaction system including
the cDNA was homologous to multiple ESTs, mRNAs and
2 µL cDNA products. The reaction mixture was subjected to
genomic sequences of rice with high identities (Table 1).
one cycle of denaturation at 94℃ for 5 min, annealing at 40℃
Interestingly, the homologous ESTs are mostly isolated from
for 4 min and extension at 72℃ for 2 min; 15 cycles of
the induced tissues and organs of rice, for example, the
denaturation at 94℃ for 45 s, annealing at 54℃ for 2 min and
CI491559 came from rice being induced by ACC, CI368508
extension at 72℃ for 1 min; and 20 cycles of denaturation at
and CI366717 were identified in the callus, the other ESTs
94℃ for 45 s, annealing at 60℃ for 2 min and extension at 72℃
were reported in the stem treated by γ ray. This suggested that
for 1 min; and final extension at 72℃ for 7 min.
the cDNA was a AD-67 inducible gene in rice.
The PCR products were denatured with formamide and
xylene at 95℃ for 10 min, then 4 µL products were separated on 6% denaturing polyacrylamide gels. After silver staining ,
the bands showing differences between the control and
induction treatment were excised and recovered by boiling. The
cDNA fragments were re-amplified using the same PCR
conditions and primers as above, and recovered with agarose
gel electrophoresis. The purified fragments were linked to
Fig. 1. The result of differential display (silver staining).
Pmd18-T (TaKaRa) vector, and the positive clones were
The arrow shows the differential display band ADT3-8. The
induction time was from 0 h (CK) to 5 days.
TTCCAATCACTATCCTTCTTTCAGCATCATGTGCTTCTCTAATATATACTTGCCTTCTTTGTACTTCTGGTCCTCACCAAAGCTT CGGCA 3’ Fig. 2. Sequence of the differentially expressed fragment.
Black italic shows the primers of PCR, 5’ is the anchor primer and 3’ is the arbitrary primer.
YIN De-suo, et al. Identification of a Herbicide Safener AD-67 Inducible cDNA in Rice 237
Table 1. Nucleotide sequences homologous with ADT3-8 in rice. Fig. 3. RT-PCR analysis of inducible gene.
A. Yippee gene, the left is Marker DL2000; B. actin gene as
database can provide mass bioinformation to scientists, which
promote the rice research and release the researchers from the
laborious experimental work to the efficient computational
By BLASTX program, protein sequence analysis showed
Yippee is an intracellular protein in Drosophila, which is
that many Yippee genes in different species including yeastand
conserved in eukaryote according to the reports . This
Tetrahymena thermophila shared the same function domain
suggested that Yippee gene is an important gene. The gene
with the cDNA (Table 2). Thus, we identified the mRNA
expressed when the Drosophila was infected by pathogens,
(GenBank accession No. XM_478909) as the transcript of the
functioning as an immune related protein. The Yippee gene of
ADT3-8 according the homologous sequences of the fragment.
rice we identified according to the cDNA fragment is induced
by safener AD-67, this is consistent with the results of previous
studies, which concluded that safener could induce the
Using the primers designed for the mRNA of rice Yippee
expression of R gene . Therefore, we deduced that the
gene, RT-PCR result proved that the gene transcripts increased
mechanisms of immune responses in rice and Drosophila might
after induced by AD-67 (Fig. 3). The significant increase was
found at 6 h after treatment, with maximum at 12 h after
Although the function of Yippee gene was discussed in
treatment. At 5 days after treated by AD-67, the expression
some studies in Drosophila, its respondent in rice is still
could also be detected. Moreover, sequencing of the RT-PCR
unknown and should be studied. Moreover, if there is a
products showed the nucleotide sequence of the 731 bp PCR
regulator element in the 5’ upstream of the gene will also need
product was identical to the mRNA (GenBank accession No.
This study was supported by the National High
Rice is the staple food crop, as well as the valuable model
Technology Research and Development Program of China
plant in cereal crops. The genome draft sequence of rice had
(Grant No. 2003AA212030), and the Program for Changjiang
been completed early in 2002, and the whole genome fine map
Scholars and Innovative Research Team in University (PCSIRT)
had also been published by different groups. The rice genome
(Grant No. IRT0453). The authors thank to Dr. XU Zhengjun at
Table 2. Protein sequences homologous with ADT3-8.
the Rice Research Institute, Sichuan Agricultural University for
in mammalian cells and transgene mice. Proc Natl Acad Sci, USA,
his suggestions and directions on the experiment.
1996, 93: 3346-3351.
Zuo J, Niu Q W, Chua N H. An estrogen receptor-based
transactivator XVE mediate highly inducible gene expression in
transgenic plants. Plant J, 2000, 24: 265-273.
Hershey H P, Stoner T D. Isolation and characterization of cDNA
Ren H W, Xiao F Z, Xing Z W. Chemically regulated
clones for RNA species induced by substituted benzene-
expression systems and their applications in transgenic plants.
sulfonamides in corn. Plant Mol Biol, 1991, 17: 679-690. Transg Res, 2003, 12: 529-540.
Hu T Z. The chemical-inducible expression system of plant genes.
Gatz C. Chemical control of gene expression. Ann Rev Plant Mol Plant Breeding, 2003, 1(5): 731-736. Physiol Plant Mol Biol, 1997,48: 89-108.
Gatz C, Quail P H. Tn10-encoded tet repressor can regulate an
12 Gatz C, Lenk I. Promoters that respond to chemical inducers.
operator-containing plant promoter. Proc Natl Acad Sci, USA,
Trends Plant Sci, 1998, 3: 352-358.
1988, 85: 1394-1397.
13 Hatzios K K. Herbicide safeners: Effective inducers of plant
Gatz C, Frohberg C, Wendenburg R. Stringent repression and
defense gene-enzyme systems. Phytoparasitica, 2003, 31: 3-7.
homogeneous de-repression by tetracycline of a modified
14 de Veylder L, van Montagu M, Inze D. Herbicide safener-
CaMV35S promoter in intact transgenic tobacco plants. Plant J,
inducible gene expression in Arabidopsis thaliana.Plant Cell
1992, 2: 397-404. Physiol, 1997, 38(5): 568-577.
5 Granger C L, Cyr R J. Characterization of the yeast
Wang F G, Zhao J R, Guo J L, Sun S X. Review of research into
copper-inducible promoter system in Arabidopsis thaliana. Plant
establishing a DNA fingerprint database of new Chinese maize
Cell Rep,2001, 20: 227-234.
cultivars. Chinese Bull Bot, 2005, 22(1): 121-128. (in Chinese
Yves D, Alexis P, Gethin R, et al. The ethanol switch: A tool for
tissue-specific gene induction during plant development. Plant J,
16 Roxstrom-Lindquist K, Faye I. The Drosophila gene reveals a
2003, 36: 918-930.
T,Chua N H. A glucocorticoid-mediated transcriptional
novel family of putative zinc binding proteins highly conserved
induction system in transgenic plants. Plant J, 1997, 11(3): 605-612.
among eukaryotes. Insect Mol Biol,2001, 10: 77-86.
No D, Yao T P, Evans R M. Ecdysone-inducible gene expression
The Centre For Women’s Reproductive Care Harvey Ward MB ChB, BSc(Med), FCOG (SA), MMed (O&G), FRANZCOG Obstetrician & Gynaecologist WHEN YOUR DOCTOR PRESCRIBES CLOMID You have been advised to take Clomid (Clomiphene by thickening mucus in the cervix and impairing citrate) also sold as Serophene. This is a mild sperm penetration. Recent studies suggest a fertility drug,
FIORENZO CONTI 1. PERSONAL HISTORY Department of Experimental & Clinical Medicine Section of Neuroscience & Cell Biology Università Politecnica delle Marche Via Tronto 10/A, Torrette di Ancona I-60020 Ancona (Italy) Phone 0039 071 220 6056 Faz [email protected] www.fiorenzocontigroup.it Birth Date and Place August 13, 1955 Villerupt (France) Current position