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Application of Proteomic Technology in Gene Functional Study Using
Rice Mutant
Wang, A. Z., Chou, C. Y., Chen, S. H., Tseng, T. H., and Wang, C. S.*
Laboratory of Molecular Genetics, Department of Agronomy, Taiwan Agricultural Research
Institute, Council of Agriculture, Taiwan, ROC. *E-mail: cswang@wufeng.tari.gov.tw
Abstract
Mutants are good materials for studying functional genomics especially when the whole
rice genome has been sequenced. A novel mutation pool containing more than 3,000 mutants
with wide variations on the same genetic background of the TNG67 rice variety has been
developed by sodium azide mutagenesis at the Department of Agronomy, Taiwan Agricultural
Research Institute.
In the present report, we demonstrate the integration of two-dimensional
(2-D) electrophoresis, mass spectrometry and bioinformatics to identify and characterize the
function of protein in the early senescence mutant, SA0401. In our system, more than 1000
protein spots can be identified on a pI 4-7 2-D gel and several mutant specific proteins have
been identified by LC MS/MS (ESI-Q-TOF). Several proteins were identified and found to be
mutant-specific and corresponding to their characteristics. About 50 major leaf proteins
commonly present in all leaf materials are found to involve in photosynthetic pathway
providing the protein markers for construction of a basic protein picture for functional study
of mutants. A senescence associate protein, OSSAP1, and its corresponding gene, a
germin6-like gene, are specifically expressed in the recessive early senescence mutant,
SA0401. By using the mutant and taking the advantages of rice genomic resources we are be
able to study the relationship among gene, protein, function, and phenotype, this
mutant-proteomic approach provides a new vision for the study of rice functional genomics.
Introduction
Rice (Oryza sativa L.) is the staple food for over half of the world’s population, and
provide Asian more than 80% of food supply per year (cited from FAO Statistical Databases,
FAOSTAT).Because of its economic importance, the analysis of rice genome will have a great
contribution to the improvement of rice production. Of particular importance is the emerging
role of rice as a model system among the cereal crops in view of its very small gen ome size
(430Mb), besides, the rice genetic maps and genomic sequences will become relevant across
the cereal crop species. Under the active investigation, the genome of rice has been entirely
sequenced by the International Rice Genome Sequencing Project (IRGSP) consortium and
private companies (Sasaki and Burr, 2000; Goff et al., 2002; Yu et al., 2002). The whole
genome sequence of rice was published in December 2002 that is the achievement of the rice
structural genomics. Further efforts should be focused on functional study of the genes
1
including the number of genes in the rice genome and when, where and how a gene is
expressed in rice. Because most of the annotated rice genes were predicted according to
sequences homology comparison by computational tools and most of their functions have not
been experimentally proved. For instance, in September 2004, only 41.6% [4332 annotated
genes/ (10113 small gene models, partial genes and pseudogenes plus 312 published genes)]
of BAC/PAC predictions on chromosome 3 and 10 of rice have been annotated (The Institute
For Genomic Research, TIGR, http://www.tigr.org/tdb/e2k1/osa1/). Following structural
genomics, the next coming challenge will be directed towards understanding the function of
all genes as well as proceeding rice functional genomic analysis. The achievements in rice
functional genomics will also provide valuable information to the functional analysis of other
cereals.
Transcriptomic tools, such as DNA microarray and Serial Analysis of Gene Expression
(SAGE), permit simultaneous examination of thousands of transcripts. However, the complex
regulatory routes or networks, from post-translational modification to protein turnover, cannot
be studied at the cDNA level. The focus of functional genomics must be transferred from gene
to protein in order to determine more complex biological reaction. Thus, the proteomic
approach is facilitated to resolve the questions of functional genomics. Since its description in
1975 (Klose, 1975; O’Farrell, 1975), the two-dimensional electrophoresis (2-DE) has become
a powerful technique in the separation and analysis of proteins. The 2-DE analysis allows to
examine the expression of hundreds of genes and to compare the patterns ob tained from
different genotypes, conditions or developmental stages. In recent years, consisted of 2 -DE
analysis, mass spectrometry (Fenn et al., 1989; Karas et al., 1989) and bioinformatics,
proteomic tools provide the feasibility and sensitivity to identify and characterize proteins of
various materials. The proteomic approach is the most promising way to identify tissue
specificity, diversity, regulation and post-translational modification of proteins. Therefore, it
will play a major role in functional study of rice genes in the post genomic era.
The aim of this paper is to develop an approach to display rice proteins on 2-D gel and to
construct a 2-D gel database for mutants of a novel rice mutation pool derived by sodium
azide mutagenesis on the same genetic background of the Tainung 67 (TNG67) variety (Wang
et al., 2002a). After examining protein patterns of different mutants and wild type, the
mutant-specific proteins are identified by mass spectrometry. In this report, a senescence
associated protein of the early senescent mutant, SA0401, is identified and its corresponding
gene is cloned and characterized. We demonstrate an efficient system for gene functional
study from phenotype to genotype using mutant by taking the advantage of rice genomic
resources.
2
Material and Method
1. Material preparation
A novel mutation pool of TNG67 variety was developed by sodium azide mutagenesis at
the Department of Agronomy, Taiwan Agricultural Research Institute. After more than 10
generations of self-crossing, selection, and purification by pedigree method, the mutation pool
contains more than 3,000 mutants on the same genetic background of TNG67 variety (Wang
et al., 2002a). So far, the identified mutants are diversified with characteristics including
pathogen (Magnaporthe grisea, Xanthomonas orazae pv. oryza) and insect (brown
planthopper, white-back hopper, rice leaf folder) resistance; herbicide (bentazon, glufosinate,
glyphosate) （Wang et al., 2002b）and stress (UV, chilling, drought) tolerance; plant hormone
(gibberellin, GA) mutants; pericarp coloration, shape, chemical composition (starch, storage
proteins, aroma), and grain eating quality; hundreds of visible mutations in morphology, such
as plant type, leaf shape, leaf color, panicle type; even growth stage, grain development, and
yield capability (Wang et al., 2002a). Genetic analysis shows that most morphological
mutations are recessive, however the traits for disease, insect resistance and aroma of grain
are dominant.
Genetic analyses have been conducted on the several mutants with significant differences
in leaf phenotypic characteristics as compared with their origin TNG67. The priority of
mutants is used to facilitate genetic analysis with the origin TNG67 var. All the materials
chosen for this project are either single dominant or recessive traits (Wang, C. S. unpublished
data). The protein profiles of mutants were compared with the wild type in order to study the
relationship among phenotype, protein, and gene. All the mutants were field grown according
to traditional managements. Leaf materials were harvested at the proper time, frozen with
liquid nitrogen and stored at -80℃ for proteomic analysis. Samples for nucleotide extraction
were lyophilized and stored at -20℃ before processing.
2. Protein extraction
Protein extraction method is modified from Hurkman and Tanaka (1986). A portion
(500mg) of the rice leaf blades was ground into powder in a mortar with seasand and liquid
nitrogen. Homogenized extract with extraction buffer [0.7M sucrose, 0.5M Tris-base, 50mM
EDTA, 0.1M KCl, 30mM HCl, 2% v/v β-mercaptoethanol (β-ME), 10% insoluble
polyvinylpolypyrolidone (PVPP), 1mM phenylmethyl sulfonyl fluoride (PMSF)] Then the
mix extract was centrifugated (20min, 4℃, 15,000 rpm) to remove the insoluble material. The
supernatant was completely mixed with water-saturated phenol and proceeded phase exchange
with extraction buffer twice (20min, 4℃, 15,000 rpm). The supernatant was precipitated with
3 volumes 0.1M ammonium acetate/methanol for more than 4hours at -20℃. The protein
pellet was collected through centrifugation (20min, 4℃, 15,000 rpm), and washed the pellet
3
with 0.1M ammonium acetate/methanol (-20℃) twice and followed with acetone (-20℃) once.
Then, dried the pellet with Speed-Vac (Servant-model), and suspended the pellet in lysis
buffer [9.5M urea, 4% w/v CHAPS, 40mM Tris-base, 15mM 1,4-dithiothreitol (DTT)].
Protein quantification was determined by using Bradford method (Bradford, 1976).
3. Two-dimensional gel electrophoresis (2-DE)
The 2-DE facilities of Amersham Biosciences Co. were used in this experiment for
two-dimensional electrophoresis according to the public protocol (Berkelman and Stenstedt,
1998; Görg et al., 1999) with little modifications.
(1) Isoelectric focusing (IEF) electrophoresis
Extracted protein (200ug) was redissolved in rehydration buffer (8M urea, 2% w/v
CHAPS, 15mM DTT, 0.5% v/v IPG buffer pH3-10 or pH4-7, and a trace of bromophenol
blue). Then the 11cm pH3-10 or pH 4-7 IPG dry strip was soaked in rehydration buffer in
Regular Strip Holder and conducted at 30V for 12hours, followed by 200V for
200Volt-hours, 500V for 500 Volt-hours, 1,000V for 1,000 Volt-hours and 8,000V for
20,000 Volt-hours on IPGphor (Amersham Biosciences) in order for protein isoelectric
focusing separation. After IEF, the IPG strips were placed in individual glass tubes and
soaked with equilibration buffer [50mM Tris-base (pH8.8), 6M urea, 30% v/v glycerol,
2% w/v sodium dodecyl sulfate (SDS), and a trace of bromophenol blue] on a rocker for
15min twice. First equilibration was performed with 1% DTT, and second equilibration
was performed without DTT.
(2) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
After equilibration, the IPG strip was imbedded on separating gel [12.5% acrylamide,
0.375M Tris-base (pH8.8), 0.2% SDS, 0.05% APS, 0.0357% TEMED] and sealed with
0.1% agarose solution to prevent it from movement or floating in the electrophoresis
buffer. Assembled the electrophoresis unit (Hoefer SE600) and added SDS electrophoresis
buffer (25mM Tris, 0.2M glycine, 0.1% SDS), and then started the electrophoresis. The
SDS-PAGE gels were ran at 17℃ and conducted one gel at a constant current of 1.25watt
for 15min, followed by 40watt for 3hours. The electrophoresis was stopped when tracking
dye ran to approximately 1mm from the bottom of the gel.
(3) Silver staining
Protein spots on gel were visualized by silver staining according as modification
of Rabilloud et al. (1988). After termination of the second-dimension electrophoresis
(SDS-PAGE), removed gels from gel cassette and soaked in fixing solution (30%
alcohol, 10% acetic acid) for one hour with gentle shaking. Gels were soaked in 30%
alcohol for 20 min twice, and then washed with desalted water for 2min twice. Used
4
0.0025% sodium dithionite solution 1min, then washed with desalted water for 1min
twice. Stained with silver impregnation solution (0.1% silver nitrate, 0.003%
formaldehyde) for 20min with gentle shaking, then soaked in desalted water for 45 sec
and rinsed briefly with desalted water. Developed in solution (3% sodium cabonate,
0.0005% sodium thiosulphate pentahydrate, 0.0185% formaldehyde) until visualization
of spots and appropriate coloration, then add 3.5% acetic acid quickly in order to
terminate develop reaction. The gels were washed with lot of desalted water for 10min
twice to clean develop solution. In the end, soaked the gels in storage solution (20%
methanol, 10% glycerol) and stored at 4℃.
4. Gel scanning and computer analysis
The images of the gels were scanned with ImageScanner and ImageMaster
LabScan software (Amersham Biosciences) after visualization. The protein spots on the
gel were analyzed automatically with either ImageMaster 2D Elite or Ettan Progenesis
software (Amersham Biosciences).
5. Protein identification
Protein spots of interest were excised and subjected to reduction, pyridylethylation,
and tryptic digestion (Tsay et al., 2000). Multiple peptide sequences were determined in
a single run by capillary reverse phase chromatography (Waters) directly coupled to an
ESI-Q-TOF MS/MS (Micromass) equipped with a standard electrospray source.
Interpretation of the resulting mass spectra was facilitated by database correlation with
the algorithm (Mascot: MS/MS Ions Search), and matched the fit protein or gene.
6. RNA extraction, reverse-transcription polymerase chain reaction (RT-PCR), and
RT-PCR product cloning
Rice leaf total RNA was extracted by the phenol/chloroform method as described
by (McCarty, 1986). For RT-PCR reaction (Promega Corp.), primers Germ6-1
(5’-CKCMGCCRCCRGRGAARCMG)
and
Germ6-3R
(5’-TAATACCAATGTTTTCCTTTATTA) were designed according to the cDNA
sequences of the germin-like protein 6 (GER6, AF032976). The reaction solution
(nuclease-free water, 1X reaction buffer, 0.2mM dNTP Mix, 1mM magnesium sulfate,
1ng RNA template, 1uM downstream primer, and 1uM upstream primer) was mixed
completely on ice, then heated at 70℃ for 10min, cooled down at least 3min on ice. Add
0.1u/ul AMV reverse transcriptase and 0.1u/ul Tfl DNA polymerase to reaction solution,
and then placed on the PCR Thermocycler (Biometra). The RT-PCR were conducted at
the program as: 48℃ for 45min, 94℃ for 2min to the synthesis of the first strand of
cDNA. Then, the reactions were proceeded at 94℃ for 30sec, 57℃ for 1min, 68℃ for
5
2min for 32 cycles, then 68℃ for 7min for PCR amplification, and finally the reaction
was maintained at 4℃. The RT-PCR products were separated in 1.2% agarose gel at
100volt, stained by ethidium bromide, visualized on UV box and photographed. DNA
fragments of interest after RT-PCR amplification were cloned into pGEM-T Easy vector
(Promega Corp.) and sequenced according to the protocols in Molecular Cloning.
7. Northern hybridization and probe preparation
Total RNA (20ug) was mixed with 5.25ul 37% formaldehyde, 15ul 37%
formamide, 6.75ul 5X MOPS, and 1.5ul ethidium bromide. The RNA mixture was
denatured at 70℃ for 10min, then stored on ice quickly. The RNA was separated in
1.2% agarose gel with formaldehyde at 100volt visualized on UV box and photographed.
Tnen, northern blotting was conducted by capilliary force to transfer RNA the onto NC
membrane (MSI?) as of Wang and Vodkin (1994). The blotted RNA was cross-linked to
membrane by UV-crosslinker (Spectronic Corp.). DNA fragment for hybridization (0.?
kb, OSSAP1) was synthesized by random primer reaction using Prime-It II (Promega
Corp.) kit.
The blotted membrane was pre-hybridized with hybridization solution at
42℃ for 1hour, then hybridized with denatured probe at 42℃ for 16hours. The
hybridized membrane was washed with low stringency solution
for 30min twice, then washed with high stringency solution
at room temperature
at 55℃ for 40min. The
hybridized membrane was sealed with plastic warp and pasted on X-ray film in order
for autoradiography processing.
6
Results
Our final goal is to establish the protein 2-DE profile for tissues at various
developmental stages and collect the basic information for the TNG67 rice variety. This basic
dataset will provide reference gels for not only mutant-wild type comparison but also
mutant specific proteins isolation. Our preliminary result shows that most of leaf protein spots
are distributed in a quite narrow range between pI 4.6 and pI 7.2, and a wide range of
molecular weight 27.7kDa to 225.2kDa. These proteins could not be clearly separated on a
pI.3 to pI10 IEF gel strip (Fig. 1a). Therefore, we narrowed down the isoelectric point range
and used the pI 4 to pI 7 gel strip in order to increase the resolution of proteins display. In our
typical 2-D gel (pI 4-7, 11cm IPG strip), about 1,000 leaf protein spots can be detected by
image analysis (Fig. 1b).
Many mutants with leaf morphological mutations are available in our novel mutation
pool (Wang et al, 2002) and six of them are shown in Fig. 2a. Besides, the mutant-specific
protein spots(red circles), many constitutive protein spots (green circles) are aso detected (Fig.
2b). Through ESI Q-TOF MS/MS identification, some constitutive spots are found to be
involved in basic metabolism, including GAPDH, hydrolase, cysteine synthase, glycine
decarboxylase subunit, fructose-bisphosphate aldolase, β-1,3-glucanase, triosephosphate
isomerase, nucleoside diphosphate kinase, superoxidase dimutase, protein disulfide isomerase,
malate dehydrogenase and peptide methionine sulfoxide reductase. The photosynthetic related
proteins account for the largest group of identified proteins including ribulose bisphosphate
carboxylase large subunit, ribulose bisphosphate carboxylase small subunit, RuBisCO subunit
binding-protein α-subunit, photosystem II (PSII) oxygen evolving-protein, oxygen-evolving
enhancer protein 1, PSII protein D1, PS Q (B) protein and L-ascorbate peroxidase. We also
observed some the dnaK-type molecular chaperone HSC70-9 group proteins that belong to the
heat shock protein family with 70kDa molecular weight (Hsp70). However, several
proteins
???
could not be grouped, such as ferritin, which is a major non-heme iron
storage protein.
With these standardlized gels of TNG67 variety, we will be able to study the differential
expression of proteins in the specific mutant by using the 2-DE technology and the proteomic
approach. All the mutants chosen for leaf protein 2-DE analysis have been genetically
confirmed by crossing to its wild type TNG67. We have established and identified the mutant
specific proteins by analyzing mutant-specific spot patterns (hot spot) on 2-DE gels. For
example, an early leaf senescence mutant, SA0401, shows a significant senescence symptom
when its leaves are fully expand at 40 days after transplanting (Fig. 3). Several differentially
expressed protein spots named as rice senescence associated proteins (OSSAP), only appear
on the gel prepared from early senescent leaves (Fig. 4c) of SA0401 mutant, but are absent in
the normal green leaves (Fig. 4b) of SA0401 and leaves of TNG67 variety (Fig. 4a). The
7
senescence specific protein spots (OSSAP1) of SA0401 mutant was isolated, purified, and
identified with ESI Q-TOF MS/MS. Of this protein spot, three peptides are found to match a
germin-like protein 6 (AAC04837, Fig. 5). The germin-like protein 6 was reported to be
related to the oxalate oxidase of wheat and barley (Membre and Bernier, 1998), but its
function is still unknown. We name it as the rice senescence associated protein 1 (OSSAP1).
The amino acid sequences of the OSSAP1 are deduced from the mRNA of a japonica rice
gene GER6 (AF032976) by blast analysis of NCBI. In addition to AF032976, there are five
similar genes, AF032971, AF032972, AF032973, AF032974, and AF032975 of oxalate
oxidase/germin-like protein family are found in the rice genome. The AF032976 were
reported and located in the BAC clone b6015 [AL117264 of Oryza sativa indica (GLA4)
genomic DNA, chromosome 4; Fig. 6] of rice.
Many proteins such as 33kDa PSII oxygen evolving protein、PSII oxygen-evolving
complex protein 1, PSII oxygen-evolving complex protein 2, PSII protein D1,
nucleoside-diphosphate kinase, glycine decarboxylase subunit, pathogenesis–related protein
10 are also found to be specific to the early senescence leaf of SA0401 mutant.(此段考慮刪
除)
To study the difference in gene expression, primer Germ6-1 and Germ6-3R, of
germin-like protein 6 (AAC04837) were designed for RT-PCR using RNA prepared from
leaves of SA0401 and TNG67. The result shows that a specific cDNA fragment was
specifically amplified in the senescence leaf of the SA0401 mutant with the expected size,
about 0.9kb (Fig. 7). This specific fragment can be amplified in neither the normal leaf of
SA0401 nor any leaves of TNG67. The 0.9kb senescence specific gene fragment was cloned,
sequenced, and blast analyzed. The result shows that the length of the ORF of OSSAP1 is
943bp and is homologous to the germin-like protein 6 (GER6) mRNA (AF032976) with 99%
identity, data not shown). The expression of OSSAP1 gene was further analyzed by northern
hybridization using the cloned fragment as a probe. The result shows that the OSSAP1 gene is
only expressed in the early senescent leaves of SA0401 mutant(Fig. 8).
The genomic DNA of SA0401 and TNG67 were used as templated to amplify the SAP
related genes the result shows that a 1.1kbDNA fragment was amplified in both SA0401 and
TNG67 sugesting that the OSSAP1 gene is presented in both SA0401 and TNG67 genomes.
Results of cloning, sequencing and blast analyzing the DNA fragment derived from SA0401
shows that the genomic DNA of OSSAP1 gene is 1121bp in length and with 99% identity to
the germin-like protein 6 (GER6) mRNA (AF032976). Digestion the genomic DNA of
SA0401 and TNG67 by different restriction enzyme combinations and hybridized with the
1.1kb fragment as a probe, the result shows that germin-like gene family is presented as a
multiple gene family in the rice genome (Fig. 8).
8
Discussion
Mutagenesis is usually applied to induce mutants with new trait for breeding new variety
or for general research. Generally, mutations are of very little economic value and difficult to
keep and hence they were ignored or discarded by breeders in the past. In long term rice
studies, hundreds of mutants have been reported and registered in Rice Genetic News Letter
(http://www.shigen.nig.ac.jp/rice/oryzabase/rgn/newsletter.jsp). Most of these mutants were
collected from various sources and of very diversed genetic backgrounds. For instance, the
website of Oryzabase (http://www.shigen.nig.ac.jp /rice/oryzabase/) in Japan has a collection
contains 13,396 mutations which were derived from many mutation methods or mutagens, and
were obtained from more than 10 different rice varieties. Though therein, many near-isogenic
mutants were derived from a same genetic background of Taichung 65 (T65 or TC65) variety
by at least five to six backcrosses. Wang et al (2002a) reported a novel rice mutation pool
containing more than 3,000 mutants derived by sodium azide mutagenesis from a sigle rice
variety TNG67. To our knowledge, this is also a mutation pool in rice contains the largest
number of mutants on a same genetic background.
Because this mutation pool is derived from the same genetic background of TNG67
variety, we will be able to locate or map several mutated genes at one time by using the
advanced molecular techniques to isolate the genes and annotate the function of genes with
these mutants.
Plant senescence, especially rice leaf senescence has been investigated for years, and
many senescence-related genes and proteins, such as
have been reported (Murchie et al.,
1999; Lee et al., 2001; Cha et al., 2002). But the hottest spot in this report, OSSAP1
(germin-like protein 6) has not been reported to be correlated with plant senescence. In the
NCBI database, the germin-like protein 6 was annotated to be similar to wheat and barley
oxalate oxidase that was conducted most likely by sequence comparison.
So far, no report
on either protein function or molecular study was conducted on the germin-like protein 6 in
rice.
In the present report, we demonstrate the functional annotation for the putative
germin-like protein 6 of ice through proteomic approach using mutant. In our result, most of
the senescence-related proteins of SA0401 are related with to the PSII oxygen-evolving
proteins or PSII proteins. Except different subunits or complexes, same proteins may be with
different molecular weight and isoelectric point. Actually, we identify many isoforms of a
same protein in the constitutive protein list. It seems that proteomic tools either permit
qualitative and quantitative analysis or display post-translational modification, in which
transcriptomic tools are lacking.
Although, the biological function of germin-like protein 6 is still unknown, our result
support that the rice senescence associated protein, OSSAP1, and its gene is only specifically
expressed in the senescence leaf of SA0401. Further experiment will be conducted to study
9
the relationship between leaf senescence symptom and the proteins in the early senescence
mutant,SA0401. Besides, transgenic approach will be applied to study whether the early
senescence is determined by the OSSAP1. A high priority goal of our program is to establish
gene-protein-function-phenotype network.
Through our examination, we ensure proteomic tools not only detect protein molecular
marker but also directly respond to physiological and genetic properties, and open a new area
for plant functional genomic research. The functions of proteomic tools are either gene
detection or post-translational modification identification, and beyond transcriptomic tools.
This novel rice mutation pool can serve a good resource for traditional studies on genetics,
breeding, physiology and plant-microbe interaction. Following completion of the entire rice
genome sequence and development of genomics, transcriptomics, proteomics, metabolomics,
and silico-genomics, rice mutants can be provided more than before, especial for gene cloning
and function assay. After established the protein database of mutants by 2-DE analysis, we use
this database to screen mutation proteins in order to exercise proteomic approach and clone
corresponding genes, furthermore, permit functional genomic approach.
Acknowledgement
The authors thank the financial support to Chang-sheng Wang from National Program for
Agricultural Biotechnology, National Science Council, Taiwan ( NSC 91-3112-P-005-001-Y).
References
Berkelman, T., and Stenstedt T. 1998. 2-D Electrophoresis - using immobilized pH gradient:
Pincoples and Methods. Amersham Biosciences, Uppsala, Sweden.
Bradford, M. A rapid and sensitive method for the quantitation of microgram quantities of
protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976. 72:248-254.
Cha, K.W., Lee, Y.J., Koh, H.J., Lee, B.M., Nam, Y.W., and Paek, N. C. 2002. Isolation,
characterization, and mapping of the stay green mutant in rice. Theor Appl Genet.
104 :526-532.
Chittum, H. S., Lane, W. S., Carlson, B. A., Roller, P. P., Lung, F. D., Lee, B. J. and Hatfield,
D. L. 1998. Rabbit beta-globin is extended beyond its UGA stop codon by multiple
suppressions and translational reading gaps. Biochemistry 37:10866-10870.
Eng, J. K., McCormick, A. L., and Yates, J. R. III. 1994 An approach to correlate tandem mass
spectral data of peptides with amino acid sequences in a protein database. J. Am. Soc. Mass
Spectrom. 5:976-989.
FAO Statistical Databases (FAOSTAT) Retrieved from http://apps.fao.org/default.jsp
Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. and Whitehouse, C. M. 1989. Electrospray
10
ionization for mass spectrometry of large biomolecules. Science 246:64-71.
Goff, S. A., D. Ricke, T. H. Lan, G. Presting, R. Wang, M. Dunn, J. Glazebrook, A. Sessions, P.
Oeller, H. Varma, D. Hadley, D. Hutchison, C. Martin, F. Katagiri, B. M. Lange, T.
Moughamer, Y. Xia, P. Budworth, J. Zhong, T. Miguel, U. Paszkowski, S. Zhang, M.
Colbert, W. L. Sun, L. Chen, B. Cooper, S. Park, T. C. Wood, L. Mao, P. Quail, R. Wing, R.
Dean, Y. Yu, A. Zharkikh, R. Shen, S. Sahasrabudhe, A. Thomas, R. Cannings, A. Gutin, D.
Pruss, J. Reid, S. Tavtigian, J. Mitchell, G. Eldredge, T. Scholl, R.M. Miller, S. Bhatnagar,
N. Adey, T. Rubano, N. Tusneem, R. Robinson, J. Feldhaus, T. Macalma, A. Oliphant, and
S. Briggs. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. japonica).
Science 296(5565): 92-100.
Görg, A., Obermaier, C., Boguth, G., Weiss, W. 1999. Recent developments in
two-dimensional gel electrophoresis with immobilized pH gradients: wide pH gradients up
to pH 12, longer separation distances and simplified procedures. Electrophoresis
4-5:712-717.
Hurkman W. J. and Tanaka, C. K. 1986. Solubilization of plant membrane proteins for
analysis by two-dimensional gel electroforesis. Plant Physiology 81, 802–806.
Karas, M., Bahr, U., Ingendoh, A. and Hillenkamp, F. 1989. Laser desorption-ionization mass
spectrometrie of proteins with masses 100,000 to 250,000 dalton. Angew. Chem. Int. Ed.
Engl. 28:760-761.
Klose, J. 1975. Protein mapping by combined isoelectric focusing and electrophoresis of
mouse tissues. A novel approach to testing for induced point mutations in mammals.
Humangenetik 26:231-243.
Lee, R-H., Wang, C-H., Huang, L-T., and Chen, S-C. G. 2001. Leaf senescence in rice plants:
cloning and characterization of senescence up-regulated genes. 5 J Exp Bot. 2:1117-21.
Mascot:
MS/MS
Ions
Search
Retrieved
http://www.matrixscience.com/cgi/index.pl?page=/search_ form_select.html
from
McCarty, D. R. 1986. A simple method for extract of DNA from mazie tissue. Maize Genet.
Crop Newsl. 60:61.
Membre, N. and Bernier, F. 1998. The rice genome expresses at least six different genes for
oxalate oxidase/germin-like proteins (Accession Nos. AF032971, AF032972, AF032973,
AF032974, AF032975, and AF032976) (PGR98-021) Plant Physiol. 116 (2):868.
Murchie, E. H., Chen, Y-Z., Hubbart, S., Peng, S., and Peter Horton, P. 1999. Interactions
between Senescence and Leaf Orientation Determine in Situ Patterns of Photosynthesis and
Photoinhibition in Field-Grown Rice. Plant Physiol. 119: 553–564.
O’Farrell, P. H. 1975. High resolution two-dimensional electrophoresis of proteins. J. Biol.
Chem. 250:4007-4021.
Oryzabase Retrieved from http://www.shigen.nig.ac.jp/rice/oryzabase/
Rabilloud, T., Carpentier, G., and Tarroux, P. 1998. Improvement and simplification of
low-background silver staining of proteins by using sodium dithionit. Electrophoresis
9:288-291.
Rice Genetic News Letter Retrieved from http://www.shigen.nig.ac.jp/rice/oryzabase/
rgn/newsletter.jsp
11
Sasaki, T. and B. Burr. 2000. International Rice Genome Sequencing Project: the effort to
completely sequence the rice genome. Curr. Opin. Plant Biol. 3(2): 138-41.
The Institute For Genomic Research (TIGR) Retrieved from http://www.tigr.org/tdb/
e2k1/osa1/
Tsay, Y.-G., Wang, Y.-H., Chiu, C.-M., Shen, B.-J., and S.-C, Lee. 2000. A strategy for
identification and quantitation of phosphopeptides by liquid chromatography/tandem mass
spectrometry. Anal. Biochem. 287:55-64.
Wang et al. (1994)
Wang, C. S., T. H. Tseng, and C. Y. Lin. 2002a. Rice biotech research at the Taiwan
Agricultural Research Institute. APBN. 6：950-956.
Wang, C. Y., Tseng, T. H., and Wang, C. S. 2002b. Bentazone herbicide tolerant mutants in
rice (Oryza sativa L.). J. Genet. Mol. Biol. 13: 284-294.
Yu, J., S. Hu, J. Wang, G. K Wong., S. Li, B. Liu, Y. Deng, L. Dai, Y. Zhou, X. Zhang, M. Cao,
J. Liu, J. Sun, J. Tang, Y. Chen, X. Huang, W. Lin, C. Ye, W. Tong, L. Cong, J. Geng, Y.
Han, L. Li, W. Li, G. Hu, X. Huang, W. Li, J. Li, Z. Liu, L. Li, J. Liu, Q. Qi, J. Liu, L. Li, T.
Li, X. Wang, H. Lu, T. Wu, M. Zhu, P. Ni, H. Han, W. Dong, X. Ren, X. Feng, P. Cui, X. Li,
H. Wang, X. Xu, W. Zhai, Z. Xu, J. Zhang, S. He, J. Zhang, J. Xu, K. Zhang, X. Zheng, J.
Dong, W. Zeng, L. Tao, J. Ye, J. Tan, X. Ren, X. Chen, J. He, D. Liu, W. Tian, C. Tian, H.
Xia, Q. Bao, G. Li, H. Gao, T. Cao, J. Wang, W. Zhao, P. Li, W. Chen, X. Wang, Y. Zhang, J.
Hu, J. Wang, S Liu., J. Yang, G. Zhang, Y. Xiong, Z. Li, L. Mao, C. Zhou, Z. Zhu, R. Chen,
B. Hao, W. Zheng, S. Chen, W Guo., G. Li, S. Liu, M. Tao, J. Wang, L. Zhu, L. Yuan, and H.
Yang. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science
296(5565): 79-92.
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Fig. 1. Dendrograms: 1a, pI 3-10 and 1b, pI 4-7 of two dimensional gel electrophoresis (11cm
IPG strip) for leaf proteins of TNG67 rice variety.
13
Fig. 2. Leaf morphological mutants (2a) derived from TNG67 variety were used for proteomic
study, and their corresponding two dimensional gel electrophoreses (pI 4-7) (2b) for
proteins extracted from leaves at 40 days after transplanting (DAP).
14
15
Fig. 3. Leaves of TNG67 (3a) and its early senescence mutant SA0401 (3b) at 40 days after
transplanting. Senescence symptom appears when leaves are fully expanded.
16
Fig. 4. The partial enlarged two dimensional gel electrophoresis for leaf of the wild type,
TNG67, (4a) and the normal leaf (4b), and the senescent leaf (4c) of the early
senescence mutant SA0401. The rice senescence associated protein (OS SAP) are red
circled, especially the OSSAP1 is marked with an arrow.
17
>gi|5852087|emb|CAB55394.1| zwh0010.1 [Oryza sativa (indica
cultivar-group)]
MASRAFAAVF AAVALVVCSS VLPRVLASDP SQLQDFCVAD KLSAVFVNGF VCKNPKQVTA
NDFFLPKALG VPGNTVNAQG SAVTPVTVNE LPGLNTLGIS FARIDFAPNG QNPPHTHPRA
TEILTVLQGT LLVGFVTSNQ PGGGNLQFTK LLGPGDVFVF PQGLIHFQLN NGAVPAVAIA
ALSSQNPGVI TIANAVFGST PPILDDVLAK AFMIDKDQVD WIQAKFAAPP AASGGGGGFI
GGGGGGGFPG GGAP
Fig. 5. Three peptides (red letters) were identified in the hottest protein spot , OSSAP1, by
using ESI Q-TOF MS/MS. This protein was matched to the germin-like protein 6
(AAC04837). A putative oxalate oxidase in wheat and barley and was named as rice
senescence associated protein 1 (OSSAP1).
18
Fig. 6. The RT-PCR result was amplified from total RNA of normal leaf (N) and senescence
leaf (S) of TNG67 and the early senescence mutant, SA0401. The primers were
designed from germin-like protein 6 (AAC04837). A 0.9kb band is specifically
amplified from the senescent leaf of SA0401 mutant by RT-PCR.
19
Fig. 7. The northern hybridization of total RNA of normal leaf (N) and senescence leaf (S)
from TNG67 and SA0401 hybridized with the isotope labeled GER6 clone. The
germin-like gene (about 1.1kb) is only expressed in the senescent leaf of SA0401
mutant.
20
Fig. 8. The southern hybridization of digested genomic DNA of TNG67 (1) and SA0401 (2)
hybridized with the isotope labeled GER6 genomic clone (about 1.1kb). B: Bam HI, E:
Eco RI, H: Hind III, N: Nde I, S: Ssp I.
21
Legends
Fig. 1. Dendrograms: 1a, pI 3-10 and 1b, pI 4-7 of two dimensional gel electrophoresis (11cm
IPG strip) for leaf proteins of TNG67 rice variety.
Fig. 2. Leaf morphological mutants (2a) derived from TNG67 variety were used for proteomic
study, and their corresponding two dimensional gel electrophoreses (pI 4-7) (2b) for
proteins extracted from leaves at 40 days after transplanting (DAP).
Fig. 3. Leaves of TNG67 (3a) and its early senescence mutant SA0401 (3b) at 40 days after
transplanting. Senescence symptom appears when leaves are fully expanded.
Fig. 4. The partial enlarged two dimensional gel electrophoresis for leaf of the wild type,
TNG67, (4a) and the normal leaf (4b), and the senescent leaf (4c) of the early
senescence mutant SA0401. The rice senescence associated protein (OS SAP) are red
circled, especially the OSSAP1 is marked with an arrow.
Fig. 5. Three peptides (red letters) were identified in the hottest protein spot, OSSAP1, by
using ESI Q-TOF MS/MS. This protein was matched to the germin-like protein 6
(AAC04837). It was a putative oxalate oxidase of wheat and barley and was named as
rice senescence associated protein 1 (OSSAP1).
Fig. 6. The RT-PCR result was amplified from total RNA of normal leaf (N) and senescence
leaf (S) of TNG67 and the early senescence mutant, SA0401. The primers were
designed from germin-like protein 6 (AAC04837). A 0.9kb band is specifically
amplified from the senescent leaf of SA0401 mutant by RT-PCR.
Fig. 7. The northern hybridization of total RNA of normal leaf (N) and senescence leaf (S)
from TNG67 and SA0401 hybridized with the isotope labeled GER6 clone. The
germin-like gene (about 0.9kb) is only expressed in the senescent leaf of SA0401
mutant.
Fig. 8. The southern hybridization of digested genomic DNA of TNG67 (1) and SA0401 (2)
hybridized with the isotope labeled GER6 genomic clone (about 1.1kb). B: Bam HI, E:
Eco RI, H: Hind III, N: Nde I, S: Ssp I.
22