Genome analyses have been conducted in higher organisms including rice
(Yu et al. 2002). The post-genome era has already begun for rice.
We analyzed differential electrophoresis of rice using radiation mutants
which have been obtained since 1962 (Tanaka 1967) because expressed protein
pattern of mutants was expected to be different from normal cultivar.
For the identification of mutant gene, the conventional two-dimensional
electrophoresis and mass spectrometry (MS), a proteomic approach, was
employed to the identification of mutant gene. In this paper, we attempted
mutant analysis of the ribulose bisphosphate carboxylase oxygenase large
subunit (RuBisCO/LS) genes, the key enzyme of photosynthesis, which were
identified by a combination of 2D-PAGE, MS, single strand conformation
polymorphism (SSCP), and nucleotide sequencing.
Electrophoresis of rice leaf proteins has sometimes been hindered by unknown
substances which were thought to be polyphenol and salts. Thus an acetone
precipitation was applied prior to the separation of the first dimension
on 2D-PAGE. The lysis buffer used in this experiment did not contain any
detergents (Kajiwara and Tomooka 1999) to avoid the overlapping of proteins
on 2D-gel and the RuBisCO/LS that existed in the soluble fraction (Kajiwara
and Kaneko 2002). Prominent spots were analyzed by MS/MS (Kaneko et
al 2002) to identify the gene encoding the protein by Mascot searching
(Perkins et al. 1999).
Many differences were observed in 250 radiation mutant rice lines which
had yellowish leaf against normal cultivars, Nihonmasari and Norin-22.
They showed differences in pIs, molecular weights, and/or the amounts
of expressed proteins which might be caused by mutations in the gene or
post-translational modifications on the protein (Data not shown). Another
possibility was that the expression might be changed through the effects
of protein-protein interaction networks. We thus focused on the RuBisCO/LS
because it is the key enzyme in photosynthesis.
Based on the first screening of radiation mutant lines by 2D-PAGE, five
lines, i.e. Nos. 29, 169, 180, 238, and 255, were assumed to be undergoing
mutation on RuBisCO/LS (Data not shown). The changes in pI might
have been caused by the replacement of amino acids caused by mutations
of the gene, or by post-translational modifications of the proteins. We
hypothesized that the changed pI of the proteins was related to
Theoretically, the amplified DNA of normal rice RuBisCO/LS should be 1,926
bp (Shimada and Sugiura 1991). The amplified DNA from both normal cultivars
and mutant lines was 1.9 kb in agarose gel electrophoresis; no difference
was observed (Data not shown).
Some of the five analyzed mutant lines showed differences in the SSCP
analysis (Fig. 1). All of the PCR products were digested by the sets of
AccII, AluI, HhaI, HaeIII or Cfr13I,
MboI, MspI, XspI restriction enzymes. Polymorphism
in the SSCP analysis digested by the AccII series was observed
in lines 180, 205, and 238 in the electrophoresis at 23C and 8C. SSCP
analysis also showed pattern changes in the digested PCR product of the
Cfr13I series (Data not shown). These were assumed to be changes
in restriction sites or in the secondary structure of ssDNA caused by
We identified some mutations by DNA sequencing of RuBisCO/LS gene in mutant
line No. 180 (Data not shown) in detail. In the upstream and downstream
regions of the noncoding sequence, there are several insertions, deletions,
and substitutions of nucleotides. Though their meanings are not known,
these mutations in the non-coding regions might be causing the decrease
of RuBisCO/LS gene product in mutant rice line No. 180.
The nucleotide sequence of the coding region of the RuBisCO/LS gene of
mutant rice line
180 was conserved more than that of the non-coding region.
In all, 14 substitutions of nucleotides were found in the coding region
of the No. 180 RuBisCO/LS gene. Six amino acids in RuBisCO/LS were predicted,
based on the changes from normal cultivars; they were K14Q, D94E, P176Q,
L178W, K183I, and S228A. These mutational substitutions which caused changes
in charges, and thus in the electrophoresis pattern were considered to
be changed in the first dimension. Though we do not have direct evidence
of the functional changes caused by these substituted amino acids in RuBisCO/LS
activities, some mutations, i.e. P176Q, L178W, and K183I, accumulated
in the region of the RuBisCO activase binding region.
RuBisCO/LS makes a complex with RuBisCO/SS for the fixation of carbon
dioxide. Additionally, RuBisCO is required for the Bsd2 gene product
(Brutnell et al. 1999) and RuBisCO activase (Ott et al.
2000) for full activity. Though further analysis is required, these mutations
in the RuBisCO activase binding region might affect characters, including
leaf color, through interaction with Bsd2 gene product and the
We could identify the mutant gene from protein analysis to nucleotide
sequencing using RuBisCO gene as an example. The approach used here was
considered to be one of the effective proteomic approaches to identify
the mutant gene.
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