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ガンマ線照射によりイネに誘発された突然変異の解析と利用
誌名誌名 Gamma field symposia
ISSNISSN 04351096
巻/号巻/号 47
掲載ページ掲載ページ p. 41-46
発行年月発行年月 2010年5月
農林水産省 農林水産技術会議事務局筑波産学連携支援センターTsukuba Business-Academia Cooperation Support Center, Agriculture, Forestry and Fisheries Research CouncilSecretariat
Gamma Field Symposia, No. 47, 2008 Institute of Radiation Breeding
NlAS, Japan
41
Analysis and Application of Gamma-Ray-Induced Mutations
Ryouhei Mo阻TA
Institut巴ofRadiation Breeding, National Institute of Agrobiological Sciences
2425 Kamimurata, Hitachi-Ohmiya, Ibaraki 319-2293, Japan
Introduction
Gamma-irradiation -induc巴dmutations in higher
plants are widely used in plant breeding and research
for functional genomics. However, there has been
very little knowledge into the types of mutations that
gamma irradiation causes in higher plant g巴nomes.In
this s加dy,we used rice, the full genome sequence of
which is available, for analysis of mutations induc巴d
by gamma-irradiation.
Mutation analysis using mutants induced by
gamma irradiation
W巴 screenedrice mutants for Wx (WANG et al.,
1990), PLA (ITOH et al., 1998; KAWAKATSU et al.,
2006),巴ndospermprotein synthesis genes (TAKAIW A
et al., 1991; KRIs冊~AN et al., 1993; SHORROSH et al.,
1993), gibber巴llin-relatedgenes (SAKAMOTO et al.,
2004), and CAO (TANAKA et al., 1998)企oma popula-
tion derived from gamma-irradiation (Fig. 1). DNA
was extracted and the coding regions were amplified
by PCR. If the whole coding region is amplified, it is
judged that small mutations have occurred, so these
regions are sequenced. However, if part or all of the
coding region is not amplified, it is judged that a large
deletion or an inversion or translocation has occurred,
and detailed analysis is conducted. Investigation of a
total of 15 mutations showed that 12 were caused by
deletions, 2 were bas巴 substitutionsand 1 was an in-
version. Nine of the 12 deletions were small (1-6 bp)
and the remaining 3 were large (62.ふ129.7kbp).
There was no deletion ranging from several hundred
phenotype
Wx l CAO
PLA1
PLA2
GluA1,A2
GluB4,B5
GLB
Fig. 1 Phenotypes of mutants used in this s加dy.
z CPS,KS
KO,κAO GA30x
G101,2
42 Ryouhei MORITA
bp to several kbps. These data suggested that the mu-
tations induced by gamma-irradiation tended to be de-
letions, and that deletions of several bp or more than
several tens ofkbp tended to be readily inducible. Mu-
tations induced by ionizing radiation are thought to oc-
cur during repair of DNA double-strand breaks
(DSBs) by non-homologous enふjoining(NHEJ), a re“
pair mechanism with frequent errors (SHIKAZONO et
al., 2001; KIMURA and SAKAGUCHI, 2006). Deletions
larger than several tens of kbp are thought to be the reω
sult of 2 DSBs and the loss of the DNA fragment be-
tween them after rejoining of the strands (NAITO et
al., 2005). The fact that deletions ranging from sev-
eral hundred bp to several kbp are difficult to induce
and deletions greater than several tens of kbp are easy
to induce shows that the interval between DSBs of
DNA caused by gamma-irradiation is greater than at
least several tens of kbp.
NAITO et al. (2005) analyzed mutations derived
from gamma-irr・adiationand carbon-ion-irradiation of
pollen in Arabidopsis plants; they reported that huge
deletions of several Mbps frequently occurred in the
Ml gener・ationthat were not inherited in the M2 gen-
eration. They considered that the non幽 inheritanceof
these large deletions indicated that, as the size of the
deletions increased, the probability that the genes nec-
essary for gametogenesis or survival were deleted inω
creased too. Because these deletions are weeded out,
only deletions ranging from tens of kbp to about 100
kbp are retained in the M2 generation. In contrast, neuω
tron irradiation results in deletions ranging fr・omsev-
eral hundred bp to several kbp (SUN et al., 1992;
BRUGGEMANN et al., 1996; SALMERON et al., 1996;
Li et al., 2001). Although the mechanism for DNA
mutation by neutrons is unknown, there may be differ-
ences in the sizes of deletions that can be readily in-
duced by gamma-rays and neutrons.
Knocking out tandem-duplicated genes
In plant genomes, around 15% of the genes are
members of tandem-duplicated gene (ARABIDOPSIS
GENOME INITIATIVE, 2000; INTERNATIONAL RICE
GENOME SEQUENCING PROJECT, 2005). Because of
genetic redundancy, it is often difficult to study
tandem“duplicated gene function. B巴causegamma 1r-
radiation can induce large deletion, it is possible to
knock out tandem-duplicated gene together. An exam-
ple is the glul mutant (IIDA et al., 1997). Analysis of
this mutant, in which one of the endosper羽 proteins
(glutelin) is not synthesized, showed that it was the re-
sult of a 129.7-kbp deletion that destroyed 2 glutelin
genes, GluB4 and GluB5 (Fig. 2) (MORITA et al.,
2007). As these 2 genes are extremely similar, glute-
lin synthesis is maintained if one of them is de-
stroyed. In the glul mutant, glutelin synthesis is
thought to be lost owing to the simultaneous destruc-
tion of both genes. Thus, when the knocking out
tandem-duplicated genes is desired, the use of gamma-
rays rather than chemical mutagens may be appropri-
ate.
Isolation of genes near centromeres
Mutants showing delayed leaf yellowing during
senescence are called stay-green mutants. We
screened stay-green mutants named nyc3 from a popu-
lation derかedfrom irradiation with gamma-rays and
carbon ions. Map倫 basedcloning to isolate the NYC3
gene revealed the gene near the centromere of chro-
mosome 6. Because recombination is suppr・essednear
the centromere, mapping using 838 nyc3 homozy-
gotes only limited the candidate region to about 5600
kbp. A new approach for reducing the size of the can-
didate region was needed. We thought that mainte-
nance of very large deletions induced near the
centromere by gamma-irr・adiationwould be easier
than in other regions because of the low density of
genes near centromeres (NAGAKI et al., 2004; Wu et
al., 2004). PCR markers were prepared at about 100-
kbp intervals, and large deletions were searched for
by the absence of amplification. As expected, nyc3
and most nyc3 alleles carried deletions larger than 500
kbp. These results showed that this region can main-
tain a high 合equencyof large deletions. The smallest
deletion, about 400 kbp, was found in nyc3-3. This re“
Analysis and Application of Gamma-Ray-Induced Mutations 43
(A)
GluB5 GluB4 54280 54280 平 -R
'惨 4時十一一骨骨“時参............砂.. .... 卜
FO F1 R4 R1 F2 R2 F3 R3 R3 F3 R2 F2 R1 R4 F1 FO glu1-del-R1
(8) F1 F2 F3 FO 40866-F 54280-F F1 R1 R2 R3 R4 40866-R 54280-R 単目当坐旦1
(C)
GluB5 GluB4
YEロゴ記以;F 1 •
.J glu1-del-R1
Fig. 2 Identification of deletion in glul mutant by PCR. (A) Struc加 eof GluB5 and GluB4 and positions of primers. (B)
PCR amplification of the DNA regions around GluB5 and GluB4. (c) Structure of deletion in glul.
gion contains 15 predicted genes. We a仕巴mpteda
complementation test and confirmed that a gene
named Os06g0354700 is NYC3. (Morita et al., 2009)
Thus, gamma-iITadiation could be used to narrow th巴
size of the candidate region for genes located near the
centromere, where map-based c10ning is difficu1t.
Acknowledgments
This work was supported by grants丘omthe Min-
ishy of Agriculture, Fores仕y,and Fisheries of Japan
(Genomics for Agricultural Innovation A恥1R-0003
and GR-1003) and, in part, by the budget for nuc1ear
research of the Ministry of Education, Cul印re,
Sports, Science, and Technology of Japan.
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Analysis and Application of Gamma-Ray-Induced Mutations 45
ガンマ線照射によりイネに誘発された突然変異の解析と利用
森田竜平
農業生物資源研究所放射線育種場
〒319-2293 茨城県常陸大宮市上村田2425
放射線照射により作出された突然変異体は育種
母本として利用されるだけでなく,基礎研究リ
ソースとして遺伝子の機能解析に利用され,大き
な成果をあげている。しかし,高等植物に放射隷
を照射した掠,ゲノムにどのような突然変異が誘
発されるかという分子レベルでの知見はほとんど
存在しない。そこで,ゲノム配列が決定されてい
るイネを材料に用いて ガンマ線照射で誘発され
る突然変異の解析を行った。ガンマ線を照射した
後代集団から ,Wx遺伝子,PLA遺伝子,経乳タン
パク質合成遺伝子,ジベレリン合成および感受性
遺松子,CAO遺伝子に突然変異が生じた個体をそ
れぞれスクリーニングし PCRによるDNA変異の
解析を行った。合計15個体の突然変異体を調査し
た結果, 12個体で欠失が, 2個体で塩基置換が,
l個体で逆{立が生じていた。欠失のサイズに注目
すると, 12個体中 9個体では小さい欠失(1-6
bp)が,残りの 31障体では大きい欠失 (62.8-
129.7kbp)が生じていた。数百bpあるいは数kbpの
欠失が生じた個体は存在しなかった。以上のこと
から,イネにガンマ線を照射した際に誘発される
突然変異の多くは欠失であることと,数bpあるい
は数十kbp以上の大きさの欠失が誘発されやすい
{頃向があることが明らかとなった。
ガンマ線照射で、日大欠失が誘発されると,隣接
した捜数の遺伝子が同時に破壊される O 植物のゲ
ノム中には遺怯子の重複が頻繁に観察されるが,
ガンマ線を照射すると重複した遺{云子群をまとめ
て破壊できる可能性がある。重捜した遺伝子がま
とめて破壊された併としてglul突然変異体が挙げ
られる。膳乳タンパク質グルテリンの 1つを合成
しない突然変異体glulを解析した結果,この突然
変異体では129.7kbpの欠失により,重複した 2つ
のグルテリン遺伝子GluB4とGluB5が破壊されて
いることが明らかとなった。 2つのグルテリン遺
伝子は非常に良く似ているため,どちらか一方が
破壊された場合にはグルテリンの合成能力は保た
れると考えられる。 glul突然変異体では 2つのグ
ルテリン遺伝子が同時に破壊されたため,グルテ
リンの合成能力を失ったと考えられた。このよう
に,隣接した複数の遺伝子を同時に破壊したい場
合には,化学変異原ではなくガンマ線を変異原と
して利用することが適していると考えられた。
ガンマ線を照射した集団から,クロロフィル分
解が遅延するstaygreen突然変異体nyc3をスクリー
ニングした。マップベースクローニングによる遺
伝子単離を試みたところ,原困遺伝子は第 6染色
体のセントロメア近傍に存在することが分かっ
た。セントロメア近傍は紐み換えが抑制された領
域であるため, F2個体数を増加しでも候補領域
を5600kbp以下に絞りこむことができなかった。
そこで,欠失を利用した候補領域の絞り込み法を
考案した。ガンマ線は数十kbp以上の欠失を誘発
可能であるが,セントロメア近傍は遺伝子密度が
低いことから,他の領域よりも在大な欠失が維持
されやすいことが考えられた。そこで,約100kbp
間隔でPCRマーカーを作成し,増幅の有無により
E大欠失を探索したところ,期待遇りnyc3には約
500kbpの欠失が生じていることが分かった。ま
た,複数のアリルについて欠失の有無を調査した
ところ,多くのアリルで臣大欠失が克っかり,こ
の領域では高い頻度で在大欠失が維持されること
が分かつた。最も欠失のサイズが小さいアリルで
は約400kbpの欠失が生じていた。この400kbpに含
46 Ryouhei MORITA
まれる遺伝子数は15と予想され,相補試験により
原因遺伝子を特定することができた。 以上のこと
から,組み換えが抑制されマップベースクローニ
ングが国難なセントロメア近傍に位置する遺伝子
の候補領域の絞り込みに,ガンマ線照射が利用で
きるケースが存在すると考えられた。
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