東北大学遺伝生態研究センター年報 1998

東北大学遺伝生態研究センター年報 1998

著者

東北大学遺伝生態研究センター

発行年

1998-03

は じ め に

本｢遺伝生態研究センター｣は昭和63年に設置されて以来,平成10年3月をもっ

て10年の存続期限を迎え, 4月からは,また新たに同名の｢遺伝生態研究センター｣

として発足します｡したがって平成9年度は,現在の｢遺伝生態研究センター｣の

最後の年度として,その設置目的である｢生態系における生物種の遺伝的基礎の解

明｣の総決算を行うとともに,平成10年度からの新研究センターにおける｢遺伝生

態研究｣への足掛かりを作る年度でもありました｡

これまでの10年間を振り返ってみますと, ｢遺伝生態研究｣の旗揚げと黍明期,

展開と発展期,そして総括と将来への展望期という3期に分けて位置づけ,それぞ

れの時期の目標に沿った研究活動を計画し実行してきました｡全国の研究者との共

同研究や新たな研究の可能性を探ったワークショップの開催,国内外の研究者との

情報交換を担った｢センター通信｣や英文｢ニュースレター｣の刊行,これらはそ

れぞれの時期の｢遺伝生態研究｣の目標を達成するために無くてはならぬものであ

りました｡一方,自己点検･評価活動や外部評価を実施するなどして,現センター

の設置目的の円滑且つ効果的な達成を図るとともに,各研究者の研究活動をより効

果的に活性化することに努めてまいりました｡

毎年度の研究活動等を振り返るこの『年報』も,そうした自己点検･評価活動の

一環であります｡

存続期限の最終年度に当たる平成9年度は,市民に公開したワークショップを開

催し, ｢遺伝生態研究の台頭,発展,そして未来への展望｣及び｢地球環境が変わ

ると生物の生活はどうなるか｣について問いかけ,そして討議を行いました｡同時に,

現センターで進められている研究及び他機関の研究者との共同研究に関し, 35課題

に及ぶ公開展示発表も行いました｡また, ｢菌類における環境シグナル応答一菌学

における遺伝生態研究-｣に関して, 19名の外国人招待講演者及び9名の国内招待

講演者によるCOE国際シンポジウムを開催しました｡これらの企画はいずれも,

これまでの｢遺伝生態研究センター｣での｢遺伝生態研究｣を学問として完成させ

るための基盤となるものでありました｡

ここに現｢遺伝生態研究センター｣の最後の『年報Jを刊行するに当たり,関係

諸賢のこれまでの御協力に対し,心からお礼申し上げますとともに,新｢遺伝生態

研究センター｣におきましても,より一層の御支援と御鞭蛙を賜りますようお願い

申し上げます｡

平成10年3月

東北大学遺伝生態研究センター長

大 瀧   保

目   次

第1章 部門別研究活動

生態生理研究部門

適応生態研究部門

遺伝子生態研究部門

環境情報研究部門

臨界生態遺伝研究部門

生態システム研究部門(客貞部門)

第2章 共同利用研究

ワ ーク シ ョ ッ プ 共 同 利 用 研 究

第3章 刊行物

刊 行 物 一 覧

◎I G Eシリーズ

◎I S Kシリーズ

◎セン タ ー通信

◎ニュースレター

45 46

50

52 53

第4章 そ の 他

外国人訪問者等による特別講義及び特別研究会-･--･･･---･ 55

大学院学生等名簿

職        員

-穆命令生態生理研究部門命-令

本研究部門では,植物や微生物のそれぞれの種に特異的な遺伝子情報の発現と生態因子との関わり

を主として研究している｡すなわち,微生物から高等植物まで,すべての生物が有する特定の形態や

行動(能力)は,その生物に組み込まれている遺伝子に基礎をもつが,その発現は光や重九 さらに

温度や湿度などその生物体を取り巻いている生態因子によって大きな影響を受ける｡菌類や単細胞性

藻類などの微生物,それに野生の高等植物を材料として,これらの遺伝子発現に及ぼす生態因子の影

響とその作用機作を,主として遺伝学的また生理学的方法を用いて解析している｡平成9年度におけ

る成果は次の通りである｡

接合菌類に属し,光や重力などの外的要因に対して敏感に反応するヒゲカビヤミズタマカビを用い,

次のような成果を得た｡ (1)接合反応における細胞骨格の動向,すなわち配偶子嚢形成後に微小管の

配向に乱れが生じること,アクチンは接合過程の初期の生長の極性の変化と関係のあることを明らか

にした｡ (2)前配偶子嚢の形成においては,伸長と接合壁の形成という2つの現象が,細胞壁の構築

の異なる機構によって制御されていることを明らかにした｡ (3)いくつかの遺伝的マーカーを持った

ヒゲカどの塩素酸耐性株を単離し,野生型との間で種々の核比のヘテロカリオンを作成し,塩素酸耐

性と耐性株の核比との関係を明らかにした｡ (4)ヒゲカどの重力刺激受容体として,細胞内に含まれ

る結晶体やglobulesの関与の有無を検討した結果,結晶体が短時間内に重力刺激を感受して沈降する

ことが明らかになった｡ (5)ミズタマカどの胞子嚢柄に回転円盤上に載せて遠心力をかけ,その屈性

反応の結果からこのカどの重力屈性は胞子嚢柄の発生段階によって異なることが示唆された｡

(6)ヒゲカどの10個のキチン合成酵素遺伝子(C〃∫)ホモログを検討したところ次のことが判明した｡

①糸状生長に重要といわれるクラスⅢのホモログは見つからなかった｡ ②ノーザン解析からは,樵

定8.2kbのmRNAを転写している遺伝子の存在が明らかになった｡既知のCHS遺伝子ほぼ4kbに収

まることからその長さに興味が持たれる｡

(大瀧保,官署厚,田部茂, Christine SCHIMEK,楠井丈,堀江直司,小松京子)

黄藻多核細胞,フシナシミドロの一部を青色光で照射すると約4時間で照射域の中央から原形質の

連続した枝が発生する｡これまでに葉緑体が最初に照射域へ集合することが観察されているが,横位

置の決定から枝の発生に至る反応鎖については不明であった｡細胞骨格や核の移動と形態変化を顕徴

蛍光抗体法等を用いて観察すると共に,各種の特異的阻害剤を用いて,この光細胞形態形成反応の時

系列解析を行い,以下の結果を得た｡

葉緑体集合は蛋白リン酸化とアクチン微繊維の細網化が仲介し,枝発生に必要不可欠である｡葉緑

体におくれて,多数の核が原形質とともに照射域へ運ばれ枝予定域に集中する｡核は微小管によって

運搬される｡微小管の破壊は核の分布を乱し,原形質と核の集合を阻害し,従って枝は発生しない｡

1

-枝発生は新しい遺伝子発現を必要とする｡

どの核でどのような遺伝子が発現するのか,青色光による核分裂誘導の可能性などについてはまだ

検討中である｡多核細胞体制の成り立ちと維持機構について,広く研究していくため,マリモ,ハネモ,

粘菌など他の多核細胞を扱っている研究者と多核細胞研究会を発足させた｡ (片岡博尚,高橋文雄)

(野生シロイヌナズナの花成調節機構の遺伝学的解析)日本産の野生8系統を環境制御装置内で栽

培し,各系統の正確な開花までの日数を測定した結果, 20日から110日まで変異し,いずれの系統も

低温処理によって花成時期が早められることが明かとなった｡ FRI及びFLCの遺伝子型が決定され

ている系統(col, Ler)との交配を行い,その後代(Fl, F2)の開花までの日数を解析した｡遅咲き

が優性形質として発現し, F2で見られた開花までの日数の分離比から花成制御が2遺伝子座支配で

あることが示唆された｡両遺伝子座の近傍に位置するマイクロサテライトマーカーを用いて各F2

個体の遺伝子型を決定し,開花までの日数と遺伝子型との対応を検討した｡日本産の8系統はFRIの

み機能的な対立遺伝子を持つ遺伝子型(5系統)とFRIとFLCの両方で機能的な対立遺伝子を持つ遺

伝子型(3系統)の2つのグループに大別されることが明かとなった｡以上の結果より,日本産野生

シロイヌナズナ8系統の花成時期はFRIとFLCによって強く制御されていることが明らかとなり,

両遺伝子座による花成の制御機構が広く野生系統に存在する可能性が示唆された｡ (芝地博幸らとの

共同研究)

(ヒメムカシヨモギにおける生活史可塑性の集団比較)生活史可塑性が帰化植物の分布拡大に果た

している役割を検討するために北米原産のヒメムカシヨモギと近縁種であるオオアレチノギクを用い

て集団間における越冬型および非越冬型の比率,集団の発芽特性について検討した｡ヒメムカシヨモ

ギには二つの型が混在し北海道東部,東北内陸地域の少雪寒冷地では非越冬型個体の比率が多く,北

海道中央および南部,東北の日本海沿岸の多雪地域には越冬型の比率が高かった｡越冬型は冬季にロ

ゼットがバーナリゼーションを受けることによって,非越冬型では吸水後未発芽のままの種子が冬季

の低温を受け,未発芽バーナリゼーションという特殊な過程を経て秋の一斉開花を誘導している｡こ

れは本来二年生を示していた種の中に一年生植物の性質を付与し,より寒冷地への分布拡大を可能に

したと考えられる｡この様な分化が見られないオオアレチノギクはその分布を東北北部の多雪地域ま

でに限定されている｡発芽様式においても非越冬型個体の比率が高い地域の集団は一次休眠性が強く,

埋土種子が形成されやすいと考えられる｡ (佐野成範らとの共同研究) (石栗義雄)

- 2 -1997年1月-12月の間に刊行した論文,著書の抄録

Ootaki, T.. Y. Yamazaki, T. Noshita and S. Takahashi : Excess carotenoids disturb prospective

cell-to-cell recognition system in mating responses of Phycomyces blakesleeanus. Mycoscienee 37 (4) : 4271435,

1996.

Carotenogenic mutants of Pkyconyces, which accumulate excess β- carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated

together with a helper wild type of the same mating type against the wild type of the opposite mating

type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore

de-velopment, though the mating response was significantly activated in the early stages and abundant

zygophores were formed. Exceptional acceleration of the zygospore development under these ex-perimental conditions occurred in a regulatory albino mutant (card), which dose not accumulate excess intermediate carotenoids. Chemically･ or genetically- induced overproduction of β ･ carotene or lycopene

also inhibited the zygospore development. These results imply that the zygospore development of

Phy-Comyces is maximal when the intracellular amount of P-carotene is optimal (-wild type). and that

pher-omones act mainly ln the early stages of mating, while other factors such as the cell-to-cell recognition

system may also be involved in the later stages. IntraceHular accumulation of excess P -carotene or its

intermediates probably disturb such later- stage factors.

Kana-uchi, A., C.T. Yamashiro, S. Tanabe and T. Murayama : A rag homologue of Newospora crassa

reg-ulates morphology. Mol. Gen. Genet. 254 : 427-432, 1997.

1n order to study the role of slgnal transduction pathways in the regulation of morphology ln

NeurosL)011a CT.aSSa. We Cloned and characterized a ras homologue, termed NC- yas2. The predicted protein

product of this gene is composed of 229 amino acid residues and contains all the consensus sequences

shared by the ras protein family. The gene is located in linkage group V. An NCIYaS2 disruptant showed

morphological characteristics very similar to those of the smco7mutant, which also maps to linkage

group V. Nucleotide sequence analysis revealed that the smco7 mutant harbored a single base deletion in

the NCI YaS2 gene. which is predicted to result in the truncation of the protein product. Introduction into

the smco7mutant of an NC- ras2 clone yielded stable transformants with awild-type phenotype. The

smco7mutant exhibited very slow hyphalgrowth and the rate of conidial formation was approximately

one two-hundredth of wild type. The smco7mutation causes both the changes in the patten of hyphal growth and the defects in cell wall synthesis. Both the diameter and the length of the aplCal compartment

were shorter in the hyphae of the smco7mutant. These results suggest that NC-ras2is identical to _

smco7. and that the signal transduction pathway mediated by the Nc-ras2 protein regulates the apical

growth of hyphae, cell wall synthesis, and conidial formation in N. C71aSSa.

Miyazaki, A. and T. Ootaki : Multiple genes for chitin synthase in the zygomycetefungus Phycomyces

blakesLeeanus. J. Gen. Appl. Microbiol. 43 (6) : 333-340, 1997.

Two different newly designed primer sets were used in PCR to amplify the fragments of chitin

synthase (CHS) encoding genes (CHS) from the zygomycete fungus Phyco仇yCeS btakesleeanus, in which

the cell wall is mainly made of chitin and chitosan, deacetylated derivative from chitin. DNA sequencing

and alignment analysis of the deduced amino acid sequences showed the possible existence often

difLerent genes. Six different DNA fragments. designated PbCHSl, PbCHS2, PbCHS3, PbCHS4, PbCHS5 and PbCHS6, homologous to chitin synthase genes were identlfied in 250-bp products. From 3501bp pro-ducts, four different fragments, PbCHS7. PbCHS8, PbCHS9 and PbCHSIO, were obtained. Clustal analysis suggested that while this fungus may not have class Ⅲ-CIiS, class L,Ⅱ-and IV-CHS could be present

- 3 -in multiple forms. Only PbCHSl. PbCHS2, PbCHS7and PbCHS8genes were actively expressed - 3 -in the youPg germlings cultured in the liquid medium. TranScriptional products from PbCHS8 and PbCHSIO･ 8.2 kb long poly(A)十RNA, might suggest the existence of an unidentified type of CHS or the possibility

of a multifunctional gene including CHS function.

Yamazaki, YH A. Miyazaki and T. Ootaki : Possible involvement of chitinous extracellular fibrils on the

sexual development in the zygomycete Phycomyces blakesleeanusI Advances in Chitin Sciences Vol･ Ⅱ , ed･ by A･

Domard et al., Jacques Andre, Lyon. 133-138, 1997.

The zygomycete Phycomyces blakeslceanus, with the two mating types (+) and (-), displays a

morphologically dynamic sexual reaction The physical contact of two sexual organs is essential for the

progress and completion of mating･ Electron microscopic observation revealed･ that the two progametan一

gia of the opposite mating types were connected by extraceHular fibris･ ln in-vivo staining experiments･

the fibrils stained specifically with Calcofluor White, Congo Red, Evans Brue and fluorescein labelled

wheat germ agglutinin, suggesting that they contain chitinous materials･ Most of the stained progametan一 gia failed to develop to further stages, even after the dyes were removed. Cultivation at low (4℃) or high (25℃)temperature or under strong blue light as well as treatment with cycloheximide resulted in under-development or damage of the fibrils･ Also, the fibrils developed poorly when mutants affected in the carotene biosyn仇esis pathway were mated.

These results imply, that the extracellular fibrils are a necessary structural feature in the

mat-ing response of Pkycomyces. They may be involved in the orientation of the two progametangial cells and

also serve to strengthen the structural bond between the progametangia･

Hiroyuki, S., Y. Ishiguri and S･ Kawano : Genetic variation and relationships of Japanese populations of

oxalis co,nicuEata L. (0Xalidaceae) detected by random amplified polymorphic DNA (RAPD). plant

Species Biol. 12 : 25-34, 1997･

RAPD analysis was applied to estimate the genetic variability in Japanese populations of Oxalis comiculaEa. This species comprlSeS two floral morphs, homostyled and long- styled, with different

breed-in豆systems (i.eH autogamy in the homostyled populations andmiXed mating ip the long-styled popula-tions). The number of RAPD phenotypes tended to be larger in homostyled populations than in

long-styled ones, and genetic diversity expressed by Simpson's indices of diversity was also generally larger in former than in the latter.

To examine the effects of breeding system on the levels of genetic differentiation among popula･ tions, two- and one-level hierarchical variance partitionings were performed by the analysis of molecular

variance (AMOVA). In two level AMOVA, significant variance component between the floral morphs was observed, In one-level AMOVA, the proportions of variance partitioned among populations wlthin

each floral morph and among individuals within populations were similar in the homostyled and long-styled populations, despite the difference in breeding system･

To obtain insight into the relationships among individuals and/or poulations, principal co-｡rdinate analysis using 184 indlViduals (PCOA) , and clustering analysis of 12 populations (UPGMA)

were performed. PCOA indicated that the range of variation for homostyled individuals was more

inclu-slVe than that for long-styled ones, A UPGMA dendrogram that consisted of two clusters showed that

the long-styled populations were nested in the homostyled ones･

- 4 -その他の記事

山崎 裕,田部 茂,大瀧 保:糸状菌の生長および行動解析のための写真･ビデオ撮影法｡日本菌

学会報｡第38巻,第3号, 194-206ページ

学会等での口頭発表,展示発表

官尊 厚,田部 茂,大瀧 保:接合菌類ヒゲカどのコロニー形成変異体(col)の性質.日本菌学会

第41回大会(1997年5月24-25日,広島)

田部 茂,山崎 裕,大瀧 保:接合菌類ヒゲカどの接合過程における細胞骨格の配列｡日本菌学会

第41回大会(1997年5月24-25日,広島)

Miyazaki, A" Y. Yamazaki, T. Ootaki : Possible involvement of chitinous extracellular fibrils on sexual

development in a Zygomycetes Phycomyces blakesleeanus : 7th International Conference on Chitin and

chitosan (1997年9月3 - 5日, Lyon, France)

大瀧 保,官尊 厚:接合菌ヒゲカどの形態形成｡日本植物学会第61回大会(1997年9月18-20日,

習志野)

田部 茂,官尊 厚,大瀧 保:糸状菌ヒゲカどの生長や形態形成における温度の影響｡日本植物学

会第61回大会(1997年9月18-20日,習志野)

Miyazaki, A., T. Ootaki : Molecular cloning and characterization of chitin synthase genes of Phyco7nyCeS･

Joint Symposium of COE International Symposium of Institute of Genetic Ecology, Tohoku University

and Fifth International Symposium of the Mycological Society oHapan (1997年12月4 - 6日,仙台)

Schimek, C" T. Horie, P. Eibel, P. Galland, T. Ootaki : Organelles in the growing zone of Phycomyces and

their involvement in graviperception. Joint Symposium of COE International Symposium of Institute

of Genetic Ecology, Tohoku University and Fifth lnternational Symposium of the Mycological Society

oHapan (1997年12月4-6日,仙台)

田部 茂,官尊 厚,大瀧 保:接合菌ヒゲカどの接合過程における生長様式｡日本植物学会東北支

部第11回大会(1997年12月13-14日,盛岡)

堀江直司, SchimekChristine,大瀧 保,三原 等:接合菌ミズタマカどの胞子嚢柄における,重力

及び遠心力刺激に対する反応の解析｡日本植物学会東北支部第11回大会(1997年12月13-14日,盛

岡)

高橋文雄,菱沼 借,片岡博尚:フシナシミドロ多核細胞の青色光照射域で誘発される核分裂と分枝｡

日本植物生理学会1997年度年会(1997年3月27-29日,京都)

片岡博尚,高橋文雄:青色光によるフシナシミドロ分枝誘導の初期過程一阻害剤の効果｡日本植物学

会61回大会(1997年9月18-20日,習志野)

- 5 -川井浩史,佐々木秀明,神谷充伸,大橋洋平,片岡博尚:褐藻アミジグサ目における細胞内硫酸イオ

ン蓄積の分布｡日本植物学会61回大会(1997年9月18-20日,習志野)

高橋文雄,菱沼 佑,片岡博尚:フシナシミドロの成長点誘導における核と微小管の動態｡日本植物

学会61回大会(1997年9月18-20日,習志野)

片岡博尚,高橋文雄:青色光によるフシナシミドロ分枝誘導反応の時系列解析｡日本植物学会東北支

部11回大会(1997年12月13-14日,盛岡)

- 6 -鯛埠命令適応生態研究部門噂噛堵埠命

当研究部門では,耕地生態系を含む生態系に存在する植物の遺伝的変異を解明し,種々の環境スト

レスへの適応に有用な遺伝子資源の開発に資するとともに,これら特定環境に対する植物の反応性,

適応性を研究している｡平成9年度は研究材料に線虫も加えて分子生物学的アプローチをさらに強化

し,次の成果を得た｡

これまで,植物がストレス環境を回避する機構を解明するために,水分屈性と重力屈性の発現機構

について突然変異体を用いて解析してきた｡この屈性発現にカルシウムが関与することが示唆された

ので,植物特異的なカルシウム依存性キナ-ゼ(CDPK)遺伝子の単離を試みた｡その結果,エンド

ウの根においては,少なくとも7分子種のCDPK遺伝子が発現しており,そのうち3種は外部からの

カルシウム添加によりその発現が上昇することがわかった｡今後,それらの発現ならびに蛋白質リン

酸化活性と重力屈性や水分屈性の関係を解析する｡

水分屈性における偏差成長の制御因子として細胞壁伸展性の変化が示されたので,エンドウの根か

らエンド型キシログルカン転移酵素(EXGT)遺伝子の単難を試みた｡その結果,根で強く発現する

新しいタイプのEXGT遺伝子の全長を得ることに成功した｡この遺伝子のmRNA発現パターンを解

析した結果,根の水分屈性の発現とともにその蓄積が変動することがわかった｡また,水分屈性の発

現と関連してオーキシン制御遺伝子も変動することが認められた｡

重力屈性のメカニズムとその遺伝的制御機構を解明するため,オオムギの重力屈性突然変異体と野

生型の芽ばえに重力刺激処理を行い,それぞれからcDNAライブリーを作成し,サブトラクト法を行っ

たoその結果澄現量に差のあるクローンとしてリボゾームタンパク,シクロアルセノール合成酵素,

オリゴサッカリン転移酵素をコードする遺伝子が単離された｡

(高野 守,小沼芳和, Jnrgen Marquardt,藤井伸治,東谷篤志,高橋秀幸)

ウリ科植物の芽ばえのペグ(peg)は,重力形態形成として肱軸基部の皮層細胞の成長極性が変化

して形成される突起状組織である｡この重力形態形成にはオーキシンとエチレンが関与することが示

されたので,オーキシン制御遺伝子とエチレン受容体遺伝子をキュウリ由来のcDNAライブラリー

より単離するとともに,それらの塩基配列を決定し,ノーザンハイプリグイゼ-ション法とinsitu

法で遺伝子発現を解析した｡また,ペグ形成部位における皮層細胞の成長極性の変化がみとめられる

直前に,表層微小管の配向が変化していることがわかった｡さらに,コルヒチンの処理がペグ形成に

阻害的に作用し,ペグ形成に微小管の配向が重要な役割を果たしていることが示唆された｡

(藤井伸治,小林真由美,東谷篤志,高橋秀幸)

半乾燥地に適応した紅菅麦の探播き耐性のメカニズムを解明することを目的に,探播きとエチレン

によるコムギの第一節間伸長を解析した結果,エチレンが第-節間の伸長に不可欠であり,探播きに

よって増大するエチレン生成が第一節間伸長を促進するものと考えられたが,探播き耐性の品種間差

- 7 -異をエチレン生成量およびエチレン感受性(エチレン受容体遺伝子の発現)で説明することはできな

かった｡ -さらに,日本品種の中でもタクネコムギが,エチレンに反応して第一節間を著しく伸長させ

ることをみいだしたので,タクネコムギの育種素材となった品種のエチレン反応性を調べた｡

(西滞武明,東谷篤志,高橋秀幸)

生物が多様な環境に適応し進化してJ5た過程は,それぞれ個々の生物種が独自の遺伝的多様性を獲

得した結果である｡この多様性の獲得機構は,減数分裂過程を含む生殖細胞(配偶子)形成において

最も顕著に行われることが知られている.そこで土壌自活性の線虫(C･ elegans)を材料に用い,減数

分裂過程において高頻度に生じる遺伝子組換え機構ならびにその制御機構についての解析を始めたo

これまでのところ,遺伝子組換えに関与する大腸菌RecA様遺伝子とcheckpoint制御に関与するヒ

トat.axia telangiectasia mutated gene (ATM)のそれぞれの線虫における相同遺伝子の単牡に成功し･

｡DNAの全塩基配列決定を行った｡       (東谷篤志,高橋秀幸)

ウリ類の花の雌雄性分化とエチレンレセプター(ETRl/ERS)との関連を検討するため,キュウリ

の雌雄性分化において機能するエチレンレセプター様遺伝子群の単髄を試みた｡その結果,これまで

に単離したものに加えて新たなエチレンレセプター様遺伝子群をみいだし,それらの発現が雌雄性分

化に果たす役割を花芽の発育段階との関係から解析している○ (山崎聖司,藤井伸治,高橋秀幸)

zoysia属植物の分布については既に報告したが,本年度は･本属植物のうち自生種を対象として,

本属の塩生植物的特性の検討を行った｡本属は,

Chloridoideaeに属するが,本亜科の葉面にはmic-r｡hairsが存在し,これが排塩機能をもつsaltglandとなっている｡ Zoysiaの二,三種については,こ

れの存在が確認されているが,自生種すべてについては未だ確認されておらず,南西日本に分布する

中小型種について,次年度に検討する予定である｡       (庄司舜-)

1997年1月-12月の間に刊行した論文,著書の抄録

Takahashi, H∴ Hydrotropism : the current state of our knowledge･ J･ Plant Res･ 110 : 163-169, 1997･

The response of roots to a moisture gradient has been reexamined, and positive hydrotroplSm has been demonstrated in recent years. Agravitropic roots of a pea mutant have contributed to the studies on hydrotrop-ism. The kinetics of hydrotropic curvature, interactions between hydrotropism and gravitropism･ moisture gra-dients required for the induction of hydrotroplSm･ the sensing site for moisture gragra-dients, characteristlCS Of hyd-rotroplC Signal and differential growth, and calcium involvement in signal transduction have been subjects of these studies, This review summarizes the current state of our knowledge on hydrotropism in roots･

Takano, M., H. Takahashi and H, Suge:Calcium requirement for the induction of hydrotropism and en･

hancement of calcium-induced curvature by water stress in primary roots of pea･ Pisum sativum L･

plant Cell Physiol. 38(4):385-391, 1997.

p｡sitive hydr.tropic Curvature in the roots of the agravitropic pea (Pisum sativum L) mutant･

ageotropum, occurred when the root cap was exposed to a gradient of water potential by an asymmetric application of agar containing sorbitol lTakano et al. (1995) planta 197 : 410] ･ As previously reported

- 8 -lTakahashi and Suge (1991) physt'ol. Plant. 82:24], in this study the hydrotropic response due to un-ilateral application of sorbitol to the root cap was totally inhibited by pretreatment with ethyleneglycoト bis- (P -aminoethylether) N, N, N', N'-tetraacetic acid (EGTA). However. hydrotropic response of the EGTA-treated roots was recovered only when EGTA was replaced by a 10 mM calcium (CaCl2) Solution prior to hydrostimulation. A calcium channel blocker, lanthanum (LaCl｡), also inhibited hydrotropic

curvature of ageotrop7im roots. Whereas the hydrotropic response was affected by neither nifedipine nor

verapamil. Application of calcium ionophore, A23187, resulted in a significant promotion of hydrotropic

curvature. Furthermore, ageoEroPwm roots Curved away from a calcium source when an agar block

con-taining 10 mM calcium was asymmetrically applied to the root cap. This calcium･induced curvature was

found to be accelerated by water stress and significantly inhibited by LaC13. While the calcium- induced

curvature commenced within 1 h after application. hydrotropic curvature became visible 3 to 4 h after

an exposure to a gradient of water potential. These results indicate that apoplastic calciumand its innux through the plasmamembrane are involved in the induction of hydrotropism in roots. A gradient of water

potential in the root cap may cause a physiological change that is mediated by calcium, which ultimately

leads to the curvature in仇e elongation region associated with the hydrotropic response･

Hirasawa, T., H. Takahashi, H. Suge and K. Ishihara : Water potential, turgor and cell wall properties

in elongating tissues of the hydrotropically bending roots of pea (Pisum sativum L.). plant, Cell and

Environment 20 : 38二卜386, 1997.

The hydrotropic bending of roots of an ageotropic pea mutant, ageoEゆm, was studied in humid

air in a chamberwith a steady humidity gradient. We examined the effects of atmospheric humidity

around the root on the water status of root tissues, as well as the wall growth and the hydraulic

prop-erties of the elongating tissues. Atmospheric humidity at the surface of the root was clearly lower on the

side orientated towards the air with lower humidity than on the side orientated towards the air with higher humidity. However, there were no differences in water potential and osmotic potential between the tissues that faced air with higher and lower humidities in the elongating and mature regions. Plastic ex-tensibility was higher in the tissues that faced the air with lower humidity than in the tissues that faced the air with higher humidity. No differences in turgor pressure and yield threshold were observed

be-tween the tissues that faced air with higher and lower humidities. Therefore, the extensibility of the cell

wall appeared to be responsible for the different growth rates of tissues in root hydrotropism･ A further

probable cause of the hydrotroplCal bending of roots is changes in the hydraulic conductance in the elongating tlSSueS. Since the hydrotropic bending of roots occurred only when a root tip was exposed to a humldity gradient, hydrotropism nigh occur after perception of a difference in humidity by the root tip, With accompanying changes in cell wall extensibility and hydraulic conductance･

Takahashi, H.:Gravimorphogenesis:gravity-regulated formation of the peg in cucumber seedlings･

Planta 203 : S164-S169, 1997,

Formation and/or positioning of a protuberance. or peg, in cucumber (Cucumis sativus L.)

seedlings is controlled by gravity･ The gravisensing apparatus for this gravimorphogenesis is possibly shared with that of gravitropism. Sheath cells in the transition zone between stem and root contain sedi-mentable amyloplasts, but amylopasts in cortical cells (peg cells) do not sediment uniformly. These puta-tive statoliths appear before peg initiation becomes visible on the lower side of the transition zone･ The increased level of auxin on the lower side of the transition zone, which may occur following gravipercep-tion, could be a factor responsible for peg development･ This system may be a useful model in spacenight experiments for understanding the mechanisms underlying gravity- regulated formation of the peg･ and its interaction with gravitroplSm, auXin physiology and cell growth polarity･

- 9 -suge, HH T. Nishizawa, H･ Takahashi and K･ Takeda:Phenotypic plasticity of internode elongation

stimulated by deep- seeding and ethylene in wheat seedlings･ Plant, Cell and Environment 20 : 961-964･

1997.

Deep-seeding and ethylene were found to stimulate extension growth of the first internode of

intact wheat (Triticum aestivum L.) seedlings in darkness. Seedlings of Hon Mang Mai emerged from

much deeper in the soil than the seedlings of the other varieties used and their first internodes elongated to a much greater extent in response to ethylene･ Carbon dioxide slowed elongation of the first internode

and inhibited ethylene action. Elongation of the first internode due to deep-seeding and ethylene treat一

ment showed high heritabilities, suggesting a genetic basis underlying those traits･

Higashitani, A., N. Higashitani and KI Horiuchi : Minus･strand origin of filamentous phage versus

trapscriptional promoters in recognition ofRNApolymerase･ Ptoc･ Natl･ Acad. Sci･ USA 94 :

2909-2914, 1997.

Replication of complementary-strand DNA in filamentous phages is initiated by a primer RNA

that is synthesized at the minus-strand origin on the viral single- stranded DNA by Escherichia coli RNA

polymerase holoenzyme containing the ♂ 70 subunit･ We have demonstrated that the affinity of RNA

polymerase in viEyo to the origin is about 16-fold higher than that to the tacUV5 promoter･ We have also

shown that the temperature dependence of the primer RNA synthesis is much lower than that of lacUV5

transcription･ The high affinity of RNA polymerase to the origin depends on the single strandedness of

the "-10 region." A nucleotide sequence of the nontemplate strand in the -10 region was found to be im-portant for the function, but that of the template strand was not･ These observations suggest that ♂ 70

subunit directly interacts with the single- stranded nontemplate strand containing adenine residue(S) at the -10 region of promoter.

Higashitani, A･, Y･ Ishii, Y･ Kato and K･ Horiuchi : Functional dissection of a celトdivision inhibitor･

sulA, of Escherichia coli and its negative regulation by Lon･ Moll Gen･ Genet･ 254 : 351-357･ 1997･

sulA is induced in Eschcrichia coLi by the SOS response and inhibits ceH division

throughin-teraction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletios of SulA and a series of alanine substitution

mutants. Arginine at position 62, 1eucine at 67, tryptophan at 77 and lysine at 87･ in the central region of

sulA, Were all essential for the inhibitory activity･ Residues 3-27 and the C-terminal 21 residues were dispensable for the activity･ The mutant protein lacking N-terminal residues 3-47 was inactive･ as was that lacking the C-terminal 34 residues･ C-terminal deletions of 8 and 21 residues increased the growth

inhibiting activity in lon十cells, but not in Lwr cellsI The wild-type and mutant SulA proteins were

is0-1ated in a form fused to E. coLi maltose-binding protein, and tested in vitro for sensitivity to Lon

pro-tease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids･ but did

not degrade the derivative lacking 21 residues at the C-terminus･ Futhermore, the wild-type SulA and

the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not･

MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by

Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal

region of SulA the protein was stable in lon十cells･ These results indicate that the C-terminal 20 residues

of SulA permit recognitlOn by, and complex formation with, Lon, and are necessary･ but not sufficient･ for degradation by Lon.

-10-Suge, H., Takahashi and K. Takeda:The rate of leafemergence and its heritability in wheat.Jpn.∫.

Crop Sci. 66:6561662, 1997.

Using two varieties of wheat with genetically differlng leaf lengths, seveal parameters

concern-ing leaf growth were examined under controlled environmental conditions. Heritabilities for the para一

meters of leaf growth were calculated based on measurements of F2 generation of the hybrid between

these two varieties. The rate of leaf emergence (i. e., the speed oHeafemergence per unit time or per

unit length) was almost the same between the two parent varieties, although the average length oHeaf

and average number of days required for the emergence of one leaf were different due to the difference in leaf length. Heritabilities for the average length of leaf and for the days required for the emergence of

one leaf were 77.7 and 85.2%, respectively. In F2 generation, however, no correlation was detected

be-tween the two characteristics (r-0.152). The heritabilities for the speed of leaf emergence per unit time

and unit length were 55.5 and 45.2%, respectively, suggesting that leafgrowth rate (speed ofleaf

growth per unit timeand per unit length) can be changed by selection.

Fujii. N. : Pattern of DNA binding of nuclear proteins to the proximal Agrobacterium rhizogenes roIC

promoter is altered during somatic embryogenesis of carroL Gene 201 : 55-62, 1997･

carrot cells cultured in vitro in a medium supplemented with 2,4- D (2,4-dichlorophenoxyacetic acid) proliferate as unorganized cell clusters. Upon removal oE 2,4-D from the culture medium. these

cells undergo somatic embryo formation globular, heart.and torpedo stagges. Since the proximal1255'bp

upstream region of the roLCgene of the Ri plasmid confers somatic embryogenesis- related activation on

the uidA gene in transgenic carrot cell culture, We investigated the interaction of nuclear proteinswith

the proxima1-255 bp upstream sequences to characterize the mechanism of somatic embryogenesis-related

activation. Gel retardation experiments revealed that there were several different profiles of the relative levels of DNA binding activities in nuclear protein extracts from calli, PEMs (proembryogenic masses) ,

globular embroyos, and heart/torpedo embryos. The binding activity associated with a fragment (-203

bp t0 -92bp) of one protein (BI) was most abundant in globular embryos. Another DNA binding protein (Al一) showed the highest DNA binding activity in calli, but had low binding activities in PEMs and glo-bular embryos. Such an altered pattern of DNA binding activities of nuclear proteins may contribute to

somatic embryogenesis- related activation of the 7101C promoter. Competition experiments with oligonuc-leotides revealed that the BI protein interacts with AT- rich sequences.

西洋武明,菅  洋,高橋秀幸:深播き条件下におけるコムギの第-節間伸長とエチレン反応性の遺

伝について.日作東北支部報(TohokuJournal of Crop Science). 40:4ト43, 1997.

深播き耐性の強い紅菅麦と普通品種のハルヒカリ,ハルユタカの間で交雑を行い,雑種第二

代(FZ)の第一節間の伸長に対する深播きおよびエチレンの作用を検討したoその結果,深播きおよび､

エチレンに対する第-節間の反応性には複数の遺伝子が関与するものの,両者の遺伝力は著しく高い

値を示し,これらの性質を利用した選抜･育種が可能であることが示された｡

Marquardt, J. and E. Rhiel : The membrane･ intrinsic light-harvesting complex of the red alga Galdieria

sulPhuraria (formerly Cyanidium caldarium) : biochemical and immunochemical characterization･

Biochim. et Biophy. Acta 1320: 153-164, 1997.

The membrane-intrinsic light-harvesting complex of the red alga Galdieria sulphuraria

(formerly Cyanidium caldarium) could be isolated by gel-electrophoresis as a green bandwith an

apparent molecular mass of about 20kDa･ The band had a long-wavelength absorption maximum at 672 nm and a fluorescence maximum (77K) at 680mm and reacted with an antibody against light･harvesting proteins of higher plant Photosystem L Screening of thylakoid membranes with antisera directed against

→1日-Various chlorophyll a / b and chlorophyll a /c llght-harvesting proteins indicated the existence of at

least 4 distinct llght-harvesting polypeptides with apparent molecukar masses between 17 and 20 kDa･ Isolation of Photosystem I and of a fraction enriched in Photosystem II showed that these polypeptides are exclusively bound to Photosystem I, thus forming a holocomplex which binds at least 205 molecules

ofchlorophyll α, and 33 and 37 molecules of zeaxanthin and β-carotene, respectively･ Additionally,

there is some evidence for the existenJCe Of a second Photosystem I pool without light-harvesting

com-plexes. In-Vitro translation experiments showed that at least two of the five polypeptides which

consti-tute the membrane- intrinsic light- harvesting complex of Galdieria suLphuraria are translated from the

poly (A) -en-riched RNA fraction. They could be immunoprecipitated as preproteins being 3 to 4 kDa

larger in size than the mature polypeptides･

Marquardt, ∫., H. Senger., H. Miyashita, S. Miyachi and E･ Morschel : Isolation and characterization of

biliprotein aggregates from- Acaryochloris marina･ a Prochlwon- like proka.ryote containing mainly chlorl

ophyll d･ FEBS Letters･ 410:4281432, 1997･

phycobiliprotein aggregates were isolated from the prokaryote Acaryochloris mayiyza･ Containing chlorophyll a as major plgment. In the electron microscope the biliprotein aggregates appear as

rod-shaped structures of26･6×11･3 mm, composed oHour ring･rod-shaped subunits 5･8 nm thick and ll･7 mm

in diameter. Spectral data indicate that the aggregates contain two types of biliproteins : phycocyanin

and an allopycocyanin-type plgment.with very efficient energy transfer from the phycocyanin-to

allophycocyanin- type constituent･ The chromophore- binding polypeptides of the plgmentS have apparent

molecular masses of 16.2 and 17.4 kDa. They crossreact with antibodies against phycocyanin and

allophycocyanin from a red alga.

Rhiel E., J. Marquardt, M. Eppard. E. Morschel and W. E. Krumbein : The right harvesting system of

the diatom Cyclotella cryPtica. Isolation and characterization of the main right harvesting complex and

evidence for the existence of minor pigment proteins･ Bot･ Acta･ 110: 1091117, 1997･

The main chlorophyll a /c light harvesting comeplex of the diatom CycLotella cryL'tica was

is0-ldted by sucrose density gradient centrifugation･ It consisted of two polypFptides of Mrs 18000 and

22000. Both polypeptides and fragments thereof, obtained by formic acid treatment･ were blocked at their N- termini. An antiserum raised against the two subunits selectively lmmunOlabeled the thylakoid within th｡ ｡hl｡roplasts. The sub-units were nuclear encoded and could be immunoprecipitated from poly (A) +

RNA as precursor proteins in the Mr range of 20000 to 24000･ The existence of minor chlorophyll

pro-tein complexes and their possible function in light climate adaptation processes was investigated in cells adapted to low light and high light conditions･ Low light grown cells contained more fucoxanthin and

less β -carotene relative to chlorophyll α than high light adapted cells. The xanthophyll cycle pigments diatoxanthin and diadinoxanthln increased five- fold relative to chlorophyll α under high light condltions･

western-immunoblotting experiments with antisera raised against several chlorophyll a/b and chlorl

ophyll α〟 antenna complexes demonstrated that, beside the dominating chlorophyll α〟 light harvesting

complex.minor antenna complexes might exist, which, in part･ seem to react to the light climate applied･

M. Umeda" N. Fujii., Y. Manabe and H. Uchimiya : Molecular and biochemical characterization of a

proteasome subunit from rice and carrot cellsI Mol･ Gen･ Genetl 255 : 19-27･ 1997･

proteasomes function mainly ln the ATP- dependent degradation of proteins that have been

con-jugated with ubiquitim We isolated a CDNA clone for a rice protein that exhlbited high homology to

sub-unit CZ of proteasomes, Southern blot analysis revealed that the corresponding gene was present as a

single copy in the rice genome･ After fractionation of a crude extract from cultured cells･ a 35-kDa

pro-tein that cross-reacted with antibodies against the C2 subunlt Was recovered in the peak fraction of both

-12-20 S and 26 S complexes. The same antibodies cross-reacted with two proteins in seedlings, one of

which was the same as thatdetected in cultured cells. The level of the protein was reduced in roots under conditions of high salinity. The 35-kDa protein was not detected in the nuclei of rice or carrot cells. However, during somatic embryogenesis of carrot cells, the C2 subunit was found in the nucleus at the globular stage, and it gradually disappeared in the period from the heart to the torpedo stage. Cells at the globular stage are proliferating rapidly, thus, it is possible that proteasomes are associated with the proliferation of plant cells.

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藤井伸治:微小重力下における植物の成長と遺伝子発現に関する研究｡平成8年度宇宙基地利用基礎

実験費研究報告書｡宇宙科学研究所｡ 191-195.

高橋秀幸,三井久幸,藤井伸治,阿部清美,本河光博,茂木 巌:植物および微生物の強磁場に対す

る反応の解析｡東北大学金研強磁場超伝導材料研究センター平成8年度年次報告書｡東北大学金属

材料研究所｡ 29ト294.

学会等での口頭発表,展示発表

藤井伸治,高橋秀幸:キュウリ芽生えの重力形態形成時におけるETRl類似遺伝子の発現｡日本植物

生理学会第37回大会(1997年3月27-29日,京都)

高野 守,藤井伸治,西谷和彦,平沢 正,高橋秀幸:エンドウ根の水分屈性におけるエンド型キシ

ログルカン転移酵素(EXGT)遺伝子の発現｡日本植物生理学会第37回大会(1997年3月27-29日,

京都)

-13-西滞武明,菅   洋,高橋秀幸,武田和義:コムギ深播き適応性の品種間差異とエチレン反応性の

肺劃=_ついて｡第203回日本作物学会(1997年4月2-3日,水戸)

高橋秀幸:植物のメカノセンサー｡日本学術会議宇宙利用シンポジウム(1997年7月16-17日,東京)

高野 守,藤井伸治,西谷和彦,平沢 正,高橋秀幸:疑似微小重力下における根の屈性運動｡日本

学術会議宇宙利用シンポジウム(19J97年7月16-17日,東京)

Fujii, N., 班. Takahashi : Expression of ETR1-like genes during gravity･regulated morphogenesis in

cucumber seedlings. Plant Biology 97 (August 2-6, 1997, Vancouver, BC)

Takano, M., N. Fujii, K. Nishitani, T. Hirasawa, H. Takahashi : Possible relationship between the

ex-pression of EXGT gene and differential growth in hydrotropically responding roots of ageotroj･um

∼ pea. plant Biology 97 (August 2-6, 1997, Vancouver, BC)

西洋武明,菅  洋,高橋秀幸:探播き条件下におけるコムギ第-節間伸長とエチレン反応性の遺

伝について｡第40回日本作物学会東北支部講演会(1997年8月24-25日,北上)

Higashitani. A., K. Horiuchi : Initiation mechanisms of DNA replication in filamentous bacteriophages.

National Institute of Genetics, hternational Symposium on Gene Functions to Cell Differentiation.

(september 16-19, 1997, Shizuoka)

阿部清美,藤井伸治,茂木 巌,本河光博,高橋秀幸:植物の強磁場に対する成長反応の解析｡日本

宇宙生物科学会第11回大会(1997年9月19-20日,東京)

Marquardt, J., K. Abe, N. Fujii, H. Takahashi : Isolation of genes involved in gravitropism.日本宇宙生

物科学会第11回大会(1997年9月19-20日,東京)

藤井伸治,高橋秀幸:キュウリ由来オーキシン制御遺伝子とエチレン受容体(ETRl)類似遺伝子の構

造解析｡日本宇宙生物科学会第11回大会(1997年9月19-20日,東京)

高野 守,藤井伸治,東谷篤志,高橋秀幸,西谷和彦,平沢 正,山下雅道:微小重力下における根

の姿勢制御関連遺伝子の発現｡日本宇宙生物科学会第11回大会(1997年9月19-20日,東京)

高橋秀幸,藤井伸治,菅  洋:ウリ科植物の重力形態形成-キュウリ芽ばえのペグ形成機構につい

て｡第61回日本植物学会(1997年9月18-20日,習志野)

Takahashi, H∴ HydrotroplSm in roots : an adaptive response of plants to arid environment.

Interna-tional Symposium on Cellular Stresses (October ll-12, 1997, Seoul)

Higashitani, A., S. Sato, T. Ishihara, H. Takahashi : Search for homologs of human ATM gene in C.

ele-gans. lst 3R (Replication, Recombination and Repair) symposium. (October 13-16, 1997, Hyogo)

高野 守,藤井伸治,東谷篤志,平沢 正,西谷和彦,高橋秀幸:根の水分屈性発現時に変動する細

胞壁酵素遺伝子｡根の研究会第8回大会(1997年11月22日,金沢)

東谷篤志,佐藤修正,石原 健,高橋秀幸:線虫におけるヒトATM相同遺伝子の解析｡第20回日本

分子生物学会年会(1997年12月16-19日,京都)

ー14--命尊命遺伝子生態研究部門命令-埠

当研究部門では, ｢人為的に遺伝子を改変した生物の生態系における動態｣を主要テーマとして,

植物及び微生物を対象に研究を進めている｡本年度は以下のような研究を行った｡

今後の研究課題｢植物ゲノムの改変法の開発｣を視野にいれて,細胞融合,遺伝子導入の研究を継

続した｡本年度は,イネとオオムギの体細胞雑種を用いてcDNAを作成し,ディファレンシャルディ

スプレイ法により,体細胞雑種中で再構成された遺伝子,または,オオムギ由来の遺伝子で発現して

いるものを単離し, 8種の遺伝子断片のクローニングとシークエンスした｡今後は,これらの遺伝子

の機能について調査する予定である｡エレクトロポーション法でイネのカルスに遺伝子を直接導入す

るための基礎実験を行い,形質転換体を得ることが出来た｡

ダイズは,カルスからの植物の再分化が非常に困難な重要な作物である｡そこで,種子を用いた形

質転換系の確立を目指した｡pIG121をアグロバクテリウム液を,種子の肱に注射したところ,10-20%

の割合で形質転換体が得られることがわかった｡今後,この方法により,農業上有用な遺伝子(LEA)

を導入する予定である｡

昨年度に引き続き,アスパラガスの性の分化に研究を進めた｡ (1)花芽形態形成遺伝子群の単離:

ABCモデルのクラスC属に属し,雌ずいの形態形成に関連すると思われるAGAMOUSのホモログ遺

伝子を単離するため,昨年単離された約170bpのMADS boxの塩基配列を基に, RACE法を用いて,

3'側の塩基配列の単離を試みた｡その結果,シロイヌナズナのAGAMOUS遺伝子の728-1407bpに相

当する断片が得られ,全部で, AGAMOUS遺伝子の530-1407bpに相当する788bpの塩基配列が単離

された｡ (2)アスパラガス植物の染色体解析:核型分析及び性染色体の識別法を確立しつつ, FISH法

を用いて, 45srDNA遺伝子座の解析を行った｡ (3)アスパラガスにおける形質転換系の検討:将来,

単離した性に関わる遺伝子の発現を見るために,形質転換系の検討を行った｡ pIG121のアグロバク

テリウムを用いて形質転換を行い, 8個体の雌個体中うち, 5個体が形質転換体であった｡

(北揮絵里奈,エフェンディ,木坂広明,雀 賢美,菅野 明,亀谷寿昭)

遺伝子改変微生物の生態とその利用:遺伝子を改変して作出した非病原性軟腐病菌は微生物農薬と

して1997年7月に認可された｡この微生物農薬は植物細菌病に対する防除剤として日本で開発された

第-号である｡ 1998年春,この微生物農薬はバイオキーパーの商品名で発売の予定である｡植物細菌

病の防除剤として汎用されている銅剤との併用を可能にし減農薬に資するため,生物防除剤の銅剤耐

性化を図り,キノンド-およびZボルドーに強い耐性を持つ生物防除剤を作出することが出来た｡つ

ぎに,ハクサイ汁液中のバクテリオシン誘発因子の精製を進めた結果,単糖にアミノ基の結合した物

質であることが判明した｡なお､この物質は軟腐病菌に対しペクチナ-ゼの生産も誘導することが分

かった｡トランスポゾン挿入変異によりバクテリオシン生産能を欠失した菌株を用い,バクテリオシ

ー15-ンの生産に関わる遺伝子を解析した｡その結果,バクテリオシンの調節遺伝子を解明することができた｡

(郡司祐一,荘 敦,チレメ･アンバービン･ジェドゥ,菊本敏雄)

1997年1月-12月の間に刊行した節文,著書の抄録

Kanno, A" M. Nakazono, A. Hirai and T･ Kameya : A chloroplast derived trnHgene is expressed in the

mitochondrial genome of gramineous plants･ Plant Molecular Biology 34 : 353-356･ 1997･

We reported previously that the mitochondrial sequence that contains the chloroplast- derived

trnHgene has been highly conserved in the region around one terminus oE thejunction between

chloroplast-derived and mitochondrion-specific sequences in most of the gramineous plants analyzed [15]. The results of RTIPCR, northern hybridization･ in vityo capping and'ribonuclease protection

ex-periments show that the chloroplast- derived ErnHgene is transcribed from a putative promoter that is

located in the mitochondrion-specific sequence･ Gene expression in this region seems to be correlated with the conservation of the sequence at the junction between the chloroplast- derived fragment and the mitochondriom- specific sequence.

Kanno, AリY. Lee and T･ Kameya : The structure of the chloroplast genome in members of the genus

AsJ'aragus. Theor･ Appl･ Genet･ 95 : 119611202･ 1997･

Ⅰ｡ a previous study we constructed a physical map of the chloroplast DNA (ctDNA) of garden

asparagus (Asparagus oNiciulis L. cv 'Mary Washingron 500W': Lee et all 1996)･ In the present study

we have constructed and compared HindIII and XhoI restriction maps of the ctDNAsof eight species of

AsJ'araguS : namely, A. o/ficinalis, A. schoberioides, A･ cochchinchinensis, A･ J'lumosus･ A･ /alcatus･ A･ sprengeri, A. virgatus and A. asJ･aragoidesI The ctDNA of A･ ojficinalis has 32and 23 sites that are recog-nized by Hi7dIIl and XhoI, respectively. Taking the physical map of the ctDNA of A･ offuinalis as a

standard, we found that the ctDNAs of A jblcat7IS. A sPrengeri, and A･ asJ,aragoides each had one

addi-tional Hi7dIII site and lacked one Xhol site. We also detected two relatively large deletions of nucleotides

in the ctDNA from A. Cochinchinensis by sequencing analysis･ Both of these deletions were located in a

non-Co°ing region between the ndnC and t7mVgenes and were 95 bp and 347 bp in length･ respectively･

The regions around the deletions exhibited strong homology･ and short direct- repeat sequences were de-tected at the borders of the deletions, an indication that these deletions were the result of intramolecular recombination mediated by the direct repeats･

Kanno, A" H. Kanzaki and TI Kameya : Detailed analyses of chloroplast and mitochondrial DNAsfrom

the hybrid plant generated by asymmetric protoplast fusion between radish and cabbage･ Plant Cell

Reports 16 : 479-484. 1997t

ln a p,evious report, intergeneric somatic hybrids between red cabbage (Brassica oLeracea LI var. capiEaEa) and radish (RaPhanus sativus L. cv･ Shougoin) Were produced by protoplast fusion. Plant morphology, chromosome number, isozyme patterns･ and Smal cleavage pattern of chloroplast DNA indi-cated that the hybrid plants have the red cabbage nucleus and the radish chloroplasts･ In this report, we analyzed the organization of chloroplast and mitochondrial DNAs from this hybrid using Southern

hybr-dization. The restriction patterns of almost all regions of the chloroplast DNA from the hybrid were

similar to that of radish, except for one region near the rPs16gene, which encodes the chloroplast

ribo-somal protein S16･ In contrast to chloroplast DNA･ the restriction pattern of mitochondrial DNA from the

hybrid was quite different from that of the parents･

-16-Lee, Y., Kanno and T. Kameya : Phylogenetic relationships in the genus AsJ)wagus based on the

restl･ic-tion enzyme analysis of the chloroplast DNA. Breeding Science 47 : 375-378. 1997.

AsJ,ayagus officinalis is important horticultural species. Genus AsJ)aragus includes some ornamental species and some medicinally importantspecies. and distributed on dry land in the Old

World. To clarify the phylogenetic relationships within this genus, we compared the restriction sites of

chloroplast DNAs(ctDNAs) among ten species : A officinalis, A. cochinchinensis, A schoberioides, A.

aspwagoides, AJalcatus, A. macowanii, A. I)lumosus, A. scandens, A. virgatus and A. sprmgeri.

Phylogenetic relationships within this genus, derived from the ctDNA restriction sites, yielded values for interspecific divergence (100×p) that ranged from 0.4 to 2.4. Compared to other groups,

such as Zea (0.0 to 0.3) and Aegi妙S / Tn'ticum (0.24 to 1.0), the species of AsJwagus exhibited higher

levels of variation in their ctDNA. A phylogenetic tree showed that the AsL)ayagus species used in this

study could be divided into two clusters. The phylogenetic relationships derived from variations in the ctDNA were slightly different from those proposed by Bailey (1994) and Clifford and Conran (1987).

The ctDNAsof AJalcatus (tetraploid, 2n-40) and A. sprengeri (hex aploid, 2n-60) gave the same

res-triction pattern. indicating that these species are very closely related. Moreover. it is of considerable in-terest that the polyploid species of Aspwagus were grouped in one cluster. The three dioecious species, A. ojficinalis, A. schoberioides and A. cochinchirzensis, were also grouped into one cluster, which suggests

the possibility that dioecy in Asparagus might have had a monophyletic origin.

Fluminhan, A. and T. Kameya : Involvement of knob heterochromatin in mitotic abnormalities in

germi-nating aged seeds of maize. Genome 40 : 91-98, 1997.

Seeds of three inbred lines of maize, with contrasting numbers of heterochromatic knobs and stored under two different ageing treatments, wereanalyzed for the occurrence of abnormalities at mito-tic anaphase in root meristems 3, 7, 21, 42. and 56 days after germination, and in root meristems of their freshly harvested selfed progeny. The largest category of detectable aberrations involved breakage of

knobbed chromosome arms. We have obtained evidence that knob beterochromatin plays a central role in

the origin of primary chromosome bridges. The initial event responsible for the occurrence of breakages and lagging chromosomes was characterlZed by the nondisjunction of newly replicated sister chromatids, which was observed to occur preferentially at the knob level. Such configurations, and all the other types of abnormalities (as for example, lagging chromosomes, typical chromosome bridges, fragments, and micronuclei) , Were observed at decreasing frequencies throughout root growth. Nevertheless, we have detected the occurrence of breakage- fusion-bridge cycles that were initiated by broken chromo-somes. The relationship between late-replicating DNA in maize knob heterochromatin and the

vulner-abillty Ofsuch regions to breakage is discussed. Our observations suggest a similarity between the 一

mechanisms involved or associated with the origln Of the described abnormalities and those reported to occur in cultured maize cells.

Kisaka, H" M. Kisaka, A. Kanno and T. Kameya : Production and analysis of plants that are somatic

hybrids of barley (Hordeum vulgaye L.) and carrot (Daucus carota L.). Theor. Appl. Genet. 94 :

221-226. 1997.

In order to obtain plants that were somatic hybrids of balrley (Hordeum vulgare L.) and carrot (Daucus cayota L.). We fused protoplasts that had been isolated from 6-month101d suspension cultures of carrot cells wlth protoplasts isolated from barley mesophyll by electrofusion After culture for 1 month at25℃, the cells were cultured for 5 weeks at4℃, and were then returned t0 25℃ for cultureon a

shoot-inducing medium. Three plants (mos. 1, 2 and 3) were regenerated from the cells. The morphology

of ther egenerated plants closely resembled that of the parental carrot plants･ A cytological analysis of

eallus cultures induced from these plants indicated that most of the celts had about 24 ehromosomes.

-17-fewer than the sum of the numbers of parent chromosomes which was 32･ Southern hybridization analy-sis with fragments of the rgPl gene used as probe showed that the regenerated plants contained both

ba-rley and carrot genomic DNA. Chloroplast (ct) and mitochondrial (mt) DNAswere also analyzedwith

several probes･ The ctDNA of the regenerated plants yielded hybridization bands specific for both bar-ley and carrot when one fragmentofrice ctDNA was used as probe･ Further一more･ the regenerated

plants yielded a barley specific band and a novel band with another fragment of rice ctDNA as a probe･

one of the regenerated plants (no. 1) yielded a novel pattern ofhybridized bands of mt DNA (with an αゆ6 probe) that was not detected with either of the parents･ These results indicated that the regener-ated plants were somatic hybrids o‖)arley and carrot and that recombination of both the chloroplast

genomes and the mitochondrial genomes might have occurred･

Kikumoto, T. Ampaabeng Kyeremeh, G･ Chuang D･ Y･ and Gungi Y∴ Biological control of the soft rot

disease of Chinese cabbbage with avirulent mutant strains of EruJinia cwotovora subsp. cwotovora･

Pro-ceedings of the Fourth International Workshop on Plant Growth- Promoting Rhizobacteria Japan- OECD

Joint Workshop pp. 118-119, 1997.

By means of ethyl methanesulfonate (EMS) treatment, avlrulent bacteriocln producing mutants

of Erwinia carotovora subsp. carotovoya were isolated･ These mutant strains proved to be effective bi010-gical control agents against the soft rot disease of Chinese cabbage in the field･

Kanno, A., M. Nakazono, A. Hirai and T. Kameya : Maintenance of Chloroplast･derived sequences in

the mitochondrial DNA of Gamineae. Current Genetics 32 : 413-419, 1997.

Evidence for the transfer of DNA from the chloroplast to the mitochondrion has been reported

in many higher plants and, in most cases, the transferred chloroplant genes do not have the ability to

en-code functional products as a consequence of base substitutions and/or multiple rearrangements･ We re･

ported previously that the sequence of one end of a chloroplast-derived (Cトderived) fragment of DNA that contained the rps19 and tnlHgenes has been maintained in most gramineous plants and its presence

seems to be correlated wlth gene expression in this region. In this study, We investigated whether or not

the ct･derived sequences ln mitochondrial DNA (mtDNA) from some gra叫neous plants and species of

o7yZa are COnSerVed, and whether or not such conservation is related to gene expression in these regions･

we found that two junctlOnS between ct-derived and mitochondrial sequences were conserved among

some gramineous plants Around these regions, We found a cLderived gene for tRNA and the promoter of a mitochondrial gene on the ct-derived sequences, respectively, and these regions were transcribed through the junctions. This result indicates that the junctions and/Or regions that are transcribed and functional in mitochondria have been strongly conserved and maintained durlng their evolution. In O7yZa, some junctions between ct-derived and mitochondrial sequences were conserved and other junctions were not. These variations seem to have been caused by deletions and/Or rearrangements and appear to

be specific to the type of genome･ In the case of Oyyza･ the timing of deletions and/or rearrangements oL

ct-derived sequences is llkely to have coincided with the divergence of the various genome types

学会での口頭発表,展示発表

木坂広明,亀谷寿昭:細胞融合によるイネとオオムギの体細胞雑種植物の作出と解析｡日本植物細胞

分子生物学会(7月20-21日,熊本)

Fluminhan,A.,近江戸伸子,福井希一,亀谷寿昭:植物染色体におけるトポイソメラーゼ皿の分析o

日本育種学会(3月29-30日,東京)

-18-Fluminhan, A.,亀谷寿昭,福井希-,佐藤雅志,山口 勇:イネ長期間保存種子における有糸分裂不

安定性の分析｡日本育種学会(10月18-19日,鳥取)

Kisaka, H., T. Kameya : Production and characterization of the plants that are somatic hybris between

barley and carrot. The 8th Sabrao General Congress (Sept. 24128, Seoul, Korea)

Fluminhan, A., T. Sato, T. Kameya, i. Yamaguti : Mitotic abnormalities in tissue cultures and in

germi-nating aged seeds of Japonica rice. The 8th Sabrao General Congress (Sept. 24-28, Seoul, Korea)

Kanno, A., T. Kameya : Cloning and variation of ribosomal DNA from Asparagus onicinalis L‥

Interna-tional Asparagus Symposium Ouly 15-17, Washington, USA)

Fluminhan, A., A. Ohmido, N. Fukui, K. Mizugaki, T. Kameya : Immunofluorescence analysis of the

dis-tribution of methylated cytosine in maize and rice somatic chromosomes. The 5 th Plant and Animal

Genome Conference Oanuary 20-23, Sam Diego, USA)

Fluminhan, A., A. Ohmido, I. Fukui, T. Kameya : The distribution of topoisomerase II on maize somatic

chromosomes, The 39th Annual Maize Genetics Conference (Maech 13-16, Clearwater, Florida,

UsA)