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植物の育種においては、育種目標の高度化に伴って、実用作物に有用な遺伝形質を提供する遺伝子供給源の範囲が拡大してきた。この範囲を、作物との有性生殖によって利用できる植物よりさらに大きく広げる方法として、細胞融合が開発された。細胞融合は雑種化の範囲を拡大するだけでなく、葉緑体ゲノムとミトコンドリアゲノムの新しい組合わせを生じる等の利点を有する。本研究では、シロイヌナズナとキャベツの細胞融合によって得られた体細胞雑種を出発点として、Brassica oleracea に属するカイランを主たる花粉親に用いた連続戻し交雑の経過と、その結果得られた雄性不稔個体の特性をとりまとめた。雄性不稔はBC8 世代以降固定し、不稔の原因はシロイヌナズナとキャベツの間で組換ったミトコンドリアゲノムの構造にあると推定された。一方、体細胞雑種後代の種子稔性は、世代の経過とともに向上した。

アブラナ科作物においては、近年細胞質雄性不稔を利用したF1品種が急速に普及している。従来、アブラナ科のF1育種にはオグラ型細胞質が用いられてきたが、それ以外の雄性不稔細胞質を見出そうとする試みが世界的に進められ、新たにDCGMS型細胞質や野生種が持つミトコンドリアのorf108遺伝子が発見され、詳しく研究された。それらの研究の過程で、雄性不稔細胞質と稔性回復遺伝子の複雑な対応の存在が明らかになってきた。そこで、オグラ型のorf138遺伝子、DCGMS型のorf463遺伝子および野生種のorf108遺伝子に焦点を当て、これらと稔性回復遺伝子の関係についての最近の研究をレビューした。

植物ゲノム科学研究センターでは、将来の作物育種への応用を目指して、核および細胞質ゲノムの解析と新しいバイオ技術の開発を進めてきた。本研究センターのこの研究プロジェクトはさらに2年間延長して継続される。この機会に過去5年間の研究業績をリストとして紹介する。

植物ゲノム科学研究センターでは、「核と細胞質のゲノム情報を活用した新しいバイオ技術の開発と細胞質ゲノムの構造解析」と題する5ヵ年計画のプロジェクトを実施している。このプロジェクトに含まれる実験課題は多岐にわたり、大きく、1）葉緑体の遺伝子組換え、2）ミトコンドリアゲノムの構造解析、3）その他、の3つに分けられる。また、それぞれのカテゴリーの中で、基盤的な研究、さらには萌芽的な研究がなされている。今年度は主として以下の課題が実地された。本稿では、このうち第7の課題について研究成果を報告する。また当研究センターが主催した国際シンポジウムおよびセンターのスタッフの業績について報告する。 1．有用遺伝子を葉緑体ゲノムに持つ組換えレタスならびにトマトの作出 2．パンコムギにおける葉緑体形質転換系の開発 3．斑入りを示す組換えタバコの葉緑体ゲノムの構造変異 4．イネ科およびアブラナ科植物のミトコンドリアゲノムの解読 5．タマネギのミトコンドリアゲノムの解読 6．ナスの稔性回復遺伝子の単離 7．ダイコンにおける雄性不稔遺伝子と稔性回復遺伝子の対応 8．ゲノム編集による新しい遺伝子改変技術の検討

細胞質雄性不稔（CMS）は，核とミトコンドリアの相互作用により，正常な花粉の形成機能が失われてしまう母性遺伝形質である。ダイコンのオグラ型CMSはミトコンドリアゲノムにコードされているorf138の発現によって引き起こされるが，核に存在する稔性回復遺伝子がORF138タンパク質の発現を減らすことでCMSは抑制される。著者らは，野生のハマダイコン（Raphanus sativus）から，orf138 mRNAの5’プロセシングに関わる稔性回復遺伝子Rftを単離するため，マップベースド・クローニングを行なった。フォスミドクローンのシークエンスと全ゲノムシークエンスの解析データをあわせ，Rftに近接するDNAマーカーを作製した。このマーカーを用いて分離集団でジェノタイピングを行なった結果，Rftは68kbのゲノム領域内に限定された。この領域の構造を解析したところ，Rftはミトコンドリアへの移行シグナル配列を持った，PPR（Pentatrico-Peptide Repeat）タンパク質をコードしていることが示唆された。, Cytoplasmic male-sterility（CMS）is a maternally inherited trait that prevents plants from producing functional pollen. CMS is caused by nuclear-mitochondrial interactions, and Ogura CMS found in Japanese radish is associated with ORF138 protein encoded by the mitochondrial gene orf138. The male sterile phenotype is controlled by nuclear fertility restorer（Rf）genes that reduce the amount of ORF138 protein. One of the Rf genes, termed Rft, was previously identified in the Japanese wild radish. Rft was shown to reduce the amount of ORF138 protein by the processing of 5’ end of orf138 mRNA. In order to clone Rft, a map-based cloning was conducted. Combining the data from fosmid clone sequencing and whole genome shotgun sequencing, we have developed DNA markers that were closely located to the Rft locus. As a result of genotyping of the 403 segregating plants, we physically delimited Rft locus to a 68 kb region. Analysis of this region suggested that the Rft gene encoded the Pentatrico-Peptide Repeat（PPR）protein with a predicted mitochondrial targeting pre-sequence., 10, KJ00009319339, P, 研究ノート, Research Note

ダイコンのオグラ型細胞質雄性不稔性は，アブラナ科野菜のF1育種に重要な形質である。この雄性不稔は，ミトコンドリアゲノムにコードされているorf138遺伝子の働きによって引き起こされる。一方で，核ゲノムにコードされている稔性回復遺伝子（Rf）は，orf138遺伝子の発現を抑制することで，正常な機能をもった花粉を形成させる。著者らは，野生のハマダイコン（Raphanus sativus）から，orf138遺伝子転写産物の5’プロセシングに関与する稔性回復遺伝子であるRftを単離・同定することを目指して，F₂BC₁分離集団を用いて，Sequence Tagged Site（STS）マーカーの作製を行なった。マッピングの結果，Rftはマーカー間で0.9cMの領域内に設定された。さらに，Rft遺伝子から0.6cMの位置にマップされたSTSK17マーカー，およびこのマーカー配列をもとにして作製されたRft遺伝子と完全連鎖するK17LF2R1マーカーは，いずれもRf遺伝子様のPPR（pentatricopeptide repeat）タンパク質をコードする配列の一部を有することから，RftもこのようなPPRタンパク質ファミリーに属する遺伝子である可能性が示唆された。, Ogura male-sterile cytoplasm is widely distributed in Japanese wild radish. The cytoplasm has been used in the F1 hybrid seed production in Cruciferous crops. The male-sterility is associated with the mitochondrial protein encoded by the gene orf138, whereas nuclear fertility restorer（Rf ）genes prevent accumulation of ORF138 protein. One of the Rf genes, Rft, was previously identified in the Japanese wild radish. Rft was shown to reduce the amount of ORF138 protein by processing 5’ end of orf138 mRNA. In order to isolate Rft, we have developed the Sequence Tagged Site（STS）markers surrounding the Rft locus using an F2BC1 segregating population. As a result of molecular mapping, Rft was located between two markers the distance of which was estimated to be 0.9 cM. Since nucleotide sequences of STSK17 marker（0.6 cM）and K17LF2R1 marker（0.0 cM）contain the sequence characteristic to the Rf -like PPR（pentatricopeptide repeat）protein genes, it is likely that the gene Rft also encodes a member of the PPR protein family., 11, KJ00008753479, P, 研究ノート, Note

This study was designed to record some physiological responses to research relationship between skin temperature, heat discharge and behavioral thermoregulation during sleeps. Changes in skin temperature, heat discharge and behavioral thermoregulation were recorded for 7 hours from 0:00 to 7:00. Changes in skin temperature and heat discharge show that during 0:00～1:30, they rise as time passes in all experimental conditions. One side, average behavioral thermoregulation occurs during 0:00～1:30 more than that of during 0:00～7:00. When average heat discharge during 0:00～1:30 was less than that of during 0:00～7:00,average behavioral thermoregulation during 0:00～1:30 occurs more than that of during 0:00～7:00. Results shows that the less the body loses its heat during 0:00～1:30, the more behavioral thermoregulation occurs.

This study was designed to record some psychological and physiological responses to evaluate the effects of such responses by a convective air conditioning system in a hospital on the quality of sleep. Changes in skin temperature, degree of relaxation, movement in sleep and feeling of sleep were recorded. Results of skin temperature and autonomic nervous system showed sleep is composed 3 periods,24:00-2:00, 2:00-5:00 and 2:00-5:00. During first period of 24:00-2:00, 26°C was found to be better than 28°C and 30°C. As for second and third periods of 2:00-7:00, 26-28°C is superior to 30°C for sleep in a hospital.

In medical facilities, the aim of room temperature is based on past studies for postures of standing and chair sitting, and on epidemiological knowledge. Studies aimed to clarify an evaluation of change of thermal environment based on psychological and physiological reaction for a posture of supine are needed. This report aims to exam an evaluation of change of thermal environment based on psychological and physiological reaction for hospitals. As a result, mean skin temperature for thermal neutralization became 33.7〜33.8℃ and for maximum thermal comfort became 34.1〜34.3℃ regardless of sex and cooling system almost equally to the value. Based on amount of physiological unit and psychological slope ratio, the radiant cooling system and convection cooling system has almost the same performance for hospitals.

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1. 1. Propagation of impulse is studied electrophysiologically in the heart of an adult armed wrestler beetle. 2. 2. The velocity of propagation shows extensive range of values, for example, from 5.2 cm to 2100m/sec in a specimen at a temperature of 26.5°C. 3. 3. The propagation is considered to be the result of the alignment behavior of pacemakers as represented by a phase-response curve. 4. 4. The propagation may be derived from sequence of impulses generated spontaneously and successively from one pacemaker to an adjacent one with a short delay, the length of which may vary widely.

2002年度大会 (北陸) 学術講演梗概集

1. 1. Innervation of the heart by the cardio-inhibitory nerve was electrophysiologically examined in Ligia exotica. 2. 2. Three kinds of cardio-regulatory impulses, one inhibitory and two acceleratory, were identified in each of the two anterior cardiac nerves. 3. 3. The inhibitory impulses induced inhibitory postsynaptic potentials (IPSPs) in all the six neurons comprising the cardiac ganglion and suppressed their spontaneous burst activities. 4. 4. The inhibitory impulses induced inhibitory junctional potentials (IJPs) in the myocardial cells and hyperpolarized their membrane potentials. 5. 5. We concluded that the cardio-inhibitory nerve innervates directly both the cardiac ganglion cells and the myocardial cells.

1. 1. Effects of evoked impulses in cardioregulatory nerves on the heart rate were observed in the crab, Sesarma dehaani. 2. 2. The nerve m has two regulatory fibers; one is inhibitory and another is excitatory. After longitudinal splitting of the nerve m, the stimulation of anterior part shows a decrease of the heart rate and that of the posterior part increases it. 3. 3. Stimulation of the nerve I only increases the heart rate. 4. 4. The nerve fibers could be categorized physiologically by different thresholds for the intensity of stimulation required for activation or by the change in stimulated portion of the nerve. 5. 5. Conversely, by the stimulation of single fibers, the nerve were analyzed in relation to the stimulation frequency and the resulting heart rate.

1. 1. Coordination of activities between the auricle and ventricle was studied in the heart of oysters. 2. 2. The auricles and ventricle possess different rates of intrinsic rhythms, and auriculo-ventricular (A-V) delay varies among preparations. 3. 3. The ventricular beat, depending on its moment in the auricular beating cycle, has prolonging or shortening effects on the beat interval of the auricle and vice versa. 4. 4. Stretching is suggested to be the mediating factor of the coordination by which auricle and ventricle accelerate and inhibit each other according to the beat moments in the beating cycle, to obtain a coordinated beat.

1. 1. Mediating factors of co-ordination of rhythms between the auricle and ventricle and the effect of a brief stretch on the electrical activity of the myocardium were studied in the heart of oyster. 2. 2. The co-ordination was maintained even in the state where only mutual stretching between the auricle and ventricle was kept and no electrical connection was found between them. Thus it was assumed that the co-ordination is mediated by mutual stretching without direct interactions in their electrical activities. 3. 3. A brief stretch altered the periodic membrane potential changes of the myocardium and caused prolongation or shortening of the beat interval depending on the moment of its application. 4. 4. Mechanism of the co-ordination of rhythms mediated by mutual stretching between the auricle and ventricle was discussed in relation to the positive and negative chronotropic effects on the beat interval by the brief stretch.

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1. 1. The Ligia cardiac ganglion which is composed of six similar-sized neurons has been studied electrophysiologically. 2. 2. Intracellular recordings from the ganglion cells show periodic burst activity with conspicuous pacemaker potentials between bursts. 3. 3. All of the ganglion cells burst almost simultaneously and produce compound action potentials in the ganglionic trunk and the ganglionic nerve branches. 4. 4. The burst discharge is not always initiated in the same cell, but changes during spontaneous activity. 5. 5. When isolated, any of the ganglion cells could produce periodic burst activity with pacemaker potentials. 6. 6. Intracellular stimulation applied to one of the ganglion cells changed the burst interval and reset the burst rhythm of all ganglion cells. 7. 7. In Ligia heart, all ganglion cells have the pacemaker activity and may be mononeurons to the heart. These are compared to the properties of neurogenic hearts of some other crustaceans and Limulus.

Vertical distribution of dissolved oxygen and chlorophyll in summer-autumn season were determined in the offshore of Osaka in Lake Suwa. In mid-summer, when whole the surface of the lake was covered with blooming Microcystis, dissolved oxygen in the surface water layer could attain over-saturation to the utmost extent (160%). Late in August the amount of dissolved oxygen markedly decreased less than 90% saturation, in spite of a large amount of Microcystis existed. It might be related to the declining of the photosynthetic activity of the alga. Moreover, if drifting of the upper water containing a great part of Microcystis by wind (Susu-mizu) occurred, serious decrease of dissolved oxygen (less than 50% saturation) was observed. Wind and waves usually were not favorable to increase oxygen into the surface water. In such a situation carps cultured in the floating nets may be exposed to clanger of asphyxia unless a large amount of dissolved oxygen is supplied from surrounding water outside of the nets during the night. There was a relationship between the pH value and the saturation percentage of dissolved oxygen in the water of 0 to 2 m depth., Article, 信州大学理学部紀要 2(2): 139-148(1968)

Predation of mosquito larvae, Culex pipiens, by the guppy, Lebistes reticulatus PETERS, which has been recently acclimatized in hot springs in Japan, was studied under various experimental conditions. 1. Guppies could not survive in water of 35℃ and over, or at 10℃ and less. 2. At 25℃ guppies showed maximum predation of mosquito larvae. Adult guppies ate 21-91 mg of fourth instar larvae in 24 hours. These amounts correspond to 10-36 larvae in number when the larvae average 2.5 mg in weight per individual. At 30℃ the amounts of mosquito larvae eaten by adult guppies decreased from that at 25℃, but the decrease was less than that at 20℃. At 15℃ guppies ate few larvae and almost ceased growing. As a rule the quantity of larvae eaten by male guppies was less than that by femals. 3. Guppies could eat mosquito larvae in darkness as well as in light, though some decrease in amount of predation was observed. 4. Guppies ate mosquito larvae even when daphnids were available. There was slightly greater preference for daphnids than for mosquito larvae., Article, 信州大学理学部紀要 1(2): 79-92(1967)