詳細

築山　拓司 （ツキヤマ　タクジ）

  • 農学部 農業生産科学科　准教授
Last Updated :2024/04/25

コミュニケーション情報 byコメンテータガイド

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    「動く遺伝子」トランスポゾンが生み出す遺伝的多様性（＝個性）を研究しています。得られた知見を基に、作物の品種改良に有用な変異創成技術の開発を目指しています。

  • 報道関連出演・掲載一覧

    ＜報道関連出演・掲載一覧＞ ●2023/8/23 　毎日新聞 　コーヒー抽出残渣を用いた循環型農業の共同研究について

研究者情報

学位

  • 博士(農学)（京都大学）

ホームページURL

J-Global ID

研究キーワード

  • 転移因子   キチナーゼ   育種   イネ   

現在の研究分野（キーワード）

    「動く遺伝子」トランスポゾンが生み出す遺伝的多様性（＝個性）を研究しています。得られた知見を基に、作物の品種改良に有用な変異創成技術の開発を目指しています。

研究分野

  • 環境・農学 / 遺伝育種科学
  • ライフサイエンス / 遺伝学

経歴

  • 2019年04月 - 現在  タキイ研究農場付属園芸専門学校講師
  • 2018年04月 - 現在  吉備国際大学地域創成農学部非常勤講師
  • 2016年04月 - 現在  近畿大学農学部准教授
  • 2007年04月 - 2016年03月  京都大学大学院農学研究科助教
  • 2005年11月 - 2007年03月  京都大学大学院農学研究科助手

学歴

  • 2001年04月 - 2005年03月   京都大学大学院   農学研究科   農学専攻 博士課程
  • 1999年04月 - 2001年03月   京都大学大学院   農学研究科   農学専攻 修士課程
  •         - 2001年   京都大学   Graduate School, Division of Agriculture
  • 1994年04月 - 1998年03月   近畿大学   農学部   農学科
  •         - 1998年   Kinki University   Faculty of Agriculture

所属学協会

  • 日本遺伝学会   日本農芸化学会   日本育種学会   Japanese Society of Breeding   

研究活動情報

論文

  • Sakura Hayashi; Takuji Tsukiyama; Atsuo Iida; Masato Kinoshita; Akihiko Koga
    Genome 65 3 183 - 187 2022年03月 [査読有り]
     
    The majority of DNA-based transposable elements comprise autonomous and nonautonomous copies, or only nonautonomous copies, where the autonomous copy contains an intact gene for a transposase protein and the nonautonomous copy does not. Even if autonomous copies coexist, they are generally less frequent. The Tol2 element of medaka fish is one of the few elements for which a nonautonomous copy has not yet been found. Here, we report the presence of a nonautonomous Tol2 copy that was identified by surveying the medaka genome sequence database. This copy contained three local sequence alterations that affected the deduced amino acid sequence of the transposase: a deletion of 15 nucleotides resulting in a deletion of 5 amino acids, a base substitution causing a single amino acid change, and another base substitution giving rise to a stop codon. Transposition assays using cultured human cells revealed that transposase activity was reduced by the 15-nucleotide deletion and abolished by the nonsense mutation. This is the first example of a nonautonomous Tol2 copy. Thus, Tol2 is in an early stage of decay in the medaka genome, and is therefore a unique element to observe an almost complete decay process that progresses in natural populations.
  • Evaluation of diamide sensitivity in rice (Oryza sativa L.)
    Syusei Tahara; Yuichiro Yoshinaka; Eiji Tanesaka; Takuji Tsukiyama
    J. Crop Res. 66 7 - 11 2021年10月 [査読有り]
  • Hiroki Saito; Yutaka Okumoto; Takuji Tsukiyama; Chong Xu; Masayoshi Teraishi; Takatoshi Tanisaka
    Plants (Basel, Switzerland) 8 12 2019年11月 [査読有り]
     
    The photoperiod-insensitivity allele e1 is known to be essential for the extremely low photoperiod sensitivity of rice, and thereby enabled rice cultivation in high latitudes (42-53° north (N)). The E1 locus regulating photoperiod-sensitivity was identified on chromosome 7 using a cross between T65 and its near-isogenic line T65w. Sequence analyses confirmed that the E1 and the Ghd7 are the same locus, and haplotype analysis showed that the e1/ghd7-0a is a pioneer allele that enabled rice production in Hokkaido (42-45° N). Further, we detected two novel alleles, e1-ret/ghd7-0ret and E1-r/Ghd7-r, each harboring mutations in the promoter region. These mutant alleles alter the respective expression profiles, leading to marked alteration of flowering time. Moreover, e1-ret/ghd7-0ret, as well as e1/ghd7-0a, was found to have contributed to the establishment of Hokkaido varieties through the marked reduction effect on photoperiod sensitivity, whereas E1-r/Ghd7-r showed a higher expression than the E1/Ghd7 due to the nucleotide substitutions in the cis elements. The haplotype analysis showed that two photoperiod-insensitivity alleles e1/ghd7-0a and e1-ret/ghd7-0ret, originated independently from two sources. These results indicate that naturally occurring allelic variation at the E1/Ghd7 locus allowed expansion of the rice cultivation area through diversification and fine-tuning of flowering time.
  • Allelic Differentiation at the E1/Ghd7 Locus Has Allowed Expansion of Rice Cultivation Area
    Hiroki, Saito, Yutaka Okumoto,; Takuji Tsukiyama,; Chong Xu,; Masayoshi Teraishi and Takatoshi Tanisaka
    Plants 8 550  2019年11月 [査読有り]
  • 明日の育種学を考える
    谷坂隆俊; 築山拓司; 西尾剛; 矢野昌裕; 小林麻子; 加屋隆士; 坂本理沙; 齋藤希; 矢部志央理
    育種学研究 21 2 157 - 168 2019年11月 [査読有り][招待有り]
  • Compatibility of three bacterial strains in Agrobacterium-mediated transformation of monokaryotic mycelia of Flammulina velutipes.
    Tanesaka Eiji; Mori Misuzu; Tsuji Kenya; Tsukiyama Takuji
    J. Crop Res. 63 31 - 33 2018年12月 [査読有り]
  • QTL analysis of VGI (Vigorously growing plant in IM294) mutants related traits in rice
    Chong Xu; Takuji Tsukiyama; Masayoshi Teraishi; Takatoshi Tanisaka; Yutaka Okumoto
    Journal of Crop Research 63 1 15 - 19 2018年11月 [査読有り]
  • Yohei Koide; Atsushi Ogino; Takanori Yoshikawa; Yuki Kitashima; Nozomi Saito; Yoshitaka Kanaoka; Kazumitsu Onishi; Yoshihiro Yoshitake; Takuji Tsukiyama; Hiroki Saito; Masayoshi Teraishi; Yoshiyuki Yamagata; Aiko Uemura; Hiroki Takagi; Yoriko Hayashi; Tomoko Abe; Yoshimichi Fukuta; Yutaka Okumoto; Akira Kanazawa
    Proceedings of the National Academy of Sciences of the United States of America 115 9 E1995 - E1962 2018年02月 [査読有り]
     
    Understanding the genetic basis of reproductive barriers between species has been a central issue in evolutionary biology. The S1 locus in rice causes hybrid sterility and is a major reproductive barrier between two rice species, Oryza sativa and Oryza glaberrima. The O. glaberrima-derived allele (denoted S1g) on the S1 locus causes preferential abortion of gametes with its allelic alternative (denoted S1s) in S1g/S1s heterozygotes. Here, we used mutagenesis and screening of fertile hybrid plants to isolate a mutant with an allele, S1mut, which does not confer sterility in the S1mut/S1g and S1mut/S1s hybrids. We found that the causal mutation of the S1mut allele was a deletion in the peptidase-coding gene (denoted “SSP”) in the S1 locus of O. glaberrima. No orthologous genes of SSP were found in the O. sativa genome. Transformation experiments indicated that the introduction of SSP in carriers of the S1s allele did not induce sterility. In S1mut/S1s heterozygotes, the insertion of SSP led to sterility, suggesting that SSP complemented the loss of the functional phenotype of the mutant and that multiple factors are involved in the phenomenon. The polymorphisms caused by the lineage-specific acquisition or loss of the SSP gene were implicated in the generation of hybrid sterility. Our results demonstrated that artificial disruption of a single gene for the reproductive barrier creates a “neutral” allele, which facilitates interspecific hybridization for breeding programs.
  • Kum Rise; Tsukiyama Takuji; Inagaki Haruka; Saito Hiroki; Teraishi Masayoshi; Okumoto Yutaka; Tanisaka Takatoshi
    GENES & GENETIC SYSTEMS 90 6 408  2015年12月 [査読有り]
  • 河西恵; 築山拓司; 寺石政義; 奥本裕; 谷坂隆俊
    作物研究 60 1 55 - 57 近畿作物・育種研究会 2015年11月 [査読有り]
     
    イネ品種銀坊主の種子にガンマ線照射して得られた細粒突然変異系統IM294は,イネユビキチン様タンパク質遺伝子Rurm1(Rice ubiquitin-related modifier-1)の機能を喪失している.RURM1と高い相同性を示す酵母のUrm1pは,酸化ストレス応答タンパク質Ahp1pの機能を修飾することで酸化ストレス応答に関与することから,Rurm1もイネの酸化ストレス応答に関与している可能性があるがその詳細は未解明である.本研究は,イネの酸化ストレス応答におけるRurm1の役割を明らかにするために,酸化ストレス誘導薬剤であるメチルビオロゲン(MV)に対する銀坊主およびIM294の耐性を調査した.MV処理によるクロロフィルa,b含量の減少程度を調べたところ,処理濃度が高くなるにつれて銀坊主およびIM294いずれにおいてもクロロフィル含量は大きく低下した.しかし,クロロフィル含量の減少程度に関しては,銀坊主・IM294間に有意差はなかった.このことから,イネにおいて,Rurm1はMVによって誘導される酸化ストレスに対する防御機構には関与しないことが示唆された.
  • Rise Kum; Takuji Tsukiyama; Haruka Inagaki; Hiroki Saito; Masayoshi Teraishi; Yutaka Okumoto; Takatoshi Tanisaka
    MOLECULAR BREEDING 35 8 159  2015年08月 [査読有り]
     
    Posttranscriptional RNA processing inclusive of alternative splicing and alternative polyadenylation, as well as transcriptional regulation, plays important regulatory roles in eukaryotic gene expression. In eukaryotic genomes, transposable elements can alter gene expression at both transcriptional and posttranscriptional levels. Miniature Ping (mPing) is an active miniature inverted-repeat TE discovered in the rice genome, and its insertion renders adjacent genes stress inducible. In this study, we examined the effect of mPing insertion into coding sequences on RNA processing. The 30 RACE (rapid amplification of cDNA ends) analysis of mutant alleles, each harboring an mPing insertion, revealed that mPing induced various alternative splicing events. Furthermore, it was found that mPing induced alternative polyadenylation within its sequence. In the mutant allele, the body region of mPing was heavily methylated, whereas the mPing-flanking regions were moderately methylated. These results indicate that mPing alters transcript structures posttranscriptionally via induction of alternative splicing that most likely depends on DNA methylation. Based on these results, we discuss the availability of mPing as an insertional mutagen in rice.
  • Yoshihiro Yoshitake; Takayuki Yokoo; Hiroki Saito; Takuji Tsukiyama; Xu Quan; Kazunori Zikihara; Hitomi Katsura; Satoru Tokutomi; Takako Aboshi; Naoki Mori; Hiromo Inoue; Hidetaka Nishida; Takayuki Kohchi; Masayoshi Teraishi; Yutaka Okumoto; Takatoshi Tanisaka
    SCIENTIFIC REPORTS 5 7709  2015年01月 [査読有り]
     
    Plants commonly rely on photoperiodism to control flowering time. Rice development before floral initiation is divided into two successive phases: the basic vegetative growth phase (BVP, photoperiod-insensitive phase) and the photoperiod-sensitive phase (PSP). The mechanism responsible for the transition of rice plants into their photoperiod-sensitive state remains elusive. Here, we show that se13, a mutation detected in the extremely early flowering mutant X61 is a nonsense mutant gene of OsHY2, which encodes phytochromobilin (P Phi B) synthase, as evidenced by spectrometric and photomorphogenic analyses. We demonstrated that some flowering time and circadian clock genes harbor different expression profiles in BVP as opposed to PSP, and that this phenomenon is chiefly caused by different phytochrome-mediated light signal requirements: in BVP, phytochrome-mediated light signals directly suppress Ehd2, while in PSP, phytochrome-mediated light signals activate Hd1 and Ghd7 expression through the circadian clock genes' expression. These findings indicate that light receptivity through the phytochromes is different between two distinct developmental phases corresponding to the BVP and PSP in the rice flowering process. Our results suggest that these differences might be involved in the acquisition of photoperiod sensitivity in rice.
  • Siviengkhek Phommalath; Masayoshi Teraishi; Takanori Yoshikawa; Hiroki Saito; Takuji Tsukiyama; Tetsuya Nakazaki; Takatoshi Tanisaka; Yutaka Okumoto
    BREEDING SCIENCE 64 4 409 - 415 2014年12月 [査読有り]
     
    Black soybeans have been used as a food source and also in traditional medicine because their seed coats contain natural phenolic compounds such as proanthocyanidin and anthocyanin. The objective of this research is to reveal the genetic variation in the phenolic compound contents (PCCs) of seed coats in 227 black soybean cultivars, most of which were Japanese landraces and cultivars. Total phenolics were extracted from seed coats using an acidic acetone reagent and the proanthocyanidin content, monomeric anthocyanin content, total flavonoids content, total phenolics content, and radical scavenging activity were measured. The cultivars showed wide genetic variation in PCCs. Each of the contents was highly correlated with one another, and was closely associated with radical scavenging activity. PCCs were also moderately associated by flowering date but not associated by seed weight. Cultivars with purple flowers had a tendency to produce higher PCCs compared with cultivars with white flowers, suggesting that the W1 locus for flower color can affect phenolic compound composition and content. Our results suggest that developing black soybean cultivars with high functional phenolic compounds activity is feasible.
  • Shota Teramoto; Takuji Tsukiyama; Yutaka Okumoto; Takatoshi Tanisaka
    PLOS GENETICS 10 6 e1004396  2014年06月 [査読有り]
     
    Miniature inverted-repeat transposable elements (MITEs) are numerically predominant transposable elements in the rice genome, and their activities have influenced the evolution of genes. Very little is known about how MITEs can rapidly amplify to thousands in the genome. The rice MITE mPing is quiescent in most cultivars under natural growth conditions, although it is activated by various stresses, such as tissue culture, gamma-ray irradiation, and high hydrostatic pressure. Exceptionally in the temperate japonica rice strain EG4 (cultivar Gimbozu), mPing has reached over 1000 copies in the genome, and is amplifying owing to its active transposition even under natural growth conditions. Being the only active MITE, mPing in EG4 is an appropriate material to study how MITEs amplify in the genome. Here, we provide important findings regarding the transposition and amplification of mPing in EG4. Transposon display of mPing using various tissues of a single EG4 plant revealed that most de novo mPing insertions arise in embryogenesis during the period from 3 to 5 days after pollination (DAP), and a large majority of these insertions are transmissible to the next generation. Locus-specific PCR showed that mPing excisions and insertions arose at the same time (3 to 5 DAP). Moreover, expression analysis and in situ hybridization analysis revealed that Ping, an autonomous partner for mPing, was markedly up-regulated in the 3 DAP embryo of EG4, whereas such up-regulation of Ping was not observed in the mPing-inactive cultivar Nipponbare. These results demonstrate that the early embryogenesis-specific expression of Ping is responsible for the successful amplification of mPing in EG4. This study helps not only to elucidate the whole mechanism of mPing amplification but also to further understand the contribution of MITEs to genome evolution.
  • Quan Xu; Hiroki Saito; Ikuo Hirose; Keisuke Katsura; Yoshihiro Yoshitake; Takayuki Yokoo; Takuji Tsukiyama; Masayoshi Teraishi; Takatoshi Tanisaka; Yutaka Okumoto
    MOLECULAR BREEDING 33 4 813 - 819 2014年04月 [査読有り]
     
    Flowering time is closely associated with grain yield in rice (Oryza sativa L.). In temperate regions, seasonal changes in day length (known as the photoperiod) are an important environmental cue for floral initiation. The timing of flowering is important not only for successful reproduction, but also for determining the ideal balance between vegetative growth and reproductive growth duration. Recent molecular genetics studies have revealed key flowering time genes responsible for photoperiod sensitivity. In this study, we investigated the effect of three recessive photoperiod-insensitive alleles, se13, hd1 and ghd7, on yield components in rice under Ehd1-deficient genetic background conditions to ensure vegetative growth of each line. We found that se13-bearing plants had fewer panicles, hd1-bearing plants showed decreased grain-filling percentage, and ghd7-bearing plants appeared to have fewer grains per panicle and fewer secondary branches. Our results indicate that the pleiotropic effects of photoperiod-insensitive genes on yield components are independent of short vegetative growth. This will provide critical information which can be used to create photoperiod-insensitive varieties that can be adapted to a wide range of latitudes.
  • Takayuki Yokoo; Hiroki Saito; Yoshihiro Yoshitake; Quan Xu; Takehito Asami; Takuji Tsukiyama; Masayoshi Teraishi; Yutaka Okumoto; Takatoshi Tanisaka
    PLOS ONE 9 4 e96064  2014年04月 [査読有り]
     
    Floral transition from the vegetative to the reproductive growth phase is a major change in the plant life cycle and a key factor in reproductive success. In rice (Oryza sativa L.), a facultative short-day plant, numerous flowering time and flower formation genes that control floral transition have been identified and their physiological effects and biochemical functions have been clarified. In the present study, we used a Se14-deficient mutant line (HS112) and other flowering mutant lines to investigate the photoperiodic response, chromosomal location and function in the photoperiod sensitivity of the Se14 gene. We also studied the interactive effects of this locus with other crucial flowering time genes. We found that Se14 is independent of the known photoperiod-sensitive genes, such as Hd1 and Ghd7, and is identical to Os03g0151300, which encodes a Jumonji C (JmjC) domain-containing protein. Expression analysis revealed that the expressions of RFT1, a floral initiator known as a "florigen-like gene", and Ehd1 were up-regulated in HS112, whereas this up-regulation was not observed in the original variety of 'Gimbozu'. ChIP assays of the methylation states of histone H3 at lysine 4 (H3K4) revealed that the trimethylated H3K4 in the promoter region of the RFT1 chromatin was significantly increased in HS112. We conclude that Se14 is a novel photoperiod-sensitivity gene that has a suppressive effect on floral transition (flowering time) under long day-length conditions through the modification of chromatin structure by H3K4me3 demethylation in the promoter region of RFT1.
  • Induced mutations will help mold the future of food
    Tsukiyama T; Saito H; Naito K; Teraishi M; Monden Y; Tanisaka T
    Gamma Field Symposia 50 1 - 19 2014年03月
  • Tsukiyama Takuji; Teramoto Shota; Yasuda Kanako; Horibata Akira; Mori Nanako; Okumoto Yutaka; Teraishi Masayoshi; Saito Hiroki; Tanisaka Takatoshi
    GENES & GENETIC SYSTEMS 88 6 358  2013年12月 [査読有り]
  • Kanako Yasuda; Makoto Ito; Tomohiko Sugita; Takuji Tsukiyama; Hiroki Saito; Ken Naito; Masayoshi Teraishi; Takatoshi Tanisaka; Yutaka Okumoto
    MOLECULAR BREEDING 32 3 505 - 516 2013年10月 [査読有り]
     
    Transposable elements (TEs) are DNA fragments that have the ability to move from one chromosomal location to another. The insertion of TEs into gene-rich regions often affects changes in the expression of neighboring genes. Miniature Ping (mPing) is an active miniature inverted-repeat TE discovered in the rice genome. It has been found to show exceptionally active transposition in a few japonica rice varieties, including Gimbozu, where mPing insertion rendered adjacent genes stress-inducible. In the Gimbozu population, it is highly possible that several genes with modified expression profiles are segregating due to the de novo mPing insertions. In our study, we utilized a screening system for detecting de novo mPing insertions in the upstream region of target genes and evaluated the effect of mPing on the stress response of the target genes. Screening for 17 targeted genes revealed five genes with the mPing insertion in their promoters. In most cases, the alteration of gene expression was observed under stress conditions, and there was no change in the expression levels of those five genes under normal conditions. These results indicate that the mPing insertion can be used as a genetic tool to modify an expression pattern of a target gene under stress conditions without changing the expression profiles of those under natural conditions.
  • Kanako Yasuda; Takuji Tsukiyama; Shanta Karki; Yutaka Okumoto; Masayoshi Teraishi; Hiroki Saito; Takatoshi Tanisaka
    Euphytica 192 1 17 - 24 2013年07月 [査読有り]
     
    Miniature Ping (mPing) is the first active miniature inverted-repeat transposable element to be identified in rice, and its mobilization is activated by stress treatments. We have examined the mobilization of mPing in four NERICA (New Rice for Africa) lines and 13 interspecific lines. All 17 lines are inbred progenies derived from crosses between Oryza sativa variety WAB56-104 as the recurrent parent and the O. glaberrima variety CG14 as the donor parent. We found that 16 of the 17 lines studied inherited mPing together with its autonomous partner, Pong, from WAB56-104. Transposon display of mPing disclosed polymorphic banding patterns among these lines. Most importantly, seven of the lines displayed clear polymorphic banding patterns for mPing, indicating that mPing might have been mobilized in these lines. Locus-specific PCR analysis also confirmed the mobilization of mPing. These results signify that interspecific hybridization may activate the transposition of mPing. Based on these results, we discuss the potential use of the mPing system as an efficient tool for gene tagging in interspecific hybrid rice. © 2012 Springer Science+Business Media Dordrecht.
  • Takuji Tsukiyama; Shota Teramoto; Kanako Yasuda; Akira Horibata; Nanako Mori; Yutaka Okumoto; Masayoshi Teraishi; Hiroki Saito; Akiko Onishi; Kanako Tamura; Takatoshi Tanisaka
    Molecular Plant 6 3 790 - 801 2013年 [査読有り]
     
    Miniature inverted-repeat transposable elements (MITEs) are widespread in both prokaryotic and eukaryotic genomes, where their copy numbers can attain several thousands. Little is known, however, about the genetic factor(s) affecting their transpositions. Here, we show that disruption of a gene encoding ubiquitin-like protein markedly enhances the transposition activity of a MITE mPing in intact rice plants without any exogenous stresses. We found that the transposition activity of mPing is far higher in the lines harboring a non-functional allele at the Rurm1 (Rice ubiquitin-related modifier-1) locus than in the wild-type line. Although the alteration of cytosine methylation pattern triggers the activation of transposable elements under exogenous stress conditions, the methylation degrees in the whole genome, the mPing-body region, and the mPing-flanking regions of the non-functional Rurm1 line were unchanged. This study provides experimental evidence for one of the models of genome shock theory that genetic accidents within cells enhance the transposition activities of transposable elements.© 2012 The Author.
  • Yuki Hamamoto; Takuji Tsukiyama; Yoshihiro Yoshikake; Masayoshi Teraishi; Yutaka Okumoto; Takatoshi Tanisaka
    Bioscience, biotechnology, and biochemistry 77 12 2480 - 2 2013年 [査読有り]
     
    We describe a transient dual-luciferase assay combined with a glucocorticoid-inducible system for rice protoplasts. Luciferase genes were efficiently induced by adding 0.1 µM of dexamethasone to the protoplast suspension, the activity of the luciferases reaching a maximum 6 h after induction. This assay system is applicable to studying the translation efficiency of rice by using the luciferase gene harboring tandem copies of an interesting codon at the 5' end.
  • Yasuda Kanako; Tsukiyama Takuji; Saito Hiroki; Naito Ken; Teraishi Masayoshi; Tanisaka Takatoshi; Okumoto Yutaka
    GENES & GENETIC SYSTEMS 87 6 431  2012年12月 [査読有り]
  • Ayesha A; Yoshitake Y; Yokoo T; Kamal M; Teraishi M; Tsukiyama T; Okumoto Y
    Journal of Crop Research 57 33 - 38 近畿作物・育種研究会 2012年 [査読有り]
     
    農業上重要な遺伝子を同定するための相補性検定において,形質転換効率の改善は重要である.しかしながら,アグロバクテリウムを介したイネ形質転換効率において大きな品種間差異が存在する.銀坊主には,非常に活性化された転移因子mPingが内在しており,価値の高い研究材料である.我々は,mPing SCARマーカーおよびmPingタギングシステムを利用し,銀坊主に誘発された突然変異遺伝子を同定することに成功してきた.しかしながら,銀坊主の非常に低い形質転換効率は,候補遺伝子の相補性検定および機能解析の妨げとなっている.本報では,銀坊主に適した効果的なアグロバクテリウム形質変換法を報告する.銀坊主は,アグロバクテリウム感染に対する耐性が極めて弱く,アグロバクテリウムとの共存培養後に活性のあるカルスを再生することが困難であった.これを克服するために,感染には非常に低濃度のアグロバクテリウムを,一次スクリーニングでは高濃度のメロペンを使用した.また,共存培養時に感染したカルスを紙タオルで水分を拭き取って乾燥した状態を保つことも重要であった.その結果,一度に十分量の形質転換体を獲得することが可能となった.形質転換効率は14.3%であり,再生効率は90%以上であった.
  • Hiroki Saito; Yutaka Okumoto; Yoshihiro Yoshitake; Hiromo Inoue; Qingbo Yuan; Masayoshi Teraishi; Takuji Tsukiyama; Hidetaka Nishida; Takatoshi Tanisaka
    THEORETICAL AND APPLIED GENETICS 122 1 109 - 118 2011年01月 [査読有り]
     
    In rice (Oryza sativa), a short-day plant, photoperiod is the most favorable external signal for floral induction because of the constant seasonal change throughout the years. Compared with Arabidopsis, however, a large part of the regulation mechanism of the photoperiodic response in rice still remains unclear due mainly to the lack of induced mutant genes. An induced mutant line X61 flowers 35 days earlier than its original variety Gimbozu under a natural photoperiod in Kyoto (35A degrees 01'N). We attempted to identify the mutant gene conferring early heading to X61. Experimental results showed that the early heading of X61 was conferred by a complete loss of photoperiodic response due to a novel single recessive mutant gene se13. This locus interacts with two crucial photoperiod sensitivity loci, Se1 and E1. Wild type alleles at these two loci do not function in coexistence with se13 in a homozygous state, suggesting that Se13 is an upstream locus of the Se1 and E1 loci. Linkage analysis showed that Se13 is located in a 110 kb region between the two markers, INDEL3735_1 and INDEL3735_3 on chromosome 1. A database search suggested that the Se13 gene is identical to AK101395 (=OsHY2), which encodes phytochromobilin synthase, a key enzyme in phytochrome chromophore biosynthesis. Subsequent sequence analysis revealed that X61 harbors a 1 bp insertion in exon 1 of OsHY2, which induces a frame-shift mutation producing a premature stop codon. It is therefore considered that the complete loss of photoperiodic response of X61 is caused by a loss of function of the Se13 (OsHY2) gene involved in phytochrome chromophore biosynthesis.
  • Takanori Yoshikawa; Yutaka Okumoto; Daisuke Ogata; Takashi Sayama; Masayoshi Teraishi; Masakazu Terai; Toshiya Toda; Katsushige Yamada; Kazuhiro Yagasaki; Naohiro Yamada; Takuji Tsukiyama; Toshiaki Yamada; Takatoshi Tanisaka
    BREEDING SCIENCE 60 3 243 - 254 2010年09月 [査読有り]
     
    Soybean (Glycine max (L) Merr.) isoflavones have attracted considerable attention for their diverse effects on human health. To determine the genetic factors that contribute to the high isoflavone contents of the soybean varieties 'Peking' and 'Tamahomare', we conducted QTL analyses for the total content of daidzein derivatives (DAC), genistein derivatives (GEC) and glycitein derivatives (GLC), and for the total content of isoflavones (TIC) using recombinant inbred lines (RILs) derived from the cross between 'Peking' and 'Tamahomare'. Ninety six RILs were planted in Kyoto, Osaka and Nagano in 2003 and in Osaka and Nagano in 2004. Transgressive segregation for TIC was detected in all the environments tested. Composite interval mapping for TIC revealed four QTLs: qIso1, qIso2, qIso3 and qIso4, located on LG-A1 (Chr.5), LG-A2 (Chr.8), LG-C1 (Chr.4) and LG-D2 (Chr.17), respectively. The high-isoflavone alleles were derived from Peking at qIso1 and qIso4 and from Tamahomare at qIso2 and qIso3. Several other groups have already reported the former two QTLs, but qIso2 and qIso3 are new discoveries. Our results indicate that the large variation in TIC measurements observed in the RILs could have resulted from the combined effects of alleles at the four QTLs derived from distantly related varieties.
  • Takuji Tsukiyama; Jongwon Lee; Yutaka Okumoto; Masayoshi Teraishi; Takatoshi Tanisaka; Kuniyo Inouye
    BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY 74 2 430 - 432 2010年02月 [査読有り]
     
    A rice ubiquitin-related modifier-1 (Rurm1) gene was cloned and transformed in Escherichia coli. We successfully expressed the RURM1 protein as a glutathione S-transferase (GST)-fusion protein by cultivating the E. coli cells at 16 degrees C for 16 h. After cleavage of GST, we obtained a single protein of 12 kDa. This protein was identified as the RURM1 protein by western blot analysis.
  • 加藤 舞; 西中 未央; 奥本 裕
    作物研究 54 137 - 142 近畿作物・育種研究会 2009年11月 [査読有り]
  • ASAMI Takehito; OKUMOTO Yutaka; SAITO Hiroki; YUAN Qingbo; MONDEN Yuki; TERAISHI Masayoshi; TSUKIYAMA Takuji; TANISAKA Takatoshi
    作物研究 54 85 - 89 2009年11月 [査読有り]
  • Ken Naito; Feng Zhang; Takuji Tsukiyama; Hiroki Saito; C. Nathan Hancock; Aaron O. Richardson; Yutaka Okumoto; Takatoshi Tanisaka; Susan R. Wessler
    NATURE 461 7267 1130 - U232 2009年10月 [査読有り]
     
    High-copy-number transposable elements comprise the majority of eukaryotic genomes where they are major contributors to gene and genome evolution(1). However, it remains unclear how a host genome can survive a rapid burst of hundreds or thousands of insertions because such bursts are exceedingly rare in nature and therefore difficult to observe in real time(2). In a previous study we reported that in a few rice strains the DNA transposon mPing was increasing its copy number by similar to 40 per plant per generation(3). Here we exploit the completely sequenced rice genome to determine 1,664 insertion sites using high-throughput sequencing of 24 individual rice plants and assess the impact of insertion on the expression of 710 genes by comparative microarray analysis. We find that the vast majority of transposable element insertions either upregulate or have no detectable effect on gene transcription. This modest impact reflects a surprising avoidance of exon insertions by mPing and a preference for insertion into 5' flanking sequences of genes. Furthermore, we document the generation of new regulatory networks by a subset of mPing insertions that render adjacent genes stress inducible. As such, this study provides evidence for models first proposed previously(4-6) for the involvement of transposable elements and other repetitive sequences in genome restructuring and gene regulation.
  • Shanta Karki; Takuji Tsukiyama; Yutaka Okumoto; Govinda Rizal; Ken Naito; Masayoshi Teraishi; Tetsuya Nakazaki; Takatoshi Tanisaka
    BREEDING SCIENCE 59 3 297 - 307 2009年09月 [査読有り]
     
    The distribution and proliferation among Oryza species of miniature Ping (mPing), the only miniature inverted-repeat transposable element (MITE) that is active in all organisms, were investigated through analysis of 78 accessions encompassing nine of the 10 genomic constitutions of the genus Oryza. The mPing family transposons, mPing, Ping, and Pong, were detected in only two of the AA genome species, Oryza saliva and its direct ancestor O. rufipogon, whereas the inactive rice MITE Kiddo was detected in the AA, BB, CC, BBCC and CCDD genome species. Furthermore, the insertion sites and copy numbers of mPing were considerably different among O. rufipogon accessions. A phylogenetic analysis showed that the O. rufipogon accessions used in this study could be grouped into four clusters which were not associated with the copy number of mPing or the presence of Ping. These results suggest that the mPing family was present in the ancestor of O. rufipogon and that mPing proliferated independently in each accession. Based on these results, one possible evolutionary history of the distribution and proliferation of mPing family in O. rufipogon is discussed.
  • Qingbo Yuan; Hiroki Saito; Yutaka Okumoto; Hiromo Inoue; Hidetaka Nishida; Takuji Tsukiyama; Masayoshi Teraishi; Takatoshi Tanisaka
    THEORETICAL AND APPLIED GENETICS 119 4 675 - 684 2009年08月 [査読有り]
     
    A late heading-time mutant line, HS276, which was induced by gamma-irradiation of seeds of the japonica rice (Oryza sativa L.) variety Gimbozu, exhibits an extremely long basic vegetative growth phase (BVP). A genetic analysis using the F(2) population from the cross between HS276 and Gimbozu revealed that the late heading of HS276 is governed by a single recessive mutant gene. The subsequent analysis on heading responses of HS276 and Gimbozu to four photoperiods (12, 13, 14, and 15 h) and to the photoperiodic transfer treatment from a short photoperiod to a long photoperiod revealed that the mutant gene confers an extremely long BVP and increases photoperiod sensitivity under long photoperiod (14 and 15 h). The BVP durations of HS276 and Gimbozu were estimated at 30.1 and 16.0 days, respectively; the mutant gene, compared with its wild type allele, elongates the duration of BVP by 14 days. Linkage analysis showed that the mutant gene is located in the 129 kb region between the two INDEL markers, INDELAP0399_6 and INDELAP3487_2, on the distal part of the short arm of chromosome 6. None of the other BVP genes are located in this region; therefore, we declared this a newly detected mutant gene and designated it ef7. A recently established program to breed rice suitable for low latitudes, where short photoperiodic conditions continue throughout the year, aims to develop varieties with extremely long BVPs and weak photoperiod sensitivities; the mutant gene ef7, therefore, will be quite useful in these programs because it confers an extremely long BVP and little enhances photoperiod sensitivity under short photoperiod.
  • Hiroki Saito; Qingbo Yuan; Yutaka Okumoto; Kazuyuki Doi; Atsushi Yoshimura; Hiromo Inoue; Masayoshi Teraishi; Takuji Tsukiyama; Takatoshi Tanisaka
    THEORETICAL AND APPLIED GENETICS 119 2 315 - 323 2009年07月 [査読有り]
     
    A recently established rice breeding program in low latitudes aims to develop varieties with extremely long basic vegetative growth (BVG) periods and weak photoperiod sensitivities. The Taiwanese japonica variety Taichung 65 (T65) harbors a recessive allele ef1 at the Ef1 (Early flowering 1) locus, thereby exhibiting an extremely long BVG period. The previous reported functional allele Ehd1 (Early heading date 1), located on chromosome 10, encodes a B-type response regulator, thereby shortening the BVG period, whereas its nonfunctional allele ehd1 greatly prolongs the BVG period. A conventional analysis using F(2) and F(3) populations and a subsequent CAPS analysis based on the amino acid sequences of Ehd1 and ehd1 showed that Ef1 and Ehd1 were at the same locus. The CAPS analysis also indicated that the Taiwanese japonica varieties with extremely long BVG periods all harbor ef1, but that ef1 does not exist among indica and japonica varieties in the low latitudes. Since ef1 has not been found in any japonica varieties outside Taiwan, this allele might have originated in Taiwan. Sequence analysis revealed that the mutant allele ef1-h, which prolongs the BVG period even more than ef1 does, harbors an mPing insertion in exon 2, which causes the complete loss of gene function. Our results indicate that both ef1 or ef1-h alleles can be used as new gene sources in developing rice varieties with extremely long BVG periods for low latitudes.
  • Jiayu Wang; Tetsuya Nakazaki; Shuqian Chen; Wenfu Chen; Hiroki Saito; Takuji Tsukiyama; Yutaka Okumoto; Zhenjin Xu; Takatoshi Tanisaka
    THEORETICAL AND APPLIED GENETICS 119 1 85 - 91 2009年06月 [査読有り]
     
    The breeding of japonica varieties with erect-pose panicle (EP) has recently progressed in the northern part of China, because these varieties exhibit a far higher grain yield than the varieties with normal-pose panicle (NP). A genetic analysis using the F(2) population from the cross between Liaojing5, the first japonica EP variety in China, and the Japanese japonica NP variety Toyonishiki revealed that EP is governed by a single dominant gene EP. Based on previous studies, map-based cloning of EP locus was conducted using Liaojing5, Toyonishiki, their F(2) population, and a pair of near-isogenic lines for EP locus (ZF14 and WF14) derived from the cross between the two varieties; consequently, the STS marker H90 was found to completely cosegregate with panicle pose. The H90 is located in the coding sequence AK101247 in the database, and the AK101247 of Liaojing5 has a 12 bp sequence in exon 5 replaced with a 637 bp sequence of its wild type allele. It was therefore considered that the AK101247 encodes the protein of the wild type allele at EP locus, and that the sequence substitution in exon 5 of Liaojing5 is crucial for expression of the EP phenotype. The effects of EP gene on agronomic traits were investigated using two pairs of near-isogenic lines (ZF6 vs. WF6 and ZF14 vs. WF14) derived from the cross between the two varieties. Experimental results showed that EP gene markedly enhanced grain yield, chiefly by increasing number of secondary branches and number of grains on the secondary branch. EP gene also produced a remarkable increase in grain density.
  • Takashi Sayama; Tetsuya Nakazaki; Goro Ishikawa; Kazuhiro Yagasaki; Naohiro Yamada; Naoko Hirota; Kaori Hirata; Takanori Yoshikawa; Hiroki Saito; Masayoshi Teraishi; Yutaka Okumoto; Takuji Tsukiyama; Takatoshi Tanisaka
    PLANT SCIENCE 176 4 514 - 521 2009年04月 [査読有り]
     
    In soybean (Glycine max [L.] Merr.), varieties with seed-flooding tolerance at the geminating stage are desirable for breeding in countries with much rainfall at sowing time. Our study revealed great intervarietal variation in seed-flooding tolerance as evaluated by germination rate (GR) and normal seedling rate (NS). Pigmented seed coat and small seed weight tended to give a positive effect on seed-flooding tolerance. Subsequently, QTL analysis of GR and NS were performed and a total of four QTLs were detected. Among them, Sft1 on the linkage group H (LG_H) exhibited a large effect on GR after a 24 h treatment; however, Sft2 near the I locus on LG_A2 involved in seed coat pigmentation exhibited the largest effect on seed-flooding tolerance. Sft1, Sft3 and Sft4 were independent of seed coat color and seed weight. Based on the results, we discussed the physiological effects of genetic factors responsible for seed-flooding tolerance in soybean. (C) 2009 Elsevier Ireland Ltd. All rights reserved.
  • Yuki Monden; Ken Naito; Yutaka Okumoto; Hiroki Saito; Nobuhiko Oki; Takuji Tsukiyama; Osamu Ideta; Tetsuya Nakazaki; Susan R. Wessler; Takatoshi Tanisaka
    DNA RESEARCH 16 2 131 - 140 2009年04月 [査読有り]
     
    Although quantitative traits loci (QTL) analysis has been widely performed to isolate agronomically important genes, it has been difficult to obtain molecular markers between individuals with similar phenotypes (assortative mating). Recently, the miniature inverted-repeat transposable element mPing was shown to be active in the japonica strain Gimbozu EG4 where it had accumulated more than 1000 copies. In contrast, most other japonicas, including Nipponbare, have 50 or fewer mPing insertions in their genome. In this study we have exploited the polymorphism of mPing insertion sites to generate 150 PCR markers in a cross between the closely related japonicas, Nipponbare x Gimbozu (EG4). These new markers were distributed in genic regions of the whole genome and showed significantly higher polymorphism (150 of 183) than all other molecular markers tested including short sequence repeat markers (46 of 661). in addition, we performed QTL analysis with these markers using recombinant inbred lines derived from Nipponbare x Gimbozu EG4, and successfully mapped a locus involved in heading date on the short arm of chromosome 6. Moreover, we could easily map two novel loci involved in the culm length on the short arms of chromosomes 3 and 10.
  • In Silico Survey of Transposable Elements in Soybean.
    Teraishi, M; Y. Ito; K. Yano; T. Nakazaki; T. Tsukiyama; Y. Okumoto; H. Saito; A. Kitajima; T. Tanisaka
    J. Crop. Res 54:71-74 2009年 [査読有り]
  • QTL analysis of seed-flood tolerance of the yellow soybean variety ‘Enrei’.
    Hirata, K; Yoshikawa, T; Teraishi, M; Komatsu, K; Takahashi, M; Hirota, N; Nakazaki, T; Sayama, T; Tsukiyama, T; Okumoto, Y; Tanisaka, T
    Journal of Crop Research 2009年 [査読有り]
  • High isoflavone content mutants induced from the soybean variety ‘Tambaguro’ with gamma irradiation.
    Yoshikawa, T; Okumoto, Y; Terai, M; Yamada, K; Teraishi, M; Tsukiyama, T; Tanisaka, T
    Journal of Crop Research 2009年 [査読有り]
  • mPingを含んで転写される遺伝子の同定
    稲垣春香; 築山拓司; 門田有希; Shanta Karki; 奥本裕; 中崎鉄也; 寺石政義; 谷坂隆俊
    作物研究 54 75 - 80 2009年 [査読有り]
  • Nobuhiko Oki; Kentaro Yano; Yutaka Okumoto; Takuji Tsukiyama; Masayoshi Teraishi; Takatoshi Tanisaka
    GENES & GENETIC SYSTEMS 83 4 321 - 329 2008年08月 [査読有り]
     
    Transposable elements (TEs) have played important roles in the evolution of genes and genomes of higher eukaryotes. Among the TEs in the rice genome, miniature inverted-repeat transposable elements (MITEs) exist at the highest copy number. Some of MITEs in the rice genome contain poly(A) signals and putative cis-acting regulatory domains. Insertion events of such MITEs may have caused many structural and functional changes of genomes. The genome-wide examination of MITE-derived sequences could elucidate the contribution of MITEs to gene evolution. Here we report on the MITEs in the rice genome that have contributed to the emergence of novel genes and the expansion of the sequence diversity of the genome and mRNAs. Of the MITE-derived sequences, approximately 6000 were found in gene regions (exons and introns) and 67,000 in intergenic regions. In gene regions, most MITEs are located in introns rather than exons. For over 300 protein-coding genes, coding sequences, poly(A) sites, transcription start sites, and splicing sites overlap with MITEs. These sequence alterations via MITE insertions potentially affect the biological functions of gene products. Many MITE insertions also exist in T-untranslated regions (UTRs), 3'UTRs, and in the proximity of genes. Although mutations in these non-protein coding regions do not alter protein sequences, these regions have key roles for gene regulation. Moreover, MITE family sequences (Tourist, Stowaway, and others) are unevenly distributed in introns. Our findings suggest that MITEs may have contributed to expansion of genome diversity by causing alterations not only in gene functions but also in regulation of many genes.
  • Kosonh Xayphakatsa; Takuji Tsukiyama; Kuniyo Inouye; Yutaka Okumoto; Testuya Nakazaki; Takatoshi Tanisaka
    ENZYME AND MICROBIAL TECHNOLOGY 43 1 19 - 24 2008年07月 [査読有り]
     
    Class II chitinase CHT11 is one of the 12 chitinases identified in rice (Oryza sativa L.). In order to elucidate its enzymatic properties, the Cht11 gene was cloned and overexpressed in Escherichia coli BL21 cells as a glutathione-S-transferase (GST) fusion protein. The optimal culture temperature and the optimal time after induction were found to be 16 degrees C and 12 h, respectively. In the process of purification, a non-specific 70-kDa protein bound to the fusion protein was successfully removed from recombinant CHT11 by adding 3 mM ATP-Mg in wash buffer. In the culture conditions and purification procedures presented in this study, the final yield of recombinant CHT11 from 800 ml of the culture supernatant was 4.1 mg with 11.5-fold purification. The optimal temperature and pH of recombinant CHT11 were determined to be 35-40 degrees C and 5.5-6.5, respectively. It inhibited the hyphal growth of both avirulent and virulent strains of Trichoderma viride at 100 and 300 mu g in medium, but the growth inhibition zone was subtle even when 300 mu g of protein was applied. This indicates that the antifungal activity of recombinant CHT11 is considerably lower than other chitinases. (C) 2008 Elsevier Inc. All rights reserved.
  • OKI Nobuhiko; OKUMOTO Yutaka; TSUKIYAMA Takuji; NAITO Ken; NAKAZAKI Tetsuya; TANISAKA Takatoshi
    近畿作物・育種研究 52 39 - 43 2007年07月 [査読有り]
  • T. Nakazaki; T. Tsukiyama; Y. Okumoto; D. Kageyama; K. Naito; K. Inouye; T. Tanisaka
    GENOME 49 6 619 - 630 2006年06月 [査読有り]
     
    Rice (Oryza sativa L.) pathogenesis-related (PR)-3 chitinases, like other PR proteins, are each coded by one of the genes of a multigene family in the plant genome. We assembled the database information about rice PR-3 chitinase sequences. A total of 12 PR-3 chitinase loci (Cht1 to Cht12) were found deployed in the rice genome. Some of the loci were occupied by 2 or more alleles. For all the loci expect Cht4, Cht5, Cht6, and Cht11, the amino acid sequence was polymorphic between japonica and indica varieties of rice, but glutamic acid acting as a catalytic residue was completely conserved in all the loci expect Cht7. All the genes except Cht7, which was not tested in this study, were transcripted in some organs (leaf, sheath, root, and meristem) of rice plants. These results suggest that chitinase proteins encoded by the genes at these loci have important biological effects, at least antifungal activities, on rice plants. We also proposed a new classification of rice PR-3 chitinases based on their domain structures. This classification was consistent with the results of phylogenetic analysis of rice chitinases.
  • GST融合イネPR-3キチナーゼの精製とその作用特性の解析
    築山拓司; 中﨑鉄也; 寺地徹; 田中千尋; 奥本裕; 谷坂隆俊
    近畿作物・育種研究2002） 47号: 51-54,48号: 47-51 2003年 [査読有り]
  • イネユビキチン様タンパク質遺伝子Rurm1がメチルビオロゲンによって誘導される酸化ストレス応答におよぼす効果
    河西恵; 築山拓司; 寺石政義; 奥本裕; 谷坂隆俊
    作物研究 60 1 55 - 57 1998年11月 [査読有り]

書籍

  • 初めての酵素化学
    築山 拓司 (担当:分担執筆範囲:第7章 酵素の遺伝子工学／第9章 酵素の作用ーキチナーゼー)シーエムシー出版 2016年12月

講演・口頭発表等

  • Transposon-mediated alternative splicing in the rice genome  [通常講演]
    Tsukiyama T; Kum R; Inagaki H; Saito H; Teraishi M; Okumoto Y; Tanisaka T
    International Symposium"Establishing Next-Generation Genetics" 2015年 ポスター発表
  • Utilization of active MITE mPing as a novel genetic tool for modification of stress response in rice  [招待講演]
    Yasuda K; Tsukiyama T; Saito H; Naito K; Teraishi M; Tanisaka T; Okumoto Y
    7th International Rice Genetic Symposium 2013年11月 口頭発表（招待・特別）
  • Loss-of-function of an ubiquitin-related modifier RURM1 promotes the mobilization of the active MITE mPing  [通常講演]
    Tsukiyama T; Teramoto S; Yasuda K; Horibata A; Mori N; Okumoto Y; Teraishi M; Saito H; Onishi A; Tamura K; Tanisaka T
    7th International Rice Genetic Symposium 2013年11月 ポスター発表
  • Gene expression analysis and characterization of rice (Oryza sativa L.) class II chitinase CHT11  [通常講演]
    Tsukiyama T; Inouye K; Kosonh X; Okumoto Y; Nakazaki T; Tanisaka T
    International Symposium on Frontier in Plant Proteome Research -Contribution of proteomics technology in creation of useful plants- 2008年 ポスター発表
  • Differential expression of 11 PR-3 chitinase genes in rice (Oryza sativa L.) plants during rice blast infection and signal molecule treatments  [通常講演]
    Tsukiyama T
    The 6th Kyoto University International Symposium “Plant sciences in Japan and China -from genetics to breeding” 2005年 ポスター発表
  • Different roles of PR-3 chitinase isozymes in rice  [通常講演]
    Tsukiyama T; Okumoto Y; Inouye K; Terachi T; Tanaka C; Nakazaki T; Tanisaka T
    5th International Rice Genetics Symposium and 3rd International Rice Functional Genomics Symposium 2005年 ポスター発表
  • Biological functions of PR-3 chitinase isozymes in rice (Oryza sativa L.)  [通常講演]
    Tsukiyama T; Inouye K; Nakazaki T; Terachi T; Tanaka C; Mikami B; Okumoto Y; Tanisaka T
    World Rice Research Conference 2004 2004年 ポスター発表

MISC

共同研究・競争的資金等の研究課題

  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 種坂 英次; 築山 拓司; 白澤 健太
     
    菌床培地において、正常株nは全ての栽培瓶で子実体を形成した。変異株d3とd4は一部の栽培瓶で菌床表面の一部のみから子実体を形成したことから、両株は子実体形成に関して極めて不安定であるものの、完全な子実体形成不能ではないと判断した。一方、変異株d5は菌床面に子実体原基の様な凹凸は観察されたが、それ以上の分化はみられなかったことから、完全な子実体形成不能株であると判断した。フラスコ内のMYS液体培地において、正常株nでは25℃で培養した栄養菌糸体を15℃の低温下に培養温度を変更することで子実体を形成したが、変異株d3、d4、d5では低温下でも子実体は形成されなかった。 RNA-seq解析において、エノキタケの子実体形成期に特異的に発現することが知られているFDS遺伝子やエノキタケを含む数種の菌類でも子実体形成期に発現するhyd1遺伝子が、低温刺激を与えた正常株でのみ誘導される遺伝子としてピックアップされた。RT-qPCRによる子実体形成関連遺伝子の発現解析において、Fvhyd1遺伝子とFvFDS1遺伝子は低温刺激を与えた、変異株d3、d4では発現せず、正常株nの原基形成期に特異的に発現していた。しかし、低温刺激を与えた、変異株d5では両遺伝子の発現が確認されたことから、この2つの遺伝子は子実体原基形成において、必要条件であるが十分条件では無いと考えられた。また、両遺伝子の発現はすべての菌株で連動していたことから、これらの発現を制御する共通の転写調節因子の存在が考えられる。本研究によって、これまで野生株を用いた子実体形成期に発現すると観察されていたFDS1やhyd1遺伝子の特性が、子実体形成不全株を用いることで、より明確に示された。
  • 転移因子の活性を制御するエピゲノムリプログラミング機構の解明
    日本学術振興会：科学研究費補助金(基盤研究(C))
    研究期間 : 2021年04月 -2024年03月 
    代表者 : 築山拓司; 吉川貴徳; 谷坂隆俊
  • 転移因子による選択的スプライシングを制御るスプライシング暗号の解明（継続）
    公益財団法人 G-7奨学財団：研究開発助成
    研究期間 : 2022年04月 -2023年03月
  • 転移因子による選択的スプライシングを制御るスプライシング暗号の解明（継続）
    公益財団法人 G-7奨学財団：研究開発助成
    研究期間 : 2021年04月 -2022年03月 
    代表者 : 築山拓司
  • コーヒー抽出残渣での栽培に適したヒラタケ属きのこの品種開発とその廃菌床の農業利用
    一般社団法人ヤンマー資源循環支援機構：研究助成（一般）
    研究期間 : 2021年04月 -2022年03月 
    代表者 : 築山拓司
  • 転移因子による選択的スプライシングを制御するスプライシング暗号の解明
    公益財団法人 G-7奨学財団：研究開発助成
    研究期間 : 2020年04月 -2021年03月 
    代表者 : 築山拓司
  • 日本学術振興会：科学研究費助成事業 基盤研究(C)
    研究期間 : 2018年04月 -2021年03月 
    代表者 : 築山 拓司; 谷坂 隆俊; 奥本 裕
     
    イネ熱帯ジャポニカ品種Oiranは、mPingが転移しているイネ品種銀坊主と同じSNPタイプのPingが存在するにもかかわらず、Pingは発現しておらず、mPingも転移していない。今年度はまず、Oiranとイネ温帯ジャポニカ品種日本晴（Pingは発現している）のPingのプロモーター領域のDNAメチル化を調査した。その結果、日本晴と比較して、OiranではPingのプロモーター領域が高度にメチル化されていた。このことから、OiranではPingの発現を全身的に抑制する因子によってmPingの転移が抑制されていると考えられた。そこで、Oiranと日本晴の交雑後代（ON系統）を作出し、Pingの発現とメチル化程度を調査した。その結果、ON系統F1個体では、Pingのプロモーター領域のメチル化が低下しており、Pingの発現量が日本晴と同程度であった。このことから、OiranにおけるPing発現抑制因子は日本晴にも対立遺伝子がある場合は劣性であるか、日本晴には別の座にPing活性化因子が存在する可能性が示唆された。Oiran由来のPingのみをホモで有するON系統F2個体群において、Ping発現抑制因子をもたない個体が現れるのではないかと期待されたが、Pingの発現量が日本晴以上あるいは同程度の個体は得られなかった。Pingが抑制されていたON系統F2個体では、プロモーター領域が高度にメチル化されていた。ON系統F3個体群においては、Pingの発現は抑制されていた。今年度は、OiranにおけるPing発現抑制因子を同定することはできなかったものの、イネの亜種間交雑によってPingのメチル化が低下し、発現が回復することを見出した。また、亜種間交雑によって低下したメチル化は、自殖によって世代を経るごとに再度メチル化することを明らかにした。
  • 日本学術振興会：科学研究費助成事業
    研究期間 : 2017年04月 -2020年03月 
    代表者 : 種坂 英次; 築山 拓司
     
    単核性発茸特性を示すエノキタケ系統（bmHY4）について、アグロバクテリウムを介した形質転換（AMT）の効率化を進めた。アグロバクテリウム株LBA4404とC58C1ではEHA105と比較して4～5倍の形質転換効率を得た (Tanesaka et al. 2018)。一方、トランスポゾンの自律性転移による変異体の作出を試みた。真菌類Fusarium oxysporum由来の自立性転移因子impalaの全長を含むpUC-imp160領域をバイナリベクター（pPZP-HYG2）のT-DNA領域に導入し（pPZP-imp）、pPZP-impを用いたAMTによるエノキタケ形質転換体を作出した。
  • 活性型転移因子を利用したゲノムショック育種法の開発
    文部科学省：科学研究費補助金(基盤研究(A))
    研究期間 : 2014年 -2018年 
    代表者 : 奥本 裕; 寺石政義; 築山拓司; 齊藤大樹; 小出陽平
  • イネ活性型転移因子mPingの活性を制御する遺伝子の単離
    文部科学省：科学研究費補助金(基盤研究(C))
    研究期間 : 2014年 -2017年 
    代表者 : 谷坂 隆俊
  • 未利用酵素資源の食品工業、医薬品工業および農業科学への利用
    ワキ製薬株式会社：共同研究
    研究期間 : 2015年04月 -2016年03月 
    代表者 : 築山 拓司
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2013年 -2016年 
    代表者 : 築山 拓司; 奥本 裕; 寺石 政義; 齊藤 大樹
     
    本研究は、イネ活性型転移因子mPingが宿主ゲノムのエピゲノムにおよぼす効果を解析するとともに、mPingの転移と宿主のエピジェネティックな制御の関係を解明することで、転移因子を用いたエピゲノム育種を提案しようとするものである。イネ品種銀坊主は1,000コピー以上のmPingを有している。25年度は、Post-bisulfite Adaptor Tagging (PBAT)法でmPing配列特異的なライブラリーを作成し、次世代シーケンサー(NGS)を用いてコピー間のメチル化状態の網羅的解析を試みた。その結果、mPing内部のシトシンの平均メチル化程度は、CG、CHGおよびCHHサイトにおいてそれぞれ95%、58%および31%であった。本研究結果を基に、これまでの研究で明らかになっている16箇所のmPing挿入位置のメチル化程度を比較解析したところ、活性型mPingではほとんど全てのシトシンのメチル化が低下したのに対して、不活性型mPingではメチル化の低下はほとんどみられなかった。このことから、メチル化程度の減少がmPingを活性化する要因であることが確かめられた。しかし、本年度は、ライブラリーの調整が不十分であったため、コピー間のメチル化程度を比較することはできなかった。ユビキチン様タンパク質をコードするRurm1を機能喪失した突然変異系統IM294においてmPingは、メチル化の変化を伴わず、活発に転移している。これまでの研究から、IM294においては、コドン特異的にタンパク質翻訳が低下することが明らかになっている。IM294におけるmPing活性化機構を明らかにするために、iTRAQを用いた定量プロテオーム解析によって、IM294特異的にタンパク質量が低下している遺伝子の同定を試みた。結果の詳細は、解析中である。
  • 文部科学省：科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2014年 -2015年 
    代表者 : 築山 拓司
     
    本研究では、イネStowawayファミリー転移因子・Pyongの再活性化を試みた。日本晴を含む温帯ジャポニカ品種には、Pyongは存在せず、染色体5および10を除く全ての染色体にPyongと高い相同性を示す23のPyong様因子が座乗することが明らかになった。しかし、これらは、DNA脱メチル化処理やガンマ線照射では再活性化しなかった。バイオインフォマティクス解析によって、トウモロコシから推定自律性因子Zm-aPyong (Zea mays active Pyong, 3084bp)を同定した。これらの成果によって、StowawayファミリーMITEの転移を解析することが可能になった。
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2010年 -2013年 
    代表者 : 奥本 裕; 寺石 寺石; 築山 拓司; 齊藤 大樹
     
    イネトランスポゾンmPingは、プロモーター領域に転移することで下流の遺伝子の発現に低温や塩ストレス応答性を付与することから、新たな発現プロファイルをもつ遺伝子の創出に繋がると期待される。本研究では、まず、mPingが活発に転移しているイネ品種銀坊主を用い、mPing挿入によって生じる変異遺伝子の選抜系を構築した。次いで、プロモーター領域にmPing挿入を有するストレス応答性遺伝子の発現プロフィルとDNAメチル化程度を解析した。その結果、mPingは、既存のプロモーター領域のメチル化程度を変えることなく、近傍のストレス応答性遺伝子のストレス応答性を多様に改変することが明らかになった。
  • 文部科学省：科学研究費補助金(挑戦的萌芽研究)
    研究期間 : 2011年 -2012年 
    代表者 : 築山 拓司
     
    トランスポゾン mPing が活発に転移しているイネ細粒突然変異系統IM294 の自殖後代に出現した強勢変異体 VGI の強勢発現機構を解析した。その結果、強勢発現には mPing の爆発的転移が不可欠であり、VGI1 においては mPing 挿入を内部もしくは近傍に有する遺伝子が 20 個存在することが明らかになった。本研究の結果は、トランスポゾンが転移によって生物進化を牽引することを実験的に示唆したものである。
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2009年 -2012年 
    代表者 : 谷坂 隆俊; 築山 拓司; 築山 拓司; 奥本 裕; 寺石 政義; 谷坂 隆俊; 築山 拓司
     
    イネ MITE・mPing は、品種銀坊主において今なお活発に転移している。本研究において、(1)mPing は近傍遺伝子の発現に正もしくは中立な効果を付与すること、(2)銀坊主には mPing 転移を制御する遺伝的要因が存在すること、および(3)ユビキチン様タンパク質 RURM1 の機能喪失が mPing 転移に起因する遺伝的多様性の拡大に有効であることが明らかになった。これらの成果によって、mPing を用いた高効率トランスポゾンタギング法の基盤が確立できた
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2006年 -2007年 
    代表者 : 奥本 裕; 谷坂 隆俊; 中崎 鉄也; 築山 拓司
     
    mPingの起源を明らかにするためにOryza属19種50系統におけるmPingの分布を調査した。その結果、mPing配列は供試した全てのOryza種に分布し、遠縁種間でもmPingの配列は極めて高度(99%以上)に保存されていた。野生イネに見いだされたmPing配列は3種に大別でき、銀坊主で最初に見出された430bpのもの(I型)、242-252にかけて9塩基の置換を有するもの(II型)、および239-249にかけての11塩基が欠失しているもの(III型)があった。後2者の分布がAAゲノムのO.sativa、O.rufipogonに限定されているのに対して、I型は一部のAAゲノム種を除く全ての種に見出された。したがって、mPing配列の起源はOryza属の種分化以前に遡ると考えられた。また、PongはO.sativaおよびO.rufipogonの全ての系統に認められたにも関わらず、Pingの分布は一部の系統に限られていた。このことは、Pongの起源はO.rufipogonが他のAAゲノム種から分化するときに遡り、PingはPongの後から起源したことを示唆している。銀坊主はmPingならびにPingの数が極めて多い特異な遺伝子型をもつ。とりわけmPingは、既に1000コピー以上に達しているにもかかわらず、毎世代増幅を続けている。銀坊主においてmPingの転移が抑制されない原因を解明するため、mPing転移が抑制されている日本晴と銀坊主との交雑後代F_4個体別F_5系統を用いて系統毎に親個体にはない新規挿入数を調査した。さらに、銀坊主と日本晴との間のmPing挿入多型を利用して作製した遺伝子地図に基づいたmPing活性に関するQTL解析を実施した結果、銀坊主のmPing転移活性に関与するQTLを染色体1および4に検出した。
  • 文部科学省：科学研究費補助金(基盤研究(B))
    研究期間 : 2005年 -2007年 
    代表者 : 谷坂 隆俊; 奥本 裕; 中崎 鉄也; 寺石 政義; 築山 拓司
     
    MITEsは、転移に伴って遺伝子の構造やその発現を変えることから、生物の進化に大きく関わっていると考えられる。非自律性トランスポゾンmPingは、動植物を通じて初めて同定された転移活性を有するイネのMITEである。これまでの研究から、mPingは、イネ品種銀坊主において通常の栽培条件下でも高い転移活性を有しており、遺伝子近傍に転移しやすいことが明らかとなっている。mPingの転移には、自律性因子PingおよびPongがコードする転移酵素が不可欠であり、それらのコピー数は品種間で異なる。本研究では、まず、銀坊主におけるPingの座乗位置の調査の過程において、Tc1/marinerスパーファミリーに属する新規トランスポゾンPyongを同定した。データベース検索の結果、品種日本晴の染色体1および12にもPyongと90%以上の高い相同性を示す配列がに存在することが明らかとなった。このことは、Pyong因子は、近年、イネゲノム中で増加したことを示唆している。次いで、銀坊主、日本晴および細粒突然変異系統IM294のcDNAを用いたトランスポゾンディスプレイ法によって、mPing配列を含んで転写される遺伝子の同定を試みた。その結果、mPing配列を含んで転写されている11の領域が同定された。これらのうち、6つは遺伝子の転写領域、5つは遺伝子をコードしない領域であり、供試品種・系統間にはこれら領域にmPing挿入多型があることが明らかとなった。また、mPing挿入がある遺伝子のmPing挿入位置より上流および下流の転写量を調査したところ、mPing挿入位置より下流の転写量が顕著に低下していることが明らかとなった。これらのことは、mPingの挿入が、ゲノムの構造のみならず、近傍配列の転写を増進および抑制していることを示唆している。
  • アブシジン酸によって誘導されるイネクラスⅡキチナーゼCHT11が植物体の老化および成長・発育に及ぼす効果の解析
    京都大学：若手研究者スタートアップ研究費
    研究期間 : 2005年04月 -2006年03月 
    代表者 : 築山 拓司
  • イネにおけるPR-3キチナーゼを介したストレス防御および生育・分化機構の解明
  • Analysis of stress defense- and growth and development-mechanism mediated PR-3 chitinase in rice (Oryza sativa L.)

委員歴

  • 2017年01月 - 現在   ナント種苗株式会社   組換えDNA実験安全委員会 外部学識経験者委員
  • 2020年04月 - 2022年03月   日本育種学会   代議員
  • 2016年04月 - 2020年03月   日本育種学会   運営委員（集会）
  • 2014年04月 - 2016年03月   日本育種学会   幹事
  • 2010年04月 - 2012年03月   日本育種学会   幹事
  • 2008年04月 - 2010年03月   日本育種学会   常任幹事

担当経験のある科目

  • 植物分子生物学近畿大学
  • 植物育種学近畿大学

その他のリンク

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