マイクロアレイを用いた長期低温遭遇時のウメ休眠芽のトランスクリプトーム解析 Custom Microarray Analysis for Transcript Profiling of Dormant Vegetative Buds of Japanese Apricot during Prolonged Chilling Exposure

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Abstract

永年性作物において越冬芽の休眠は,環境に適応するために進化した成長制御機構のひとつであり,かつ翌年の成長を左右する重要な農業形質のひとつである.芽の休眠には多くの遺伝的要因が関わるばかりでなく,多岐にわたる環境要因や樹勢,樹齢の影響もうけており,多くの遺伝子の発現制御が休眠に関与していると考えられる.そこで本研究では,休眠関与候補遺伝子の単離を目的に,カスタムマイクロアレイを用いたウメ休眠芽のトランスクリプトーム解析をおこなった.まず,我々が先行研究で獲得したウメ芽 EST 配列情報をもとに,ゲノム全体をできるだけ偏りなく網羅するように選びだした 58539 ユニジーンに相当するプローブを搭載したマイクロアレイを構築した.次いで,休眠覚醒を誘導する長期の低温処理を施したウメ休眠芽の遺伝子発現変動を調査した.その結果,有意に 2 倍以上発現が上方制御あるいは下方制御されるプローブが 2345 個あるいは 1059 個同定された.これらの変動遺伝子のなかには季節的な発現同調性がみられるものがあった.下方制御される遺伝子のなかには,先行研究で休眠への関与が示唆されている <i>DORMANCY ASSOCIATED MADS-box </i>が含まれており,上方制御される遺伝子にはリポキシゲナーゼが含まれていた.Parametricanalysis of gene set enrichment 解析の結果,長期の低温遭遇によってジャスモン酸やオキシリピン生合成・代謝の GO タームが有意に上昇し,概日リズムの GO タームが有意に減少した.これらの GO タームに含まれる遺伝子について定量 RT-PCR を行った結果,マイクロアレイで検出された発現変動パターンとほぼ同様であった.以上の結果より,本研究で構築したマイクロアレイはウメ休眠芽の網羅的な遺伝子発現解析に有効であることが示された.本実験では,ウメの休眠覚醒に関与する遺伝子ネットワークあるいは新規候補遺伝子の探索に活用した.

Bud dormancy is a critical developmental process for perennial plant survival, and also an important physiological phase that affects the next season's growth of temperate fruit trees. Bud dormancy is regulated by multiple genetic factors, and affected by various environmental factors, tree age and vigor. To understand the molecular mechanism of bud dormancy in Japanese apricot (<i>Prunus mume</i> Sieb. et Zucc.), we constructed a custom oligo DNA microarray covering the Japanese apricot dormant bud ESTs referring to the peach (<i>P. persica</i>) genome sequence. Because endodormancy release is a chilling temperature-dependent physiological event, genes showing chilling-mediated differential expression patterns are candidates to control endodormancy release. Using the microarray constructed in this study, we monitored gene expression changes of dormant vegetative buds of Japanese apricot during prolonged artificial chilling exposure. In addition, we analyzed seasonal gene expression changes. Among the 58539 different unigene probes, 2345 and 1059 genes were identified as being more than twofold up-regulated and down-regulated, respectively, following chilling exposure for 60 days (<i>P</i> < 0.05). Cluster analysis suggested that the expression of the genes showing expression changes by artificial chilling exposure were coordinately regulated by seasonal changes. The down-regulated genes included <i>P. mume DORMANCY-ASSOCIATED MADS-box</i> genes, which supported previous quantitative RT-PCR and EST analyses showing that these genes are repressed by prolonged chilling exposure. The genes encoding lipoxygenase were markedly up-regulated by prolonged chilling. Our parametric analysis of gene-set enrichment suggested that genes related to jasmonic acid (JA) and oxylipin biosynthesis and metabolic processes were significantly up-regulated by prolonged chilling, whereas genes related to circadian rhythm were significantly down-regulated. The results obtained from microarray analyses were verified by quantitative RT-PCR analysis of selected genes. Taken together, we have concluded that the microarray platform constructed in this study is applicable for deeper understanding of the molecular network related to agronomically important bud physiology, including dormancy release.

Journal

  • Journal of the Japanese Society for Horticultural Science

    Journal of the Japanese Society for Horticultural Science 83(1), 1-16, 2014

    THE JAPANESE SOCIETY FOR HORTICULTURAL SCIENCE

Codes

  • NII Article ID (NAID)
    130004510808
  • Text Lang
    ENG
  • ISSN
    1882-3351
  • Data Source
    J-STAGE 
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