成果報告書詳細
管理番号20110000000474
タイトル*平成22年度中間年報 新エネルギー技術研究開発 バイオマスエネルギー等高効率転換技術開発(先導技術開発) 糖化酵素を高度に蓄積するバイオ燃料用草本植物の開発
公開日2011/6/7
報告書年度2010 - 2010
委託先名株式会社ホンダ・リサーチ・インスティチュート・ジャパン 国立大学法人京都大学
プロジェクト番号P07015
部署名新エネルギー部
和文要約和文要約等以下本編抜粋:1.研究開発の内容及び成果等 (1)小胞体に糖化酵素を高度に蓄積するバイオ燃料用草本植物の開発(担当:ホンダ・リサーチ・インスティチュート・ジャパン)(1)-1.糖化酵素の微生物発現 酵母発現系により調製した標準糖化酵素を利用して、蛍光基質を用いた酵素活性測定法とそこからの酵素蓄積濃度算出法を確立した。 (1)-2.植物発現ベクターの構築 合成した糖化酵素遺伝子および単離した小胞体蓄積関連因子をもとに、PCR法を用いて計10種以上の植物発現ベクターを作製した。
英文要約Title: New Energy Technology Development. Development of Technology for High-efficiency Conversion of Biomass and Other Energy (Development of Preparatory Basic Bioenergy Technologies). Development of Herbaceous Biofuel Feedstocks that Store Cellulolytic Enzymes (FY2009-FY2012) FY2010 Annual Report
An objective of this research is to store cellulolytic enzymes in subcellular organelles of herbaceous biofuel feedstocks to enable a simultaneous supply of cellulosic biomass and cellulolytic enzymes. Our goal is to produce modified switchgrass that stores 1% of its dry weight in cellulolytic enzymes thereby enabling all of the plant’s cellulose to be saccharified.
(1) Storage of a Large Amount of Cellulolytic Enzymes in Endoplasmic Reticulum. Expression cassettes that contained the cellulolytic enzyme gene and genetic elements related to endoplasmic reticulum (ER) storage were constructed, introduced into binary vectors and then used for Agrobacterium-mediated transformation of rice. More than 10 transgenic plants introduced with these expression constructs were obtained and subjected to enzyme activity assay and immunoblot analysis. Transgenic lines, which formed novel ER-derived storage organelles, accumulated up to 3.5% (total soluble protein) of cellulolytic enzyme and these concentrations were higher than that observed in transgenic lines used conventional expression systems. Formation of novel ER-derived storage organelles were confirmed in GFP-expressed transgenic plants by fluorescent microscopic analysis.
(2) Storage of a Large Amount of Cellulolytic Enzymes in Vacuole. Expression cassettes that contained the cellulolytic enzyme gene and the isolated gene responsible for efficient vacuolar storage were constructed, introduced into binary vectors to generate transgenic Arabidopsis plants. More than 30 transgenic plants with these expression constructs were obtained and then subjected to assay of the enzyme activity and to immunoblot analysis. The accumulation level of vacuole-targeted cellulolytic enzyme was three times higher than the reported level of the apoplast-targeted cellulolytic enzyme. Furthermore, we found that the accumulation level of exogenous cellulolytic enzyme in the vacuoles can be increased by genetically reducing the endogenous vacuolar protein levels.
(3) Genetic Modification of Switchgrass. More than 500 callus lines were obtained from independent mature seeds, and over 50 lines showed plant regeneration capacity. Three lines, which showed high regeneration capacity, were used for Agrobacterium-mediated transformation, and transgenic plants were successfully recovered from all lines.
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