成果報告書詳細
管理番号20120000000171
タイトル*平成23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2015/12/4
報告書年度2011 - 2011
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
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) FY2011 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. The ultimate objective is to produce modified switchgrass that stores 1% of its dry weight (6.5% of total soluble protein) 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/green fluorescent protein (GFP) gene and genetic elements related to endoplasmic reticulum (ER) storage were constructed, introduced into binary vectors and then used for Agrobacterium-mediated transformation of switchgrass. Approximately 10 transgenic plants introduced with these expression constructs were obtained and subjected to microscopic analysis and enzyme activity assay. Formation of novel ER-derived storage organelles was confirmed in GFP-expressed transgenic switchgrass plants by fluorescent microscopic analysis. Formation of novel ER-derived storage organelles was also confirmed in cellulolytic enzyme-accumulating transgenic rice plants by immunostaining with anti-cellulolytic enzyme antibody.
(2) Storage of a Large Amount of Cellulolytic Enzymes in Vacuole. We generated the expression cassettes that contain both the genes encoding the cellulolytic enzyme/fluorescent protein and an inducible promoter-driven vacuole storage-related genetic element. They were introduced into binary vectors and then used for Agrobacterium-mediated transformation of switchgrass. Approximately 10 transgenic plants introduced with these expression constructs were obtained and subjected to biochemical and histological analyses. Both fluorescence microscopic analysis and immunoblot analysis confirmed that the accumulation level of the fluorescent protein in transgenic switchgrass increased several fold after the induction treatment. Transgenic switchgrass that formed novel storage vacuoles accumulated more than 6.5% (total soluble protein) of cellulolytic enzyme after induction treatment.
(3) Genetic Modification of Switchgrass. Elite callus lines, which have been established in this project and showed high regeneration capacity, were used for Agrobacterium-mediated transformation. Highly efficient transformation method is successfully established by optimization of various transformation stages.
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