成果報告書詳細
管理番号20100000001517
タイトル*平成21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) 遺伝子組換え技術による新規なミスカンサス育種素材の創出
公開日2010/9/9
報告書年度2009 - 2009
委託先名国立大学法人北海道大学北方生物圏フィールド科学センター
プロジェクト番号P07015
部署名新エネルギー技術開発部
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)ミスカンサスにおける形質転換植物作製技術の開発 1)内容 ミスカンサスの中で豊富な遺伝資源を有するススキ(Miscanthus sinensis)を用いてアグロバクテリウム法やパーティクルガン法を用いた遺伝子組換え技術を確立する。実験当初はレポーター遺伝子として緑色蛍光タンパク質(GFP)遺伝子を用いて最適な実験諸条件を明らかにする。寒地型イネ科牧草から単離したフルクタン(多糖)合成酵素遺伝子の導入を図る。ススキに導入することにより、フルクタンが液胞内に蓄積され、その結果、細胞内の糖含量を増加させるとともに、生長が促進され、バイオマスを増加させる効果が期待できるとともに組換え植物体中リグニン含量の低下が予想される。
英文要約Title : Development of Novel Miscanthus Breeding Materials through Genetic Modification (FY2009-2010) FY 2009 Annual Report.
The perennial herbaceous plant, Miscanthus genus, is a suitable feedstock for biofuel production because of its high biomass production and sustainability. It is necessary, however, to develop a novel Miscanthus variety through genetic improvement that has valuable characteristics that would make it a more suitable and reliable feedstock. In this context, the development of high biomass yielding Miscanthus breeding strains with high efficiency of saccharification, is being attempted through genetic modification. We have two strategies for the molecular breeding of Miscanthus. One of them is obtaining fructan-accumulating transgenic Miscanthus plants by the introduction of fructosyltransferase genes isolated from C3 grass, perennial ryegrass. Another one is transgenic down-regulation of enzymes involved lignin biosynthesis.
An in vitro culture system has been established in susuki (Miscanthus sinensis). A high concentration of 2,4-D (5 mg/L) in combination with a low concentration of BA (0.01 mg/L) was proved to be suitable for callus induction. Shoot regeneration occurred using 1 mg/L of TDZ together with a lower concentration of auxin, IAA (0.5 mg/L). Miscanthus shoots were directly subcultured into MS medium to develop roots. In an accession derived from ‘Tanegashima’ high efficiency of shoot regeneration was observed. We have used the callus line from ‘Tanegashima’ in the following transgensis experiment.
Transgenesis by particle bombardment has been carried out using embryogenic callus of Miscanthus sinensis. We have established the protocol for transformation of reporter gene (GFP gene) by a particle bombardment. We have obtained several hundred putative transgenic plants with GFP gene. The expression of GFP in leaflets was observed. Confirmation of transgenesis is now underway.
We constructed vectors containing fructosyltransferase genes from perennial ryegrass. Transformation of these genes into embryogenic callus of Miscanthus sinensis is now underway.
Five partial cDNAs encoding one of enzymes involved lignin biosynthesis, COMT (caffeic acid O-methyltransferase) were isolated from the Miscanthus plant. Sequences of these partial COMT genes were highly similar to COMT genes from other perennial herbaceous species such as tall fescue and perennial ryegrass. We will construct an RNAi vector using sequence information of the genes to induce the down-regulation of COMT.
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