Attempts of plant mitochondrial genome modification via mitochondria-targeting TALENs

2018 Fiscal Year Final Research Report

• Project/Area Number: 17K19256
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Research Field: Agricultural and Environmental Biology and related fields
• Research Institution: Tohoku University
• Principal Investigator: Kazama Tomohiko 東北大学, 農学研究科, 助教 (30431464)
• Research Collaborator: TORIYAMA kinya ; ITO masako ; IKEDA kenichiro
• Project Period (FY): 2017-06-30 – 2019-03-31
• Keywords: 植物ミトコンドリア / ゲノム改変

Outline of Final Research Achievements

In this study, we attempted to develop a method of a mitochondrial genome modification. (1) We tried to develop a method for an insertion of a transgene, gfp gene, via homologous recombination. Because we have found that a double strand break caused by mitochondria-targeted TALEN (mitoTALEN) is repaired by homologous recombination. To replace an orf79 in BT-type CMS mitochondrial genome, we constructed atp6-gfp donner vector. This vector contains 2-kb upstream and downstream sequence of orf79 and the orf79 sequence is replaced with gfp gene. To deliver atp6-gfp vector to mitochondria, we used particle bombardment. However, we could not obtain gfp-inserted plants. This indicates that the particle bombardment is not effective for a gene delivery to mitochondria. (2) We tried to modify an orf314, previously termed orf288, using mitoTALEN. Although we obtained orf314-lost transgenic Taichung 65, we could not obtain orf314-modified plants.

Free Research Field

植物分子遺伝学

Academic Significance and Societal Importance of the Research Achievements

植物ミトコンドリアのゲノム上には，呼吸に関与する因子のみでなく，農業上重要な形質である細胞質雄性不稔性の原因遺伝子もコードしている．さらに，機能不明のorfも多く存在していることが知られていが，機能不明のorfについての解析も進んでいないのが現状である．そこで，人工制限酵素TALENにミトコンドリア移行シグナルを付加したmitoTALENを用いて切断箇所へのGFP遺伝子の挿入を試みた．成功すれば，世界で初めてのミトコンドリア遺伝子導入技術として学術的な意義がある．一方，雄性不稔の原因遺伝子を導入する事が可能となれば，多収育種の基盤を提供することが可能となるため，社会的意義も大きいと考えられる．