2023-01-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/653259嘉德麗雅蘭 (Cattleya) 之切花及盆花之壽命皆偏短，不及產業需求。嘉德麗雅蘭為更年性花卉之一，對乙烯非常敏感。ETHYLENE INSENSITIVE 2 (EIN2) 為乙烯訊息傳導蛋白質，負責調控乙烯反應。本計畫擬利用群聚且規律性間隔的短回文重複序列 (clustered regularly interspaced short palindromic repeats, CRISPR)/Cas9系統之基因組定點編輯 (targeted genome editing) 技術，針對嘉德麗雅蘭參與乙烯訊息傳導的ETHYLENE INSENSITIVE 2 (EIN2) 基因進行編輯，使EIN2 基因喪失功能，以阻斷嘉德麗雅蘭之乙烯訊息傳導，使嘉德麗雅蘭無法感受乙烯，達到延長花期的效果。前一年度已完成嘉德麗雅蘭 EIN2 基因序列 sgRNA 之設計及合成，並組裝 sgRNA 與 Cas9 核糖核蛋白複合物測試，確認導引序列可正確引導 Cas9 切割目標序列。今年度將進一步構築植物用表現質體，並進行嘉德麗雅蘭葉片及類原球體之農桿菌媒介基因轉殖，期望經基因編輯的嘉德麗雅蘭中乙烯訊息傳導受阻，取得延長切花壽命的嘉德麗雅蘭。 Both cut and potted flowers of Cattleya have shorter shelf-life than industrial need. Cattleya is one of the climacteric flower crops whose flowers are very sensitive to ethylene. In this project, genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was employed to knockout ETHYLENE INSENSITIVE 2(EIN2) gene in Cattleya to prolong the shelf-life of Cattleya flowers by interrupting ethylene signal transduction. EIN2 gene had been cloned and its sequence had been analyzed for candidate guide sequences of single-guide RNA (sgRNA). Single-guide RNA (sgRNA) transcribed by T7 promoter in vitro and mixed with Cas9 protein to form ribonucleoprotein (RNP) had been validated its function by in vitro digestion last year. In this year, the guide sequence will be constructed to form plant genome editing-expression plasmid. The editing-expression construct will be introduced into Cattleya leaf discs and protocorm-like body (PLB) by Agrobacterium-mediated transformation. Transformed leaf discs or PLBs will be screened on selection medium containing antibiotics. Hopefully, the longevity of cut flowers of edited Cattleya could be prolonged.嘉德麗雅蘭;基因編輯;群聚且規律性間隔的短回文重複序列/CRISPR 關聯蛋白質;Cattleya;Gene Editing;clustered regularly interspaced short palindromic repeats; CRISPR)/Cas9