2018-01-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/664379摘要：&#160;文心蘭為臺灣第一外銷切花，在採後處理及分級選別過程中容易拉扯花枝，導致 大量花朵脫落或花藥蓋脫落，數小時後會產生大量乙烯，使花朵壽命減少，即使經 燻蒸處理加上保鮮劑處理，文心蘭切花瓶插壽命之延長效果還是有限。本計畫將以 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，針對文心蘭 EIN2 基因進行編輯，使 EIN2 基因喪失功能 ，阻斷乙烯訊息傳導，使文心蘭無法感受乙烯，達到延長文心蘭切花花期之目的。 首先將利用文心蘭 EIN2 基因之專一性區域合成寡核&#33527;酸引子，利用 T7 啟動子於 試管內合成專一性 sgRNA，與 Cas9 核糖核酸蛋白複合物 (ribonucleoprotein, RNP) 進行組裝，形成核糖核酸蛋白複合物。接著採用聚乙二醇 (polyethylene glycol, PEG) 轉殖法，將 Cas9 RNP 核糖核酸蛋白複合物，進行文心蘭原生質體之 核轉染，之後進行基因編輯頻率檢測。基因編輯的頻率之檢測，將利用可辨認並切 除非完全互補異型雙股 DNA (mismatched heteroduplex DNA) 的 T7 endonuclease I，檢測突變股與對照股之雜合情形。進一步將上述構築進行文心蘭基因轉殖，期望 轉殖文心蘭中乙烯訊息傳導受阻，取得延長切花壽命的文心蘭。<br> Abstract: &#160;Oncidesa is an important ornamental crop whose cut flower is with the highest export value in Taiwan. During postharvest and classification ethylene is induced by pollinia cap dislodgment and petals become wilting. The effect of fumigation by fresh-keeping agent is still not good enough. Genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) will be employed to knockout EIN2 gene in Oncidesa to prolong the shelf-life of Oncidesa cut flowers by interrupting ethylene signal transduction. First of all, single-guide RNA (sgRNA) will be transcribed by T7 promoter in vitro and mixed with Cas9 protein to form ribonucleoprotein (RNP). Then the Cas9 RNP will be introduced into Oncidesa protoplasts by polyethylene glycol (PEG)-mediated transfection. The editing efficacy will be investigated by T7 endonuclease I assay, polymerase chain reaction, and sequencing. Later on, both functional sgRNA and Cas9 gene will be constructed in the binary vector for Agrobacterium -mediated transformation into embryogenic callus of Oncidesa . Hopefully, the longevity of cut flower of edited Oncidesa could be prolonged.文心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OncidesaGene Editingclustered regularly interspaced short palindromic repeatsCRISPR/Cas9