2020-01-012024-05-15https://scholars.lib.ntu.edu.tw/handle/123456789/662074摘要：文心蘭花色鮮豔、花型優美，目前已成為臺灣外銷的第一大切花，然在其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。為了設計文心蘭 EIN2 基因專一的 CRISPR 序列，首先進行文心蘭 EIN2 cDNA 之選殖，並分析可供編輯之不同專一性區域，分別合成寡核&#33527;酸引子，完成基因編輯表現質體之構築。接著採用聚乙二醇 (polyethylene glycol, PEG) 轉殖法，將基因默化構築，進行文心蘭葉片或癒傷組織來源之原生質體的核轉染，經高解析熔解曲線分析 (high resolution melting curve assay) 及 T7 endonuclease I 分析，顯示有鹼基突變，經次選殖後定序分析，顯示編輯位點產生單一核甘酸多型性單，編輯效率為 3.3%。另外，上一年度完成之基因編輯農桿菌轉殖用表現質體，已利用基因槍法及農桿菌媒介法，轉殖至文心蘭癒傷組織，今年度將進行基因編輯文心蘭細胞之篩選及再生，期望取得基因編輯成功之細胞，經增殖及再生為延長花期的文心蘭。<br> Abstract: Oncidesa orchid one of the most importnat cut flowers in Taiawn. Currently, about 80% of Oncidesa cut flowers are exported, with the biggest export value in cut flower industry. During harvest and postharvest ethylene production is induced by pollinia cap dislodgment and makes petals wilt. The effect of fumigation by fresh-keeping agent is still not good enough. In this project, genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) were employed to knockout EIN2 gene in Oncidesa to prolong the shelf-life of Oncidesa cut flowers by interrupting ethylene signal transduction. As a first step of this project, EIN2 cDNA was cloned and its sequence was analyzed for candidate target sequences of single-guide RNA (sgRNA). Synthetic oligonucleotides were annealed and constructed to obtain genome editing-expression plasmid. After the expression plasmid was introduced into Oncidesa protoplasts by polyethylene glycol (PEG)-mediated transfection the editing efficacy was investigated by high resolution melting curve assay, T7 endonuclease I assay, and PCR-sequencing. Single-nucleotide polymorphisms were detected around the editing site with 3.3% efficiency. On the other hand, both functional sgRNA and Cas9 gene cassettes were constructed in the binary vector for Agrobacterium-mediated transformation last year. The final construct was transferred into embryogenic calli of Oncidesa via particle bombardment and Agrobacterium method. In this year, the embryogenic calli of Ondidesa will be selected for the presence of green fluorescent protein gene and further regenerated into Oncidesa seedlings. Hopefully, cut flowers of edited Oncidesa could last longer in a vase.文心蘭基因編輯群聚且規律性間隔的短回文重複序列及其關聯蛋白質OncidesaGene Editingclustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)