利用CRISPR/Cas9系統編輯文心蘭EIN3基因延緩花瓣老化

Abstract

文心蘭為我國最大宗外銷切花。文心蘭在採收及分級過程中，易因花藥蓋脫落而加速內生乙烯生成，進而造成花瓣老化喪失商品價值。ETHYLENE INSENSITIVE 3 (EIN3)為乙烯訊息傳導途徑之正調控因子，可促進許多不同反應乙烯以及逆境的基因表現，剔除EIN3基因功能，可阻斷乙烯訊息傳導，達到延長花期的效果。已知於文心蘭花苞中表現的EIN3基因OgEIL1及OgEIL2，並於花多開始萎凋時達最大表現量，故本研究利用CRISPR/Cas9基因編輯系統，剔除文心蘭OgEIL1或OgEIL2基因功能。首先於OgEIL1及OgEIL2基因序列，依sgRNA之鹼基偏好、GC含量、位置等條件，各挑選2個區域作為sgRNA的目標序列，合成寡核苷酸後構築至通用表現載體。分別將10、20、30 μg表現質體DNA經由聚乙二醇 (polyethylene glycol, PEG) 轉殖法導入文心蘭原生質體，各培養24、48、72小時後，萃取基因組DNA以高解析熔解曲線分析 (high resolution melting curve analysis, HRM) 及T7核酸內切酶I (T7 endonuclease I, T7E1) 分析，檢測結果顯示，目標序列已受到CRISPR/Cas9編輯而產生突變，再經定序比對未轉殖細胞之目標序列，編輯效率為3.3%，基因序列為單一核苷酸突變，造成胺基酸由精胺酸改變為麩醯胺酸。另外以農桿菌真空滲入法基因轉殖至文心蘭葉片，進行短暫表現分析，尚未偵測到突變的目標序列。分別以距離6、9、12公分對文心蘭癒傷組織進行基因槍轟擊，以6公分處理所觀察到的GFP螢光表現量最多。利用基因槍法以及農桿菌媒介法轉殖CRISPR/Cas9表現質體至癒傷組織，進行穩定表現基因轉殖，透過抗生素篩選，擬轉殖細胞將經分子檢測及再生，獲得EIN3基因突變之轉殖株，期望將來透過自交，去除外來基因，獲得非基因轉殖之突變後代，並可有效延緩文心蘭之花瓣老化。

Oncidesa is the largest export cut flower in Taiwan. Loss the pollinia cap during harvest and classification induces production of endogenous ethylene in Oncidesa. ETHYLENE INSENSITIVE 3 (EIN3) is a positive regulator of the ethylene signaling pathway that promotes the expression of many different ethylene and stress genes. In this study, the CRISPR/Cas9 gene editing system was used to knock-out the gene function of OgEIL1 or OgEIL2 gene, which all two expressed abundantly EIN3 genes in Oncidesa flower. First of all, two target sequence from both OgEIL1 and OgEIL2 genes were selected according to the base preference, GC content and location in the gene to synthesis oligonucleotides and constructed into the universal expression vector. Ten, twenty, thirty μg of CRISPR/Cas9 expression vectors were introduced into protoplasts via polyethylene glycol (PEG), and then genomic DNA was extracted for high-resolution melting after 24, 48, 72 hours of culture. Results of high resolution melting curve analysis (HRM) and T7 endonuclease I (T7E1) assay showed that the target sequences had been edited by CRISPR/Cas9 as a single nucleotide mutation with 3.3% editing efficiency. In addition, the expression vectors were transferred to the leaves of Oncidesa by vacuum-assisted agroinfiltration, and no target sequence was edited. particle bombardment was performed at 6, 9, 12 cm target distance to Oncidesa callus, and the GFP fluorescence was observed at 6, 9 cm. Callus transformed with CRISPR/Cas9 expression vectors using particle bombardment or Agrobacterium-mediated method were selected by antibiotics. After regeneration and confirmation of genome editing, the transgenic plants will be self-pollinated to obtain self-life prolonged mutant progeny without foreign sequence.