以CRISPR/Cas9技術編輯水稻EPSPS基因

Abstract

CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats-associated protein 9)是能在多種生物間有效率地進行基因編輯的創新技術。本試驗利用辨識PAM (protospacer adjacent motif)序列為NGAN之Cas9變種VQR蛋白，修飾水稻EPSPS (5-enolpyruvylshikimate-3-phosphate synthase)基因，使其第177個胺基酸proline改為serine或threonine(對應DNA序列為CCA置換為TCA或ACA)，產生抗嘉磷塞水稻。將欲發生置換位置設計於PAM上游第4個序列，以農桿菌法轉形至水稻癒傷組織，並透過PCR分析PAM上游20個鹼基對內是否發生單一鹼基對置換。結果共獲得25個轉殖系，每個轉殖系分析12個位點，皆沒有發現鹼基對置換，其中測試結果為假陽性的有1.6%。以嘉磷塞進行抗性測試，最終篩選濃度建議為5 mM。轉殖系癒傷組織將繼續培養發根，直至收取T1種子。

Clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) has emerged as the most powerful technology for efficient genomic modification in many organisms. To obtain glyphosate resistant rice, VQR variant of Cas9 requires the sites containing NGAN PAM (protospacer adjacent motif) was expressed and expected to mutate one-point of rice EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), of which the codon CCA for the glycine residue at position 177 of the protein was changed to encode a serine or threonine residue (DNA sequences are TCA or ACA, respectively). The position of the target base was 4 bases upstream PAM. Rice calli were transformed using Agrobacterium. Single-nucleotide substitution was detected by PCR technique between 20 bases within the protospacer. Analyzing 12 positions of each transgenic plants of 25 independent lines showed that there was no substitution; 1.6% of the tested results were pseudo. The concentration of glyphosate selection criteria was 5 mM in this study. Transgenic seedlings were regenerated until T1 seeds which are collected for further research.