蝴蝶蘭定點突變及抗病毒之基因轉殖

Abstract

蝴蝶蘭(Phalaenopsis spp.)是臺灣外銷最大宗花卉作物，受蕙蘭嵌紋病毒(Cymbidium mosaic virus, CymMV)及齒舌蘭輪斑病毒(Odontoglossum ringspot virus, ORSV)等病毒性病害嚴重影響品質。本論文期望運用基因編輯及核糖核酸干擾(RNA interference)技術，取得抗病毒之蝴蝶蘭。首先建立蝴蝶蘭之基因編輯平台，選取蝴蝶蘭八氫番茄紅素去飽和酶(phytoene desaturase, PDS)基因，以CRISPR/Cas9系統進行突變；此基因若喪失功能，會導致白化表觀。經分析P. amabilis PDS cDNA序列及選殖部分PDS基因片段，選擇位於第二個及第三個顯子的20 bp作為標靶序列，構築於表現載體，以農桿菌注射法及聚乙二醇法，分別轉入P. amabilis之葉片及原生質體。轉殖後抽取基因組DNA，進行高解析熔解曲線分析及次世代定序，結果顯示農桿菌注射法之葉片及聚乙二醇法之原生質體中，均造成標靶序列發生單一核苷酸之置換，表示標靶序列具有功能。穩定性轉殖採用花粉管導入法，分別將基因編輯表現質體及雙重病毒基因默化質體轉殖至蝴蝶蘭，得到之原球體於GUS活性組織化學染色後皆有藍點之表現；轉殖基因編輯表現質體得到之原球體具白化表觀，顯示PDS基因編輯成功。

Phalaenopsis orchids are the largest export floral crops in Taiwan. Infections by viruses such as Cymbidium mosaic virus (CymMV) and Odontoglossum ringspot virus (ORSV) dramatically reduce the value of Phalaenopsis. In this study, genome editing and RNA interference (RNAi) technology were used to develop virus resistant Phalaenopsis. In order to establish a genome editing platform in Phalaenopsis, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) was used to mutate the phytoene desaturase (PDS) gene. Loss-of-function of PDS leading to the bleaching phenotype indicates the editing event happened. After the analysis of PDS cDNA sequence and cloning of partial PDS gene, two 20-nucleotide guide sequences located at the second and third exons, respectively, were selected to construct into the expression vector. The gene editing constructs were transformed into leaves and protoplasts of P. amabilis via agroinfiltration and polyethylene glycol (PEG)-mediated transformation, respectively. Genomic DNAs extracted from transformed explants were subjected to the high-resolution melting curve (HRM) analysis and next generation sequencing (NGS). Single nucleotide substitutions were detected in the guide sequence of edited cells. For stable transformation, gene editing expression plasmids and the virus dual gene silencing plasmid were transformed into Phalaenopsis via pollen tube pathway. Putative transgenic protocorms showed blue spots after the GUS assay. Several putative transgenic protocorms with albino phenotype indicated that PDS gene had been edited in these transformants successfully.