- 學位論文
應用RNA干擾技術抑制香蕉ACC氧化酶基因表現之研究
Studies on RNA Interference-Mediated Inhibition of Banana 1-Aminocyclopropane-1-Carboxylate Oxidase Genes
摘要
RNA干擾技術（RNA interference）係造成基因默化（gene silencing）的主要策略之一，目前已廣泛應用於基因功能的探討。本研究首先以CaMV 2X 35S啟動子，香蕉ACC氧化酶基因（1-aminocyclopropane-1-carboxylate oxidase）MAO1的第一個隱子為spacer，構築RNA干擾基因默化之通用載體pRNAi。 接著以GUS作為報導基因，並構築完整GUS順向基因-隱子（ intron）-反向基因，及反向基因-隱子-順向基因質體，另以GUS 3’端1220∼1806個核苷酸長587 bp片段構築反向基因片段-隱子-順向基因片段之質體，及GUS 3’端第1406~1430個核苷酸特有25 bp構築反向基因片段-隱子-順向基因片段之質體，以基因槍轉殖法共轉殖至蝴蝶蘭花瓣圓片，測試質體默化GUS表現能力，顯示反向基因-隱子-順向基因之構築較順向基因-隱子-反向基因默化效果為佳，可達85.98%基因默化效果；利用GUS 3’端 587 bp片段構築反向基因片段-隱子-順向基因片段之質體，則有最佳基因默化能力，能抑制GUS活性98.52%；而使用GUS特有之25 bp也能默化基因表現，能抑制82.91% GUS活性。以默化效果最佳之GUS 3’端 587 bp片段構築反向基因片段-隱子-順向基因片段之質體pR3GAnS，與含有報導基因GUS之質體pBI221共轉殖12小時後分析，GUS仍有37.05%的活性，而共轉殖後表達48小時的GUS活性較其他表達時間為低，顯示RNAi質體於此時可以發揮最強的基因默化效果。表達時之溫度亦會影響RNAi的能力，40℃環境下GUS 3’端 587 bp片段構築反向基因片段-隱子-順向構築默化基因表現的能力為95.16%，25℃處理為81.70%，而10℃處理會使得基因默化能力只剩下20.53%。基因默化質體默化目標基因的能力，以蝴蝶蘭花瓣圓片進行測試，對較弱或較強的啟動子效果較佳，即以香蕉重泛素基因啟動子引導GUS基因作為報導質體之基因默化能力為97.17%，以及利用香蕉β-1,3-葡聚糖酶基因啟動子引導GUS基因作為報導質體之基因默化能力為98.52%，比含CaMV 35S啟動子的pBI221作為報導質體之默化效率91.45%為高。 另一方面，為應用RNA干擾技術抑制香蕉果實過程之乙烯生成，比對香蕉ACC氧化酶MAO1及MAO2之cDNA核苷酸序列，利用二序列間相似度最低的區域，構築於pRNAi載體，分別為順、反向及反、順向結構，並利用農桿菌轉殖法進行轉殖，目前已取得具有MAO2專一RNA干擾反、順向構築之香蕉擬轉殖株，日後可研究MAO1及MAO2基因表現對香蕉果實後熟的影響，並分析RNA干擾抑制香蕉果實乙烯生成量之效果。
並列摘要
The most important strategy in gene silencing is RNA interference（RNAi）, which has been successfully used for studying the function of genes. In this study, CaMV 2X 35S promoter with the spacer from intron1 of banana ACC oxidase gene MAO1 was chosen to construct RNAi vector. β-glucuronidase（GUS） gene was used to test the silencing effect. Full length of GUS in sense-intron-antisense or antisense-intron-sense orientation, and coding regions from nucleotide 1220 to 1806（587 bp）and 1406 to 1430（25 bp）of GUS in antisense-intron-sense orientation were constructed as RNAi effector plasmids. After co-bombardment with reporter and effector plasmids, petal discs from Phalaenopsis were analyzed for GUS activity. The inhibition for GUS expression of RNAi effector plasmid with antisense-intron-sense construct was 85.98%, which was higher than the sense-intron-antisense one. Moreover, both the 587 bp and 25 bp of GUS regions in RNAi construct could reduce 98.52% and 82.91% of GUS activity, respectively. According to the results of time point analysis for RNAi efficiency, the proper time point for analysis of RNAi effect was 48 hr after transformation. Low temperature may affect RNAi efficiency. In this study of low temperature treatment at 10℃ could reduce RNAi efficiency by about 4-fold. Higher RNAi efficiency was revealed by promoters from banana polyubiquitin or β-1,3-glucanase genes, whereas CaMV 35S promoter showed a slightly decreased silencing effect. On the other hand, RNAi was used as strategy to inhibit genes involved in banana ripening. According to the analysis of nucleotide sequences, unique regions specific for banana ACC oxidase genes MAO1 and MAO2 were constructed into antisense-intron-sense and sense-intron-antisense orientation to study RNAi response. RNAi plasmids were introduced into banana suspension cells through Agrobacterium-mediated transformation system. Putative transgenic plants with MAO2 antisense-intron-sense RNAi construct expressed GUS activity upon staining with X-Gluc.