菸草抗胡瓜嵌紋病毒基因轉殖之研究

Abstract

胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)，感染宿主範圍廣泛，導 致園藝作物產值及產量上的損失，香蕉(Musa spp.) 為其宿主之一，目前 尚未發展出直接且有效的防治方法。本論文採用RNA 干擾(RNA interference) 策略提升作物對胡瓜嵌紋病毒之抗性，將默化CMV 外鞘蛋白 基因之質體以農桿菌媒介基因轉殖法進行邊沁菸草(Nicotiana benthamiana) 及香蕉轉殖。基因默化轉殖殖體，分別含有CMV 外鞘蛋白 基因全長及五個保守區域組合之反向重複序列，並在正義與反義序列中間 以香蕉ACC 氧化酶(ACC Oxidase) 基因Mh-ACO1 第一個隱子作區隔， 期待在菸草及香蕉轉殖株中，表現出具默化效果的髮夾型雙股結構RNA， 進一步達到抗病毒目的。二個構築之擬轉殖邊沁菸草各取得12 個轉殖系， 經GUS 活性組織化學染色(Histochemical GUS assay) 及聚合酶連鎖反應 (polymerase chain reaction)，證實轉殖事件的發生，並透過T1 種子後裔分 析，已確認具單一拷貝及雙拷貝轉殖基因之轉殖株。另以7 種不同香蕉品 種之果手原體為材料，進行農桿菌媒介法基因轉殖，以超音波震盪及真空 抽氣輔助，轉殖後經抗生素篩選具抗性之不定芽，期望獲得帶有默化質體 之香蕉不定芽。

Cucumber mosaic virus (CMV) is one of the most important viruses in plantae and with wide range of host. The infection of CMV causes enormous losses of production and economics in various horticultural crops. Banana (Musa spp.) is one of the hosts of CMV. Till now, no effective method has been developed to manage CMV. To raise the resistance against CMV, RNA interference was used as the strategy in genetic transformation. Inverted repeats of the full length and the fragment containing five 25 bp conserve regions of CMV coat protein gene separated by Mh-ACO1 intron 1 were constructed respectively. Silencing expressing constructs were transformed into Nicotiana benthamiana to evaluate the silencing efficiency of CMV coat protein gene. Twelve transgeinic lines of Nicotiana benthamiana in each construct had be confirmed by GUS ( β -Glucuronidase) staining and polymerase chain reaction According to the results of segregation test of tobacco T1 progeny, transgenic lines carrying single-copy and double copy transgene have been identified. Meanwhile, hand primordia from 7 different cultivars of banana were transformed with silencing constructs via Agrobacterium-mediated assisted by sonication or vacuum infiltration, several antibiotic-resistant adventitious buds were regenerated.