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標題: 利用人工改造微小RNA研發廣泛抗多種番茄斑萎病毒屬病毒之不具選擇標幟的轉基因作物
Development of Marker-Free Transgenic Crops Conferring Broad-Spectrum Resistance against Different Tospoviruses through Artificial Microrna Approach
作者: 葉錫東
關鍵字: 技術發展;marker free;生物技術, 植物保護類;不具選擇性標幟基因;廣泛抗性;轉基因植物;人工改造微小RNA;broad-spectrum resistance;transgenic plant;artificial mircoRNA
番茄斑萎病毒屬病毒 (tosopovirus) 寄主範圍廣並在世界各地造成作物生產的重大損失。利用轉基因的策略可使作物產生對抗不同病毒的抗性。然而作物在自然界總是面臨多種病毒的感染,因此需有一種轉基因策略能快速使一作物產生廣泛的抗不同病毒的性狀。抗抗生素或抗殺草劑的基因常被用來當作植物基因轉殖過程中的選擇性標幟,然而基於環境安全及人類健康的顧慮,發展出不具選擇性標幟基因的轉基因作物是新的趨向。微小RNA(microRNA, miRNA)在動植物細胞中可鎖定特定的mRNA 加以分解,為調控生長及分化的重要因子,其為大小介於20-24 個核苷酸之間單股RNA。本實驗室與洛克斐勒大學合作,已發展出一種創新的人工改造微小RNA (artificial miRNA, amiRNA)的方法,其終產物為病毒的互補序列,在阿拉伯芥的轉殖株中,可以很有效的對抗植物病毒感染,且無任何副作用。本計畫擬利用這種創新的策略,建構出不具選擇標幟且又能廣泛抗病毒的轉基因作物。研究將以miRNA159 的前趨物為基本骨架,進行人工改造,將miRNA 的片段分別取代成番茄斑萎病毒屬病毒(tospoviruses)的L、NSs、Gn/Gc基因高度保留區的互補序列,以構築出其相對的amiRNAs。這些不同amiRNAs 將被構築成雙重或三重聚合體,並置於具有兩個T-DNA 的二元載體 (binary vector)中,再以農桿菌在菸草上進行轉殖,將選擇性標幟及標的amiRNA 插入兩個不同的位點。選擇性標幟基因的分離將在R1 或R2 子代中進行分析篩選。再透過雜交的方式,可將不同的amiRNAs 結合在一起,以獲得對不同tospoviruses 之廣泛性抗性。一旦利用菸草的系統將最佳amiRNAs 的組合篩選出來後,相同的構築策略將立即應用於番茄作物上。本研究為迅速提供廣泛性抗不同tospoviruses 性狀的新策略,所產生不具篩選標幟並且抗大多數tospoviruses 的轉基因作物具有低生物安全風險的特性,並具全球應用價值。

Tospoviruses are the important plant viruses having a wide host range and causingeconomically important diseases to many crops world wide. Transgenic technology offersthe possibility for development of genetically modified crops with transgenes conferringresistance to different plant viruses. However, plants conferring resistance to single virus iseconomically limited since a crop is normally infected by more than one virus. Hence, thedevelopment of transgenic plants with broad-spectrum resistance to different viruses is verycrucial. Antibiotic or herbicide resistance genes are commonly used as selectable markers forregeneration of transgenic plants during plant transformation and become unnecessary andundesirable in the later stage, regarded as an environmental safety issue and human healthproblem. Thus, the development of marker-free transgenic plants is a critical approach toease these concerns. Recently, microRNAs (miRNAs) have been identified as importantregulators of gene expression in both plants and animals. These miRNAs are single strandedRNAs, 20-24 nucleotides (nt) in length, generated from processing of longer pre-miRNAprecursors by DCL1 in Arabidopsis thaliana. Our laboratory, through the collaboration withRockefeller University, has developed an innovative artificial miRNA approach which hasproven to be highly effective against Turnip Mosaic virus (TuMV) and Turnip yellow mosaicvirus (TYMV) in Arabidopsis. In this proposal, artificial miRNA (amiRNA) will be used asan innovative strategy for the development of marker-free transgenic crops throughAgrobacterium-mediated transformation. To explore this possibility, 273 bp backbone ofpre-miRNA159a will be used to develop artificial pre-miRNAs159 (pre-amiRNAs159)containing sequences complementary to the conserved regions of L, NSs and Gn/Gc genesof tospoviruses. Dimeric and trimeric artificial microRNAs harboring different motifs of LRNA together will be constructed. The same strategy will also be followed for NSs andGn/Gc genes for targeting at different conserved regions. These pre-amiRNAs will beintroduced into a marker-free binary vector using the gateway recombination system. Thesegregation of the selection marker will be analyzed at the progeny level at R1 and R2generations. Pyramiding of these genes in the generated marker-free lines will be carried outby crossing the selective lines with different amiRNAs to obtain high degrees ofbroad-spectrum resistance to different tospoviruses. Once the best combinations ofamiRNAs are selected from tobacco system the same constructs will be applied immediatelyto tomato. The marker-free crops generated through this innovative approach withbroad-spectrum resistance to most tospoviruses are globally beneficial with less biosafetyconcerns.
其他識別: NSC97-2313-B005-036-MY3
Appears in Collections:動物科學系

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