Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36018
標題: 利用竹嵌紋衛星核酸載體表現竹嵌紋病毒複製酵素功能區
Expression of Functional Domains of Bamboo Mosaic Virus Replicase by BaMV Satellite-Based Expression Vector
作者: 蔡宜珍
Tsai, Yi-chen
關鍵字: Bamboo Mosaic Virus;竹嵌紋病毒;BaMV Satellite;methyltransferase;RNA helicase-like;RNA-dependent RNA polymerase;竹嵌紋衛星核酸;轉甲基化酵素;核酸解螺旋酵素;核酸聚合酵素
出版社: 生物科技學研究所
摘要: 
竹嵌紋病毒(Bamboo mosaic virus, BaMV)為長絲狀植物病毒,屬於potexvirus group 的一員,具有單股正意的RNA基因體,5’端具有cap構造,3’端則有poly(A),不包括poly(A)全長6366核苷酸,含有五個主要轉譯區(open reading frame, ORF)。ORF1轉譯出的155 kDa 蛋白和病毒的複製有關,其N端為轉甲基化酵素(methyltransferase),中間區段為核酸解螺旋酵素(helicase),而C端核苷酸聚合酵素(RNA-dependent RNA polymerase)。在竹嵌紋病毒的分離株中,有些另含有與病毒之基因體無同源性的衛星核酸(satellite RNA; satBaMV RNA),不包括poly(A)其全長為836核苷酸,含有一個轉譯區,可以表現一個非結構性的20 kDa蛋白質(P20),此轉譯區非複製所必須,可為外源基因取代,以發展成載體系統。至目前為止BaMV的複製酵素尚無法在植物體內偵測到,推測其原因可能是表現量太低。因此,為增加複製酵素的表現量,將ORF1上的三個功能蛋白區域分別選殖在竹嵌紋病毒衛星核酸載體上,所構築的質體命名為pF4-MT、pF4-Hel、pF4-Pol以及pF4-M.H的構築體,分別表現轉甲基化酵素、核酸解螺旋酵素、核酸聚合酵素以及轉甲基化酵素和核酸解螺旋酵素的融合體,希望能在植物內有較多量的複製蛋白表現,以利進行複製蛋白於植物細胞內存在位置的偵測,並且亦可同時觀察較大量的複製蛋白是否會影響竹嵌紋病毒衛星核酸的複製能力。將這些構築體進行生體外轉錄所得的轉錄體和BaMV的基因體,共同接種菸草(N. benthamiana),再經由北方墨點法的偵測,證實pF4-MT 、pF4-Hel、pF4-Pol 和pF4-M.H可在竹嵌紋病毒的幫助之下進行複製,但其複製效率較野生型satBaMV差。另外,在竹嵌紋病毒基因體的第一個轉譯區上的三個功能區間分別作突變,再分別將這些基因體突變株和含有相對的功能區間的重組衛星核酸(chimeric satBaMV RNA)去共同接種植物,以探討此三功能區在植物體內是否是可被分開來執行其功能。

Bamboo mosaic virus (BaMV), a member of the potexvirus group, contains a single-stranded, positive sense RNA genome with a 5' Cap structure and a 3'poly (A) tail. The entire nucleotide sequence comprises 6366 nts [excluding the 3'poly (A) tail], with five conserved open reading frames (ORFs). ORF1 contains three functional domains, a methyltransferase domain, an RNA helicase-like domain and an RNA-dependent RNA polymerase domain, from the N to the C terminus. A satellite RNA (satBaMV) associated with BaMV is a linear RNA molecule of 836 nts [excluding the poly (A) tail] containing an ORF which encodes a protein of 20 kDa (P20). SatBaMV has been developed to be an effective foreign gene expression vector system. The replicase of BaMV has remained undetectable in planta up to date, possibly because of the low expression level or transient expression of the replicase. In order to enhance expression of the replicase, the three functional domains of ORF1 were cloned into satBaMV replicon, designated as pF4-MT, pF4-Hel, pF4-Pol, and pF4-MH to express the methyltransferase, the RNA helicase-like, the RNA-dependent RNA polymerase, and the methyltransferase RNA helicase-like fusion domains, repectively. The in vitro transcripts of these constructs were coinoculated into N. benthamiana with helper BaMV. All four chimeric satellites were replicating in plant as shown by Northern blot assay, but the replication was less efficient than that of the wild-type satBaMV. In order to study the possibility for the three motifs of ORF1 to function independently in planta. Mutations that disrupt the function of each domain of ORF1 protein were created and used in a functional complementation study by coinoculation with the chimeric satBaMV RNA carrying the corresponding wild type proteins.
URI: http://hdl.handle.net/11455/36018
Appears in Collections:生物科技學研究所

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