Expression of Functional Domains of Bamboo Mosaic Virus Replicase by BaMV Satellite-Based Expression Vector

• Main Authors: Yi-chen Tsai, 蔡宜珍
• Other Authors: Yau-Heiu Hsu
• Format: Others
• Language: zh-TW
• Published: 2004
• Online Access: http://ndltd.ncl.edu.tw/handle/47752372086580868857

碩士 === 國立中興大學 === 生物科技學研究所 === 92 === 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.