The Promoter Analysis of Bamboo Mosaic Potexvirus Plus-Sense Genomic RNA Synthesis by

• Main Authors: Hsiao-Ning Chiu, 邱曉寧
• Other Authors: Ching-Hsiu Tsai
• Format: Others
• Language: zh-TW
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/29444016047246424999

碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === Bamboo mosaic potexvirus (BaMV) consists of a single-stranded positive-sense RNA genome. The 3¢ untranslated region (UTR) of BaMV genomic RNA was identified to form a tertiary structure and recognized as template by viral RNA-dependent RNA polymerase (RdRp) complex to synthesize the minus strand RNA. However the promoter on the minus strand RNA for plus strand genomic RNA synthesis is not clear. The secondary structure of (-)77RNA was predicated by MFOLD program and confirmed by specific enzymatic cleavage. Single-strand unpaired nucleotides were probed with ribonuclease A (specific on pyrimidine), T1 (specific on Guanine), T2 (on all four nucleotides) and the paired or stacked nucleotides were cleaved by ribonuclease V1. The probing results indicated that (-)77RNA could form two stable stem-loops in which the major stem-loop contained lower and upper stems. In order to identify the promoter for the plus strand RNA synthesis, we have prepared three different sizes of the minus strand RNA transcripts with co-3¢ end ((-)39, (-)77, and (-)173RNA) as templates for an in vitro RdRp replication assay. We found that (-)39RNA has the lowest replication efficiency, and (-)77RNA has similar replication efficiency with (-)173RNA. This result suggested that the promoter sequence for plus strand RNA synthesis might be located on the last 77 nucleotides of the minus strand RNA, especially the major stem-loop. Mutations were introduced to abolish or revert the major stem structures. Results showed that both sequence and structure of lower stem are necessary for RNA replication and maintaining the upper stem is far more important than the sequence requirement.