Studies on the origin and repair mechanism of satellite RNAs associated with Cucumber mosaic virus

• Main Authors: Huei-Ling You, 游慧玲
• Other Authors: 胡仲祺
• Format: Others
• Language: zh-TW
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/04807273308412405351

碩士 === 中興大學 === 生物科技學研究所 === 95 === Cucumber mosaic virus (CMV), the type member of the Cucumovirus, is a plant virus with a tripartite, single-stranded positive-sense RNA genome. Some CMV strains contain small RNAs that have been demonstrated to be satellite RNAs (satRNAs), which are dependent on CMV for replication, encapsidation, and transmission, but share little or no sequence similarity with CMV. Previous studies have shown that satRNAs are often found in experimental hosts infected with CMV, and deletion mutants of satRNAs are usually automatically repaired to the wild type. However, the origin and repair mechanism of satRNA remain to be elucidated. The purpose of this study is to explore the possible origin and repair mechanism of CMV associated satRNAs through comprehensive database searches and artificial evolution experiments under environmental stresses. The databases explored include plant, bacteria and invertebrate genomes, using highly sensitive algorithms of SSearch and FASTA. For artificial evolution experiments, the constructs used to inoculate are based on CMV RNA 3 driven by double 35S promoter, with most part of coat protein sequence replaced by those of satRNA variants, including positive control, satRNA-NdeI (pd35S-R3-satNdeI), and test construct, satM26 (pd35S-R3-satM26), a mutant with the nucleotides from 124 to 237 deleted, and 12 additional nucleotides that are not related to satRNAs. Co-inoculation of the constructs with CMV to Nicotiana benthamiana would result in the continuous production of satRNAs for as a start point for evolution or repair. Current database searches revealed several possible candidates, which are subject to multiple alignment analysis. The result revealed that these homologous sequences also shared substantial similarities in their predicted secondary structure models. This finding supported the possibility that satRNAs might have evolved from host genomes, and might be recognized by the viral replicases and assembled into functional replicons during certain rare transposition events. Repeated in plant inoculation tests indicated that satRNAs were not efficiently generated. To test the replication ability and the activity of the subgenomic promoter of the constructs, enhanced green fluorescent protein (EGFP) were used as a reporter and assayed in N. benthamiana protoplasts. EGFP was detected by western blot analysis 48 hrs after inoculation, suggesting that the CMV RNA 3 vector could be used to express foreign genes downstream of the subgenomic promoter. These results presented nucleotide sequence and structure evidence for the possible origins and repair mechanism of CMV satRNAs, and proved possible tools to test these hypotheses. If the EGFP have been detected in protoplast, this vector could be used to test the satRNA repair mechanism and study of origin.