Mutational studies on the replication of satellite RNAs associated with cucumber mosaic cucumovirus

• Main Authors: Yi-Fang Hung, 洪怡芳
• Other Authors: 胡仲祺
• Format: Others
• Published: 2002
• Online Access: http://ndltd.ncl.edu.tw/handle/78795951894544150965

碩士 === 國立中興大學 === 農業生物科技學研究所 === 90 === Abstract Cucumber mosaic virus (CMV), the type member of the Cucumovirus, is a plant virus with a tripartite genome. Some CMV strains contain a small RNA that has been demonstrated to be a satellite RNA (satRNA). SatRNAs are dependent on helper virus for replication, encapsidation, and transmission, but share little or no sequence similarity to the helper virus. The specific aim of this study is to elucidate the minimum required sequence signals on satRNAs for high efficiency replication to gain further insight into the biological functions of satRNAs. Deletion mutations of satRNA were created around the BamHI and NcoI sites. The transcripts of mutants were synthesized in an in vitro transcription system and were used to inoculate Nicotiana tabacum and Nicotiana benthamiana with the helper virus, CMV-NT9, which is free of satRNAs. The viability of mutants was analyzed by double-stranded RNA analyses. The nucleotide sequences of the mutant satRNAs progenies were investigated using RT-PCR followed by DNA sequencing. The first-generation progenies that contain mutations near the BamHI site could maintain 3 to 20 nucleotides deletions. The mutants with 31 nucleotides deletions near the NcoI site maintained the replication ability. It is found that 30%~70% of the first-generation progenies have reversed to wild type, while the others still maintain deletion sites between nucleotide positions 47 to 55. For the second and third-generation progenies, 30%~100% have reversed to wild type and 3 to 4 different nucleotide insertions were found between nucleotide positions 45 to 47, indicating that the nucleotides between positions 47 to 57 are not required for efficient replication with in N. tabacum. However, from N. benthamiana inoculation test, the first-generation progenies maintained similar deletion sites of the inoculum, and it is found that 55%~100% of progenies contained exactly the same mutation sites. The longest deletions allowed were 87 nucleotides around BamHI site and 31 nucleotides around NcoI site. These results suggested that the host plants played an important role in the evolution and selection of satRNA quasispecies. By mutation and recombination, satRNA may adapt to different hosts. These results might be applied in the understanding of helper virus replication and development of satRNA-based transient expression vector systems.