CLONING AND SEQUENCE ANALYSIS OF TWO SATELLITE RNAs FROM CUCUMBER MOSAIC VIRUS P1 ISOLATE

Cucumber mosaic virus (CMV) is a type member of the genus Cucumovirus. CMV have divided genomes consisting of three positive single-strands RNAs and a subgenomic RNA 4, which is generated by transcription from RNA 3 and serves as messenger RNA for the viral capsid protein. Some isolates of CMV contain abundant amounts of a small RNA (satellite RNA), which is dependent on viral genomic RNA for its replication and spread, and has no appreciable nucleotide sequence similarity with the viral RNA. CMV can infect over 800 species of plants and is an economically important pathogen of field-grown vegetables in many areas of the world.In this study, the original cucumber mosaic virus (CMV) PI isolate has been caused very mild symptoms on many plant species, after serial passages of inoculation in 5 years, it was found that CMV P1 caused moderate symptoms on several tobacco cultivars and tomato.The dsRNA purification product were used for electrophoresis in agarose gel and the result show that the amount of satellite RNA is much higher than genomic RNAs and subgenomic RNA. Primers were synthesized based on reported CMV satellite RNA sequences, and a specific band of approximately 350 bases in length was found.In cloning and screening we found more than 20 white clones and only two were selected for sequence analysis. CMV PI sat-1 was 100% homology with a sequence reported in 1995, and the other one (CMV PI sat-2) was a new satellite. CMV P1 sat-1 is a mild satellite with about 335nt and CMV PI sat-2 is a new satellite (necrotic satellite) with 394nt, it is not a mutation from CMV P1 sat-1 because there are several deletions. The two satellites shared about 90% sequence homology.The two sequenced satellite RNAs have at least one open reading frame (ORF). CMV P1 sat-1 share an ORF starting at nucleotide 11 and terminating at nucleotide 169. CMV P1 sat-2 has an ORF starting at residue 369 and terminating at residue 394, a second one starting at nucleotide 11 and terminating at nucleotide 73.The secondary structure of the two satellite RNAs shows that sequences of two satellites have very high base pairing.