Molecular cloning and characterization of Basella rugose mosaic virus and the isolate infecting Anredera cordifolia

• Main Authors: Hong-Jyun Jhu, 朱鴻鈞
• Other Authors: 張雅君
• Format: Others
• Language: en_US
• Published: 2005
• Online Access: http://ndltd.ncl.edu.tw/handle/34308325033019238926

碩士 === 國立臺灣大學 === 植物病理與微生物學研究所 === 94 === Anredera cordifolia planted in Sindian city exhibited virus-like ringspot symptom on leaves. Leaf saps were mechanically inoculated on indicator plant Chenopodium amaranticolor and C. quinoa. Systemic symptom developed on C. quinoa but only local lesions appeared on inocualted C. amaranticolor. Through three times of successive single lesion isolation on C. amaranticolor, a virus isolate N was obtained. In host range test, in addition to C. amaranticolor and C. quinoa, the virus isolate can only infect Nicotiana benthamiana and Tetragonia expansa and develope mosaic and chlorotic local lesion on leaves, respectively, in 20 species of 8 families plants. When performing backinoculations, newly formed leaves of A. cordifolia exhibit chlorotic foliar symptom. With electron microscopy inspection, the virus particles were found to be filamentous and about 650 nm in length. Indirect ELISA indicated the viruses in the symptomatic leaves reacted with anti-potyvirus monoclonal antibody. Subsequently, the degenerate primers designed for detecting potyviruses were used for further confirmation. The amplified fragments, about 1.8 kb in size, were cloned and sequenced. The sequencing data were analysed with BLAST program with other viruses in the database. The result clearly indicated that the amplified fragments were derived from a potyvirus and contained partial NIb gene, coat protein gene and 3''UTR. Furthermore, these virus clones share 98% nucleotide identity with Basella rugose mosaic virus (BaRMV) previously identified by our laboratory. Therefore, the virus isolate was named as BaRMV-N. By means of RT-PCR or RACE method utilizing potyvirus degenerate primers and BaRMV-N specific primers designed from sequencing data, the remaining genomic fragments of BaRMV-N were cloned and sequenced. Because of the high identity between the two virus isolates, the full length of BaRMV-J genome sequence was cloned and sequenced by appropriate BaRMV-N primers. Both BaRMV-N and BaRMV-J are composed of 9804 nucleotides, excluding the 3’ terminal poly(A) tail, and contain an open reading frame of 9237 nt, encoding a polyprotein of 3079 amino acids. Nine putative proteinase cleavage sites were predicted by analogy with genome arrangements of other potyviruses. The nine mature viral proteins and the amino acid identity between BaRMV-N and BaRMV-J are P1 (95%), HC-Pro (98%), P3 (97%), 6K1 (96%), CI (97%), 6K2 (96%), NIa (98%), NIb (99%) and coat protein (98%). The amino acid identity of viral proteins, polyprotein and the identity of complete nucleotide sequence when comparing with 38 different species of potyvirus were calculated. Additionally these data were used for constructing phylogenetic trees. The highest amino acid identities of CP and highest nucleotide sequence identity of whole genome of BaRMV-N and other potyviruses were 68% and 54%, respectively. Therefore, in terms of species demarcating criteria in the genus Potyvirus, BaRMV is considered as a new potyvirus and the most closely related virus is Beet mosaic virus (BtMV). In detection by ELISA, there were about 80% sample positively reacting with BaRMV antiserum so we presumed that BaRMV exist in Taiwan commonly.