Characterization of a new Cactus virus X isolate from pitaya and the construction of its infectious cDNA clone
Date Issued
2007
Date
2007
Author(s)
Lu, Yu-Chi
DOI
zh-TW
Abstract
Pitaya (Hylocereus spp.) is a perennial herbaceous plant in the family Cactaceae originated from the rainforest in tropic America. In Taiwan, there are about 1000-ha pitaya plantation mainly in the middle and southwestern areas. In 2005, we surveyed the pitayas grown in the experimental farm at National Taiwan University by indirect enzyme-linked immunosorbent assay (ELISA) using the polyclonal antiserum specific against Cactus virus X (CVX). The result indicated that up to 68% of tested samples were infected by CVX. Virus particles were purified from infected pitaya and then inoculated to Chenopodium quinoa and C. amaranticolor. The symptoms of the infected plants were different from those caused by CVX-Hu, a previous studied virus isolate in Taiwan. This result suggested there may be a new strain of CVX. To further characterize the biological and molecular features, a virus isolate named NTU was obtained after three successive single lesion isolations in C. amaranticolor. Host range and size of capsid protein of NTU isolate were determined. Virus distribution in pitaya fruit was also studied by tissue blot immunoassay. Specific primers designed according to CVX-Hu sequence were used to amplify the full-length genome of NTU isolate by RT-PCR. To determine the sequences of 5’ and 3’ ends of NTU isolate, RACE and RT-PCR were performed, respectively. When the complete genomic sequence of NTU isolate was obtained and compared with that of CVX-Hu, the amino acid sequence identities of RdRp and CP genes are 85% and 86%, respectively. Therefore, NTU isolate is considered as a new CVX isolate and thus designated as CVX-NTU. Multiple sequence alignment and phylogenetic analysis of CVX-NTU and other completely sequenced potexviruses demonstrated that CVX-NTU has a close relationship with potexviruses infecting plants in the family Cactaceae. To determine the infection frequency of CVX-NTU and CVX-Hu in pitaya, samples collected from Yangmingshan orchard were detected by ELISA and one tube multiplex RT-PCR. Up to 95% of tested samples were infected with CVX-NTU and 35% contained CVX-Hu. In addition, an previously unreported virus, Zygocactus virus X (ZVX), was detected in 50% of collected samples. The results clearly showed pitaya plants are prevalently infected by potexviruses in Taiwan. Another part of the thesis is to construct full-length cDNA clones of CVX. RNA transcripts were synthesized in vitro under the control of T7 promoter at the CVX cDNA clones. Biological activities of the clones were tested by protoplast and plant inoculation assays in C. quinoa. The screening experiment of infectious cDNA clones of CVX is still going on. Hopefully, a CVX-based vector can be developed and applied to many research areas in the future.
Subjects
紅龍果
仙人掌病毒X
感染性選殖株
pitaya
Cactus virus X (CVX)
infectious cDNA clone
Type
other
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