Coat Protein Sequence Analysis, Serological Properties and Development of Detection Tools for Two Filamentous Viruses Isolated from Sweet Potato

• Main Authors: Li-Yuan Wang, 王麗媛
• Other Authors: 葉錫東
• Format: Others
• Language: en_US
• Published: 2007
• Online Access: http://ndltd.ncl.edu.tw/handle/86431256432569931332

博士 === 中興大學 === 植物病理學系所 === 95 === Three kinds of single lesions with different size and morphology shows in indicator plant Chenopodium quinoa after mechanical inoculation and single lesions transfer from sweet potato with symptoms of yellow spots, vein mottling or mosaic. It is difficult to isolate and purify virus particles from sweet potato because the content of starch and phenolic compounds and the absent of the propagation host. In this study, using the RNA extract from sweet potato cloning and sequencing to search the molecular characteristics of the virus. Comparing the sequences with potyviruses genome in GeneBank evidence that the sequences are Sweet potato latent virus (SPLV) and Sweet potato feathery mottle virus (SPFMV). For antiserum preparation, the complete reading frame of the coat protein (CP) gene of SPLV and SPFMV were amplified from the total RNA extracted from virus-infected leaves of C. quinoa by RT-PCR with the cp-gene specific primers. The amplified DNA fragment was cloned, sequenced, and subcloned into the bacterial expression vector pET-32a(+) vector. For studying the influence of mix infection and distribution of viruses in field, back inoculation is necessary, but to infect sweet potato by mechanical inoculation is difficult. Therefore, the method of root dipping of sweet potato in the crude extracts of virus infected C. quinoa. Detection of the root dipping plants by western blotting indicated that these filamentous viruses can infect sweet potato through roots and the mix infection virus can be separated effectively. The C. quinoa shows necrotic local lesions 7 dpi isolated from sweet potato with yellow spots symptoms were used as source for total RNA extraction. A 2.0-kb product was amplified from the total RNA extracted from virus-infected leaves of C. quinoa by RT-PCR with oligo(dT) and pot2 primers. The cDNA fragment reflected 1931 nucleotides (nts) corresponding to the 3''-terminal region of potyviruses was obtained. The deduced amino acid sequence contained 578 residues encoding part of the 3’-terminal region of NIb gene (285 residues) and the complete sequence of coat protein (CP) gene (293 residues). A 197 nts of non-coding region (NCR) was found located at the 3’-terminal region of the DNA. A genetic code for aphid transmissibility of potyviruses, DAG triplet, was found at the 7-9 residues from the N-terminus of CP gene. Compared to the known sequences of strains of SPLV, the percentage of nucleotide identities of the CP gene and the NCR with SPLV-T were 96.5% and 100%, respectively. Using directional cloning, a 55 kDa fusion protein containing a complete CP sequence of SPLV and a partial sequence encoded by the expression vector plasmid (pET-32a, Novagen) was expressed and purified from cell cultures of Escherichia coli. The antiserum prepared against this fusion protein showed high sensitivity in the serological detection of infected tissue of sweet potato. According to the coat protein gene of SPLV, the specific promers Lcp1, Lcp2 were designed for amplification of coat protein by bacterial expression system. The titer of prepared SPLV antiserum determined by double diffusion test is 1/8. Two different size of chlorotic spots were shown in C. quinoa 10-14dpi. after mechanical inoculation from mottling disease symptom of sweet potato. The total RNA was extracted from mottling sweet potato, a 1.3 kb and a 1.2 kb DNA fragments were amplified by using PotI and PotII primers and RT-PCR. After cloning and sequencing and comparing to the known potyvirus in the GenBank. Specific primer was designed from the 3''-terminal of the two DNA fragments, the whole length CP and the 3''-NCR were sequenced. Total length including 1249 nts in SPFMV-CY1, the identity and the similarity were 80.6% and 86.3% comparing with CP gene of the two DNA sequences that shown they were in different strain, named SPFMV-CY1 and SPFMV-CY2, respectively. The similarity was 50-76% comparing to the 18 potyviruses with their CP amino acid sequences. The identity was highest between SPFMV-CY1 and strain C by comparing to the 56 known systems with their CP nt sequences and the similarity was highest between SPFMV-CY2 and strain O. There were 939 nts amplified by devising special primers, FM 86 and FM 1006, from both terminals of the CP gene of SPFMV-CY1 and through RT-PCR. In the proceeding of western blot, the cross reaction was found between the multi-antiserum of the two viruses of SPFMV and SPLV, and the monoclonal antibody was produced by using the fusion protein of SPFMV-CY1.