| Summary: | 碩士 === 國立高雄師範大學 === 生物科技系 === 95 === Mosaic and yellowing symptoms on leaves of Petunia hybrida seedlings were found in Taiwan. It showed positive reaction in indirect ELISA test with TMV (Tobacco mosaic virus) antibody. TMV was isolated through three times single local lesion isolation on Chenopodium quinoa. The virus caused local lesions symptom on C. quinoa, C. amaranticolor, Nicotiana benthamiana, N. rustica, Lycopersicum esculentum, Solanaceae melongena, Capsicum annuum, Gomphrena globosa and Celosia argentea by mechanical inoculation. TMV was purified from N. benthamiana with a white circular band in the centrifuge tube 3.8 cm below the surface of solution. The purified virus had maximal and minimal absorptions at 267 nm and 246 nm, respectively. Amax/A min and A280/A260 rations were 1.23 and 0.89, respectively. Rigid rod particles similar that of in Tobamovirus genus in length about 300 nm were observed under electron microscope in negatively stained dip preparation. Electrophoretic analysis revealed that a 6.4 kb size of viral RNA was observed. The molecular weight of coat protein of the virus was estimated 17.5 kDa in the SDS-PAGE and Western blotting assay. The cDNAs of TMV genome were amplified by cDNA synthesis technology and PCR, then sequenced and analyzed. Nearly full length of viral genome were 6349 nucleotides and the base compositions were 1883 adenine (29.65%); 1218 cytosine (19.18%); 1495 guanine (23.54%); 1753 uracil (27.61%), respectively. From 5' to 3' end, four open reading frames (ORFs) were recognized as RdRp/replicase protein, RdRp/readthrough protein, MP and CP genes. RdRp/replicase gene was 3,351 bp which encoded a protein of 126.1 kDa (1,116 aa), RdRp/readthrough gene was 4,851 bp which encoded a protein of 183.3 kDa (1,616 aa), MP gene was 777 bp which encoded a protein 29.0 kDa (258 aa) and CP gene was 480 bp which encoded a protein of 17.5 kDa (159 aa), respectively. Two proteins of RdRp were initiated at the same site. There are several overlaps between RdRp and MP genes. In conclusion, the results of comparison of nucleotide and amino acid sequences of TMV pet-TW (accession number: EF392659) with other TMV strains reported in database of NCBI revealed that it was a strain of TMV. Multiple alignments of nucleotide and amino acid sequences among TMV strains displayed that TMV pet-TW had a closer relationship with B (accession number: AJ011933), variant 1 (V01408), NC 82 (X68110), Fujian (AF39512
7) and Rakkyo (D63809). However, The nucleotide and amino acid
sequences of MP gene of TMV pet-TW were different from other TMV strains in length.
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