Please use this identifier to cite or link to this item:
Coat Protein Sequence Analysis, Serological Properties and Development of Detection Tools for Two Filamentous Viruses Isolated from Sweet Potato
|關鍵字:||Sweet Potato Virus;甘藷長絲狀病毒;Coat Protein Sequence;Serological;鞘蛋白基因核酸序列;血清學性狀||出版社:||植物病理學系所||引用:||Abad, J. A., Conkling, M. A., and Moyer, J. W. 1992. Comparison of the capsid protein cistron from serologically distinct strains of Sweetvpotato feathery mottle virus (SPFMV). Arch. Virol. 126: 147-157. Abad, J. A., and Moyer, J. W. 1992. Detection and distribution of Sweet potato feathery mottle virus in sweet potato by in vitro-transcribed RNA probes (Riboprobes), membrane immunobinding assay direct blotting. Phytopathology 82: 300-305. Adams, M. J., Antoniw, J. F., and Fauquet, C. M. 2005. Molecular criteria for genus and species discrimination within the family Potyviridae. Arch. Virol. 150: 459-479. Allison, R., Johnston, R. E., and Dougherty, W. G., 1986. The nucleotide sequence of the coding region of Tobacco etch virus genomic RNA: evidence for the synthesis of a single polyprotein. Virology 154: 9-20. Alconero, R. 1972. Effects of plant age, light intensity and leaf pigments on symptomatology of virus-infected sweet potato. Plant Dis. Rep. 56: 501-504. Alvarez, V., Ducasse, D. A., Biderbost, E., and Nome, S. F. 1997. Sequencing and characterization of the coat protein and 3'' non- coding region of a new sweet potato potyvirus. Arch. Virol. 142: 1635-1644. Ateka, E. M., Barg, E., Njeru, R. W., Lesemann, D. E., and Vetten, H. J. 2004. Further characterization of ‘sweet potato virus 2'': a distinct species of the genus Potyvirus. Arch. Virol. 149: 225-239. Ateka, E. M., Barg, E., Njeru, R. W., Thompson, G., and Vetten, H. J. 2007. Biological and molecular variability among geographically diverse isolates of sweet potato virus 2. Arch. Virol. 152: 479-488. Boltovets, P. M., Boyko, V. R., Kostikov, I. Y., Dyachenko, N. S., Snopok, B. A., and Shirshov, Y. M. 2002. Simple method for plant virus detection: effect of antibody immobilization technique. J. Virol. Methods 105: 141-146. Cadena-Hinohosa, M. A., and Campbell, R. N. 1981a. Serologic detection of feathery mottle virus strains in sweet potatoes and Ipomoea incarnata. Plant Dis. 65: 412-414. Cadena-Hinojosa, M. A., and Campbell, R. N. 1981b. Characterization of isolates of four aphid-transmitted sweet potato viruses. Phytophology 71: 1086-1089. Campbell, R. N., Hall, D. H., and Mielinis, N. M. 1974. Etiology of sweet potato russet crack disease. Phytopathology 64: 210-218. Carrington, J. C., and Dougherty, W. G. 1987a. Processing of the Tobacco etch virus protein abolishes aphid transmissibility of a potyvirus. Virology 178: 161-165. Carrington, J. C., and Dougherty, W. G. 1987b. Small nuclear inclusion protein encoded by a plant potyvirus genomes is a protease. J. Virol. 61: 355-362. Cali, B. B., and Moyer, J. W. 1981. Purification, serology, and particle morphology of two russet crack strains of Sweet potato feathery mottle virus. Phytophothology 71: 302-305. Chen, J., Chen, J., and Adams, M. J. 2001. A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family. Arch. Virol. 146: 757-766. Chen, C. C., Hsiang, T., Chiang, F. L., and Chang C. A. 2002. Molecular characterization of Tuberose mild mosaic virus and preparation of its antiserum to the coat protein expressed in bacteria. Bot. Bull. Acad. Sin. 43: 13-20. Chung, M. L., Hsu, Y. H., Chen, M. J., and Chiu, R. J. 1986. Virus deseases of sweet potato in Taiwan. In “Plant Virus Diseases of Horticultural Crops in the Tropics and Subtropics. FFTC Book series 33.”, pp. 84-90. Food and Fertilizer Technology Center for the Asian and Pacific Region, Taipei, Taiwan, ROC. Chung, M. L., Liao, C. H., and Li, L. 1981. Effect of virus infection on the yield and quality of sweet- potatoes. Plant Prot. Bull. 23: 137-142. Chung, M. L., Liao, C. H., Chen, M. J., and Chiu, R. J. 1985. The isolation, transmission and host range of sweet potato leaf curl disease agent in Taiwan. Plant Prot. Bull. 27: 333-342. Cohen, J., Loebenstein, G., and Spiegel, S. 1988. Infection of sweet potato by Cucumber mosaic virus depends on the presence of Sweet potato feathery mottle virus. Plant Dis. 72: 583-585. Cohen, J., Milgram, M., Antignus, Y., Pearlsman, M., Lachman, O., and Loebenstein, G. 1997. Ipomoea crinkle leaf caused by a whitefly-transmitted Gemini-like virus. Ann. Appl. Biol. 131: 273-282. Colinet, D., and Kummert, J. 1993. Identification of a Sweet potato feathery mottle virus isolate from China (SPFMV-CH) by the polymerase chain reaction with degenerate primers. J. Virol. Methods 45: 149-159. Colinet, D., Kummert, J., Lepoivre, P., and Semal, J. 1994. Identification of distinct potyviruses in mixedly-infected sweetpotato by the polymerase chain reaction with degenerate primers. Phytopathology 84: 65-69. Colinet, D., Kummert, J., and Lepoivre, P. 1997. Evidence for the assignment of two strains of SPLV to the genus Potyvirus based on coat protein and 3'' non-coding region sequence data. Virus Res. 49: 91-100. Colinet, D., Nguyen, M., Kummert, J., Lepoivre, P., and Xia, F.-Z. 1998. Differentiation among potyviruses infecting sweet potato based on genus- and virus-specific reverse transcription polymerase chain reaction. Plant Dis. 82: 223-229. Difeo, L., Nome, S. F., Biderbost, E., Fuentes, S., and Salazar, L. F. 2000. Etiology of sweet potato chlorotic dwarf disease in Argentina. Plant Dis. 84: 35-39. Dougherty, W. G., and Carrington, J. C. 1988. Expression and function of potyviral gene products. Annu. Rev. Phytopathol. 26: 123-143. Dougherty, W. G., Cary, S. M., and Parks, T. D. 1989. Molecular genetic analysis of a plant virus polyprotein cleavage site: a model. Virology 171: 356-364. Esbenshade, P. R., and Moyer, J. W. 1982. Indexing system for Sweet potato feathery mottle virus in sweet potato Ipomoea batatas using enzyme-linked immuno sorbent assay. Plant Dis. 66: 911-913. Gibbs, A. J., Mackenzie, A. M., and Gibbs, M. J. 2003. The ‘potyvirid primers' will probably provide phylogenetically informative DNA fragments from all species of Potyviridae. J. Virol. Methods 112: 41-44. Gibson, R. W., Mpembe, I., Alicai, T., Carey, E. E., Mwanga, R. O. M., Seal, S. E., and Vetten, H. J. 1998b. Symptoms, aetiology and serological analysis of sweet potato virus disease in Uganda. Plant Pathology 47: 95-102. Gibson, R. W., Mwanga, R. O. M., Kasule, S., Mpembe, I., and Carey, E. E. 1997. Apparent absence of viruses in most symptomless field-grown sweet potato in Uganda. Ann. Appl. Biol. 130: 481-490. Gibson, R. W., Aritua, V., Byamukama, E., Mpembe, I., and Kayongo, J. 2004. Control strategies for sweet potato virus disease in Africa. Virus Res. 100: 115-122. Green, S. K., Kuo, Y. J., and Lee, D. R. 1988. Uneven distribution of two potyviruses (Sweet potato feathery mottle virus and Sweet potato latent virus) in sweet potato and its implication on virus indexing of meristem derived plants. Tropical Pest Management 34: 298-302. Green, S. K., and Luo, C. Y. 1989. Elimination of Sweet potato yellow dwarf virus SPYDV by meristem tip culture and by heat treatment. Z. Pflanzenkr Pflanzenschutz 96: 464-469. Green, S. K., Luo, C. Y., and Lee, D. R. 1989. Elimination of Sweet potato latent virus from Nicotiana benthamiana with ribavirin. Plant Prot. Bull. 31: 310-315. Green, S. K., Luo, C. Y., and Wu, S. F. 1992a. Elimination of leaf curl virus of sweet potato by meristem tip culture, heat and ribavirn. Plant Prot. Bull. 34: 1-7. Green, S. K., Tsou, S. C. S., and Wu, S. F. 1992b. The effect of meristeming on yield, quality and on virus reinfection of sweet potato. Plant Prot. Bull. 34: 192-201. Hahn, S. K. 1979. Effects of viruses sweet potato virus disease on growth and yield of sweet potato Ipomoea batatas. Exp. Agric. 15: 253-256. Hsu, Y.-H., and Lee, C.-W. 1994. Sweet potato virus and virus-like diseases. Root crop yield improment, proceeding and utilization: 197-207. IsHak, J. A., Kreuze, J. F., Johansson, A., Mukasa, S. B., Tairo, F., Abo El-Abbas, F. M., and Valkonen, J. P. T. 2003. Some molecular characteristics of three viruses from SPVD - affected sweet potato plants in Egypt. Arch. Virol. 148: 2449-2460. Joseph, J., and Savithri, H. S. 1999. Determination of 3''-terminal nucleotide sequence of pepper vein banding virus RNA and expression of its coat protein in Escherichia coli. Arch. Virol. 144: 1679-1687. Johansen, E., Rasmussen, O. F., Heide, M., and Borkhardt, B. 1991. The complete nucleotide sequence of pea seed-borne mosaic virus RNA. J. Gen. Virol. 72: 2625-2632. Karyeija, R. F., Gibson, R. W., and Valkonen, J. P. T. 1998. The significance of Sweet potato feathery mottle virus in subsistence sweet potato production in Africa. Plant Dis. 82: 4-15. Karyeija, R. F., Kreuze, J. F., Gibson, R. W., and Valkonen, J. P. T. 2000. Synergistic interactions of a potyvirus and a phloem-limited crinivirus in sweet potato plants. Virology 269: 26-36. Kemp, W. G., and Collin, G. H. 1976. Feathery mottle virus of sweet potato in Ontario. Can Plant Dis. 56: 33-34. Kennedy, G. G., and Moyer, J. W. 1982. Aphid Aphis gossypii transmission and separation of 2 strains of Sweet potato feathery mottle virus from sweet potato Ipomoea batatas. J. Econ. Entomol. 75: 130-133. Kokkinos, C. D., and Clark, C. A. 2006a. Interactions among Sweet potato chlorotic stunt virus and different potyviruses and potyvirus strains infecting sweet potato in the United States. Plant Dis. 90: 1347-1352. Kokkinos, C. D., and Clark, C. A. 2006b. Real-time PCR assays for detection and quantification of sweet potato viruses. Plant Dis. 90: 783-788. Kreuze, J. F., Karyeija, R. F., Gibson, R. W., and Valkonen, J. P. T. 2000. Comparisons of coat protein gene sequences show that East African isolates of Sweet potato feathery mottle virus form a genetically distinct group. Arch. Virol. 145: 567-574. Kreuze, J. F., Savenkov, E. I., and Valkonen, J. P. T. 2002. Complete genome sequence and analyses of the subgenomic RNAs of Sweet potato chlorotic stunt virus reveal several new features for the genus Crinivirus. J. Virol. 76: 9260-9270. Li, R., Cai, S., and Salazar, L. F. 1990. Serological detection of viruses on sweet potato in China. Acta Phytopathologica Sinica 20: 189-194. Liao, C.-H., Chien, I.-C., Chung, M.-L., Chiu, R.-J., and Han, Y.-H. 1979. A study of sweet potato virus disease in Taiwan I: Sweet potato yellow spot virus disease. J. Agri. Res. China 28: 127-138. Liao, C.-H., and Chung, M.-L. 1979. Shoot tip culture and virus indexing in sweet potato. J. Agri. Res. China 28: 139-144. Liu, W.-T. 1999. Studies on meliorating flooding injury of sweet potato (Ipomoea batatas L.) by potassium Fertilizer. National Chung Hsing University, M.S. Thesis. Taichung, Taiwan. Lo, S.-F., and Liao, C.-H. 1994. Propagation of virus-free sweet potatoes through shoot-tip culture. Root crop yield improment, proceeding and utilization: 247-254. Loebenstein, G., and Harpaz, I. 1960. Virus diseases of sweet potatoes in Israel. Phytopathology 50: 100-104. López, R., Asensio, C., Guzman, M. M., and Boonham, N. 2006. Development of real-time and conventional RT-PCR assays for the detection of Potato yellow vein virus (PYVV). J. Virol. Methods 136: 24-29. Lotrakul, P., Valverde, R. A., Clark, C. A., Sim, J., and De La Torre, R. 1998. Detection of a geminivirus infecting sweet potato in the United States. Plant Dis. 82: 1253-1257. Meng, Q., Zhang, H., Zhang, X., Yang, Y., and Song, B. 1994. Isolation, purification of Sweet potato feathery mottle virus. Acta Phytopathologica Sinica 24: 227-232. Mori, M., Usugi, T., Hayashi, T., and Nishiguchi, M. 1994. Nucleotide sequence at the 3''-teminal region of Sweet potato feathery mottle virus (Ordinary strain, SPFMV-O) RNA. Bioscience Biotechnology and Biochemistry 58: 965-967. Moyer, J. W. 1986. Serological detection of Sweet potato feathery mottle virus in sweet potato. (Abstr.) Phytopathology 76: 1133. Moyer, J. W. 1986. Variability among strains of Sweet potato feathery mottle virus. (Abstr.) Phytopathology 76: 1126. Moyer, J. W., and Cali, B. B. 1985. Properites of Sweet potato feathery mottle virus RNA and capsid protein. J. Gen. Virol. 66: 1185-1189. Moyer, J. W., Cali, B. B., Kennedy, G. G., and Abou-Ghadir, M. F. 1980. Identification of two Sweet potato feathery mottle virus strains in North Carolina. Plant Dis. 64: 762-764. Moyer, J. W., and Kennedy, G. G. 1978. Purification and properties of Sweet potato feathery mottle virus. Phytopathology 68: 998-1004. Moyer, J. W., and Salazar, L. F. 1989. Viruses and viruslike diseases of sweet Potato. Plant Dis. 73: 451-455. Mukasa, S. B., Rubaihayo, P. R., and Valkonen, J. P. T. 2003. Sequence variability within the 3''-proximal part of the Sweet potato mild mottle virus genome. Arch. Virol. 148: 487-496. Mukasa, S. B., Tairo, F., Kreuze, J. F., Kullaya, A., Rubaihayo, P. R., and Valkonen, J. P. T. 2003. Coat protein sequence analysis reveals occurrence of new strains of Sweet potato feathery mottle virus in Uganda and Tanzania. Virus Genes 27: 49-56. Nakashima, J. T., Salazar, L. F., and Wood, K. R. 1993. Sweet potato feathery mottle potyvirus (C1 isolate) virion and RNA purification. J. Virol. Methods 44: 109-116. Nicolas, O., and Laliberte, J. F. 1992. The complete nucleotide sequence of Turnip mosaic potyvirus RNA. J. Gen. Virol. 73: 2785-2793. Nishiguchi, M., Mori, M., Suzuki, F., Nagata, R., Morishita, T., Sakai, J.-I., Hanada, K., and Usugi, T. 1995. Specific detection of a severe strain of Sweet potato feathery mottle virus (SPFMV-S) by reverse transcription and polymerase chain reaction (RT-PCR). Annals of the Phytopathological Society of Japan 61: 119-122. Onuki, M., and Hanada, K. 1998. PCR amplification and partial nucleotide sequences of three Dicot-infecting geminiviruses occurring in Japan. Ann. Phytopathol. Soc. Jan. 64: 116-118. Puurand, U., Makinen, K., Paulin, L., and Saarma, M. 1994. The nucleotide sequence of potato virus A genomic RNA and its sequence similarities with other potyviruses. J. Gen. Virol. 75: 457-461. Purcifull, D. E., Edwardson, J. R., Hiebert, E., and Gonaslves, D. 1984. Papaya ringspot virus. CMI/AAB Description of Plant Virus. No. 292. Ryu, K. H., Kim, S. J., and Park, W. M. 1998. Nucleotide sequence analysis of the coat protein genes of two Korean isolates of Sweet potato feathery mottle potyvirus. Arch. Virol. 143: 557-562. Sakai, J., Mori, M., Morishita, T., Tanaka, M., Hanada, K., Usugi, T., and Nishiguchi, M. 1997. Complete nucleotide sequence and genome organization of Sweet potato feathery mottle virus (S strain) genomic RNA: The large coding region of the P1 gene. Arch. Virol. 142: 1553-1562. Schaefers, G. A., and Terry, E. R. 1976. Insect transmission of sweet potato disease agents in Nigeria. Phytopathology 66: 642-645. Sheffield, F. M. L. 1957. Virus diseases of sweet potato in East Africa I. Identification of the viruses and their insect vectors. Phytopathology 47: 582-590. Souto, E. R., Sim, J., Chen, J., Valverde, R. A., and Clark, C. A. 2003. Properties of strains of Sweet potato feather mottle virus and two newly recognized potyviruses infecting sweet potato in the United States. Plant Dis. 87: 1226-1232. Tairo, F., Mukasa, S. B., Jones, R. A. C., Kullaya, A., Rubaihayo, P. R., and Valkonen, J. P. T. 2005. Unravelling the genetic diversity of the three main viruses involved in sweet potato virus disease (SPVD), and its practical implications. Mol. Plant Pathol. 6: 199-221. Usugi, T., Nakano, M., Onuki, M., Maola, T., and Hayashi, T. 1994. A new strain of Sweet potato feathery mottle virus that causes russet crack on fleshy roots of some Japanese cultivars of sweet potato. Ann. Phytopath. Soc. Japan 60: 545-554. Usugi, T., Nakano, M., Shinkai, A., and Hayashi, T. 1991. Three filamentous viruses isolated from sweet potato in Japan. Ann. Phytopathol. Soc. Jpn. 57: 512-521. Valverde, R. A., Sim, J., and Lotrakul, P. 2004. Whitefly transmission of sweet potato viruses. Virus Res. 100: 123-128. Vance, V. B., Moore, D., Turpen, T. H., Bracker, A., and Hollwell, V. C. 1992. The complete nucleotide sequence of Pepper mottle virus genomic RNA: Comparison of the encoded polyprotein with those of other sequenced potyviruses. Virology 191: 19-30. Winter, S., Purac, A., Leggett, F., Frison, E. A., Rossel, H. W., and Hamilton, R. I. 1992. Partial characterization and molecular cloning of a closterovirus from sweet potato infected with the sweet potato virus disease complex from Nigeria. Phytopathology 82: 869-875. Yang, I. L. 1972. Transmission of the feathery mottle complex of sweet potato in Taiwan. Taiwan Agric. Q. 8: 123-134. Yeh, S. D., Jan, F. J., Chiang, C. H., Doong, T. J., Chen, M. J., Chung, P. H., and Bau, H. J. 1992. Complete nucleotide sequence and genetic organization of Papaya ringspot virus RNA. J. Gen. Virol. 73: 2531-2541. Yeh, S. D., and Gonsalves, D. 1985. Translation of papaya ringspot virus RNA in vitro : detection of a possible polyprotein that is processed for capsid protein cylindrical-inclusion protein, and amorphous inclusion protein. Virology 143: 260-270. Zhu, Z., and Xue, Q. 1993. Purification and serology of Sweet potato feathery mottle virus. Virologica Sinica 8: 84-88.||摘要:||
自田間呈現黃斑、斑駁或嵌紋病徵的甘藷病株，以機械接種到指示植物奎藜 (Chenopodium quinoa)，經單斑分離後可得3種大小、型態不同的病斑。因甘藷富含澱粉及酚化物等抑制物質，又無適當繁殖寄主，故分離純化不易。因此，本研究從核酸層次著手，進行解序及分析，以了解其分子特性。經與登錄於GenBank之potyvirus基因比對，得知其分別為甘藷潛伏病毒(Sweet potato latent virus, SPLV)及甘藷羽狀斑駁病毒(Sweet potato feathery mottle virus, SPFMV)。另外由鞘蛋白基因設計專一性引子對後，利用細菌載體大量表現蛋白，快速製備抗血清，供檢測用。為探討兩種病毒在田間分布情形及其複合感染對植株生長影響，需將病毒回接甘藷植株，但利用磨擦接種不易成功。本研究嘗試將甘藷株根部浸潤於病毒粗汁液，可令病毒感染成功獲得單獨感染之病株。並比較單獨感染與複合感染病株於植株中之分布，以了解於田間分布及評估與其對產量影響。
自呈現黃斑病徵甘藷病株機械磨擦接種到奎藜7天後出現壞疽病斑。由呈現壞疽病徵奎藜以簡併式引子Pot I及Oligo(dT)，利用RT-PCR可增幅出2.0 kb核酸片段，經選殖及解序得全長含1930個核苷酸，與己發表之potyvirus作基因比對，得知為甘藷潛伏病毒 (SPLV)，即由5''端起為831nt 細胞核內含體b (nuclear inclusion b, NIb)，879 nt之全長度鞘蛋白 (coat protein, CP)基因及197 nt之3''端非轉譯區 (3'' non-coding region, 3''-NCR)及poly A尾端。與己登錄於GenBank之甘藷潛伏病毒系統 (strains)比對，結果與SPLV-TW之CP及3''-NCR之核苷酸序列相同度分別為96.5%及100%。於鞘蛋白基因兩端設計專一性引子，Lcp 1及Lcp 2兩個專一性引子，利用RT-PCR增幅出1057 nt核酸片段，經解序確定後，將其專殖到細菌表現載體pET-32a(+)中，並使其於E. Coli寄主內大量表現融合性甘藷潛伏病毒鞘蛋白。以親合性色層分析法回收純化，可得約38 kDa的融合蛋白，經免疫注射製成多元抗體。以瓊脂雙向擴散反應法測定血清力價為1/8。以間接酵素聯結抗體免疫吸附法檢測融合蛋白、壞疽病徵奎藜及斑駁或黃斑之甘藷病株，抗血清稀釋48,000倍仍可測得。以稀釋6,000倍抗血清配合西方轉漬法檢測不同病徵之田間甘藷病株，可測得33 kDa反應條帶，顯示製備之多元抗體可檢測田間罹病植物。
自呈現斑駁病徵甘藷機械磨擦接種到奎藜10-14天可出現兩種不同大小之灰白色單斑。以斑駁藷葉抽取總量核糖核酸，以簡併式引子Pot I、Pot II利用RT-PCR方法可增幅出1.3 kb及1.2 kb兩個核酸片段，經選殖及解序後與己發表之potyvirus作基因比對，得知為甘藷羽狀斑駁病毒，兩個核酸片段分別由3''端設計專一性引子將全長度鞘蛋白及3''非轉譯解序完成。解得SPFMV-CY1全長含1249個核苷酸，即比對兩條核酸序列鞘蛋白基因之相同度 (identity)為80.6%，相似度為86.3%顯示其為不同的系統 (strain)，分別命名為SPFMV-CY1，SPFMV-CY2。與potyvirus屬中18個病毒比對鞘蛋白胺基酸序列，分別有50-76%相似度。與已知56個系統比對鞘蛋白核苷酸序列，SPFMV-CY1與strain C相同度最高，SPFMV-CY2則與strain O相同度最高。由SPFMV-CY1鞘蛋白基因兩端設計專一性引子FM86、FM1006，利用RT-PCR增幅出939 nt，經解序確定後，將其專殖到細菌表現載體PET-32a(+)中，並使其於E. Coli寄主內大量表現融合性甘藷潛伏病毒鞘蛋白。以親合性色層分析法回收純化，可得約35 kDa的融合蛋白，經免疫注射製成多元抗體。以間接酵素聯結抗體免疫吸附法檢測融合蛋白、灰白斑病徵奎藜及不同病徵甘藷病株，抗血清稀釋48,000倍仍可測得。以稀釋6,000倍抗血清配合西方轉漬法檢測不同病徵之田間甘藷病株，可測得35 kDa反應條帶，顯示所製備之多元抗體可檢測田間罹病植物。
於西方轉漬檢測過程中，發現SPFMV及SPLV兩個病毒之多元抗血清易呈現交互反應 (cross reaction)，乃以SPFMV-CY1之融合蛋白進一步製備單元抗體。
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.
|Appears in Collections:||植物病理學系|
Show full item record
TAIR Related Article
Items in DSpace are protected by copyright, with all rights reserved, unless otherwise indicated.