利用轉錄後基因沉寂發展具有多重病毒抗性之轉基因西瓜

Development of transgenic watermelon with multiple viral resistance via post transcriptional gene silencing

10.6845/NCHU.2009.00575

江宜樺

西瓜 ； 病毒 ； 基因沉寂 ； watermelon ； virus ； PTGS

中興大學農藝學系所學位論文

2009年

碩士

古新梅

繁體中文

台灣位處亞熱帶地區，適合許多種類的葫蘆科作物(Cucurbitaceae)生長，而西瓜[Citrullus lanatus (Thunb.) Matsum & Nakai ]為其中重要經濟作物之一。然而，西瓜的產值常因病蟲害威脅造成嚴重損失；其中病毒病害會造成西瓜生長勢弱、瓜果畸形、裂果等情況，而導致產量降低對西瓜栽種影響甚大。目前已知對西瓜造成嚴重危害之病毒種類有九種，其中除了矮南瓜黃化嵌紋病毒(Zucchini yellow mosaic virus, ZYMV)外，胡瓜嵌紋病毒(Cucumber mosaic virus, CMV)、胡瓜綠斑嵌紋病毒(Cucumber green mottle mosaic virus, CGMMV)與西瓜嵌紋病毒(Watermelon mosaic virus, WMV)亦是造成西瓜減產的重要病毒種。為了發展出可抗多種病毒的轉基因西瓜，本實驗室先前的研究已利用轉錄後基因沉寂(post-transcriptional gene silencing, PTGS)策略，以農桿菌轉殖法將CMV、CGMMV及WMV三種不同病毒鞘蛋白基因的轉殖載體pGA482G-CGW轉殖到西瓜栽培種慧玲(cv. Feeling)中，並獲得了2個抗CMV、CGMMV及WMV的轉基因西瓜R0株系(lines)；但是因為早期研究尚未完成子代抗病的分析，因此本研究中，將此2抗病株系以自交方式獲得R1子代，利用南方墨點法確認轉基因的存在，並挑戰接種上述病毒分析植株抗性。R1子代挑戰接種結果顯示，分別有49%和56%子代對CMV呈現抗病性狀，有36%和49%的子代對WMV具有抗病性。研究中亦用北方墨點法檢測接種前抗病轉殖株系的mRNA 和siRNA (small interfering RNA)表現量，結果顯示抗病植株的mRNA表現量明顯比感病植株低，而抗病植株中亦測得siRNA的累積，證明在轉基因植物中siRNA是由PTGS機制產生，進一步提供植物對病毒的抗病性，亦顯示此抗性具有遺傳性。另外本實驗室亦發展可產生無篩選標示基因之轉殖載體，來解決篩選標示基因對於環境污染、超級雜草、基因重組等問題，故進一步利用此系統將木瓜輪點病毒西瓜系統(Papaya ringspot virus, type W isolate, PRSV-W)、CMV及ZYMV三種不同病毒鞘蛋白基因構築載體pGA2T-WoPCZ，利用農感菌轉殖法將pGA2T-WoPCZ導入西瓜栽培種慧玲中，目前得到11個轉基因株系同時含有NPT II基因及目標基因，未來將挑戰接種病毒，以獲得無篩選標示基因的抗病轉基因西瓜。

Watermelon [Citrullus lanatus (Thunb.) Matsum & Nakai] is one of the economically important crops grown in Taiwan, where the cultivation of cucurbitaceous crops is more prominent. However, the production of watermelon is limited due to various diseases, especially viral diseases which cause fruit malformation and deformation leading to reduced yield. Watermelon is affected by nine virus species including Zucchini yellow mosaic virus (ZYMV), Cucumber mosaic virus (CMV), Cucumber green mottle mosaic virus (CGMMV) and Watermelon mosaic virus (WMV) which cause severe yield loss leading to reduction in productivity. We have established a transgenic system for the management of the dreadly viral diseases of watermelon in our laboratory. Earlier, watermelon cv. Feeling was transformed with a binary vector pGA482G-CGW harbouring the viral coat protein genes of CMV, CGMMV and WMV. These transgenic watermelon lines provided broad spectrum resistance against CMV, CGMMV and WMV (Ho et al., 2007). In the present study, two independent disease-resistant transgenic watermelon lines, R0 6 and R014, were self-pollinated to obtain R1 seeds. The copy number of the target genes in the R1 lines was detected by Southern blot analysis and the lines harbouring the gene of interest were inoculated with the CMV, CGMMV and WMV. Some of the lines viz., R1 6 and R114 displayed 49% and 56% of resistance to CMV and 36% and 49% to WMV respectively. Subsequently, the expressions of mRNA and small interfering RNA (siRNA) were detected by Northern blot to verify the certain degree of resistance of transgenic plants in this study. Molecular analysis revealed that the expression of viral mRNA is low in the transgenic lines coincidence with the increase of siRNA expression. These observations suggest that multiple virus resistance observed in these resistant lines is due to PTGS. In addition, a marker-free system has been developed in our laboratory to reduce the possible risk of environmental pollution and health hazards caused by the markers in the transgenic plants. A marker free binary vector pGA2T-WoPCZ harboring the coding region of Papaya ringspot virus type W (PRSV-W), CMV and ZYMV coat protein genes under control of the 35S CaMV promoter have been constructed in this study. Watermelon cv. Feeling were transformed with the marker free constructs using Agrobacterium-mediated transformation system. Eleven independent transgenic lines were developed in this study possessing a NPTII gene and the target genes of interest. These transgenic lines will be challenged with PRSV-W, ZYMV and CMV and marker free plants resistant against these viruses will be obtained.

1. 沈白奎、鄧汀欽、余志儒、林俊義。2002。西瓜栽培管理。行政院農業委員會農業試驗所特刊第103號。
   連結：
2. 何琇銀。2007。抗胡瓜嵌紋、胡瓜綠斑嵌紋與西瓜嵌紋病毒轉基因之研發。國立中興大學農藝系碩士論文。台中。
   連結：
3. 林俊義、安寶貞、張清安、羅朝村、謝廷芳。2004。作物病害之非農藥防治。行政院農業委員會農業試驗所特刊第110號。
   連結：
4. 林學詩、全中和。2000。小型冬瓜「花蓮一號」（吉豐）的育成與推廣。花蓮區農業改良場農技報導49: 1-3。
   連結：
5. 陳明昭。1995。大胡瓜新品種台農一號成果示範。高雄區農業專訊第14期: 15。　
   連結：
6. 陳慶忠。2000。台灣番茄班萎病之發生與防治。台中區農業技術專刊 155 期。　
   連結：
7. Ainsworth, G. C. 1935. Mosaic disease of the cucumber. Ann. Appl. Biol. 22: 55-67.
   連結：
8. Avgelis, A. D., Manios, V. I., Balis, C., de Bertoldi, M., Ferrero, G. L., Maniow, V., and Kapetanios, E. 1992. Elimination of cucumber green mottle mosaic tobamovirus by composting infected cucumber residues. Acta Hortic. 302: 311-314.
   連結：
9. Baulcombe, D. C. 1996. Mechanisms of pathogen-derived resistance to viruses in transgenic plants. Plant Cell 8: 1833-1844.
   連結：
10. Chang, Y. M., Hsaio, C. H., Yang, W. Z., Hseu, S. H., Chao, Y. J., and Huang, C. H. 1987. The occurrence and distribution of five cucurbit viruses on melon and watermelon in Taiwan. J. Agri. Res. China 369: 389-397.
    連結：
11. Chen, T. C., Lu, Y. Y., Cheng, Y. H., Chang, C. A., and Yeh, S. D. 2008. Melon yellow spot virus in watermelon: a first record from Taiwan. Plant Pathol. 57: 765.
    連結：
12. Chen, W. S., Chiu, C. C., Liu, H. Y., Lee, T. L., Cheng, J. T., Lin, C. C., Wu, Y. J., and Chang, H. Y. 1998. Gene transfer via pollen-tube pathway for anti-fusarium wilt in watermelon. Biochem. Mol. Biol. Int. 46: 1201-1209.
    連結：
13. Cheng, Y. H., Liao, J. Y., Deng, T. C., Tsai, C. H., and Hu, C. C. 2009. Prevalence of Squash leaf curl Philippines virus occurred on melon plants in south of Taiwan. Plant Pathol. Bull. 18: 78.
    連結：
14. Choi, P. S., Soh, W. Y., Kim, Y. S., Yoo, O. J., and Liu, J. R. 1994. Genetic transformation and plant regeneration of watermelon using Agrobacterium tumefaciens. Plant Cell Rep. 13: 344-348.
    連結：
15. Clough, G. H., and Hamm, P. B. 1995. Coat protein transgenic resistance to watermelon mosaic and zucchini yellows mosaic virus in squash and cantaloupe. Plant Dis. 79: 1107-1109.
    連結：
16. Cooper, B., Lapidots, M., Heick, J. A., Dodds, J. A., and Beachy, R. N. 1995. A defective movement protein of MVT in transgenic plant confer resistance to multiple viruses whereas the function analog increase susceptibility. Virology 206: 307.
    連結：
17. Deng, T. C., Tsai, C. H., Chen, Y. F., and Chang. C. A. 1997. First report of cucurbit aphid-borne yellows luteovirus in Taiwan. Plant Prot. Bull. 39: 395-396.
    連結：
18. Deng, T. C., Tsai, C. H., and Liao J. Y. 2005. Seed transmissibility of viruses in Cucurbits. Seed & Nursery 7: 1-19.
    連結：
19. Ellul, P., Rios, G., Atares, A., Roig, L. A., Serrano, R., and Moreno, V. 2003. The expression of the Saccharomyces cerevisiae HAL1 gene increases salt tolerance in transgenic watermelon [Citrullus lanatus (Thunb.) Matsun. & Nakai.]. Theor. Appl. Genet. 107: 462-469.
    連結：
20. Fang, G., and Grumet, R. 1993. Genetic engineering of potyvirus resistance using constructs derived from the zucchini yellow mosaic virus coat protein gene. Mol. Plant-Microbe Interact. 6: 358-367.
    連結：
21. Fuchs, M., and Gonsalves, D. 1995. Resistance of transgenic hybrid squash ZW-20 expressing the coat protein genes of zucchini yellow mosaic virus and watermelon mosaic virus 2 to mixed infections by both potyviruses. Bio/Technology 13: 1466-1473.
    連結：
22. Fuchs, M., Tricoli, D. M., Carney, K. J., Schesser, M., McFerson, J. R., and Gonsalves, D. 1998b. Comparative virus resistance and fruit yield of transgenic squash with single and multiple coat protein genes. Plant Dis. 82: 1350-1356.
    連結：
23. Fulton, T. M. 1995. Microprep protocol for extraction of DNA from tomato and other herbaceous plants. Plant Mol. Biol. Rep. 13: 207-209.
    連結：
24. Gaba, V., Zelcer, A., and Gal-On, A. 2004. Cucurbit biotechnology-the importance of virus resistance. In Vitro Cell. Dev. Biol. Plant. 40: 346-358.
    連結：
25. Gonsalves, D., Chee, P., Provvidenti, R., Seem, R., and Slightom, J. 1992. Comparison of coat protein-mediated and genetically derived resistance in cucumber to infection by cucumber mosaic virus under field conditions with natural challenge inoculations by vectors. Bio/Technology 10: 1562-1570.
    連結：
26. Grant, S. R. 1999. Dissecting the mechanism of posttranscriptional gene silencing : divide and conquer. Cell 96: 303-306.
    連結：
27. Grumet, R. 1994. Development of virus resistant plant via genetic engineering. Plant Breed. Rev. 12: 47-79.
    連結：
28. Hseu, S. H., Huang, C. H., Chang, C. A., Yang, W. Z., Chang, Y. M., and Hsiao, C. H. 1987. The occurrence of five viruses in six cucurbit in Taiwan. Plant Prot. Bull. 29: 233-244.
    連結：
29. Hseu, S. H., Wang, H. L., and Huang, C. H. 1985. Identification of a zucchini yellow mosaic virus from Cucumis astivus. J. Agri. Res. China 34: 87-95.
    連結：
30. Huang, C. H., Chang, L., and Tsai, J. H. 1993. The partial characterization of melon vein-banding mosaic virus, a newly recognized virus infecting cucurbits in Taiwan. Plant Pathol. 42: 100-107.
    連結：
31. Jan, F. J., Fagoaga, C., Pang, S. Z., and Gonsalves, D. 2000a. A minimum length of N gene sequence in transgenic plants is required for RNA-mediated tospovirus resistance. J. Gen. Virol. 81: 235-242.
    連結：
32. Jan, F. J., Fagoaga, C., Pang, S. Z., and Gonsalves, D. 2000b. A single chimeric transgene derived from two distinct viruses confers multi-virus resistance in transgenic plants through homology-dependent gene silencing. J. Gen. Virol. 81: 2103-2109.
    連結：
33. Jan, F. J., Pang, S. Z., Tricoli, D. M., and Gonsalves, D. 2000. Evidence that resistance in squash mosaic comovirus coat protein-transgenic plants
    連結：
34. Kamachi, S., Mochizuki, A., Nishiguchi, M., and Tabei, Y. 2007. Transgenic Nicotiana benthamiana plants resistant to cucumber green mottle mosaic virus based on RNA silencing. Plant Cell Rep. 26: 1283-1288.
    連結：
35. overtime of Zucchini yellow mosaic virus (ZYMV) and Watermelon
    連結：
36. Kobayashi, T., Chappell, J. D., Danthi, P., and Dermody, T. S. 2006. Gene-specific inhibition of reovirus replication by RNA interference. J. Virol. 80: 9053-9063.
    連結：
37. Krubphachaya, P., Jurícek, M., and Kertbundit, S. 2007. Induction of RNA-mediated resistance to Papaya Ringspot Virus Type W. J. Biochem. Mol. Biol. 40: 404-411.
    連結：
38. Lecoq, H., Lisa, V., and Dellavalle, G. 1983. Serological identity of Muskmelon yellow stunt and zucchini yellow mosaic virus. Plant Dis. 67: 824-825.
    連結：
39. Liao, J. Y., Chung, C. H., Lin, T, K., Chang, C. A., and Deng, T. C. 2007. Identification of Squash leaf curl Philippines virus on Benincasa hispida in Taiwan. Plant Pathol. Bull. 16: 11-18.
    連結：
40. Lin, S. S., Hou, C. H., Huang, C. H., and Yeh, S. D. 1998. Characterization of zucchini yellow mosaic virus isolates collected form Taiwan by host reactions, serology and RT-PCR. Plant Prot. Bull. 40: 163-176.
    連結：
41. Lin, C. Y., and Jan, F. J. 2005. Current development of the strategies for generating marker- free transgenic plants. Plant Pathol. Bull. 14: 159-176.
    連結：
42. Lisa, V., Boccarod, G., D'Agostino, G., Dellavalle, G., and DAquili, M. 1981. Characterization of a potyvirus that causes zucchini yellow mosaic virus. Phytopathology 71: 667-672.
    連結：
43. Lomonossoff, G. P. 1995. Pathogen-derived resistance to plant viruses. Annu. Rev. Phytopathol. 33: 323-343.
    連結：
44. Lovisolo, O. 1981. Virus and viroid disease of cucurbits. Acta. Hortic. 88: 33-82.
    連結：
45. Nameth, S. T., Dodds, J. A., Paulsu, A. O., and Laemmlen, F. F. 1986. Cucurbit viruses of California : An ever-changing problem. Plant Dis. 70: 8-11.
    連結：
46. Napoli, C., Lemieux C., and Jorgenson, R. 1990. Introduction of a chimeric chalcone synthase gene into petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2: 279-289.
    連結：
47. Niu, Q. W., Lin, S. S., Reyes, J. L., Chen, K. C., Wu, H. W., Yeh, S. D., and Chua, N. H. 2006. Expression of artificial micro-RNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat. Biotechnol. 24: 1420-1428.
    連結：
48. Mello, C.C., and Conte, Jr. D. 2004. Revealing the world of RNA interference. Nature 431: 338-342.
    連結：
49. Milne, K. S., Grogan, R. G., and Kimble, K. A. 1969. Identification of viruses infecting cucurbits in California. Phytopathology 59: 819-828
    連結：
50. Palukaitis, P., Roossinck, M. J., Dietzgen, R. G., and Francki, R. I. 1992. Cucumber mosaic virus. Adv. Virus Res. 41: 281-348.
    連結：
51. Pang, S. Z., Jan, F. J., and Gonsalves. D. 1997. Nontarget DNA sequences reduce the transgene length necessary for RNA-mediated tospovirus resistance in transgenic plants. Proc. Natl. Acad. Sci. USA 94: 8261-8266.
    連結：
52. squash mosaic comovirus in transgenic squash plants expressing its coat
    連結：
53. Pang, S. Z., Slightom, J. L., and Gonsalves, D. 1993. Different mechanisms protect transgenic tobacco against tomato spotted wilt and impatiens necrotic spot tospoviruses. Bio/Technology 11: 819-824.
    連結：
54. Park, S. M., Lee, J. S., Jegal, S., Jeon, B. Y., Jung, M., Park, Y. S., Han, S. L., Shin, Y. S., Her, N. H., Lee, J. H., Lee, M. Y., Ryu, K. H., Yang, S. G., and Harn, C. H. 2005. Transgenic watermelon rootstock resistant to CGMMV (cucumber green mottle mosaic virus) infection. Plant Cell Rep. 24: 350-356.
    連結：
55. Peters, D., Wijkamp, L., van de Wetering, F., and Goldbach, R. 1996. Vector relations in the transmission and epidemology of tospoviruses. Acta. Hortic. 431: 29-42.
    連結：
56. Quemada, H., Sieu, L. C., Sieminiak, D. R., Gonsalves, D., and Slightom, J. L. 1990. Watermelon mosaic virus II and zucchini yellow mosaic virus: cloning of the 3'-terminal regions, nucleotide sequences, and phylogenetic comparisions. J. Gen. Virol. 71: 1451-1460.
    連結：
57. Roossinck, M. J. 2002. Evolutionary history of Cucumber mosaic Virus deduced by phylogenetic analyses. J. Virol. 76: 3382-3387.
    連結：
58. Sanford, J. C., and Johnston. S. A. 1985. The concept of parasite-derived resistance - deriving resistance genes from the parasite's own genome. J. Theor. Biol. 113: 395-405.
    連結：
59. Sambrook, J., and Russell, D. W. 2001. Molecular Cloning: A Laboratory Manual, 3th ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
    連結：
60. and function. In: The Potyviridae. CAB International, Cambridge. pp.
    連結：
61. Smith, N. A., Singh, S. P., Wang, M. B., Stoutjesdijk, P. A., Green,
    連結：
62. Tricoli, D. M., Carney, K. J., Russell, P. F., McMaster, J. R., Groff , D. W., Hadden, K. C., Himmel, P. T., Hubbard, J. P., Boeshore, M. L., Reynolds, J. F., and Quemada, H. D. 1995. Field evaluation of transgenic squash containing single or multiple virus coat protein gene constructs for resistance to cucumber mosaic virus, watermelon mosaic virus 2, and/or zucchini yellow mosaic virus. Bio/Technology 13: 1458-1465.
    連結：
63. Tsai, W. S., Shih, S. L., Green, S. K., and Jan, F. J. 2007. Occurrence and molecular characterization of Squash leaf curl Philippines virus in Taiwan. Plant Dis. 91: 907.
    連結：
64. Ugaki, M., Tomiyama, M., Kakutani, T., Hidaka, S., Kiguchi, T., Nagata, R., Sato, T., Motoyoshi, F., and Nishiguchi, M. 1991. The complete nucleotide sequence of cucumber green mottle mosaic virus (SH strain) genomic RNA. J. Gen. Virol. 72: 1487-1495.
    連結：
65. van Kammen, A., Henstre, S., and Le, T. S. 1966. Morphology of tomato spotted wilt virus. Virology 30: 574-575.
    連結：
66. Wang, H., and Stubbs, G. 1994. Structure determination of cucumber green mottle mosaic virus by X-ray fiber diffraction. J. Mol. Biol. 239: 371-384.
    連結：
67. Wang, S. M., and Chen, M. J. 1985. New strain of cucumber mottle mosaic virus causing mosaic symptoms on bottle gourd in Taiwan. Plant Prot. Bull. 27: 105-110.
    連結：
68. Ward, C.W., and Shukta, D. D. 1991. Taxonomy of potyviruses: Current problems and some solutions. Intervirology 32: 269-296.
    連結：
69. Wu, H. W., Yu, T. A., Raja, J. A. J., Wang, H. C., and Yeh, S. D. 2009. Generation of transgenic oriental melon resistant to Zucchini yellow mosaic virus by an improved cotyledon-cutting method. Plant Cell Rep. 28: 1053-1064.
    連結：
70. Yang, W. Z., Hsiao, C. H., Huang, C. H., and Liou, P. C. 2002. Breeding of the vegetable sponge F1 hybrid caltivar “Taiaung No. 1”. J. Agric. Res. China 51: 37-48.
    連結：
71. Yeh, S. D., and Gonsalves, D. 1984. Purification and immunological analyses of cylindrical-inclusion protein induced by papaya ringspot virus and watermelon mosaic virus Ι. Phytopathology 74: 1273-1278.
    連結：
72. Yeh, S. D., Lin, Y. C., Cheng, Y. H., Jih, C. L., Chen, M. J., and Chen, C. C. 1992. Identification of tomato spotted wilt-like virus on watermelon in Taiwan. Plant Dis. 76: 835-840.
    連結：
73. Yoshioka, K., Hanada, K., Harada, T., Minobe ,Y., and Oosawa, K. 1993. Virus resistance in transgenic melon plants that express the cucumber mosaic virus coat protein gene and in their progeny. Jpn. J. Breed. 43: 629-634.
    連結：
74. Zhu, C. X., Song, Y. Z., Yin, G. H., and Wen, F. J. 2009. Induction of RNA-mediated multiple virus resistance to Potato virus Y, Tobacco mosaic virus and Cucumber mosaic virus. J. Phytopathol. 157: 101-107.
    連結：
75. 李青梅。2005。西瓜銀斑病毒核鞘蛋白與矮南瓜黃化嵌紋病毒及木瓜輪點病毒西瓜系統鞘蛋白轉基因西瓜之構築。大葉大學分子生物科技學系碩士論文。台中。
76. 施傑仁。2002。利用基因沉寂機制發展抗多種病毒病害之轉基因茄科植物。中興大學植物病理學系碩士論文。台中。
77. 黃玉瓊、葉瑩。1995。瓜類作物病蟲害防治現況。瓜類作物保護技術研討會專刊: 17-21。
78. 楊景富。2007。抗多重病毒之轉基因西瓜構築及溫室抗病評估。大葉大學分子生物科技學系碩士論文。台中。
79. Arce-Ochoa, J. P., Dainello, F., Pike, L. M., and Drews, D. 1995. Field performance comparison of two transgenic summer squash hybrids to their parental hybrid line. HortScience 30: 492-493.
80. Bucher, E., Lohuis, D., van Poppel, P. M. J.A., Geerts-Dimitriadou, C.,
81. Goldbach, R., and Prins, M. 2006. Multiple virus resistance at a high
82. frequency using a single transgene construct. J. Gen. Virol. 87: 3697-3701.
83. Buttner, C., Marquardt, K., and Schickedanz, F. 1995. Studies on Transmission of cucumber Mosaic Virus (CMV) and cucumber Green Mottle Mosaic Virus (CGMMV) by nutrient solutions of ebb and flow irrigation systems. Gartenbauwissenschaft. 60: 109-114.
84. Chen, C. C., Ho, H. M,. Chang, T. F., Chao, C. H., and Yeh, S. D. 1995. Characterization of a tospovirus-like virus isolated from wax gourd. Plant Prot. Bull. 37: 117-131.
85. Chen, C. C., Ko, W. F., Pai, K. F., and Yeh, S. D. 2004. Ecology of Watermelon silver mottle virus disease on watermelon in Taiwan. Plant Pathol. Bull. 13: 317-328.
86. Fuchs, M., McFerson, J. R., Tricoli, D. M., McMaster, J. R., Deng, R. Z., Boeshore, M. L., Reynolds, J. F., Russell, P. F., Quemada, H. D., and Gonsalves, D. 1997. Cantaloupe line CZW-30 containing coat protein genes of cucumber mosaic virus, zucchini yellow mosaic virus, and watermelon mosaic virus-2 is resistant to these three viruses in the field. Mol. Breed. 3: 279-290.
87. Fuchs, M., Klas, F. E., McFerson, J. R., Gonsalves, D. 1998a.Transgenic melon and squash expressing coat protein genes of aphid-borne viruses do not assist the spread of an aphid non-transmissible strain of cucumber mosaic virus in the field. Trans. Res. 7: 449-462.
88. Gonsalves, C., Xue, B., Yepes, M., Fuchs, M., Ling, K., Namba, S., Chee, P., Slightom, J.L., and Gonsalves D. 1994. Transferring the cucumber mosaic virus-white leaf strain coat protein gene into Cucumi melo L. and evaluating transgenic plants for protection against infections. J. Amer. Soc. Hort. Sci. 119: 345-355.
89. Hollings, M., Komuro, Y., and Tochihara, H. 1975. Cucumber green mottle mosaic viruses. No. 154. In: Descriptions of Plant Viruses. Kew, Surrey, England. C.M.I./A.A.B.
90. Hsu, H. T., Lawson, R. H., Yeh, S. D., and Chiu, R. J. 1996. Serological relationship of a high temperature-recovered tospovirus in the USA and watermelon silver mottle virus in Taiwan. Phytopathology 86: 843.
91. Huang, C. H., and Chang. L. 1989. Characterization of a type W variant of papaya ringspot virus in Taiwan. Plant Prot. Bull. 31: 412.
92. Jan, F. J. 1998. Roles of nontarget DNA and viral gene length in influencing multi-virus resistance through homology-dependent gene silencing. Ph. D. Dissertation, Department of Plant Pathology, Cornell University.
93. is affected by plant developmental stage and enhanced by combination of transgenes from different lines. J. Gen. Virol. 81: 2299-2306.
94. Jan, F. J., Shih, J. R., Yeh, S. D., and Gonsalves, D. 2002. Development of transgenic plants resistant to multiple viruses via gene silencing. Page 14 in: Abstract book of XII International Congress of Virology, July 27-August 1, 2002, Paris, France.
95. Klas, F. E., Fuchs, M., and Gonsalves, D. 2006. Comparative spatial spread
96. mosaic virus (WMV) in fields of transgenic squash expressing the coat protein genes of ZYMV and WMV, and fields of nontransgenic squash. Trans. Res. 15: 527-541.
97. Pang, S. Z., Jan, F. J., Tricoli, D. M., Russell, P. F., Carney, K. J., Hu, J. S., Fuchs, M., Quemada, H. D., and Gonsalves, D. 2000. Resistance to
98. protein genes. Mol. Breed. 6: 87-93.
99. Purcifull, D. E., Edwardson, J. R., Hebert, E., and Gonsalves, D. 1984a. Papaya Ring sopt Virus. CMI/AAB Descriptions of Plant Virus. No. 292.
100. Purcifull, D., Hiebert, E., and Edwardson, J. 1984b. Watermelon mosaic virus 2. CMI/AAB descriptions of plant viruses No. 293.
101. Reddy, D. V. R., and Wightman, J. A. 1988. Tomato spotted wilt virus: Thrips transmission and control. Adv. Dis. Vector Res. 5: 203-220.
102. Shukla, D. D., Ward, C.W., and Brunt, A. A. 1994. Genome structure, variation
103. 74-112.
104. A. G., and Waterhouse, P. M. 2000. Total silencing by intron spliced
105. Hairp in RNAs. Nature 407: 319-320.
106. Wako, T., Terami, F., Hanada, K., and Tabei, Y. 2001. Resistance to Zucchini
107. yellow mosaic virus (ZYMV) in transgenic cucumber plants (Cucumis sativus L.) harboring the coat protein gene of ZYMV. Bull. Natl. Res. Inst. Veg. Ornam. Plants Tea Jpn. 16: 175-186.
108. Wang, J. J., and Yeh, S. D. 1998. Characterization of the papaya ringspot virus W type isolates collected form different areas of Taiwan by host reactions, immuneodiffusion tests and RT-PCR. Plant Prot. Bull. 40: 383-395.
109. Wesley, S. V., Helliwell, C. A., Smith, N. A., Wang, M-B., Rouse, D. T., Liu, Q., Gooding, P. S., Singh, S. P., Abbott, D., Stoutjesdijk, P. A., Robinson, S. P., Gleave, A. P., Green, A. G., and Waterhouse, P. M. 2001. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J. 27: 581-590.
110. Zitter, T. A. 1986. New virus threatens cucurbits. Am. veg. grow. 34: 13-17.

1. 李佳華(2011)。抗三種蝴蝶蘭病毒轉基因植物之研發。中興大學植物病理學系所學位論文。2011。1-55。