臺灣博碩士論文加值系統

中文摘要
本實驗室之前的研究中已成功培育出木瓜輪點病毒 (Papaya ringspot virus, PRSV) 鞘蛋白 (coat protein, CP) 轉基因木瓜，並確定其對PRSV具明顯之抵抗能力。最近發現此轉基因木瓜會被木瓜畸葉嵌紋病毒 (Papaya leaf-distortion mosaic virus, PLDMV) 所感染，因此當此轉基因木瓜未來應用於田間時，PLDMV將可能對其造成嚴重威脅。為能有效控制PRSV及PLDMV兩病毒，本研究將PRSV及PLDMV部分CP基因以非轉譯性的策略構築在同一載體上，再利用農桿菌為媒介 (Agrobacterum-mediated) 將此基因轉入泰國品種木瓜中。在75個轉型成功的株系中，於溫室條件下分別接種PRSV及PLDMV，一個月後有38個株系無病徵表現，故歸類為抗病株系 (resistant lines)。分子分析實驗中，南方墨點法 (Southern blotting) 及北方墨點法 (northern blotting) 分析結果顯示受測的感病株系中皆屬僅含有一個轉基因插入套數且有高量的轉錄體表現，而抗病株系則均含有兩個或兩個以上的轉基因插入套數且沒有轉錄體的表現。將兩個抗病株系泰國種木瓜 (12-4與15-5) 與日陞種木瓜雜交，將子代葉柄置於含康黴素 (kanamycin) 培養基中分析，顯示此兩個株系之轉基因於子代分離率皆為3:1，推測這兩個抗病株系的轉基因分別坐落在基因體的兩個基因座 (loci) 上。綜合以上結果，轉基因品系雙重抗病毒的性狀與轉基因插入套數及後轉錄基因沉寂 (post-transcriptional gene silencing) 有關。此外，為了能夠有效率且靈敏的檢測PLDMV，本研究中利用pET32a基因表現載體於大腸桿菌 (Escherichia coli) 中大量表現 PLDMV DL 之鞘蛋白，並進一步加以純化。以此蛋白免疫的老鼠，獲得一個會分泌PLDMV CP抗體之融合瘤細胞株 (hybridoma cell line) 145G4B1，由其所製備之腹水，在間接酵素連結免疫分析法（Indirect-ELISA）下其有效反應稀釋濃度可達64000倍。以西方墨點法分析此單元抗體及多元抗體As59對四個不同地區之PLDMV分離株和PRSV YK之反應，結果顯示145G4B11比As59更能專一的偵測到PLDMV鞘蛋白之存在。總結以上研究成果，我們的R0及R1轉基因木瓜株系皆具有雙重抗病毒能力，認為其應用於田間將有很大的潛力以用來防治PRSV及PLDMV，而PLDMV單株抗體的製備對於偵測田間的PLDMV應是非常有效的血清學工具。

Abstract
Transgenic papaya lines carrying Papaya ringspot virus (PRSV) coat protein (CP) gene were previously generated in our laboratory to confer resistance against PRSV infection. Recently, it was found that the resistance was overcome by Papaya leaf-distortion mosaic virus (PLDMV) that might be serious threat when the transgenic lines are practically applied in Taiwan. In this study, for the effective control of PRSV and PLDMV, an untranslatable chimeric construct containing truncated PRSV YK CP and PLDMV DL CP genes was transferred into papaya (Carica papaya cv. Thailand) via Agrobacterum-mediated transformation. A total of 75 transgenic lines was obtained and separately challenged with PRSV YK and PLDMV DL by mechanical inoculation under greenhouse conditions. Among them, 38 transgenic lines showed no symptoms one month after inoculation and were classified as resistant lines. Molecular analyses by Southern and northern blottings indicated that four sensitive lines have one insert of the construct and high amount of transgene transcript was detected, whereas the resistant lines have two or multiple inserts and no transgene transcript detected. A 3:1 ratio for the segregation of the transgene of resistant lines TPY16 12-4 and TPY16 15-5 were revealed by kanamycin assay using petioles of R1 plants derived from crossing with non-transgenic Sunrise papaya, indicating that the transgene of both lines located at two loci of chromosome. The results indicated that double resistance of transgenic lines is resulted from double or multiple copies of the insert and RNA-mediated post-transcriptional gene silencing. Furthermore, for specific and effective detection of PLDMV, one hybridoma cell line 145G4B11 secreted monoclonal antibody (MAb) to PLDMV CP was selected by immunizing mice with pET-32a(+) expressed antigen from E. coli. The titer of ascitic fluid of this MAb (145G4B11) to PLDMV CP was 64,000 as determined by indirect ELISA. In western blotting, 145G4B11 was more specific to PLDMV CP than As59, a polyclonal antibody against PLDMV. In summary, our R0 and R1 transgenic lines with double resistance are considered having a great potential to control both PRSV and PLDMV. In addition, the MAb produced 145G4B11 is an efficient serological tool for detection of PLDMV in diseased samples from field.

Content
Abstract in English…………………………………………………….……….…… 1
中文摘要………………………………………………………………………............3
第一部分 木瓜輪點病毒及木瓜畸葉嵌紋病毒雙重抗性轉基因木瓜之育成..........5
Section I Development of transgenic papaya resistant to Papaya ringspot virus and Papaya leaf-distortion mosaic virus………………………..............5
前言……………………………………………………………………………………6
Introduction……………………………………………………………………........10
Materials and methods………………………………………………………...……16
Construction of an untranslatable chimeric transgene………………………...…16
Generation of transgenic papaya lines………………………………………..….17
DNA extraction and Polymerase Chain Reaction……………………….………18
Inoculation of transgenic lines with PRSV YK or PLDMV DL…………..….…18
Inoculation with different geographic PRSV strains……………………….……19
Sourthern blot analysis……………………………………………………..……20
Northern blot analysis……………………………………………………...…….21
Segregation of analysis the transgene in R1 progeny……………………………..22
Inoculation of R1 progeny with PLDMV and PRSV…………………….……….22
Results……………………………………………………………………………….24
Construction of an untranslatable chimeric constract……………………………24
Establishment of transgenic lines………………………………………….……..24
Inoculation of transgenic lines with PRSV YK or PLDMV DL…………...…….25
Resistance assay against different geographic PRSV strains…………………….25
Sourthern blot analysis of R0 transgenic lines……………………….…………..26
The expression level of cherimeic CP transgene…………………………………27
Segregation of the the chimeric construct in R1 progeny………………...………27
Inoculation of R1 progeny with PLDMV and PRSV ……………………………28
Discussion……………………………………………………………………………29
References…………………………………………………………………………..35
Tables and Figures…………………………………………………………….……39
第二部分 木瓜畸葉嵌紋病毒單株抗體之製備……………………………………47
Section II Production of monoclonal antibody to Papaya leaf distortion-mosaic virus ………………………………………………………………...…..47
前言…………………………………………………………………………….……48
Introduction…………………………………………………………………………51
Materials and Methods…………………………………………………..………...54
Virus source and propagation………………………………………………….....54
Construction of the reading frame of the CP gene of PLDMV in pET32a vector 54
Expression and purification of PLDMV coat protein……………………...…….56
Production of monoclonal antibody …………………………………………….57
Detection of the titers of the MAb by indirect-ELISA……………………….…59
Western blot analysis…………………………………………………………….60
Detection of the different PLDMV isolates by indirect-ELISA…………...……..61
Results…………………………………………………………………………...…..63
Construction of the CP reading frame of PLDMV in pET32a vector……………63
Expression and purification of bacteria-expressed PLDMV coat protein………..63
Screening of hybridoma lines and production of MAb…………………………..64
The analysis of the reactions of MAb with bacteria-expressed PLDMV CP…….64
Detection of the titers of the MAb with crude antigens from infected plants by indirect-ELISA ……………………………………………………………….….65
Western blot analysis …………………………………………………………..…65
Detection of the different PLDMV and PRSV isolates with MAb indirect-ELISA
……………………………………………………………………………………66
Discussion…………………………………………………………………………....68
References………………………………………………………………………...…72
Figures……………………………………………………………………………….74

References (Section I)
Abel, P. P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T., and Beachy, R. N. 1986. Delay of disease development in transgenic plants that express the tobacco mosaic virus coat protein gene. Science 232:738-43.
Abel, P. P., Nelson, R. S., De, B., Hoffmann, N., Rogers, S. G., Fraley, R. T., and Beachy, R. N. 1986. Delay of disease development in transgenic plants that express the Tobacco mosaic virus coat protein gene. Science 232:738-743.
Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., and Vance, V. B. 1998. A viral suppressor of gene silencing in plants. Proc Natl Acad Sci U S A 95:13079-13084.
Bau, H. J. 2000. Studies on the resistance of transgenic papaya conferred by the coat protein gene of Papaya ringspot virus. In Ph. D. Dissertation, Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan. pp107-125.
Bau, H. J., Cheng, Y. H., Yu, T. A., Yang, J. S., and Yeh, S. D. 2003. Broad-spectrum resistance to different geographic strains of Papaya ringspot virus in coat protein gene transgenic papaya. Phytopathology 93:112-120.
Bau, H. J., Cheng, Y. H., Yu, T. A., Hsiao, C. H., and Yeh, S. D. 2004. Field evaluation of transgenic papaya lines carrying the coat protein gene of Papaya ringspot virus in Taiwan. Plant Dis. 88:594-599.
Baulcombe, D. C. 1996. RNA as a target and an initiator of post-transcriptional gene silencing in transgenic plants. Plant Mol. Biol. 32:79-88.
Cheng, Y. H., Yang, J. S., and Yeh, S. D. 1996. Efficient transformation of papaya by coat protein gene of Papaya ringspot virus mediated by Agrobacterium following liquid-phase wounding of embryogenic tissues with carborundum. Plant Cell Rep. 16:127-132.
Conover, R. A. 1976. A program for development of papaya tolerant to the distortion ringspot virus. Proc. Fla. State Horitic. Soc. 89:229-231.
Conover, R. A., and Litz, R. E. 1978. Progress in breeding papaya with tolerance to Papaya ringspot virus. Proc. Fla. State Horitic. Soc. 91:182-184.
English, J. J., Mueller, E., and Baulcombe, D. C. 1996. Suppression of virus accumulation in transgenic plants exhibiting silencing of nuclear genes. Plant Cell 8:179-188.
Fitch, M. M. M., Manshardt, R. M., Gonsalves, D., Slightom, J. L., and Sanford, J. C. 1990. Stable transformation of papaya via microprojectile bombardment. Plant Cell Rep. 9:189-194.
Fitch, M. M. M., Manshardt, R. M., Gonsalves, D., Slightom, J. L., and Sanford, J. C. 1992. Virus resistance papaya plants derived from tissues bombarded with the coat protein gene of Papaya ringspot virus. Bio/Technology 10:1466-1472.
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.
Fuchs, M., Tericoli, D. M., Carney, K. J., Schesser, M, McFerson, J. R. , and Gonsalves, D. 1998. Comparative virus resistance and fruit yeild of transgenic squash with single and multiple coat protein genes. Plant Dis. 82:1350-1356.
Gonsalves, D., and Ishii, M. 1980. Purification and serology of Papaya ringspot virus. Phytopathology 70:1028-1032.
Jan, F. J., Fagoaga, C., Pang, S. Z., and Gonsalves, D. 2000. 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.
Kasschau, K. D., and Carrington, J. C. 1998. A counterdefensive strategy of plant viruses: suppression of posttranscriptional gene silencing. Cell 95:461-470.
Kawano, S., and Yonaha, T. 1992. The occurence of Papaya leaf-distortion mosaic virus in Okinawa. Tech. Bull. of FFTC 132: 13-23, Food and Fertilizer Technology Center for the Asian and Pacific Regions, Taipei, Taiwan, R. O. C.
Maoka, T., Kashiwazaki, S., Tsuda, S., Usugi, T., and Hibino, H. 1996. Nucleotide sequence of the capsid protein gene of Papaya leaf-distortion mosaic potyvirus. Arch. Virol. 141:197-204.
Mueller, E., Gilbert, J., Davenport, G., Brigneti, G., and Baulcombe, D. C. 1995. Homology-dependent resistance: transgenic virus resistance in plants related to homology-dependent gene silencing. Plant J. 7:1001-1013.
Murashige, T., and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant 15:437-497.
Osbourn, J. K., Watts, J. W., Beachy, R. N., and Wilson, T. M. 1989. Evidence that nucleocapsid disassembly and a later step in virus replication are inhibited in transgenic tobacco protoplasts expressing TMV coat protein. Virology 172:370-373.
Purcifull, D. E., Edwardson, J. R., Hiebert, E., and Gonsalves, C. 1984. Papaya ringspot virus. CMI/AAB Descriptions of Plant Viruses, No. 84.
Rogers, S. G., Horsh, R. B., and Fraley, R. T. 1986. Gene transfer in plants: Production of transformed plants using Ti-plasmid vectors. Methods in Enzymology 118:627-640.
Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., Mullis, K. B., and Erlich, H. A. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239:487-491.
Sanford, J. C., and Johnson, S. A. 1985. The concept of parasite-derived resistance. Deriving resistance genes from the parasites own genome. J. Theor. Biol. 113:395-405.
Sijen, T., Wellink, J., Hiriart, J. B., and Van Kammen, A. 1996. RNA-mediated virus resistance: role of repeated transgenes and delineation of targeted regions. Plant Cell 8:2277-2294.
Tricoli, D. M., Carney, K. J., Pussell, P. F., McMaster, J. R., Groff, D. W., Hadden, K. C., Himmel, P. T., Hubbard, J. P., Boeshore, M. L., 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, Watermalon mosaic virus 2, and Zucchini yellow mosaic virus. Bio/Technology 13:1458-1465.
Tripathi, S., Bau, H. J., Chen, L. F., and Yeh, S. D. 2004. The ability of Papaya ringspot virus strains overcoming the transgenic resistance of papaya conferred by the coat protin gene is not correlated with higher degrees of sequence divergence from the transgene. (in press).
van Regenmortel, M. H. V., Fauqunet, C. M., Bishop, D. H. L., Cartens, E. B., Estes, M. K., Lemon, S. M., Maniloff, J., Mayo, M. A., McGeoch, D. J., Pringle, C. R., and Wickner, R. B. 2000. Family Potyviridae. In Virus Taxonomy (USA:Academic Press):pp706-712.
Wang, C. H., and Yeh, S. D. 1997. Divergence and conservation of the genomic RNAs of Taiwan and Hawaii strains of papaya ringspot potyvirus. Arch. Virol. 142:271-285.
Wang, H. L., Wang, C. C., Chiu, R. J., and Sun, M. H. 1978. Preliminary study on Papaya ringspot virus in Taiwan. Plant Prot. Bull. 20:133-140.
Wang, H. L., Yeh, S. D., Chiu, R. J., and Gonsalves, D. 1987. Effectiveness of cross-protection by mild mutants of Papaya ringspot virus for control of ringspot disease of papaya in Taiwan. Plant Dis. 71:491-497.
Wisniewski, L. A., Powell, P. A., Nelson, R. S., and Beachy, R. N. 1990. Local and systemic spread of Tobacco mosaic virus in transgenic tobacco. Plant Cell 2:559-567.
Wlison, T., and Watkins, P. 1986. Influence of exogenous viral coat protein on the co-translational disassembly of Tobacco mosaic virus. Virology 149:132-135.
Yang, J. S., and Ye, C. A. 1992. Plant regeneration from petioles of in vitro regenerated papaya (Carica papaya L.) shoots. Bot.Bull. Acad. Sin. 33:357-381.
Yeh, S. D., and Gonsalves, D. 1984. Evaluation of induced mutants of Papaya ringspot virus for control by cross protection. Phytopathology 74:1086-1091.
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.
Yeh, S. D., and Gonsalves, D. 1994. Practices and perspective of control of Papaya ringspot virus by cross-protection. Adv. Dis. Vector Res. 10:237-257.
Yeh, S. D., Gonsalves, D., Wang, H. L., Namba, R., and Chiu, R. J. 1988. Control of Papaya ringspot virus by cross protection. Plant Dis. 72:375-380.
Yeh, S. D., Jan, F. J., Chiang, C. H., Doong, T. J., Chen, M. C., 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.
Yonaha, T., Yonemori, S., and Tamori, M. 1976. Relation between the flight occurrence of alate aphids and the spread of papaya virus disease in the field. Okinawa Agriculture 14:7-15.
References (Section II)
Bahadur, K., and Sinha, R. C. 1958. The influence of vitamin C (ascorbic acid), in presence of Fe++ and Mn++ ions, on the proteolytic activity of papain employing casein as the substrate. Enzymologia 19:125-9.
Baker, K. C., Taylor, M. A., Cummings, N. J., Tunon, M. A., Worboys, K. A., and Connerton, I. F. 1996. Autocatalytic processing of pro-papaya proteinase IV is prevented by crowding of the active-site cleft. Protein Eng. 9:525-529.
Bateson, M. F., Henderson, J., Chaleeprom, W., Gibbs, A. J., and Dale, J. L. 1994. Papaya ringspot potyvirus: isolate variability and the origin of PRSV type P (Australia). J Gen Virol 75:3547-53.
Bau, H. J. 2000. Studies on the resistance of transgenic papaya conferred by the coat protein gene of Papaya ringspot virus. In Ph. D. Dissertation, Department of Plant Pathology, National Chung Hsing University, Taichung, Taiwan. pp107-125.
Bau, H. J., Cheng, Y. H., Yu, T. A., Yang, J. S., and Yeh, S. D. 2003. Broad-spectrum resistance to different geographic strains of Papaya ringspot virus in Coat Protein Gene Transgenic Papaya. Phytopathology 93:112-120.
Chan, S. J. 1999. Production of antibodies to bacterial expressed P1 protein of Papaya ringspot viruses and coat protein of Papaya leaf distortion mosaic virus. In M. S. thesis., Department of plant pathology, National Chung Hsing University, Taichung, Taiwan. pp1-53.
Chen, C. H. 2002. Expression of the coat protein gene of Papaya leaf-distortion mosaic virus by bacterial plasmid or viral vector. In M. S. thesis., Department of plant pathogolgy, National Chung Hsing University, Taichung, Taiwan. pp1-57.
Cohen, L. W., Coghlan, V. M., and Dihel, L. C. 1986. Cloning and sequencing of papain-encoding cDNA. Gene 48:219-227.
Fitch, M. M. M., Manshardt, R. M., Gonsalves, D., Slightom, J. L., and Sanford, J. C. 1990. Stable transformation of papaya via microprojectile bombardment. Plant Cell Rep. 9:189-194.
Fitch, M. M. M., Manshardt, R. M., Gonsalves, D., Slightom, J. L., and Sanford, J. C. 1992. Virus resistance papaya plants derived from tissues bombarded with the coat protein gene of papaya ringspot virus. Bio/Technology 10:1466-1472.
Kawano, S., and Yonaha, T. 1992. The occurence of Papaya leaf-distortion mosaic virus in Okinawa. Tech. Bull. of FFTC 132: 13-23, Food and Fertilizer Technology Center for the Asian and Pacific Regions, Taipei, Taiwan, R. O. C.
Kiritani, K., and Su, H.-J. 1999. Papaya ring spot, banana bunchy top, and citrus greening in the Asia and Pacific Region: Occurrence and control strategy. Japn. Agri. Res. Quar 33:23-30.
Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-5.
Maoka, T., Kashiwazaki, S., Tsuda, S., Usugi, T., and Hibino, H. 1996. Nucleotide sequence of the capsid protein gene of papaya leaf-distortion mosaic potyvirus. Arch Virol 141:197-204.
Rosenberg, A. H., Lade, B. N., Chui, D. S., Lin, S. W., Dunn, J. J., and Studier, F. W. 1987. Vectors for selective expression of cloned DNAs by T7 RNA polymerase. Gene 56:125-35.
Silva, L. G., Garcia, O., Lopes, M. T., and Salas, C. E. 1997. Changes in protein profile during coagulation of latex from Carica papaya. Braz J Med Biol Res 30:615-619.
Skelton, G. S. 1962. The effect of various chemicals on papain activation. Enzymologia 24:338-40.
Studier, F. W., and Moffatt, B. A. 1986. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189:113-30.
Studier, F. W., Rosenberg, A. H., Dunn, J. J., and Dubendorff, J. W. 1990. Use of T7 RNA polymerase to direct expression of cloned genes. Methods Enzymol 185:60-89.
van Regenmortel, M. H. V., Fauqunet, C. M., Bishop, D. H. L., Cartens, E. B., Estes, M. K., Lemon, S. M., Maniloff, J., Mayo, M. A., McGeoch, D. J., Pringle, C. R., and Wickner, R. B. 2000. Family Potyviridae. In Virus Taxonomy (USA:Academic Press):pp706-712.
Yeh, S. D., and Gonsalves, D. 1994. Practices and perspective of control of Papaya ringspot virus by cross-protection. Adv. Dis. Vector Res. 10:237-257.
Yeh, S. D., and Gonsalves, D. 1984. Evaluation of induced mutants of Papaya ringspot virus for control by cross protection. Phytopathology 74:1086-1091.
Yonaha, T., Yonemori, S., and Tamori, M. 1976. Relation between the flight occurrence of alate aphids and the spread of papaya virus disease in the field. Okinawa Agriculture 14:7-15.