臺灣博碩士論文加值系統

蝴蝶蘭為全球花卉市場中具高經濟價值的花卉作物，而且在台灣花卉出口作物中，具有重要性。台灣原生種白花蝴蝶蘭（Phalaenopsis aphrodite subsp. formosana）為育種上的重要親本。細胞分裂素（cytokinin）為植物重要的荷爾蒙之一，它在植物的生長與發育的調節扮演重要的角色。而細胞分裂素氧化酶（cytokinin oxidase）為調節細胞分裂素濃度的主要酵素。本實驗希望利用 CRISPR/Cas9 編輯系統搭配 tRNA 修飾系統來針對蝴蝶蘭的3個細胞分裂素氧化酶基因進行剔除。建構5個細胞核表現載體，經轉殖蝴蝶蘭後，利用MSO（L-methionine sulfoximine）進行篩選，得到可能的轉殖植物。萃取 DNA 後，進行作用目標位點之基因型利用以及定序分析，在 pCas9-CKX1 轉殖品系的植物（1-7）、pCas9-CKX2 轉殖品系的植物（2-22）以及 pCas9-CKX3 轉殖品系的植物（3-57）中皆有發現序列的取代以及剔除的突變產生。在共122顆植物中僅發現2個有突變的可能性，因此基因編輯效率低，在對本研究的相關步驟檢視後，發現原本應該要被剪掉切除的 Cas9 intron 仍存在，推測這是導致此基因編輯系統效率低落的主要原因之一。

P. aphrodite subsp. formosana, an endemic moth orchid in Taiwan, is one of the most commonly used parental strains in commercial breeding programs. The hormone cytokinins (CKs) play an important role in the regulation of growth and development in plants. The cytokinin oxidases (CKXs) can regulate the homeostasis of active cytokinin level. Previous study have identified three CKX genes in moth orchid by blast the CKX genes of Arabidopsis against Orchidstra database. In the present study, CRISPR/Cas9 editing technology in combination of tRNA processing mechanism was applied to knock out CKX genes in moth orchid. Five Cas9/gRNA nuclear expression vectors carrying bar gene as selection marker were constructed. Transgenes were delivered into the nuclear genome of moth orchid through Agrobacteria-mediated transformation. The T0 seeds were germinated and then selected with L-methionine sulfoximine (MSO). Subsequently, MSO-resistant seedlings of 12, 13, 18, 55 and 24 were obtained from pCas9-CKX1, pCas9-CKX2, pCas9-CKX3, pCas9-CKX23 and pCas9-CKX123 transgenic lines, respectively. Sequencing analysis for the detection of the mutational status in CKX genes revealed that three transgenic plants, each from pCas9-CKX1 (no.1-7), pCas9-CKX2 (2-22), and pCas9-CKX3 (3-57) line were different in the corresponding CKX gene sequence from wild type plant. However, the multiplex CRISPR/Cas9 editing efficiency is low in this study. The RT-PCR analysis revealed the inefficient potato IV2 intron splicing of Cas9 mRNA in moth orchid, which it might explain the low efficiency of gene editing in moth orchid.

目錄
中文摘要 I
英文摘要 II
誌謝 VI
目錄 VII
表目錄 XI
圖目錄 XII
附圖目錄 XIV
縮寫表 XV
一、研究背景 1
1-1細胞分裂素對植物生理的相關調控 1
1-2細胞分裂素（Cytokinin）之形式和結構 1
1-3細胞分裂素的合成途徑 2
1-4細胞分裂素氧化酶（CKX）的作用 3
1-5細胞分裂素的訊息傳遞路徑 4
1-6細胞分裂素在蘭花上的應用 4
1-7生物資訊分析蝴蝶蘭 CKX 基因的表現情形 5
1-8阿拉伯芥及蝴蝶蘭 CKX 在細胞內的表現位置 6
1-9 CRISPR/Cas9 基因編輯工具 7
1-10 CRISPR/Cas9 在植物上的應用 7
1-11 Mutiplex CRISPR/Cas9 系統 9
1-12研究目的 10
二、材料與方法 11
2-1白花蝴蝶蘭品系與目標基因分析 11
2-2基因轉殖載體構築 12
2-3蝴蝶蘭轉殖 21
2-4蝴蝶蘭基因分析 25
2-5蝴蝶蘭綠螢光表現分析 33
三、結果 35
3-1分析比較白花蝴蝶蘭與其他蘭科植物的 CKX 基因 35
3-2構築 Cas9/ gRNA 表現載體 37
3-3蝴蝶蘭轉殖與篩選 39
3-4 PCR 偵測外源基因是否插入基因組中 40
3-5 PCR 偵測農桿菌基因是否殘留在蝴蝶蘭中 40
3-6反轉錄 PCR 偵測 Cas9 蛋白表現情形 41
3-7分析蝴蝶蘭 CKXs 基因剔除情形 42
3-8利用 T7E1 測定法確認突變情形 43
3-9定序 PCR 產物以分析轉殖蝴蝶蘭 CKXs 目標片段 44
3-10利用選殖入 TA 載體後進行定序分析突變片段 45
3-11分析蝴蝶蘭 CKXs 基因表現位置 46
3-12分析蝴蝶蘭 CKXs 基因於洋蔥表皮細胞表現位置 46
四、討論 48
4-1分析蝴蝶基因以及其蛋白質在細胞的位置 48
4-2 PTG-Cas9 表現載體的構築 49
4-3轉殖植物篩選與外源基因分析 50
4-4 CRISPR/Cas9 對 CKX 基因的編輯效率 51
4-5 CRISPR/Cas9 在蘭科植物中的應用 53
4-6分析蝴蝶蘭的 CKX 蛋白質在細胞的位置 54
4-7總結 55
參考文獻 57
圖表 66
附錄 141

陳建瑩，應用 CRISPR/Cas9 技術造成白花蝴蝶蘭 DELLA 基因的突變，國立成功大學生物科技與產業科學系碩士論文，2018a。

陳詩璇，利用誘導表現轉錄似活化因子蛋白核酸酶（TALEN）來改變菸草業率體的 DNA，國立成功大學生物科技與產業科學系碩士論文，2018b。

陳謫凡，研究細胞分裂素氧化酶基因對台灣原生種白花蝴蝶蘭生長發育的影響，國立成功大學生物科技研究所碩士論文，2017。

Allen, G. C., Flores Vergara, M. A., Krasynanski, S., Kumar, S. and Thompson, W. F. A modified protocol for rapid DNA isolation from plant tissues using cetyltrimethyl’ammonium bromide. Nature Protocols 1, 20-23, 2006.

Anusha, P., Jyoti, T. and Sarangi, B. K. Assessment of Agrobacterium transformation efficiency in a model plant system. Journal of Plant Biology Research 3, 78-86, 2014.

Argyros, D., Mathews, E., Chiang, H., Palmer, M., Thibault, M., Etheridge, N., Argyros, A., Mason, G., Kieber, J. and Schaller, E. Type B response regulators of Arabidopsis play key roles in cytokinin signaling and plant development. The Plant Cell 20, 2102-2116, 2008.

Ashikari, M., Sakakibara, H., Lin, S., Yamamoto, T., Takashi, T., Nishimura, A. and Matsuoka, M. Cytokinin oxidase regulates rice grain production. Science 309, 741-745, 2005.

Barbezier, N., Canino, G., Rodor, J., Jobet, E., Saez-Vasquez, J., Marchfelder, A. and Echeverría, M. Processing of a dicistronic tRNA-snoRNA precursor: combined analysis in vitro and in vivo reveals alternate pathways and coupling to assembly of snoRNP. Plant Physiology 150, 1598-1610, 2009.

Bartrina, I., Otto, E., Strnad, M., Werner, T. and Schmülling, T. Cytokinin regulates the activity of reproductive meristems, flower organ size, ovule formation, and thus seed yield in Arabidopsis thaliana. The Plant Cell 23, 69-80, 2011.

Belarmino, M. M., and Mii, M. Agrobacterium-mediated genetic transformation of a Phalaenopsis orchid. Plant Cell Reports 19, 435-442, 2000.

Belhaj, K., Chaparro-Garcia, A., Kamoun, S., Patron, N. J. and Nekrasov, V. Editing plant genomes with CRISPR/Cas9. Current Opinion in Biotechnology 32, 76-84, 2015.

Bilyeu, D., Cole, L., Laskey, G., Riekhof, R., Esparza, J., Kramer, D. and Morris, O. Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiology 125, 378-386, 2001.

Chai, D., Lee, S. M., Ng, J. H. and Yu, H. L-methionine sulfoximine as a novel selection agent for genetic transformation of orchids. Journal of Biotechnology 131, 466-472, 2007.

Danilova, M., Kudryakova, N., Voronin, Y., Oelmüller, R., Kusnetsov, V. and Kulaeva, O. Membrane receptors of cytokinin and their regulatory role in Arabidopsis thaliana plant response to photooxidative stress under conditions of water deficit. Russian Journal of Plant Physiology 61, 434-442, 2014.

Deveau, H., Garneau, J. E., and Moineau, S. CRISPR/Cas system and its role in phage-bacteria interactions. Annual Review of Microbiology 64, 475-493, 2010.

Feng, Z., Zhang, B., Ding, W., Liu, X., Yang, L., Wei, P., Cao, F., Zhu, S., Zhang, F., Mao, Y. and Zhu, J. K. Efficient genome editing in plants using a CRISPR/Cas system. Cell Research 23, 1229-1232, 2013.

Faiss, M., Zalubìlová, J., Strnad, M. and Schmülling, T. Conditional transgenic expression of the ipt gene indicates a function for cytokinins in paracrine signaling in whole tobacco plants. The Plant Journal 12, 401-415, 1997.

Frebort, I., Kowalska, M., Hluska, T., Frebortova, J. and Galuszka, P. Evolution of cytokinin biosynthesis and degradation. Journal of Experimental Botany 62, 2431-2452, 2011.

Gaj, T., Gersbach, A. and Barbas III, F. ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends in Biotechnology 31, 397-405, 2013.

Gu, B., Zhou, T., Luo, J., Liu, H., Wang, Y., Shangguan, Y. and Han, B. An-2 encodes a cytokinin synthesis enzyme that regulates awn length and grain production in rice. Molecular Plant 8, 1635-1650, 2015.

Haberer, G. and Kieber, J. Cytokinins. New insights into a classic phytohormone. Plant Physiology 128, 354-362, 2002.

Hiei, Y. and Komari, T. Agrobacterium-mediated transformation of rice using immature embryos or calli induced from mature seed. Nature Protocols 3, 824-834, 2008.

Hilscher, J., Bürstmayr, H. and Stoger, E. Targeted modification of plant genomes for precision crop breeding. Biotechnolgy Journal 12, 160-173, 2017.

Hirose, N., Takei, K., Kuroha, T., Kamada-Nobusada, T., Hayashi, H. and Sakakibara, H. Regulation of cytokinin biosynthesis, compartmentalization and translocation. Journal of Experimental Botany 59, 75-83, 2008.

Hsing, X., Lin, J., Tong, G., Li, J., Chen, J. and Ko, S. Efficient and heritable transformation of Phalaenopsis orchids. Botanical Studies 57, 30-52, 2016.

Hwang, I. and Sheen, J. Two-component circuitry in Arabidopsis cytokinin signal transduction. Nature 413, 383-389, 2001.

Kakimoto, T. Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases. Plant and Cell Physiology 42, 677-685, 2001.

Kende, H. and Zeevaart, J. The five Classical plant hormones. The Plant Cell 9, 1197-1210, 1997.

Kim, H.J., Ryu, H., Hong, S.H., Woo, H.R., Lim, P.O., Lee, I.C., Sheen, J., Nam, H.G. and Hwang, I. Cytokinin-mediated control of leaf longevity by AHK3 through phosphorylation of ARR2 in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America 103, 814-819, 2006.

Kui, L., Chen, H., Zhang, W., He, S., Xiong, Z., Zhang, Y. and Chen, J. Building a genetic manipulation tool box for orchid biology: identification of constitutive promoters and application of CRISPR/Cas9 in the orchid, Dendrobium officinale. Frontiers in Plant Science 7, 20-36, 2017.

Kurakawa, T., Ueda, N., Maekawa, M., Kobayashi, K., Kojima, M., Nagato, Y. and Kyozuka, J. Direct control of shoot meristem activity by a cytokinin-activating enzyme. Nature 445, 652-655, 2007.

Kruszka, K., Barneche, F., Guyot, R., Ailhas, J., Meneau, I., Schiffer, S. and Echeverría, M. Plant dicistronic tRNA-snoRNA genes: a new mode of expression of the small nucleolar RNAs processed by RNase Z. The European Molecular Biology Organization Journal 22, 621-632, 2003.

Kurepa, J., Li, Y. and Smalle, J. A. Cytokinin signaling stabilizes the response activator ARR1. The Plant Journal 78, 157-168, 2014.

Laplaze, L., Benkova, E., Casimiro, I., Maes, L., Vanneste, S., Swarup, R. and Herrera-Rodriguez, M. B. Cytokinins act directly on lateral root founder cells to inhibit root initiation. The Plant Cell 19, 3889-3900, 2007.

Lee, K., Zhu, H., Yang, B., and Wang, K. An Agrobacterium-Mediated CRISPR/Cas9 Platform for Genome Editing in Maize. In Plant Genome Editing with CRISPR Systems. Humana Press Journals 20, 121-143, 2019.

Li, M., Li, X., Zhou, Z., Wu, P., Fang, M., Pan, X., and Li, H. Reassessment of the four yield-related genes Gn1a, DEP1, GS3, and IPA1 in rice using a CRISPR/Cas9 system. Frontiers in Plant Science 7, 350-377, 2016.

Li, J. F., Zhang, D., and Sheen, J. Cas9-based genome editing in Arabidopsis and tobacco. Methods in Enzymology 546, 459-472, 2014.

Liau, C. H., You, S. J., Prasad, V., Hsiao, H. H., Lu, J. C., Yang, N. S. and Chan, M. T. Agrobacterium tumefaciens-mediated transformation of an Oncidium orchid. Plant Cell Reports 21, 993-998, 2003.

Lim, Y., Bak, S. Y., Sung, K., Jeong, E., Lee, S. H., Kim, J. S. and Kim, S. K. Structural roles of guide RNAs in the nuclease activity of Cas9 endonuclease. Nature Communications 7, 133-150, 2016.

Liu, X., Wu, S., Xu, J., Sui, C. and Wei, J. Application of CRISPR/Cas9 in plant biology. Acta Pharmaceutica Sinica B 10, 10-16, 2017.

Lu, H. C., Hsieh, M. H., Chen, C. E., Chen, H. H., Wang, H. I. and Yeh, H. H. A high-throughput virus-induced gene-silencing vector for screening transcription factors in virus-induced plant defense response in orchid. Molecular Plant Microbe Interactions 25, 738-746, 2012.

Mähönen, A. P., Higuchi, M., Törmäkangas, K., Miyawaki, K., Pischke, M. S., Sussman, M. R. and Kakimoto, T. Cytokinins regulate a bidirectional phosphorelay network in Arabidopsis. Current Biology 16, 1116-1122, 2006.

Miao, J., Guo, D., Zhang, J., Huang, Q., Qin, G., Zhang, X., Wan, J., Gu, H. and Qu, L. J. Targeted mutagenesis in rice using CRISPR-Cas system. Cell Research 23, 1233-1236, 2013.

Mok, C., Mok, W. and Armstrong, J. Differential cytokinin structure-activity relationships in Phaseolus. Plant Physiology 61, 72-75, 1978.

Mok, W. and Mok, C. Cytokinin metabolism and action. Annual Review of Plant Biology 52, 89-118, 2001.

Nambiar, N., Siang, T. C. and Mahmood, M. Effect of 6-Benzylaminopurine on flowering of a Dendrobium orchid. Australian Journal of Crop Science 6, 218-225, 2012.

Nekrasov, V., Staskawicz, B., Weigel, D., Jones, J. D. and Kamoun, S. Targeted mutagenesis in the model plant Nicotiana benthamiana using Cas9 RNA-guided endonuclease. Nature Biotechnology 31, 691-693, 2013.

Nissim, L., Perli, SD., Fridkin, A., Perez-Pinera, P. and Lu, TK. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Molecular Cell 54, 698-710, 2014.

Qi, W., Zhu, T., Tian, Z., Li, C., Zhang, W. and Song, R. High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA-processing system-based strategy in maize. BioMed Central Biotechnology 16, 58-75, 2016.

Qu, F., W, Zhai., H, Chen., L, Zhu. and T, J. Morris. Cloning, characterization and transient expression of the gene encoding a rice U3 small nuclear RNA. Gene 172, 217-220, 1996.

Ran, A., Hsu, D., Wright, J., Agarwala, V., Scott, A. and Zhang, F. Genome engineering using the CRISPR-Cas9 system. Nature Protocols 8, 22-31, 2013.

Ryan, D. E., Taussig, D., Steinfeld, I., Phadnis, S. M., Lunstad, B. D., Singh, M., and Roy, S. Improving CRISPR-Cas specificity with chemical modifications in single-guide RNAs. Nucleic Acids Research 46, 792-803, 2017.

Ryan, C., Heler, R., MacDougall, M. S., Yeo, N. C., Chavez, A., Regan, M., and Merrill, B. J. Enhanced bacterial immunity and mammalian genome editing via RNA-polymerase-mediated dislodging of Cas9 from double-strand DNA breaks. Molecular Cell 71, 42-55, 2018.

Sakakibara, H. Cytokinins: activity, biosynthesis, and translocation. Annual Review of Plant Biology 57, 431-449, 2006.

Schmulling, T., Werner, T., Riefler, M., Krupkova, E. and Manns, I. B. Structure and function of cytokinin oxidase/dehydrogenase genes of maize, rice, Arabidopsis and other species. Journal of Plant Research, 116, 241-252, 2003.

Su, L., Chao, T., Yen, H., Chen, Y., Chen, C., Chang, A. and Shih, MC. Orchidstra: an integrated orchid functional genomics database. Plant and Cell Physiology 54, 1011-1021, 2013.

Schäfer, M., Brütting, C., Meza-Canales, I. D., Großkinsky, D. K., Vankova, R., Baldwin, I. T. and Meldau, S. The role of cis-zeatin-type cytokinins in plant growth regulation and mediating responses to environmental interactions. Journal of Experimental Botany 66, 4873-4884, 2015.

Shan, Q., Wang, YP. and Li, J. Targeted genome modification of crop plants using a CRISPR/Cas system. Nature Biotechnology 31, 686-688, 2013.

Skoog, F., Hamzi, Q., Szweykowska, M., Leonard, J., Carraway, L., Fujii, T. and Loeppky, N. Cytokinins: structure/activity relationships. Phytochemistry 6, 1169-1192, 1967.

Takei, K., Ueda, N., Aoki, K., Kuromori, T., Hirayama, T., Shinozaki, K.and Sakakibara, H. AtIPT3 is a key determinant of nitrate-dependent cytokinin biosynthesis in Arabidopsis. Plant and Cell Physiology 45, 1053-1062, 2004.

Tsai, Y. C., Weir, N. R., Hill, K., Zhang, W., Kim, H. J., Shiu, S. H. and Kieber, J. J. Characterization of genes involved in cytokinin signaling and metabolism from rice. Plant Physiology 158, 1666-1684, 2012.

Tsay, S., Ho, M., Gupta, K., Wang, S., Chen, T., and Chen, F. Development of pollen mediated activation tagging system for Phalaenopsis and Doritaenopsis. Electronic Journal of Biotechnology 15, 9-19, 2012.

Veach, Y. K., Martin, R. C., Mok, D. W., Malbeck, J., Vankova, R. and Mok, M. C. O-glucosylation of cis-zeatin in maize, Characterization of genes, enzymes, and endogenous cytokinins. Plant Physiology 131, 1374-1380, 2003.

Veerabagu, M., Elgass, K., Kirchler, T., Huppenberger, P., Harter, K., Chaban, C. and Mira‐Rodado, V. The Arabidopsis B‐type response regulator 18 homomerizes and positively regulates cytokinin responses. The Plant Journal 72, 721-731, 2012.

von Schwartzenberg, K., Nunez, F., Blaschke, H., Dobrev, I., Novák, O., Motyka, V. and Strnad, M. Cytokinins in the bryophyte Physcomitrella patens: analyses of activity, distribution, and cytokinin oxidase/dehydrogenase overexpression reveal the role of extracellular cytokinins. Plant Physiology 145, 786-800, 2007.

Wang, Y., Li, L., Ye, T., Zhao, S., Liu, Z., Feng, Y. Q. and Wu, Y. Cytokinin antagonizes ABA suppression to seed germination of Arabidopsis by downregulating ABI5 expression. The Plant Journal 68, 249-261, 2011.

Werner, T., Motyka, V., Laucou, V., Smets, R., Van Onckelen, H., and Schmulling, T. Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. The Plant Cell 15, 2532-2550, 2003.

Xie, K., Minkenberg, B. and Yang, Y. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proceedings of the National Academy of Sciences of the United States of America 112, 3570-3575, 2015.

Xu, K., Huang, X., Wu, M., Wang, Y., Chang, Y., Liu, K. and Li, T. A rapid, highly efficient and economical method of Agrobacterium-mediated in planta transient transformation in living onion epidermis. Public Library of Science One 9, 55-83, 2014.

Yanai, O., Shani, E., Dolezal, K., Tarkowski, P., Sablowski, R., Sandberg, G. and Ori, N. Arabidopsis KNOXI proteins activate cytokinin biosynthesis. Current Biology 15, 1566-1571, 2005.

Yang, H., Yu, H., and Goh, J. Functional characterisation of a cytokinin oxidase gene DSCKX1 in Dendrobium orchid. Plant Molecular Biology 51, 237-248, 2003a.

Yang, S., Yu, H., Xu, Y., and Goh, J. Investigation of cytokinin‐deficient phenotypes in Arabidopsis by ectopic expression of orchid DSCKX1. Federation of European Biochemical Societies Letters 555, 291-296, 2003b.

Zhang, G. Q., Liu, K. W., Li, Z., Lohaus, R., Hsiao, Y. Y., Niu, S. C., and Yoshida, K. The Apostasia genome and the evolution of orchids. Nature 549, 379-382, 2017.

Zhu, X., Xu, Y., Yu, S., Lu, L., Ding, M., Cheng, J., and Meng, S. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system. Scientific Reports 4, 24-40, 2014.

Zubko, E., Adams, J., Macháèková, I., Malbeck, J., Scollan, C. and Meyer, P. Activation tagging identifies a gene from Petunia hybrida responsible for the production of active cytokinins in plants. The Plant Journal 29, 797-808, 2002.