本研究主要的目的是希望能找出與香妃蝴蝶蘭體株變異花色、花形相關之基因片段，藉以了解其調控機制。實驗方法分為二部分進行；第一部分為實驗室之前找到的一個與體株變異有關的候選基因：轉位酵素(transposase)。經由北方墨點轉漬分析法 (Northern blot analysis)分析之後，發現轉位酵素基因在RNA表現層次並沒有明顯差異。為確認該轉位酵素基因在蝴蝶蘭基因組位置中是否有差異，乃利用香妃蝴蝶蘭體株變異所建立的GenomeWalker基因庫以找出轉位酵素附近的基因序列。結果得到4個不同的序列，包括水稻逆轉位子的polyprotein、玉米NADPH－dependent reductase a1基因、阿拉伯芥的putative expression protein以及Anaplasma phagocytophila RNA polymerase b subunit，可知至少有4個拷貝數目(4 copies)的轉位子基因存在香妃蝴蝶蘭的基因組。由南方墨點法分析這兩種植株的轉位子數目以及是否位於染色體的不同位置，結果亦顯示有4個拷貝數目的轉位酵素基因存在於蝴蝶蘭的基因組中。其中一個條帶在正常株與變異株有強弱差別變化，經DNA選殖並建構基因組DNA微小基因庫(mini-library)，找出轉位酵素旁的基因序列，序列比對顯示為葉綠體RNA polymerase b subunit-2。以上結果顯示，香妃蝴蝶蘭之轉位子可能因為插入葉綠體RNA polymerase b subunit-2基因中而影響到花形花色，亦可能因為插入逆轉位子或NADPH－dependent reductase等基因中而產生影響，但是都還需進一步探討。
第二部分為以香妃蝴蝶蘭和其體株變異為研究材料，設計螢光標定的引子共128組，利用cDNA-AFLP方法進行大量差異性表現基因的篩選，以期找出和體株變異相關之基因。在128種引子組合中，有32組引子不適合增殖蝴蝶蘭的基因序列，其他96組總共增殖出2956個transcript-derived fragments (TDFs)，共得到25個有差異性表現的TDFs，最後回收16個TDFs，經DNA增殖、序列分析及比對共得到27種不同之序列。進一步經由半定量反轉錄聚合脢連鎖反應(Semi-quantitative RT-PCR)確認這27個序列在正常株及變異株之RNA表現上是否有差異。實驗結果顯示有4個TDFs有較明顯差異表現，在正常株中大量表現，而在變異株中表現減少，其中3個序列的註解分別為EMF2、putative casein kinase以及大麥種子幼芽的EST，另一個序列則無法比對到註解。其中EMF2是花部器官發育的抑制子，可推測EMF2與casein kinase可能和花的變異有關。

This study was to identify the somaclonal variant related genes of Phalaenopsis Hsiang Fei cv. H. F. We proceeded this study into two parts; the first: a candidate, mutator-like transposase fragment was identified. However, no differential expression of the transposase gene was detected using Northern blot analysis between P. Hsiang Fei cv. H. F. and its somaclonal variants. Thus, the flanking sequences adjacent to the mutator-like transposon were analyzed by constructing GenomeWalker libraries. Results showed that there were at least 4 different copies of mutator-like transposon in the genome of P. Hsiang Fei cv. H. F. somaclonal variants. The four flanking sequences included retroelement polyprotein in rice, NADPH-dependent reductase a1 gene in maize, a putative expression protein in Arabidopsis thaliana and Anaplasma phagocytophila RNA polymerase b subunit. Furthermore, Southern blot analysis was carried out to determine the copy number and the integration site of the mutator-like transposon in the genomes of P. Hsiang Fei cv. H. F. and its somaclonal variants. Results showed that 4 copies of mutator-like transposaon were present in both genomes of the P. Hsiang Fei cv. H. F. and its variant. The intensity of the fourth band of P. Hsiang Fei cv. H. F. somaclonal variants is stronger than that in the normal plant. This 3-kb fragment (the fourth band) was then cloned by PCR-screening mini-library. Sequence analysis of the cloned 3-kb fragment revealed that it was a chloroplast RNA polymerase b subunit-2 gene. It is possible that the mutator-like transposon may have inserted into chloroplast RNA polymerase b subunit-2, a polyprotein of a retroelement, or NADPH - dependent reductase a1 gene and caused change in flower color and morphology in somaclonal variant. However, more studies are needed to confirm the effect of disruption of these genes.
Secondly, the cDNA-amplified restriction fragment length polymorphism (cDNA-AFLP) using 128 fluorescent primer sets were carried out and the differentially expressed fragments were confirmed with semi-quantitative RT-PCR for identification of the genes involved in somaclonal variants. Among the 128 primer sets, 32 primer sets were not suitable to amplify genes of Phalaenopsis Hsiang Fei cv. H. F. A total of 2956 transcript-derived fragments (TDFs) were amplified, and 25 TDFs were identified differentially expressed between wild type and its variants. Among them, 16 differentially expressed TDFs were recovered, and sequence analysis revealed that 27 different sequences were resided in the 16 TDFs. The differential expression of these sequences was further confirmed using semi-quantitative RT-PCR. Results showed that 4 TDFs were differentially expressed with a higher level in the wild type, including EMF2, a putative casein kinase, and an EST cDNA clone of barley seeding shoot.

1.廖敏卿。1990。蝴蝶蘭栽培。

2.劉吉昌。1998。姬蝴蝶蘭花色相關基因片段知之篩選。

3.陳文輝。2002。科學月刊：351期；pp. 32-38。

4.Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D. J. 1997. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25: 3389-402.

5.Arnholt-Schmitt, B., Herterich, S., Neumann, K. H. 1995. Physiological aspects of genome variability in tissue culture. I. Growth phase-dependent differential DNA methylation of the carrot genome (Daucus carota L.) during primary culture. Theor. Appl. Genet. 91: 809-815.

6.Austin, S., Baer, M. A., Helgeson, J. P. 1985. Transfer of resistance from potato leaf roll virus from Solanum brevidens into Solanum tuberosum by somatic fusion. Plant Sci. 39: 75-82.

7.Bachem, C. W., van der Hoeven, R. S., de Bruijn, S. M., Vreugdenhil, D., Zabeau, M., Visser, R. G. 1996. Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J. 9: 745-53.

8.Bajaj, Y. P. S. 1990. Somaclonal variation in crop improvement. Cereal Res. Commun. 1: 43-48.

9.Bajaj, Y. P. S. 1986a. In vitro regeneration of diverse plants and the cryopreservation og germplasm in wheat (Triticum aestivum L.) Cereal Res. Commun. 14: 305-311.

10.Brown, P. T. H., Göbel, E., Lörz, H. 1991. RFLP analysis of Zea mays callus cultures and their regenerated plants. Mol. Gen. Genet. 223: 324-328.

11.Charng, Y. C., Pfitzner, A. J. P., Pfitzner, U. M., Charng-Chang, K. F., Tu, J. & Kuo, T. T. 2000. Construction of an inducible transposon, INAc, to develop a gene tagging system in higher plants. Molecular Breeding. 6: 353-367.

12.Chen, L., Cheng, J. C., Castle, L. and Sung Z. R. 1997. EMF Genes Regulate Arabidopsis lnflorescence Development. Plant Cell. 9: 201 1-2024.

13.Courtial, B., Feuerbach, F., Eberhard, S., Rohmer, L., Chiapello, H., Camilleri, C., and Lucas, H. 2001. Tnt1 transposition events are induced by in vitro transformation of Arabidopsis thaliana, and transposed copies integrate into genes, Mol Genet Genomics 265: 32-42.

14.Dennis, E. S., Brettell, R. I. S., Peacock, W. J. 1987. A tissue culture induced Adh1 null mutant of maize results from a single base change. Mol. Gen. Genet. 210: 181-183.

15.Diachenko, L. B., Ledesma, J., Chenchik, A. A. and Siebert, P. D. 1996. Combining the technique of RNA fingerprinting and differential display to obtain differentially expressed mRNA. Biochem. Biophys. Res. Commun. 219: 824–828.

16.Donson, J., Fang, Y., Espiritu, S. G., Xing, W., Salazar, A., Miyamoto, S., Armendarez, V. and Volkmuth, W. 2002. Comprehensive gene expression analysis by transcript profiling. Plant Mol. Biol. 48: 75-97.

17.Duncan, R. R. 1997. Tissue culture-induced variation and crop improvement. Adv. Agron. 58: 201-240.

18.Durrant, W. E., Rowland, O., Piedras, P., Hammond-Kosack, K. E., Jones, J. D. 2000 cDNA-AFLP reveals a striking overlap in race-specific resistance and wound response gene expression profiles. Plant Cell. 12: 963-77.

19.Habu, Y., Fukada-Tanaka, S., Hisatomi, Y., Iida, S. 1997. Amplified restriction fragment length polymorphism-based mRNA fingerprinting using a single restriction enzyme that recognize a 4-bp sequence. Biochem. Biophys. Res. Commun. 219: 824-828.

20.Hang, A., Bregitzer, P. 1993. Chromosome variations in immature embryo-derived calli from six barley cultivars. J. Hered .84: 105-108.

21.Haring, M. A., Rommens, C., Nijdamp, H. J. J. and Hille, J. 1991. The use of transgenic plants to understand transposition mechanisms and to develop transposon taggign strategies. Plant Mol. Biol. 16: 449-461.

22.Hershberger, R. J., Warren, C. A., Walbot, V. 1991. Mutator activity in maize correlates with the presence and expression of the Mu transposable element Mu9. Proc Natl Acad Sci USA 88:10198-10202.

23.Hirochika, H., Sugimoto, K., Otsuki, Y., Tsugawa, H., Kanda, M. 1996. Retrotransposons of rice involved in mutations induced by tissue culture. Proc. Natl. Acad. Sci. USA. 93: 7783-7788.

24.Jaligot, E., Rival, A., Beulé, T., Dussert, S., Verdeil, J. L. 2000. Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Rep. 19: 684-690.

25.Jurecic, R., Nachtman, R. G., Colicos, S. M. and Belmont, J. W. 1998. Identification and cloning of differentially expressed genes by long-distance differential display. Anal. Biochem. 259: 235-244.

26.Klimczak, L. J., Schindler, U., and Cashmore, A. R. 1992. DNA binding activity of the Arabidopsis G-box binding factor GBFl is stimulated by phosphorylation by casein kinase II from broccoli. Plant Cell 4: 87-98.

27.Larkin, P. J., Scowcroft, W. R. 1981. Somaclonal variation-a novel source of variability from cell cultures for plant improvement. Theor. Appl. Genet. 60: 197-214.

28.Leshem, B. 1986. Carnation plantlets from verified plants as a source of somaclonal variation. Hort. Sci. 21: 320-321.

29.Lievens, S., Goormachtig, S. and Holsters, M. 2001. A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward. Nucleic Acids Research. 29: 3459-3468.

30.Ling, D. H., Ma, Z. R., Chen, W. Y., Chen, M. F. 1987. Male sterile mutant from somatic cell culture of rice. Theor. Appl. Genet. 75: 127-131.

31.Mazars, G. R., Moyret, C., Jeanteur, P., and Theillet, C. G. 1991. Durect sequencing by thermal asymmetric PCR. Nucleic Acids Res. 19: 4783.

32.Meisner, H., and Czech, M. P. 1991. Phosphorylation of transcriptional factors and cell-cycle-dependent proteins by casein kinase II. Curr. Opin. Cell Biol. 3: 474-483.

33.Milioni, D., Sado, P. E., Stacey, N. J., Roberts, K., and McCann, M. C. 2002. Early gene expression associated with the commitment and differentiation of a plant tracheary element is revealed by cDNA–amplified fragment length polymorphism analysis. Plant Cell 14: 2813–2824.

35.Money, T., Reader, S., Qu, L. J., Dunford, R. P., Moore, G. AFLP-based mRNA fingerprinting. Nucleic Acids Res. 1996. 24: 2616-2617.

36.O'Neill, S. D., Nadeau, J. A., Zhang, X. S., Bui, A. Q., and Halevy, A. H. 1993. Interorgan regulation of ethylene biosynthetic genes by pollination. Plant Cell 5: 419-432.

37.Peschke, V. M., Phillips, R. L. 1991. Activation of the maize transposable element Suppressor-mutator (Spm) in tissue culture. Theor. Appl. Genet. 81: 91-97.

38.Peschke, V. M., Phillips, R. L., Gengenbach, B. G. 1987. Discovery of transposable element activity among progeny of tissue culture-derived maize plants. Science 238: 804-807.

39.Rubinelli, P., Hu, Y., and Ma, H. 1998. Identification and sequence analysis and expression studies of novel anther-specific genes of Arabidopsis thaliana. Plant Mol. Biol. 37: 607-619.

40.Siebert, P. D., Chenchik, A., Kellogg, D. E., Lukyanov, K. A., Lukyanov, S. A. 1995. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Res. 23: 1087-1088.

41.Starlinger, P. 1993. What do we still need to know about transposable element Ac? Gene 15: 251-5.

42.Sweet, H. R. 1980. The genus Phalaenopsis. The Orchid Digest Inc. U.S.A.

43.Sutter, E., Langhans, E. W. 1981. Abnormalities in chrysanthemum regenerated from long term cultures. Ann. Bot. 48: 559-568.

44.van der Biezen, E. A., Juwana, H., Parker, J. E., Jones, J. D. 2000. cDNA-AFLP display for the isolation of Peronospora parasitica genes expressed during infection in Arabidopsis thaliana. Mol Plant Microbe Interact. 13: 895-898.

45.Vos, P., Hogers, R., Bleeker, M., Reijans, M., van de Lee, T., Hornes, M., Frijters, A., Pot, J., Peleman, J., and Kuiper, M. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23: 4407-4414.