簡易檢索 / 詳目顯示

研究生: 林斯賢
Lin, Szu-Hsien
論文名稱: 長葉木薑子蟲癭轉錄體之研究
Transcriptomic profiling of insect gall in Litsea acuminata
指導教授: 孫智雯
Sun, Chih-Wen
楊棋明
Yang, Chi-Ming
學位類別: 碩士
Master
系所名稱: 生命科學系
Department of Life Science
論文出版年: 2016
畢業學年度: 104
語文別: 中文
論文頁數: 82
中文關鍵詞: 蟲癭長葉木薑子次世代定序轉錄體光合作用植物激素細胞壁植物防禦系統
英文關鍵詞: insect gall, Litsea acuminata, next generation sequencing, transcriptome, photosynthesis, plant hormone, cell wall, plant defense system
DOI URL: https://doi.org/10.6345/NTNU202203615
論文種類: 學術論文
相關次數: 點閱:96下載:3
分享至:
查詢本校圖書館目錄 查詢臺灣博碩士論文知識加值系統 勘誤回報
  • 蟲癭(insect gall)是植物經過造癭昆蟲刺激後產生不正常增生組織。至今蟲癭的形成機制仍不明瞭。先前研究著重於分類學、動物學、生態學等,隨著次世代定序發展,使得探討基因表現等轉錄體研究也越快捷。本論文研究長葉木薑子(Litsea acuminata)蟲癭之轉錄體,以了解蟲癭的形成發育機制。研究蟲癭與其宿主葉基因表現差異,一共發現1,452個差異表現基因,其中包含細胞壁、植物激素代謝、植物逆境、光合作用、二次代謝、四吡咯生合成等類群。資料顯示蟲癭組織光合作用相關基因表現量降低,推測蟲癭組織無法提供足夠養分構築蟲癭結構或是提供造癭昆蟲,營養則可能由宿主葉而來,進而顯示植物組織從葉片轉變成蟲癭組織是一種營養流的改變,即從供源(source)轉變成積儲(sink)。本研究還發細胞壁相關基因表現上升,以及植物激素如油菜素類固醇、生長素與吉貝素相關基因表現上升,並推測上述幾種植物激素在蟲癭形成中可能扮演重要角色。造癭昆蟲不但誘導蟲癭構形還啟動植物防禦系統,如致病過程相關蛋白質(pathogenesis-related proteins, PR protein)基因表現上升,此可能誘發植物系統性誘導抗病機制(systemic acquired resistance)以阻礙病原侵入。

    Insect galls are the abnormal growth of plant tissue induced by herbivore, bite or egg laying from insects. To date, the mechanisms of gall formation are still undetermined. Previous researches used taxonomical, zoological and ecological approaches to study insect galls. In this study the insect galls were investigated with the view of transcriptome. We collected cup-shaped galls on host leaves of Litsea acuminata, and characterized the differential gene expression between galls and its host leaves by using next generation sequencing and transcriptomic approaches. A total of 1,452 differentially expressed genes (DEGs) were found between insect gall and it host leaf. These DEGs are classified into functional category of cell wall, hormone metabolism, abiotic or biotic stress, photosynthesis, secondary metabolism, tetrapyrrole synthesis and so on. The data showed that the gall shift from a source (host leaves) to a sink (gall tissues) with decreased gene expression of photosynthetic-related genes. I also analyzed the cell wall synthesis and plant hormone like brassinosteroids, auxin and gibberellin. These hormones may play important role in gall development. Insects not only induce the structure of galls but also stimulate plant defense system. For example that increased expression of genes related to pathogenesis-related proteins (PR protein). They are induced as part of systemic acquired resistance (SAR) to resist disease.

    目錄 中文摘要 i Abstract ii 目錄 iv 圖目錄 vi 表目錄 vii 緒論 1 研究材料與方法 5 一、研究材料 5 二、RNA萃取、純化與轉cDNA 5 三、轉錄組定序 5 四、數據分析 6 五、即時聚合酶鏈式反應(RT-qPCR) 6 六、顯微結構分析 7 結果 8 一、重疊序列組合 8 二、序列功能及細胞位置推測 8 三、序列比對 8 四、移除重複序列 9 五、TAIR資料庫比對 9 六、蟲癭及宿主葉差異表現分析 9 (一)光合作用相關基因 10 (二)粒線體電子電鏈相關基因 11 (三)細胞壁生合成相關基因 11 (四)植物激素相關基因 12 (五)植物逆境相關基因 12 (六)水分傳輸相關基因 13 (七)葉片結構生長發育相關基因 13 七、即時聚合酶鏈式反應結果分析 13 八、顯微結構分析 14 討論 15 一、光合作用 15 二、蟲癭的生長發育 16 三、植物防禦系統 18 四、營養流 18 五、定序品質 19 六、即時聚合酶鏈式反應驗證 19 結論 20 未來展望 21 參考文獻 22 檢索表 82   圖目錄 圖1、長葉木薑子與其蟲癭 35 圖2、Contigs長度與對應到蛋白質數量分布 36 圖3、蟲癭及宿主葉間轉錄體水平變化之關係 37 圖4、Gene Ontology 38 圖5、比對14種植物蛋白質資料庫之各物種比對到基因數比例 39 圖6、MapMan基因表現 40 圖7、PageMan分析 41 圖8、光合作用基因表現 42 圖9、粒線體電子傳遞鏈基因表現 43 圖10、細胞壁相關基因表現 44 圖11、四吡咯(tetrapyrrole)生合成(葉綠素a及b)基因表現 45 圖12、油菜素類固醇生合成基因表現 46 圖13、即時聚合酶鏈式反應與轉錄體資料迴歸關係 47 圖14、光合作用相關基因之即時聚合酶鏈式反應結果 48 圖15、四吡咯相關基因之即時聚合酶鏈式反應結果 49 圖16、色素蛋白複合體用相關基因之即時聚合酶鏈式反應結果 50 圖17、粒線體電子傳遞鏈相關基因之即時聚合酶鏈式反應結果 51 圖18、細胞壁相關基因之即時聚合酶鏈式反應結果 52 圖19、油菜素類固醇相關基因之即時聚合酶鏈式反應結果 53 圖20、長葉木薑子葉片及蟲癭之光學顯微鏡分析 54 圖21、長葉木薑子葉片及蟲癭之穿透式電子顯微鏡分析 55 圖22、長葉木薑子葉片及蟲癭之類囊膜間距差異 56  表目錄 表1、重疊序列組合及Contigs組裝品質 57 表2、各植物功能類群比對基因數與差異表現基因數及所佔比例 58 表3、光合作用相關基因數與DEGs數及所佔比例 60 表4、光合作用相關DEGs及其所屬TAIR ID與倍數差異 61 表5、粒線體電子傳遞鏈相關基因數與DEGs數及所佔比例 62 表6、粒線體電子傳遞鏈相關DEGs及其所屬TAIR ID與倍數差異 63 表7、細胞壁相關基因數與DEGs數及所佔比例 64 表8、細胞壁相關DEGs及其所屬TAIR ID與倍數差異 65 表9、植物激素相關基因數與DEGs數及所佔比例 68 表10、植物激素相關DEGs及其所屬TAIR ID與倍數差異 69 表11、植物逆境相關基因數與DEGs數及所佔比例 71 表12、植物逆境相關DEGs及其所屬TAIR ID與倍數差異 72 表13、水分傳輸相關基因數與DEGs數及所佔比例 75 表14、水分傳輸相關DEGs及其所屬TAIR ID與倍數差異 76 表15、葉片結構生長發育相關基因數與DEGs數及所佔比例 77 表16、葉片結構生長發育相關DEGs及其所屬TAIR ID與倍數差異 78 表17、即時聚合酶鏈式反應之引子設計 79 表18、次世代定序數據與即時聚合酶鏈式反應資料比對 81

    林聖豐。2011。長葉木薑子葉片多型性蟲癭之造癭癭蚋生物系統分類。國立中興大學昆蟲學系碩士論文。

    梁立明、楊淑燕、楊正澤、陳明義。1999。關刀溪森林生態系變葉新木薑子與長葉木薑子蟲癭之發育。林業研究季刊。Vol. 21。75-89頁。

    陳昱潔。2016。長葉木薑子杯狀蟲癭捕光複合體分析。國立臺灣師範大學生命科學系碩士論文。

    黃盟元。2011。Daphnephila屬癭蚋蟲癭之生態生理特性研究。國立臺灣師範大學生命科學研究所博士論文。

    廖玲秀。2003。由大葉楠造癭木蝨Triozashuiliensis (Yang)探討造癭昆蟲的營養適應。國立中興大學昆蟲學系碩士論文。

    Agarrwal, R., A.P. Padmakumari, J.S. Bentur, and S. Nair. 2016. Metabolic and transcriptomic changes induced in host during hypersensitive response mediated resistance in rice against the Asian rice gall midge. Rice (New York, N.Y.). 9:5.

    Andersson, M.N., E. Videvall, K.K.O. Walden, M.O. Harris, H.M. Robertson, and C. Lofstedt. 2014. Sex- and tissue-specific profiles of chemosensory gene expression in a herbivorous gall-inducing fly (Diptera: Cecidomyiidae). BMC Genomics. 15:19.

    Bailey, S., D.M. Percy, C.A. Hefer, and Q.C. Cronk. 2015. The transcriptional landscape of insect galls: psyllid (Hemiptera) gall formation in Hawaiian Metrosideros polymorpha (Myrtaceae). BMC Genomics. 16:943.

    Baumberger, N., C. Ringli, and B. Keller. 2001. The chimeric leucine-rich repeat/extensin cell wall protein LRX1 is required for root hair morphogenesis in Arabidopsis thaliana. Genes & development. 15:1128-1139.

    Berardini, T.Z., S. Mundodi, L. Reiser, E. Huala, M. Garcia-Hernandez, P. Zhang, L.A. Mueller, J. Yoon, A. Doyle, G. Lander, N. Moseyko, D. Yoo, I. Xu, B. Zoeckler, M. Montoya, N. Miller, D. Weems, and S.Y. Rhee. 2004. Functional Annotation of the Arabidopsis Genome Using Controlled Vocabularies. Plant Physiol. 135:745-755.

    Brenner, S., M. Johnson, J. Bridgham, G. Golda, D.H. Lloyd, D. Johnson, S. Luo, S. McCurdy, M. Foy, M. Ewan, R. Roth, D. George, S. Eletr, G. Albrecht, E. Vermaas, S.R. Williams, K. Moon, T. Burcham, M. Pallas, R.B. DuBridge, J. Kirchner, K. Fearon, J.-i. Mao, and K. Corcoran. 2000. Gene expression analysis by massively parallel signature sequencing (MPSS) on microbead arrays. Nat Biotech. 18:630-634.

    Chen, T.W., R.C. Gan, T.H. Wu, P.J. Huang, C.Y. Lee, Y.Y. Chen, C.C. Chen, and P. Tang. 2012. FastAnnotator- an efficient transcript annotation web tool. BMC Genomics. 13 Suppl 7:S9.

    Depuydt, S., and Christian S. Hardtke. 2011. Hormone Signalling Crosstalk in Plant Growth Regulation. Current Biology. 21:R365-R373.

    Dorchin, N., M.D. Cramer, and J.H. Hoffmann. 2006. Photosynthesis and sink activity of wasp-induced galls in Acacia pycnantha. Ecology. 87:1781-1791.

    Draeger, C., T. Ndinyanka Fabrice, E. Gineau, G. Mouille, B.M. Kuhn, I. Moller, M.-T. Abdou, B. Frey, M. Pauly, A. Bacic, and C. Ringli. 2015. Arabidopsis leucine-rich repeat extensin (LRX) proteins modify cell wall composition and influence plant growth. BMC Plant Biol. 15:1-11.

    Dreger-Jauffret, F., and J.D. Shorthouse. 1992. Diversity of gall-inducing insects and their galls. Oxford University Press, Oxford, New York etc. 8-33 pp.

    Ellis, M., J. Egelund, C.J. Schultz, and A. Bacic. 2010. Arabinogalactan-proteins: key regulators at the cell surface? Plant Physiol. 153:403-419.

    Ewing, B., and P. Green. 1998. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 8:186-194.

    Fujioka, S., and T. Yokota. 2003. Biosynthesis and metabolism of brassinosteroids. Annual review of plant biology. 54:137-164.

    Gambino, G., I. Perrone, and I. Gribaudo. 2008. A Rapid and Effective Method for RNA Extraction from Different Tissues of Grapevine and Other Woody Plants. Phytochem. Anal. 19:520-525.

    Gao, X., J.Y. Zhu, S. Ma, Z. Zhang, C. Xiao, Q. Li, Z.Y. Li, and G.X. Wu. 2014. Transcriptome profiling of the crofton weed gall fly Procecidochares utilis. Genet. Mol. Res. 13:2857-2864.

    Giron, D., E. Huguet, G.N. Stone, and M. Body. 2016. Insect-induced effects on plants and possible effectors used by galling and leaf-mining insects to manipulate their host-plant. Journal of Insect Physiology. 84:70-89.

    Griesser, M., N.C. Lawo, S. Crespo-Martinez, K. Schoedl-Hummel, K. Wieczorek, M. Gorecka, F. Liebner, T. Zweckmair, N.S. Pavese, D. Kreil, and A. Forneck. 2015. Phylloxera (Daktulosphaira vitifoliae Fitch) alters the carbohydrate metabolism in root galls to allowing the compatible interaction with grapevine (Vitis ssp.) roots. Plant Sci. 234:38-49.

    He, L., and G.J. Hannon. 2004. MicroRNAs: small RNAs with a big role in gene regulation. Nature reviews. Genetics. 5:522-531.

    Heath, M.C. 2000. Hypersensitive response-related death. Plant Mol.Biol. 44:321-334.

    Huang, M.Y., H.M. Chou, Y.T. Chang, and C.M. Yang. 2014. The number of cecidomyiid insect galls affects the photosynthesis of Machilus thunbergii host leaves. J. Asia-Pac. Entomol. 17:151-154.

    Huang, M.Y., W.D. Huang, H.M. Chou, C.C. Chen, P.J. Chen, Y.T. Chang, and C.M. Yang. 2015. Structural, biochemical, and physiological characterization of photosynthesis in leaf-derived cup-shaped galls on Litsea acuminata. BMC Plant Biol. 15:12.

    Huang, M.Y., M.M. Yang, W.N. Jane, Y.T. Chang, and C.M. Yang. 2009. Insect-induced cecidomyiid galls deficient in light-harvesting protein complex II showing normal grana stacking. J. Asia-Pac. Entomol. 12:165-168.

    Khajuria, C., C.E. Williams, M. El Bouhssini, R.J. Whitworth, S. Richards, J.J. Stuart, and M.-S. Chen. 2013. Deep sequencing and genome-wide analysis reveals the expansion of MicroRNA genes in the gall midge Mayetiola destructor. BMC Genomics. 14:1-11.

    Kirchhoff, H., C. Hall, M. Wood, M. Herbstova, O. Tsabari, R. Nevo, D. Charuvi, E. Shimoni, and Z. Reich. 2011. Dynamic control of protein diffusion within the granal thylakoid lumen. Proc Natl Acad Sci U S A. 108:20248-20253.

    Koyama, Y., I. Yao, and S.I. Akimoto. 2004. Aphid galls accumulate high concentrations of amino acids: a support for the nutrition hypothesis for gall formation. Entomol. Exp. Appl. 113:35-44.

    Kyndt, T., S. Denil, A. Haegeman, G. Trooskens, L. Bauters, W. Van Criekinge, T. De Meyer, and G. Gheysen. 2012. Transcriptional reprogramming by root knot and migratory nematode infection in rice. The New phytologist. 196:887-900.

    Lamesch, P., T.Z. Berardini, D. Li, D. Swarbreck, C. Wilks, R. Sasidharan, R. Muller, K. Dreher, D.L. Alexander, M. Garcia-Hernandez, A.S. Karthikeyan, C.H. Lee, W.D. Nelson, L. Ploetz, S. Singh, A. Wensel, and E. Huala. 2012. The Arabidopsis Information Resource (TAIR): improved gene annotation and new tools. Nucleic Acids Res. 40:D1202-1210.

    Machida, C., A. Nakagawa, S. Kojima, H. Takahashi, and Y. Machida. 2015. The complex of ASYMMETRIC LEAVES (AS) proteins plays a central role in antagonistic interactions of genes for leaf polarity specification in Arabidopsis. Wiley interdisciplinary reviews. Developmental biology. 4:655-671.

    Maere, S., K. Heymans, and M. Kuiper. 2005. BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics. 21:3448-3449.

    Mani, M.S. 1964. Ecology of plant galls. W. Junk.

    Meyer, J. 1987. Plant galls and gall inducers. Gebruder Borntraeger Verlagsbuchhandlung, Science Publishers.

    Mortazavi, A., B.A. Williams, K. McCue, L. Schaeffer, and B. Wold. 2008. Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nature methods. 5:621-628.

    Nabity, P.D., M.J. Haus, M.R. Berenbaum, and E.H. DeLucia. 2013. Leaf-galling phylloxera on grapes reprograms host metabolism and morphology. Proc. Natl. Acad. Sci. U. S. A. 110:16663-16668.

    Oates, C.N., C. Kulheim, A.A. Myburg, B. Slippers, and S. Naidoo. 2015. The Transcriptome and Terpene Profile of Eucalyptus grandis Reveals Mechanisms of Defense Against the Insect Pest, Leptocybe invasa. Plant Cell Physiol. 56:1418-1428.

    Ono, H., K. Ishii, T. Kozaki, I. Ogiwara, M. Kanekatsu, and T. Yamada. 2015. Removal of redundant contigs from de novo RNA-Seq assemblies via homology search improves accurate detection of differentially expressed genes. BMC Genomics. 16:13.

    Pan, L.Y., W.N. Chen, S.T. Chiu, A. Raman, T.C. Chiang, and M.M. Yang. 2015. Is a Gall an Extended Phenotype of the Inducing Insect? A Comparative Study of Selected Morphological and Physiological Traits of Leaf and Stem Galls on Machilus thunbergii (Lauraceae) Induced by Five Species of Daphnephila (Diptera: Cecidomyiidae) in Northeastern Taiwan. Zool. Sci. 32:314-321.

    Price, P.W., G.W. Fernandes, and G.L. Waring. 1987. Adaptive Nature of Insect Galls. Environ. Entomol. 16:15-24.

    Price, P.W., G.L. Waring, and G.W. Fernandes. 1986. Hypotheses on the adaptive nature of galls. P Entomol Soc Wash. 88:361-363.

    Raman, A., S. Madhavan, S.K. Florentine, and K. Dhileepan. 2006. Metabolite mobilization in the stem galls of Parthenium hysterophorus induced by Epiblema strenuana inferred from the signatures of isotopic carbon and nitrogen and concentrations of total non-structural carbohydrates. Entomol. Exp. Appl. 119:101-107.

    Ronaghi, M., S. Karamohamed, B. Pettersson, M. Uhlén, and P. Nyrén. 1996. Real-Time DNA Sequencing Using Detection of Pyrophosphate Release. Anal Biochem. 242:84-89.

    Sanger, F., and A.R. Coulson. 1975. A rapid method for determining sequences in DNA by primed synthesis with DNA polymerase. J. Mol. Biol. 94:441-448.

    Schonrogge, K., L.J. Harper, and C.P. Lichtenstein. 2000. The protein content of tissues in cynipid galls (Hymenoptera : Cynipidae): Similarities between cynipid galls and seeds. Plant Cell Environ. 23:215-222.

    Schuster, S.C. 2008. Next-generation sequencing transforms today's biology. Nat Meth. 5:16-18.

    Seifert, G.J., and K. Roberts. 2007. The biology of arabinogalactan proteins. Annual review of plant biology. 58:137-161.

    Shi, C.Y., H. Yang, C.L. Wei, O. Yu, Z.Z. Zhang, C.J. Jiang, J. Sun, Y.Y. Li, Q. Chen, T. Xia, and X.C. Wan. 2011. Deep sequencing of the Camellia sinensis transcriptome revealed candidate genes for major metabolic pathways of tea-specific compounds. BMC Genomics. 12:1-19.

    Spurr, A.R. 1969. A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research. 26:31-43.

    Sreenivasulu, N., B. Usadel, A. Winter, V. Radchuk, U. Scholz, N. Stein, W. Weschke, M. Strickert, T.J. Close, M. Stitt, A. Graner, and U. Wobus. 2008. Barley grain maturation and germination: Metabolic pathway and regulatory network commonalities and differences highlighted by new MapMan/PageMan profiling tools. Plant Physiol. 146:1738-1758.

    Stintzi, A., T. Heitz, V. Prasad, S. Wiedemann-Merdinoglu, S. Kauffmann, P. Geoffroy, M. Legrand, and B. Fritig. 1993. Plant 'pathogenesis-related' proteins and their role in defense against pathogens. Biochimie. 75:687-706.

    Stone, G.N., and K. Schonrogge. 2003. The adaptive significance of insect gall morphology. Trends Ecol. Evol. 18:512-522.

    Talukder, S.K., P. Azhaguvel, S. Mukherjee, C.A. Young, Y. Tang, N. Krom, and M.C. Saha. 2015. De Novo Assembly and Characterization of Tall Fescue Transcriptome under Water Stress. Plant Genome. 8:13.

    Thimm, O., O. Blasing, Y. Gibon, A. Nagel, S. Meyer, P. Kruger, J. Selbig, L.A. Muller, S.Y. Rhee, and M. Stitt. 2004. MAPMAN: a user-driven tool to display genomics data sets onto diagrams of metabolic pathways and other biological processes. Plant J. 37:914-939.

    Xia, Z.H., H.M. Xu, J.L. Zhai, D.J. Li, H.L. Luo, C.Z. He, and X. Huang. 2011. RNA-Seq analysis and de novo transcriptome assembly of Hevea brasiliensis. Plant Mol.Biol. 77:299-308.

    Yang, C.-M., M.-M. Yang, J.-M. Hsu, and W.-N. Jane. 2003. Herbivorous insect causes deficiency of pigment-protein complexes in an oval-pointed cecidomyiid gall of Machilus thunbergii leaf. Botanical Bulletin of Academia Sinica. 33:135-140.

    Yang, C.M., M.M. Yang, M.Y. Huang, J.M. Hsu, and W.N. Jane. 2007. Time deficiency of photosynthetic pigment-protein complexes CP1, A1, AB1, and AB2 in two cecidomyiid galls derived from Machilus thunbergii leaves. Photosynthetica. 45:589-593.

    Yukawa, J. 1996. Insect and mite galls of Japan in colors = Nihon genshoku chuei zukan. Zenkoku Noson Kyoiku Kokai, Tokyo.

    Zerbino, D.R., and E. Birney. 2008. Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18:821-829.

    Zerbino, D.R., G.K. McEwen, E.H. Margulies, and E. Birney. 2009. Pebble and rock band: heuristic resolution of repeats and scaffolding in the velvet short-read de novo assembler. PLoS One. 4:e8407.

    Zhang, H., T. Dugé de Bernonville, M. Body, G. Glevarec, M. Reichelt, S. Unsicker, M. Bruneau, J.-P. Renou, E. Huguet, G. Dubreuil, and D. Giron. 2015. Leaf-mining by Phyllonorycter blancardella reprograms the host-leaf transcriptome to modulate phytohormones associated with nutrient mobilization and plant defense. Journal of Insect Physiology. 84:114-127.

    Zhu, J.Y., J. Sae-Seaw, and Z.Y. Wang. 2013. Brassinosteroid signalling. Development. 140:1615-1620.

    下載圖示
    QR CODE