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研究生: 宋雅琇
Ya-Hsiu Sung
論文名稱: 臺灣杜鵑之低變異Non-TIR-NBS-LRR基因亞家族族群遺傳與演化模式
Population genetics and molecular evolution in low genetic variation Non-TIR-NBS-LRR gene subfamily of the Rhododendron formosanum Hemsl.
指導教授: 黃士穎
Hwang, Shih-Ying
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: 2010
畢業學年度: 98
語文別: 中文
論文頁數: 72
中文關鍵詞: 抗病基因臺灣杜鵑軍備競賽正向天擇
英文關鍵詞: resistance gene, Rhododendron formosanum Hemsl., arms race, positive selection, Non-TIR-NBS-LRR
論文種類: 學術論文
相關次數: 點閱:121下載:1
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  • 植物抗病基因適合使用於分子族群遺傳的處理,以偵測並了解分子層次的天擇作用。而植物的抗病基因家族中以NBS-LRR (nucleotide-binding site plus Leucine-rich repeats)為最大成員。目前用來解釋植物與病原體交互作用之共演化的模式,以軍備競賽(arms race)以及壕溝戰(trench warfare)為主軸。這兩種模式分別是受到正向天擇(positive selection)和均衡天擇(balancing selection)影響。本研究選殖了先前研究得知為低度遺傳變異之Non-TIR-NBS-LRR基因亞家族之抗病基因成員。我們調查了臺灣杜鵑的10個野生族群在NBS譯碼區的歧異度,發現NBS序列的大多變異發生在族群內,顯示族群之間沒有明顯分化。此外利用CODEML分析位點模式的比較結果,偵測到NBS序列受到天擇的位點,這些區域顯示可能影響NBS與ATP結合能力,進而提高抵抗疾病的訊息傳遞和辨識病原的能力或效率有關。在本研究中支持了軍備競賽的假說,而非只是族群動態變化所致,因此在族群中抗病基因之變異是暫時的,對偶基因是因為selective sweeps而呈現高頻率。

    Plant disease resistance genes are promising candidates for using molecular population genetic approaches to detect and understand natural selection at the molecular level. The nucleotide-binding site plus Leucine-rich repeats (NBS-LRR) gene family is one of the major plant resistance genes. Two evolutionary models, an ‘arms race’ and a ‘trench warfare’, are often invoked to explain co-evolution of host-pathogen interactions. The selective forces act on these two models positive selection and balancing selection, respectively. We cloned NBS sequences which were considered low genetic variation in previous research. Then individuals from each of ten natural populations of plant Rhododendron formosanum Hemsl. were examined for the patterns of nucleotide diversity at NBS-encoding genes. Most variation of NBS sequences were found within populations indicated low population differentiation. Results from site model comparisons of the CODEML analyses detected positively selected codon sites. Regions with positively selected were might be related to ATP binding ability which promoted resistance signal transduction and enhanced pathogen recognition efficiency. This research supports an ‘arms race’ hypothesis, in which variation for resistance will be transient, and that alleles spread to high frequency because of selective sweeps.

    摘要 1 Abstract 2 壹、 前言 3 貳、 文獻探討 5 一、 基因重複與演化的關聯 5 二、 植物的抗病基因 6 三、 抗病基因的作用機制—基因對基因學說 8 四、 抗病基因的演化模式 9 (一) 軍備競賽演化模式 10 (二) 壕溝戰演化模式 11 參、 材料與方法 13 一、 研究材料 13 二、 實驗方法 13 (一) 基因組DNA萃取 13 (二) Non-TIR-NBS gene subfamily類抗病基因之PCR擴增 14 (三) NBS區域的基因cloning 16 三、 分析方法 18 (一) DNA序列比對與排序 18 (二) 族群遺傳分析 19 (三) 重建基因樹 21 (四) 序列之演化分析 22 肆、 結果 25 一、 臺灣杜鵑低變異之類抗病基因結果整理 25 (一) 偽基因數量 25 (二) Consensus sequence 25 二、 族群遺傳分析 26 (一) 遺傳歧異度 26 (二) 中性檢測 29 三、 低變異之類抗病基因的分群與親緣關係分析 30 四、 低變異NBS抗病基因之選汰壓力分析結果 31 (一) 偵測特殊演化支系的正向天擇作用之結果 31 (二) 偵測特殊位點的正向天擇作用之結果 32 (三) 偵測特殊演化支系之特殊位點的正向天擇作用結果 33 伍、 討論 36 一、 臺灣杜鵑NBS序列的族群遺傳 36 二、 選汰壓力對NBS區域之作用 38 三、 受到正向天擇的位點之區域 39 四、 軍備競賽的演化模式 41 陸、 參考文獻 44 柒、 表附錄 51 捌、 圖附錄 65 表目錄 表一、基因對基因學說於單一基因座的交互作用 51 表二、族群採樣點之資料 52 表三、引子序列 53 表四、PCR反應試劑 53 表六、Colony PCR反應試劑 54 表八、族群序列總表 55 表九、族群單套基因型的數量及種類 56 表十、各族群的遺傳歧異度和中性檢測總表 57 表十一、族群核苷酸和胺基酸歧異度的百分比(p-distance) 58 表十二、族群兩兩的差異(Pairwise difference),看其族群分化指數FST 59 表十三、AMOVA分析結果 60 表十四、各群間的遺傳歧異度和中性檢測總表 61 表十五、各群核苷酸和胺基酸歧異度的百分比(p-distance) 61 表十六、CODEML程式中branch model 和site model的分析和LRT比較不同模式的結果 62 表十七、CODEML的branch-site model A與LRT的結果 64 圖目錄 圖一、本研究中臺灣杜鵑之採樣族群地理位置圖。 65 圖二、NBS保守區與引子設計區域。 65 圖三、核苷酸歧異度在各族群的分布。 66 圖四、在各族群中πa/πs的分布。 66 圖五、(A)利用sliding-window 分析Tajima's D和(B)Fu and Li's D。 67 圖六、臺灣杜鵑低變異類抗病基因的核苷酸序列所建構產生的neighbor-joining tree。 68 圖七、臺灣杜鵑低變異類抗病基因的胺基酸序列所建構產生的neighbor-joining tree。 69 圖八、NJ tree標記上ω > 1的演化支系。 70 圖九、將各演化分支的Ka與Ks作圖。 71 圖十、在site model中偵測到受到正向天擇的位點之區域分布。 72 圖十一、在branch-site model中偵測到受到正向天擇的位點之區域分布。 72

    呂勝由, 曾彥學 (2002) 萬紫千紅--野生杜鵑花。發現綠色臺灣:臺灣植物專輯 (郭城孟主編), 第138-143頁。行政院農業委員會。
    陳佳鈴 (2007) 臺灣原生杜鵑抗病基因家族功能性演化研究, 中國文化大學生物科技研究所碩士論文。
    曾彥學, 呂勝由 (2003) 臺灣野生杜鵑花資源介紹。自然保育季刊, 第18頁。行政院農業委員會。
    Ashfield T, Bocian A, Held D, et al. (2003) Genetic and physical localization of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR genes. Mol Plant Microbe Interact 16, 817-826.
    Bakker EG, Toomajian C, Kreitman M, Bergelson J (2006) A genome-wide survey of R gene polymorphisms in Arabidopsis. Plant Cell 18, 1803-1818.
    Bent AF, Kunkel BN, Dahlbeck D, et al. (1994) RPS2 of Arabidopsis thaliana: a leucine-rich repeat class of plant disease resistance genes. Science 265, 1856-1860.
    Bent AF, Mackey D (2007) Elicitors, effectors, and R genes: the new paradigm and a lifetime supply of questions. Annu Rev Phytopathol 45, 399-436.
    Bergelson J, Kreitman M, Stahl EA, Tian D (2001) Evolutionary dynamics of plant R-genes. Science 292, 2281-2285.
    Borevitz JO, Hazen SP, Michael TP, et al. (2007) Genome-wide patterns of single-feature polymorphism in Arabidopsis thaliana. Proc Natl Acad Sci U S A 104, 12057-12062.
    Boyko A, Kathiria P, Zemp FJ, et al. (2007) Transgenerational changes in the genome stability and methylation in pathogen-infected plants: (virus-induced plant genome instability). Nucleic Acids Res 35, 1714-1725.
    Burland TG (2000) DNASTAR's Lasergene sequence analysis software. Methods Mol Biol 132, 71-91.
    Caicedo AL (2008) Geographic diversity cline of R gene homologs in wild populations of Solanum Pimpinellifolium (Solanceae). American Journal of Botany 95, 393-398.
    Caicedo AL, Schaal BA, Kunkel BN (1999) Diversity and molecular evolution of the RPS2 resistance gene in Arabidopsis thaliana. Proc Natl Acad Sci U S A 96, 302-306.
    Cannon SB, Zhu H, Baumgarten AM, et al. (2002) Diversity, distribution, and ancient taxonomic relationships within the TIR and non-TIR NBS-LRR resistance gene subfamilies. J Mol Evol 54, 548-562.
    Chen Y, Mant CT, Hodges RS (2002) Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix. J Pept Res 59, 18-33.
    Chisholm ST, Coaker G, Day B, Staskawicz BJ (2006) Host-microbe interactions: shaping the evolution of the plant immune response. Cell 124, 803-814.
    Couch BC, Spangler R, Ramos C, May G (2006) Pervasive purifying selection characterizes the evolution of I2 homologs. Mol Plant Microbe Interact 19, 288-303.
    Dawkins R, Krebs JR (1979) Arms races between and within species. Proc R Soc Lond B Biol Sci 205, 489-511.
    Doyle J, Doyle J (1987) A rapid DNA isolation procedure for small quantities of fresh leaf material. Phytochem Bull 19, 11-15.
    Excoffier L, Laval G, Schneider S (2005) Arlequin (version 3.0): An integrated software package for population genetics data analysis. Evol Bioinform Online 1, 47-50.
    Fay JC, Wu CI (2000) Hitchhiking under positive Darwinian selection. Genetics 155, 1405-1413.
    Flor H (1956) The complementary genic systems in flax and flax rust. Curr Advan Genet Mol Biol 8, 29-54.
    Force A, Lynch M, Pickett FB, et al. (1999) Preservation of duplicate genes by complementary, degenerative mutations. Genetics 151, 1531-1545.
    Fu YX, Li WH (1993) Statistical tests of neutrality of mutations. Genetics 133, 693-709.
    Goff SA, Ricke D, Lan TH, et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. japonica). Science 296, 92-100.
    Gos G, Wright SI (2008) Conditional neutrality at two adjacent NBS-LRR disease resistance loci in natural populations of Arabidopsis lyrata. Mol Ecol 17, 4953-4962.
    Grant MR, Godiard L, Straube E, et al. (1995) Structure of the Arabidopsis RPM1 gene enabling dual specificity disease resistance. Science 269, 843-846.
    Holub, E. B. (2001). The arms race is ancient history in Arabidopsis, the wildflower. Nat Rev Genet 2, 516-27.
    Jia Y, McAdams SA, Bryan GT, Hershey HP, Valent B (2000) Direct interaction of resistance gene and avirulence gene products confers rice blast resistance. Embo J 19, 4004-4014.
    Jones DT, Taylor WR, Thornton JM (1992) The rapid generation of mutation data matrices from protein sequences. Comput Appl Biosci 8, 275-282.
    Kimura M (1980) A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111-120.
    Kroymann J, Donnerhacke S, Schnabelrauch D, Mitchell-Olds T (2003) Evolutionary dynamics of an Arabidopsis insect resistance quantitative trait locus. Proc Natl Acad Sci U S A 100 Suppl 2, 14587-14592.
    Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105-132.
    Leister D, Kurth J, Laurie DA, et al. (1998) Rapid reorganization of resistance gene homologues in cereal genomes. Proc Natl Acad Sci U S A 95, 370-375.
    Liu JJ, Ekramoddoullah AK (2003) Isolation, genetic variation and expression of TIR-NBS-LRR resistance gene analogs from western white pine ( Pinus monticola Dougl. ex. D. Don.). Mol Genet Genomics 270, 432-441.
    Lu G, Moriyama EN (2004) Vector NTI, a balanced all-in-one sequence analysis suite. Brief Bioinform 5, 378-388.
    Lucht JM, Mauch-Mani B, Steiner HY, et al. (2002) Pathogen stress increases somatic recombination frequency in Arabidopsis. Nat Genet 30, 311-314.
    McDowell JM, Dhandaydham M, Long TA, et al. (1998) Intragenic recombination and diversifying selection contribute to the evolution of downy mildew resistance at the RPP8 locus of Arabidopsis. Plant Cell 10, 1861-1874.
    McHale L, Tan X, Koehl P, Michelmore RW (2006) Plant NBS-LRR proteins: adaptable guards. Genome Biol 7, 212.
    Meyers BC, Dickerman AW, Michelmore RW, et al. (1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. Plant J 20, 317-332.
    Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW (2003) Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15, 809-834.
    Milligan SB, Bodeau J, Yaghoobi J, et al. (1998) The root knot nematode resistance gene Mi from tomato is a member of the leucine zipper, nucleotide binding, leucine-rich repeat family of plant genes. Plant Cell 10, 1307-1319.
    Mitchell-Olds T, Schmitt J (2006) Genetic mechanisms and evolutionary significance of natural variation in Arabidopsis. Nature 441, 947-952.
    Nei M, Gu X, Sitnikova T (1997) Evolution by the birth-and-death process in multigene families of the vertebrate immune system. Proc Natl Acad Sci U S A 94, 7799-7806.
    Nei M, Hughes AL (1992) Balanced polymorphism and evolution by the birth-and-death process in the MHC loci. In: 11th Histocompatibility Workshop and Conference, eds. Tsuji, K., Aizawa, M. & Sasazuki, T., Oxford Univ. Press, Oxford.
    Nei M, Rogozin IB, Piontkivska H (2000) Purifying selection and birth-and-death evolution in the ubiquitin gene family. Proc Natl Acad Sci U S A 97, 10866-10871.
    Ohno S (1970) Evolution by gene duplication. Springer-Verlg, Brlin.
    Okuyama Y, Fujii N, Wakabayashi M, et al. (2005) Nonuniform concerted evolution and chloroplast capture: heterogeneity of observed introgression patterns in three molecular data partition phylogenies of Asian Mitella (saxifragaceae). Mol Biol Evol 22, 285-296.
    Pal A, Chakrabarti A, Basak J (2007) New motifs within the NB-ARC domain of R proteins: probable mechanisms of integration of geminiviral signatures within the host species of Fabaceae family and implications in conferring disease resistance. J Theor Biol 246, 564-573.
    Pan Q, Wendel J, Fluhr R (2000) Divergent evolution of plant NBS-LRR resistance gene homologues in dicot and cereal genomes. J Mol Evol 50, 203-213.
    Riedl SJ, Li W, Chao Y, Schwarzenbacher R, Shi Y (2005) Structure of the apoptotic protease-activating factor 1 bound to ADP. Nature 434, 926-933.
    Rose LE, Michelmore RW, Langley CH (2007) Natural variation in the Pto disease resistance gene within species of wild tomato (Lycopersicon). II. Population genetics of Pto. Genetics 175, 1307-1319.
    Rose TM, Schultz ER, Henikoff JG, et al. (1998) Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26, 1628-1635.
    Rozas J, Sanchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19, 2496-2497.
    Salmeron JM, Oldroyd GE, Rommens CM, et al. (1996) Tomato Prf is a member of the leucine-rich repeat class of plant disease resistance genes and lies embedded within the Pto kinase gene cluster. Cell 86, 123-133.
    Shang J, Tao Y, Chen X, et al. (2009) Identification of a new rice blast resistance gene, Pid3, by genomewide comparison of paired nucleotide-binding site--leucine-rich repeat genes and their pseudogene alleles between the two sequenced rice genomes. Genetics 182, 1303-1311.
    Smith JM, Feil EJ, Smith NH (2000) Population structure and evolutionary dynamics of pathogenic bacteria. Bioessays 22, 1115-1122.
    Stahl EA, Bishop JG (2000) Plant-pathogen arms races at the molecular level. Curr Opin Plant Biol 3, 299-304.
    Stahl EA, Dwyer G, Mauricio R, Kreitman M, Bergelson J (1999) Dynamics of disease resistance polymorphism at the Rpm1 locus of Arabidopsis. Nature 400, 667-671.
    Stranger BE, Mitchell-Olds T (2005) Nucleotide variation at the myrosinase-encoding locus, TGG1, and quantitative myrosinase enzyme activity variation in Arabidopsis thaliana. Mol Ecol 14, 295-309.
    Tajima F (1989) Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123, 585-595.
    Takken FL, Albrecht M, Tameling WI (2006) Resistance proteins: molecular switches of plant defence. Curr Opin Plant Biol 9, 383-390.
    Takken FL, Jakobsson M (2000) Plant resistance genes: their structure, function, and evolution. Eur. J. Plant Pathol. 106, 699-713.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24, 1596-1599.
    Tang X, Frederick RD, Zhou J, et al. (1996) Initiation of plant disease resistance by physical interaction of AvrPto and Pto kinase. Science 274, 2060-2063.
    Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res 25, 4876-4882.
    Thompson JN, Burdon JJ (1992) Gene-for-gene coevolution between plants and parasites. Nature 360, 121-125.
    Tian D, Araki H, Stahl E, Bergelson J, Kreitman M (2002) Signature of balancing selection in Arabidopsis. Proc Natl Acad Sci U S A 99, 11525-11530.
    Tiffin P, Gaut BS (2001) Molecular evolution of the wound-induced serine protease inhibitor wip1 in Zea and related genera. Mol Biol Evol 18, 2092-2101.
    Xia X, Xie Z (2001) DAMBE: software package for data analysis in molecular biology and evolution. J Hered 92, 371-373.
    Xu Q, Wen X, Deng X (2007) Phylogenetic and evolutionary analysis of NBS-encoding genes in Rosaceae fruit crops. Mol Phylogenet Evol 44, 315-324.
    Yaish MW, Saenz de Miera LE, Perez de la Vega M (2004) Isolation of a family of resistance gene analogue sequences of the nucleotide binding site (NBS) type from Lens species. Genome 47, 650-659.
    Yang S, Zhang X, Yue JX, Tian D, Chen JQ (2008) Recent duplications dominate NBS-encoding gene expansion in two woody species. Mol Genet Genomics 280, 187-198.
    Yang Z (1998) Likelihood ratio tests for detecting positive selection and application to primate lysozyme evolution. Mol Biol Evol 15, 568-573.
    Yang Z (2007) PAML 4: phylogenetic analysis by maximum likelihood. Mol Biol Evol 24, 1586-1591.
    Yang Z, Nielsen R (1998) Synonymous and nonsynonymous rate variation in nuclear genes of mammals. J Mol Evol 46, 409-418.
    Yang Z, Nielsen R (2002) Codon-substitution models for detecting molecular adaptation at individual sites along specific lineages. Mol Biol Evol 19, 908-917.
    Yoshimura S, Yamanouchi U, Katayose Y, et al. (1998) Expression of Xa1, a bacterial blight-resistance gene in rice, is induced by bacterial inoculation. Proc Natl Acad Sci U S A 95, 1663-1668.
    Zhang J (2003) Evolution by gene duplication: an update. Ecology and Evolution 18, 292-298.
    Zhang J, Nielsen R, Yang Z (2005) Evaluation of an improved branch-site likelihood method for detecting positive selection at the molecular level. Mol Biol Evol 22, 2472-2479.
    Zhang L, Peek AS, Dunams D, Gaut BS (2002) Population genetics of duplicated disease-defense genes, hm1 and hm2, in maize (Zea mays ssp. mays L.) and its wild ancestor (Zea mays ssp. parviglumis). Genetics 162, 851-860.

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