Title page for 91224002


[Back to Results | New Search]

Student Number 91224002
Author Shin-Yi Pu(蒲欣儀)
Author's Email Address y21kjoy@yahoo.com.tw
Statistics This thesis had been viewed 2982 times. Download 5 times.
Department Life Science
Year 2003
Semester 2
Degree Master
Type of Document Master's Thesis
Language zh-TW.Big5 Chinese
Title Genetic Mapping of hit1 Locus in Arabidopsis thaliana
Date of Defense 2004-07-08
Page Count 99
Keyword
  • Arabidopsis
  • heat intolerance
  • hit1
  • Abstract Abstract
    In order to understand the mechanism of plant crop heat stress, we
    screened a mutant that is difficult to tolerant heat stress called “hit1”
    (heat intolerance) inducible by chemical mutagenesis, EMS, causing a
    point mutation. Forward genetic is one of functional genetics approaches,
    and it is underlying a mutant with a desirable genetic phenotype to an
    identified mutation in a gene and to analysis specific gene’s molecular
    biology function. We use forward genetic to approach “hit1” that was a
    point mutation, so we want a cloning of sequencing mutant-define gene
    by map-based coloning (MBC). A hit1 mutant with Columbia ecotype (P1)
    and a wild type with Landsberg erect ecotype (P2) were parent to get
    recombinant inbred lines (F2). It has hit1 mutant specific phenotype lines
    of F2 generation that is a segregation population to provide hit1 genetic
    mapping. The definition of hit1 mutant phenotype was death at 37℃ in 4
    days to screen mutants from F2 generation as a segregation population
    then make hit1 gene genetic mapping, and we designed 26 pairs primers
    of simple sequence length polymorphism (SSLP) markers and 7 pairs
    primers of cleavage amplify polymorphisms (CAPS) markers for genetic
    analysis. We suggest that hit1 gene locus in AGI 18767.3kb ~ 18810.2kb,
    total 42.9kb regions, and approach the fine-scale mapping of MBC.
    Table of Content III
    目錄
    中文摘要……………………………………………………………Ⅰ
    英文摘要…………………………………………………………..Ⅱ
    目錄…………………………………………………………………Ⅲ
    圖目錄…………………………………………………………..…..Ⅶ
    表目錄……………………………………………………...……….Ⅷ
    縮寫與全名對照表…………………………………………………Ⅸ
    第一章序論…………………………….…………...1
    壹、阿拉伯芥的簡介………………………………………...1
    貳、研究基因的方法………………………………………...2
    一、Reverse genetics: ……………………………………..….2
    二、Forward genetics: ……………………………………..….3
    參、導致突變的方法…………………………………………..4
    IV
    肆、突變點的定位方法…………………………………..………7
    一、以遺傳圖譜為基礎之選殖基因法( map-based cloning, MBC ) ….........7
    二、各種標記之簡介: …………………………………………...8
    A). 標記之分類: …………………………………………........8
    B). DNA 分子標記(DNA molecular marker) ……….…………………9
    1). 限制片段長度多型性RFLP (restriction fragment length polymorphism) 11
    2). 逢機增殖多型性DNA (random amplified polymorphic DNA, RAPD) ⋯12
    3). 擴增片段長度多型性(amplified fragment length polymorphism, AFLP) ..13
    4). 簡單序列長度多型性( simple sequence length polymorphism , SSLP ) ....14
    5). 限制酵素切割擴增片段多型性( cleaved amplified polymorphic
    sequence , CAPS ) ……………………………………….16
    三、利用CELΙ 切割技術來輔助突變點的定位………………………17
    伍、研究動機與目的………………………………………..……19
    第二章材料與方法…………………………………………..22
    1. 阿拉伯芥基因體基因之粗萃取…………………………………….........22
    2. 製備挑選hit1 surpressor mutant 之M2 種子………………………………..23
    3. 挑選hit突變株……………………………………………………….23
    4. 挑選surpressor mutation …………………………………………….......24
    5. 製備挑選定位(mapping)時所需之hit1種子……………………………….25
    6. 挑選定位時所需之hit1突變株………………………………………….25
    7. 阿拉伯芥成株之培植…………………………………………………..26
    8. 突變種隱、顯性之鑑定………………………………………………...26
    9. 功能性對偶基因之互補測試(complementation assay) …………......................26
    10. 上位(epistatic) 現象之測試…………………………………………….27
    11. 卡方適合性分析(χ2•goodness-of-fit test) ………………………………...28
    12. SSLP 之操作步驟……………………………………………………....28
    V
    13. CAPS 之操作步驟………………………………………………………30
    14. CELI 之操作步驟……………………………………………………….31
    15. 設計引子所需考慮的細節…………………………………………….....32
    第三章結果……………………………………………………...34
    壹、hit1 的生長型態…………………………………………………34
    貳、hit1 基因之遺傳分析……………………………………………35
    參、hit1 基因之定位…………………………………………………36
    一、以37℃ 處理4 天後死亡之突變株(F2) 進行定位………………..37
    二、以對熱敏感之hit1 突變株(F2) 進行hit1 進行定位………………37
    三、以CEL I 酵素切割來輔助hit1 之定位………………..…………..40
    肆、hit 突變株之篩選…………………………………………………41
    一、hit1 suppressor mutant …………………………………………….41
    二、hit 突變株……………………………………………………....41
    第四章結論……………………………………………………43
    壹、阿拉伯芥功能性基因之開發……………………………………...43
    貳、hit1 基因之定位…………………………………………………....44
    參、分子標記SSLP 及CAPS 之使用………………………………...45
    肆、以CELΙ 酵素切割來輔助hit1 之定位…………………...............46
    VI
    伍、hit1 基因之遺傳分析…………………………………..…...48
    陸、hit 突變株之篩選……………………………………….....49
    第五章結論……………………………………………..52
    第六章文獻探討…………………………………………53
    圖表……………………………………………………………...….61
    附錄…………………………………………………………………82
    一、植物培養基之配製法………………………………………….. 82
    二、溶液及試劑配方……………………………………………….82
    三、藥品試劑………………………………………………………85
    四、酵素與限制酶………………………………………………… 86
    五、儀器………………………………………………………… 86
    六、hit1 基因之cDNA 序列與資料庫(NM_103933/At1g50500)之差異性。…87
    七、hit1 基因與多種物種之間保守區間之比對圖。……………………..88
    Reference Baculcombe, D. C. (1999). Fast-forward genetic based on virus-induced gene
    silencing. Curr. Opin. Plant Biol. 2, 109~113.
    Bell, C. J., and Ecker, J. R. (1994). Assignment of 30 microsatellite loci to
    the linkage map of Arabidopsis. Genomics 19, 137-144.
    Botstein, D., White, R. L., Skolnich, and Davis, R. W. (1980). Construction
    of a genetic linkage map in man using restriction fragment length
    polymorphism. Genet. Am. J. Hum. Genet. 32, 314-331.
    Breyne, P., Rombaut, D., Van Gysel, A., Van Montagu, M., and Gerats, T.
    (1999). AFLP analysis of genetic diversity within and between Arabidopsis
    thaliana ecotypes. Mol. Gen. Genet. 261, 627-634.
    Broadie, K. (1998). Forward and reverse genetic approaches to
    synaptogenesis. Curr. Opin. Neurobiol. 8, 128-138.
    Chory, J., Chatterjee, M., Cook, R. K., Elich, T., Fankhauser, C., Li, J.,
    Nagpal, P., Neff, M., Pepper, A., Poole, D., Reed, J., and Vitart, V.
    (1996). From seed germination to flowering, light controls plant
    development via the pigment phytochrome. Proc. Natl. Acad. Sci. 93,
    12066-12071.
    Chory, J., Peto, C., Feinbaum, R., Pratt, L., and Ausubel, F. (1989).
    Arabidopsis thaliana mutant that develops as a light-grown plant in the
    absence of light. Cell 58, 991-999.
    Colbert, T., Till, B. J., Tompa, R., Reynolds, S., Steine, M. N., Yeung, A. T.,
    McCallum, C. M., Comai, L., and Henikoff, S. (2001). High-throughput
    54
    screening for induced point mutations. Plant Physiol. 126, 480-484.
    Fink, A. L. (1999). Chaperon-Mediated protein Folding. Physiological
    Reviews 79, 425-442.
    Galbiati, M., Moreno, M. A., Nadzan, G., Zourelidou, M., and Dellaporta,
    S. L. (2000). Large-scale T-DNA mutagenesis in Arabidopsis for
    functional genomic analysis. Funct Integr Genomics 1, 25-34.
    Gibson, S., and Somerville, C. (1993). Isolating plant genes. Trends
    Biotechnol 11, 306-313.
    Gichner, T., Badayev, S. A., Demchenko, S. I., Relichova, J., Sandhu, S. S.,
    Usmanov, P. D., Usmanova, O., and Veleminsky, J. (1994). Arabidopsis
    assay for mutagenicity. Mutat. Res. 310, 249-256.
    Glazebrook, J., Drenkard, E., Preuss, D., and Ausubel, F. M. (1998). Use
    of cleaved amplified polymorphic sequences (CAPS) as genetic markers in
    Arabidopsis thaliana. Methods. Mol. Biol. 82, 173-182.
    Goodman, H. M., Ecker, J. R., and Dean, C. (1995). The genome of
    Arabidopsis thaliana. Proc. Natl. Acad. Sci. 92, 10831-10835.
    He, P., Li, J. Z., Zheng, X. W., Shen, L. S., Lu, C. F., Chen, Y., and Zhu, L.
    H. (2001). Comparison of molecular linkage maps and agronomic trait loci
    between DH and RIL populations derived from the same rice cross. Crop
    Science 41, 1240-1246.
    Hong, S. W., and Vierling, E. (2000). Mutants of Arabidopsis thaliana
    defective in the acquisition of tolerance to high temperature stress. Proc.
    Natl. Acad. Sci. 97, 4392-4397.
    Hong, S. W., and Vierling, E. (2001). Hsp101 is necessary for heat tolerance
    but dispensable for development and germination in the absence of stress.
    Plant J. 27, 25-35.
    55
    Hu, J.-P., Aguirre, M., Peto, C., Alonso, J., Ecker, J., and Chory, J. (2003).
    A Role for Peroxisomes in Photomorphogenesis and Development of
    Arabidopsis. Science 297, 405-409.
    Huala, E., Dickerman, A. W., Garcia-Hernandez, M., Weems, D., Reiser,
    L., LaFond, F., Hanley, D., Kiphart, D., Zhuang, M., Huang, W.,
    Mueller, L. A., Bhattacharyya, D., Bhaya, D., Sobral, B. W., Beavis, W.,
    Meinke, D. W., Town, C. D., Somerville, C., and Rhee, S. Y. (2001). The
    Arabidopsis Information Resource (TAIR): a comprehensive database and
    web-based information retrieval, analysis, and visualization system for a
    model plant. Nucleic Acids Res. 29, 102-105.
    The Arabidopsis Genome Initiative (2000). Analysis of the genome
    sequence of the flowering plant Arabidopsis thaliana. Nature 408,
    796-815.
    Izant, J. G., and Weintraub, H. (1984). Inhibition of thymidine kinase gene
    expression by anti-sense RNA: a molecular approach to genetic analysis.
    Cell 36, 1007-1015.
    Jander, G., Baerson, S. R., Hudak, J. A., Gonzalez, K. A., Gruys, K. J.,
    and L., R. (2003). Ethylmethanesulfonate Saturation Mutagenesis in
    Arabidopsis to Determine Frequency of Herbicide Resistance. Plant
    Physiol. 131, 139-146.
    Jander, G., Norris, S. R., Rounsley, S. D., Bush, D. F., Levin, I. M., and
    Last, R. L. (2002). Arabidopsis map-based cloning in the post-genome era.
    Plant Physiol. 129, 440-450.
    Konieczny, A., and Ausubel, F. M. (1993). A procedure for mapping
    Arabidopsis mutations using co-dominant ecotype-specific PCR-based
    markers. Plant J. 4, 403-410.
    56
    Koornneef, M., Alonso-Blanco, C., and Stam, P. (1998). Genetic analysis.
    Methods Mol. Biol. 82, 105-117.
    Krysan, P. J., Young, J. C., Jester, P. J., Monson, S., Copenhaver, G.,
    Preuss, D., and Sussman, M. R. (2002). Characterization of T-DNA
    insertion sites in Arabidopsis thaliana and the implications for saturation
    mutagenesis. Omics. 6, 163-174.
    Kunz, B., A. , Henson, E. S., Karthikeyan, R., Kuschak, T., McQueen, S.
    A., Scott, C. A., and Xiao, W. (1998). Defects in Base Excision Repair
    Combined with Elevated Intracellular dCTP Levels Dramatically Reduce
    Mutation Induction in Yeast by Ethyl Methanesulfonate and
    N-methyl-N*-nitro-N-nitrosoguanidine. Environmental and Molecular
    Mutagenesis 32, 173-178.
    Li, X., and Zhang, Y. (2002). Reverse genetics by fast neutron mutagenesis
    in higher plants. Funct. Integr. Genomics 2, 254-258.
    Lister, C., and Dean, C. (1993). Recombinant inbred lines for mapping RFLP
    and phenotypic markers in Arabidopsis thaliana. Plant J. 4, 745-750.
    Lukowitz, W., Gillmor, C. S., and Scheible, W. R. (2000). Positional cloning
    in Arabidopsis: why it feels good to have a genome initiative working for
    you. Plant Physiol. 123, 795-805.
    Lyttle, T. W. (1991). Segregation distorters. Annu. Rev. Genet. 25, 511-557.
    McCallum, C. M., Comai, L., Greene, E. A., and Henikoff, S. (2000).
    Targeting induced local lesions IN genomes (TILLING) for plant
    functional genomics. Plant Physiol. 123, 439-442.
    Meinke, D. W., Meinke, L. K., Showalter, T. C., Schissel, A. M., Mueller, L.
    A., and Tzafrir, I. (2003). A Sequence-Based Map of Arabidopsis Genes
    with Mutant Phenotypes. Plant Physiol. 131, 409-418.
    57
    Michelmore, R. W., Paran, I., and Kesseli, R. V. (1991). Identification of
    markers linked to disease-resistance genes by bulked segregant analysis: a
    rapid method to detect markers in specific genomic regions by using
    segregating popula-tions. Proc. Natl. Acad. Sci. 88, 9828-9832.
    Mulcahy, D. L. (1979). The rise of angiosperms : genecolical factor. Science
    206, 20-23.
    Naito, S. (2002). Arabidopsis: a genetic tool to know the unknowns.
    Tanpakushitsu Kakusan Koso 47, 1471-1475.
    Oleykowski, C. A., Bronson Mullins, C. R., Godwin, A. K., and Yeung, A.
    T. (1998). Mutation detection using a novel plant endonuclease. Nucleic
    Acids Res. 26, 4597-4602.
    Pepper, A. E., and Chory, J. (1997). Extragenic suppressors of the
    Arabidopsis det1 mutant identify elements of flowering-time and
    light-response regulatory pathways. Genetics 145, 1125-1137.
    Perry, J. A., Wang, T. L., Welham, T. J., Gardner, S., Pike, J. M., Yoshida,
    S., and Parniske, M. (2003). A TILLING reverse genetics tool and a
    web-accessible collection of mutants of the legume Lotus japonicus. Plant
    Physiol. 131, 866-871.
    Peters, J. L., Cnops, G., Neyt, P., Zethof, J., Cornelis, K., Van Lijsebettens,
    M., and Gerats, T. (2004). An AFLP-based genome-wide mapping
    strategy. Theor Appl Genet 108, 321-327.
    Peters, J. L., Cnudde, F., and Gerats, T. (2003). Forward genetics and
    map-based cloning approaches. Trends Plant Sci. 8, 484-491.
    Peters, J. L., Constandt, H., Neyt, P., Cnops, G., Zethof, J., Zabeau, M.,
    and Gerats, T. (2001). A physical amplified fragment-length
    polymorphism map of Arabidopsis. Plant Physiol. 127, 1579-1589.
    58
    Ponce, M. R., Robles, P., and Micol, J. L. (1999). High-throughput genetic
    mapping in Arabidopsis thaliana. Mol. Gen. Genet. 261, 408-415.
    Rick, C. M. (1996). Abortion of male and female ganetes in the tomato
    determined by allelic interaction. Genetics 53, 85-96.
    Schlotterer, C., and Tautz, D. (1992). Slippage synthesis of simple sequence
    DNA. Nucl. Acids Res. 20, 211-215.
    Stemple, D. L. (2004). TILLING--a high-throughput harvest for functional
    genomics. Nat. Rev. Genet. 5, 145-150.
    Tanksley, S. D., Ganal, M. W., and Martin, G. B. (1995). Chromosome
    landing: a paradigm for map-based gene cloning in plants with large
    genomes. Trends Genet. 11, 63-68.
    Tax, F. E., and Vernon, D. M. (2001). T-DNA-associated
    duplication/translocations in Arabidopsis. Implications for mutant analysis
    and functional genomics. Plant Physiol. 126, 1527-1538.
    Till, B. J., Burtner, C., Comai, L., and Henikoff, S. (2004). Mismatch
    cleavage by single-strand specific nucleases. Nucleic Acids Res. 32,
    2632-2641.
    Till, B. J., Colbert, T., Tompa, R., Enns, L. C., Codomo, C. A., Johnson, J.
    E., Reynolds, S. H., Henikoff, J. G., Greene, E. A., Steine, M. N., Comai,
    L., and Henikoff, S. (2003a). High-throughput TILLING for functional
    genomics. Methods Mol. Biol. 236, 205-220.
    Till, B. J., Reynolds, S. H., Greene, E. A., Codomo, C. A., Enns, L. C.,
    Johnson, J. E., Burtner, C., Odden, A. R., Young, K., Taylor, N. E.,
    Henikoff, J. G., Comai, L., and Henikoff, S. (2003b). Large-scale
    discovery of induced point mutations with high-throughput TILLING.
    Genome Res. 13, 524-530.
    59
    Vos, P. (1998). AFLP fingerprinting of Arabidopsis. Methods Mol. Biol. 82,
    147-155.
    Vos, P., Hogers, R., Bleeker, M., Reijans, M., Lee, T., Hornes, M., Frijters,
    A., Pot, J., Peleman, J., Kuiper, M., and Zabeau, M. (1995). AFLP: a
    new technique for DNA fingerprinting. Nucl. Acid Res. 23, 4407-4414.
    Walter, L., Stark, S., Helou, K., Flugge, P., Levan, G., and Gunther, E.
    (2002). Identification, characterization and cytogenetic mapping of a yeast
    Vps54 homolog in rat and mouse. Gene 285, 213-220.
    Welsh, J., and McClelland, M. (1990). Fingerprinting genomes using PCR
    with arbitrary primers. Nucleic Acids Res. 18, 7213-7218.
    Williams, J. G., Kubelik, A. R., Livak, K. J., Rafalski, J. A., and Tingey, S.
    V. (1990). DNA polymorphisms amplified by arbitrary primers are useful
    as genetic markers. Nucleic Acids Res. 18, 6531-6535.
    Williams, J. G., Reiter, R. S., Young, R. M., and Scolnik, P. A. (1993).
    Genetic mapping of mutations using phenotypic pools and mapped RAPD
    markers. Nucleic Acids Res. 21, 2697-2702.
    Wu, S. J., Locy, R. D., Cherry, J. H., and Singh, N. K. (2000). Mutation in
    Arabidopsis HIT1 locus causing heat and osmotic hypersensitivity. Journal
    of plant physiology 157, 543-547.
    Yang, B., Wen, X., Kodali, N. S., Oleykowski, C. A., Miller, C. G., Kulinski,
    J., Besack, D., Yeung, J. A., Kowalski, D., and Yeung, A. T. (2000).
    Purification, cloning, and characterization of the CEL I nuclease.
    Biochemistry 39, 3533-3541.
    Zolman, B. K., and Bartel, B. (2004). An Arabidopsis indole-3-butyric
    acid-response mutant defective in PEROXIN6, an apparent ATPase
    implicated in peroxisomal function. Proc. Natl. Acad. Sci. 101, 1786-1791.
    60
    Zolman, B. K., Silva, I. D., and Bartel, B. (2001). The Arabidopsis pxa1
    Mutant Is Defective in an ATP-Binding Cassette Transporter-Like Protein
    Required for Peroxisomal Fatty Acid β-Oxidation. Plant Physiol. 127,
    1266-1278.
    Zolman, B. K., Yoder, A., and Bartel, B. (2000a). Genetic analysis of
    indole-3-butyric acid responses in Arabidopsis thaliana reveals four
    mutant classes. Genetics 156, 1323-1337.
    Zolman, B. K., Yoder, A., and Bartel, B. (2000b). Genetic Analysis of
    Indole-3-butyric Acid Responses in Arabidopsis thaliana Reveals Four
    Mutant Classes. Genetics 156, 1323-1337.
    Advisor
  • Show-Jye Wu(吳少傑)
  • Files
  • 91224002.pdf
  • disapprove authorization
    Date of Submission 2004-07-15

    [Back to Results | New Search]


    Browse | Search All Available ETDs

    If you have dissertation-related questions, please contact with the NCU library extension service section.
    Our service phone is (03)422-7151 Ext. 57407,E-mail is also welcomed.