阿拉伯芥突變種(hit1)之位址定位

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蒲欣儀(Shin-Yi Pu) 查詢紙本館藏

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論文名稱

阿拉伯芥突變種(hit1)之位址定位
(Genetic Mapping of hit1 Locus in Arabidopsis thaliana)

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摘要(中)

中文摘要
為了了解植物耐受高溫的遺傳控制因子及其生理機制，我們用化學突
變劑( EMS )誘導阿拉伯芥點突變產生，並進而篩選出一株對熱缺乏耐受
性之突變種，稱之為hit1( heat intolerance ) 。這是一種典型從具遺傳性之
生理性狀找出相對應之基因及其分子作用的研究方式，亦即所謂的向前式
遺傳學分析法( forward genetics )，為功能性遺傳學研究法中的一種。因
hit1 之性狀為點突變所造成的，故其功能性基因須以遺傳圖譜為基礎來進
行定位選殖( map-based cloning, MBC ) 。其做法是先將Columbia 生態
型之hit1 突變種與Landsberg 生態型之野生種雜交，之後的F1 自交得F2
世代，F2 世代中帶有hit1 性狀之個體為hit1 定位時所需之分離族群
( segregation population )。以hit1 之突變性狀，37℃處理四天後會死亡的
原則篩選，再經簡單序列長度多型性( simple sequence length
polymorphism , SSLP )及限制酵素切割擴增片段多型性( cleaved amplified
polymorphic sequence , CAPS )兩種對生態型具專一性之分子標記進行定
位。本研究共設計了26 組SSLP 分子標記，7 組CAPS 分子標記。在檢
測過3987 株F2 世代的hit1 突變種之後，推得hit1 基因坐落在第一條染色
體， AGI map 18767.3 kb ~ 18810.2 kb 之42.9kb 範圍內，達到MBC 所需
之定位目標。

摘要(英)

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.

關鍵字(中)

★ 阿拉伯芥
★ 對熱不耐受性突變株

關鍵字(英)

★ hit1
★ heat intolerance
★ Arabidopsis

論文目次

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

參考文獻

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.

指導教授

吳少傑(Show-Jye Wu)

審核日期

2004-7-15

推文