摘要
随着模式植物拟南芥基因组全序列测定的完成,基因功能研究成为当前生命科学的前沿领域。本论文以正向遗传学的方法研究花药及花粉发育过程的基因功能,主要利用图位克隆的方法从拟南芥雄性不育突变体中克隆相应的基因。
首先对19个经化学诱变剂EMS处理得到的雄性不育突变体进行背景纯化和遗传分析,从中筛选到四个单个隐性基因控制的雄性不育突变体(EC2-157、EC1-188、EC2-115和EC2-214)。细胞学观察表明,EC2-157花药中不能形成花粉粒,EC1-188突变体中只有少量的花粉粒,但花粉粒的内容物较少;EC2-115和EC2-214突变体中能够形成花粉粒。
为了从突变体中克隆基因,我们建立了一套分子标记(共24个)用于突变基因的初定位。其中本次实验发展了18个分子标记。利用这套分子标记,将四个不育突变体EC2-157、EC1-188、EC2-115、EC2-214相应的突变基因分别定位于拟南芥的第Ⅳ,Ⅴ,Ⅴ,Ⅰ号染色体上。这套分子标记比较稳定,容易检测,目前也用于拟南芥其他突变基因的定位。
然后对突变体EC1-188的突变基因(ms188)进行了精细定位。在与ms188连锁的分子标记MC015附近设计了8个InDel分子标记,对遗传群体中2135株不育植株进行基因型分析,最后将目的基因定位于第五条染色体分子标记MDA7和K24C1之间95.8KB的区间内。该区间共有28个基因,其中一个是转录因子AtMYB103。
利用PCR的方法从突变体中扩增AtMYB103基因并进行序列分析,结果表明突变体中AtMYB103基因第二个外显子上发生了点突变,由原来编码谷氨酰胺的CAA密码子突变为终止密码子TAA。因此,我们推测,EC-188突变体可能是转录因子AtMYB103基因发生突变的结果。进一步的遗传互补以及功能分析正在进行之中。
With the completion of the model plant Arabidopsis genome sequencing in 2000, the emphasis of the life science has been into the functional genomics. In this dissertation we focused on the function of genes associated with anther and pollen development including map-based cloning of Arabidopsis genes from male sterile mutants.
From 19 Arabidopsis male sterile lines isolated from an ethyl methanesulphonate-induced (EMS-induced) population, a total of four male sterile mutants were screened with each mutant controlled by a single recessive gene. Cross-section of these male sterile mutant anthers showed that there is no pollen formation in ms157 anther while msl88 anther contains a few pollen grains with pollen shape distorted and reduced cytoplasmic content. However, pollen was formed in both anthers of ms115 and ms214.
In order to isolate these genes, we established a set of molecular markers for the first-pass mapping of Arabidopsis genes. Of the 24 InDel markers, a total of 18 were developed in this work. The mutated genes ms157, ms188, msl 15 and ms214 were mapped on chromosome IV, V, V, I, respectively, using the molecular markers developed above. This set of molecular markers is stable, and easy to use. They are also used for other gene mapping in Arabidopsis.
The ms188 gene was finely mapped. A total of 8 new InDel markers were designed to map msl88 using a segregating population with a total of 2135 male sterile progenies. MS188 was finally mapped to a region of 95.8kb between the molecular marker MDA7 and K24C1. There are a total of 28 genes in this region including a transcription factor AtMYB103.
PCR method was used to identify candidate MS 188. Sequence analysis indicated that a point mutation occurred in the second exon of the AtMYB103 gene in male sterile mutant with CAA(Gln) replaced by a stop codon TAA. Thus, we believe that AtMYB103 is the candidate gene for MS 188. The genetic complementation and gene function analysis are being undertaken.
引文
陈俊,王宗阳.2002.植物MYB类转录因子研究进展.植物生理与分子生物学学报,28(2):81-88.
陈丽.1997.植物转录因子的结构与功能.植物生理学通讯,33(6):401-409.
陈明.2003.可调控转基因植物雄性不育的研究.中国农业科学院博士研究生学位论文.
C W迪芬巴赫,G S德维克斯勒.1998.PCR技术实验指南.北京:科学出版社.
黄娟,李家洋.2001,拟南芥基因组研究进展.微生物学通报,28(3):99-101.
姜泊,张亚历,周殿元.1995.分子生物学常用实验方法.北京:科学出版社.
康定明,翟礼嘉,邓兴旺.2001.拟南芥DNA全序列测定与分析完成.植物学报,18(1):124-125.
刘强,张贵友,陈受宜.2000.植物转录因子的结构与调控作用.科学通报,45(14):1465-1474.
卢圣栋.1993.现代分子生物学实验技术.北京:高等教育出版社.
毛健民,李俐俐.2001.拟南芥——植物界的“果蝇”.生物学通报,36(12):13-14.
史桂荣,曲章义.1990.EMS与玉米诱变育种.国外农学,1:1-5.
万小荣,李玲.2002.植物的MYB蛋白.植物生理学通讯,38(2):165-170.
王年.2001.应用激活标记方法构建拟南芥突变体库.中国农业大学硕士学位论文.
王希庆,陈柏君,印莉萍.2003.植物中的MYB转录因子.生物技术通报,2:22-25.
未来十年的植物生物学研究.科技政策与发展战略,2001.6:8-11.
谢建坤.2001.水稻细胞质雄性不育育性恢复遗传机理研究.浙江大学博士学位论文.
游晴如,黄庭旭,张水金.2003.植物诱变新技术及其在水稻育种上的应用.江西农业学报,15(2):43-47.
赵淑清.武维华.2000.DNA分子标记和基因定位.生物技术通报,6:1-4.
祝丽英,池书敏,刘志增.2002.EMS对玉米花粉诱变效应的研究.河北农业大学学报,25:17-28.
朱英国.2000.水稻雄性不育生物学.武汉:武汉大学出版社.
Aarts M G, Dirkse W G, Stiekema W J, Pereira A. 1993. Transposon tagging of a male sterility gene in Arabidopsis. Nature, 363: 715-717.
Bai X F, Peisrson B N, Dong F G, Makaroff C A. 1999. Isolation and characterization of SYN1, a RAD21-like gene essential for meiosis in Arabidopsis. Plant Cell, 11: 417-430.
Bell C J, Ecker J R. 1994. Assignment of 30 microsatellite loci to the linkage map of Arabidopsis. Genomics, 19: 137-144.
Bilaud T, Koering C E, Binet-Brasselet E. 1996. The telobox, a myb-related telomeric DNA binding motif found in proteins from yeast, plants and human. Nucleic Acids Res, 24: 1294-1303.
Boulikas T. 1994. Putative nuclear localization signals (NLS) in protein transcription factors. J Cell Biochem, 55: 32-58.
Ciraudat J, Hauge B M, Valon C. 1992. Isolation of the Arabidopsis ABI3 gene by positional cloning. Pant Cell, 4: 1251-1261.
Creelman R A, Mullet J E. 1997. Biosynthesis and action of jasmonates in plants. Annu Rev Plant Physiol Plant Mol Biol, 48: 355-381.
Dehesh K, Brece W B, Quail P H. 1990. A trans-acting factor that binds to a GT-motif in a phytochrome gene promoter. Science, 250: 1397.
Foirall L, Rhodes D, Kiug A. 1986. Mapping of the sites of protection on a 5s RNA gene by the Xenopus transcription factor ⅢA, a model for the interaction. J Mol Biol, 192: 577.
Frederick. 2002. Summaries of National Science Foundation-Sponsored Arabidopsis 2010 Projects
and National Science Foundation-Sponsored Plant Genome Projects That Are Generating Arabidopsis Resources for the Community. Plant Physioi, 129: 394-437
Gaillard C, Moffatt B A, Blacker M, Laioue M. 1998. Male sterility associated with APRT deficiency in Arabidopsis thaliana results from a mutation in the gene APT1. Mol Gen Genet, 257: 348-353.
Georg J, Susan R N, Steven D R. 2002. Arabidopsis Map-Based Cloning in the Post-Genome Era. Plant Physiol, 129: 440-450.
Gilmartin P M, Memelink J, Hiratsuka K. 1992. Characterization ofa gene encoding a DNA binding protein with specificity for a light-responsive element. Plant Ceil, 4: 839.
Glover J, Grelon M, Craig S, Dennis E. 1998. Cloning and characterization of MS5 from Arabidopsis: a gene critical in male meiosis. Plant J, 15: 345-356.
Goldberg R B, Beals T P, Sanders P M. 1993. Anther development: basic principles and practical applications. Plant Cell, 5: 1217-1229.
Ishiguro S, Akiko K O, Junichi U, Okada K. 2001. The DEFECTIVE IN ANTHER DEHISCENCE1 gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis. Plant Cell, 13: 2191-2209.
Ito M. 2000. Factors controlling cyclin B expression. Plant Mol Biol, 43: 677-690.
Janny L P, Filip C, Tom G. 2003. Forward genetics and map-based cloning approaches. TRENDS in Plant Science, 8(10): 484-490.
Jackon S P, Tjian R. 1988. O-glycosylation of eukaryotic transcription factors implications for mechanism of transcriptional regulation. Cell, 55: 125.
Jin H L, Cathie M. 1999. Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol, 41: 577-585.
Kapoor S, Kobayashi A, Takatsuji H. 2002. Silencing of the tapetum-specific Zinc Finger Gene TAZ1 causes premature degeneration of tapetum and pollen abortion in Petunia. Plant Cell, 14: 2353-2367.
Kaul S, Koo H L, Fraser C M, Mayer k, White O, Mewes H W. 2000. Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature, 408: 796-815.
Koncz C, Chua N H, Schell J. 1992. Methods in Arabidopsis Research. World Scientific publishing Co Pte Ltd.
Kranz H D, Denekamp M, Greco R. 1998. Towards functional characterisation of the members of the R2R3-MYB gene family from Arabidopsis thaliana. Plant J, 16, 263-276.
Kuhn R M, Caspar T, Dehesk K. 1993. DNA binding factor GT-2 from Arabidopsis. Plant Moi Biol, 23: 337.
Li S F, Trudi H, Roger W P. 1999. A novel MYB-related gene from Arabidopsis thaliana expressed in developing anthers. Plant Cell Physiol, 40(3): 343-347.
Lin X, Kaul S, Rounsley S. 1999. Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana. Nature, 402, 761-768.
Lipsick J S. 1996. One billion years of MYB. Oncogene, 13: 223-235.
Marocco A, Wiissenbach M, Rohde W. 1989. Multiple genes are transcribed in Hordeum vulgare and Zea mays that carry the DNA-binding domain of the MYB oncoproteins. Mol Gen Genet, 216: 183-187.
Martin C, Paz-Ares J. 1997. MYB transcription factors in plants. Trends gene, 13: 67-73.
Mayer K, Wambutt R, Murphy G. 1999. Sequence and analysis of chromsome 4 of the plant Arabidopsis thaliana. Nature, 402: 769-777.
McConn M, Browse J. 1996. The critical requirement for linolenic acid is pollen development, not photosynthesis, in an Arabidopsis mutant. Plant Cell, 8: 403-416.
Meinke D W, Cherry J M, Dean C. 1998. Arabidopsis thaliana: a model plant for genome analysis. Science, 282: 662-665.
Meyerowitz E M. 1987. Arabidopsis thaliana. Annu Rev Genet, 21: 93-111.
Park J H, Halitschke R, Kim H B, Feyereisen R. 2002. A knock-out mutation in allene oxide synthase results in male sterility and defective wound signal transduction in Arabidopsis due to a block in jasmonic acid biosynthesis. Plant J, 31: 1-12.
Ralf S, Martin W, Bernd W. 2001. The R2R3-MYB gene family in Arabidopsis thaliana. Curr Opin Plant Biol, 4: 447-456.
Rhodes D, Klug A. 1986. An underlying repeat in some transcriptional control sequences corresponding to half a double helical turn of DNA. Cell, 46: 123.
Riechmann J L, Heard J, Martin G. 2000. Arabidopsis transcription factors: genome-wide comparative analysis among eukarytoes, science, 290: 2105-2110.
Romero I, Fuertes A, Benito M J. 1998. More than 80 R2R3-myb regulatory genes in the genome of Arabidopsis thaliana. Plant J, 14: 273-284.
Sabine S L, Ulrike S U, Luca E. 2003. Disruption ofArabidopsis thaliana MYB26 results in male sterility due to non-dehiscent anthers. Plant J, 34: 519-528.
Salanoubat M, Lemcke K, Rieger M. 2000. Sequence and analysis of chromosome 3 of the plant Arabidopsis thaliana. Nature, 408: 820-822.
Sambrook J, Russell D V. 2001. Molecular Cloning: A Laboratory Mannuai, 3rd ed. Cold Spring Harbor Laboratory Press.
Sanders P M, Bui A Q, Goldberg R B. 1999. Anther developmental defects in Arabidopsis thaliana male-sterile mutants. Sex Plant Repro, 11: 297-322.
Sanders P M, Lee P Y, Beals T P, Goldberg R B. 2000. The Arabidopsis DELAYED DEHISCENCE1 gene encodes an enzyme in the jasmonic acid synthesis pathway. Plant Cell, 12: 1041-1061.
Schiefthaler U, Balasubramanian S, Sieber P, Chevalier D. 1999. Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana. PNAS, 96: 11664-11669.
Schwarz S Z, Nacken W, Saedler H. 1990. Genetic control of flower development by homeotic trenes in Antirrhinum majus. Science, 250: 931.
Sorensen A M, Unte U S, Huijser P, Dekker K, Saedler H. 2003. The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor. Plant J, 33: 413-423.
Stintzi A, Browse J. 2000. The Arabidopsis male-sterile mutant, opr3, lacks the 12-oxophytodienoic acid reductase required for jasmonate synthesis. PNAS, 97: 10625-10630.
Tabata S, Kaneko T, Nakamura Y. 2000. Sequence and analysis of chromosome 5 of the plant Arabidopsis thaliana. Nature, 408, 823-825.
The Multinational Coordinated Arabidopsis thaliana Functional Genomics Project. The Multinational Arabidopsis Steering Committee, June 2002.
Theologis A, Ecker J R, Palm C J. 2000. Sequence and analysis of chromosome 1 of the plant Arabidopsis thaliana. Nature, 408: 816-820.
Trudi H, Li S F, Roger W P. 2003. AtMYB103 regulateds tapetum and trichome development in
Arabidopsis thaliana. Plant J, 35: 177-192.
Weigel D, Aloarez J, Smyth D R. 1992. LEAEY controls floral meristem identity Arabidopsis. Cell, 69: 843.
Wilson Z A, Morroll S M, Tighe P J. 2001. The Arabidopsis MALE STERILITY1(MS1) gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors. Plant J, 28(1): 27-39.
Wolfgang L, Gillmor C S, 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.
Xin Z G, Velten J P, Oliver M J, Burke J J. 2003. High-throughput DNA extraction method suitable for PCR. Biotechniques, 34: 1-6.
Yang M, Hu Y, Lodhi M, Mccombie W R, Ma H. 1999. The Arabidopsis SKP1-LIKE1 gene is essential for male meiosis and may control homologue separation. PNAS, 96: 11416-11421.
Yang S L, Xie L F, Mao H Z, Venkatesan S, De Y. 2003. TAPETUM DETERMINANT1 is required for cell specialization in the Arabidopsis anther. Plant Cell, 15: 2792-2804.
Yang W C, Ye D, Xu J, Sundaresan V. 1999. The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes Dev, 13: 2108-2117.
Yang X H, Makaroff C A, Ma H. 2003. The Arabidopsis MALE MEIOCYTE DEATH1 gene encodes a PHD-Finger protein that Is required for male meiosis. Plant Cell, 15: 1281-1295.
Zhao D Z, Wang G F, Speal B, Ma H. 2002. The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther. Genes Dev, 16: 2021-2031.