摘要
重复序列是真核生物基因组的一个重要组成部分,不仅对维持染色体的空间结构、基因的表达调控、遗传重组等都具有重要作用,而且串联重复的DNA簇往往具有种属甚至染色体的特异性。因此,克隆和分析重复序列是研究一个真核生物基因组的有效手段。大白菜(Brassica campestris L. ssp. pekinensis)是十字花科芸薹属(Brassica)中最重要的蔬菜作物之一,属二倍体物种。此外,大白菜为小染色体物种,且非同源染色体形态相似。建立有效的染色体识别方法是进行核型分析和细胞遗传学研究的重要前提。但目前为止,利用有丝分裂中期染色体,基于rDNA为探针的FISH核型分析,可把大白菜10条染色体中的6条准确区分出,但仍有4条染色体难以识别。基于此,本研究以拟南芥重复序列为引物,以大白菜自交系‘85-1’基因组DNA为模板进行PCR扩增,对大白菜端粒相关序列进行了克隆和测序分析;并以锚定大白菜不同染色体的BAC克隆为探针,在大白菜中期染色体进行FISH,结合45S rDNA、5S rDNA及着丝粒重复探针的FISH结果,构建了精确识别大白菜不同染色体的FISH标记核型图。主要研究结果如下:
1.以拟南芥的端粒重复序列(TTTAGGG)3为引物,在大白菜中扩增并克隆了一个344bp的DNA片段。序列分析和比对表明,该序列为端粒相关序列(TAS),位于大白菜2号染色体的近末端,该序列不仅含有拟南芥类型端粒重复单元,还含有其他生物的端粒重复单元。
2. 5S rDNA探针在大白菜染色体上共检测到了10个杂交信号,分别位于1-3号、5号染色体长臂的近着丝点区域和10号染色体的短臂末端;45S rDNA探针在大白菜染色体上同样检测到了10个杂交信号,但所位于的染色体及部位有所不同,分别位于1~5号染色体长臂近着丝点区域。
3.大白菜CentBr1着丝粒重复探针在大白菜中期染色体上共检测到了16个杂交信号,分别位于1号、3号、4号、6~10号染色体的着丝点区域;CentBr2着丝粒重复探针探针在大白菜10对染色体上共检测到了4个杂交信号,分别位于第2对染色体长臂近着丝点处和第5对染色体长臂的末端。
4.除A1连锁群外,其他9个连锁群筛出的BAC克隆分别在大白菜9对同源染色体上各出现了1对杂交信号,确定了大白菜染色体和连锁群的对应关系,其中A2~A10连锁群分别对应于染色体6号、2号、9号、5号、4号、7号、8号、1号和10号,建立了一个基于rDNA、CentBr1、CentBr2和染色体特异BAC克隆FISH标记的染色体核型模式图。
Repeat sequence is one of the important components in eukaryotic genomes. It is not only important to maintain chromosome structure in space, the expression regulation of genes and genetic recombinations, but also tandem repeat DNA cluster often has a specificity of genus, species or even the chromosome. Thus the cloning and analysis of repeat sequence are effective means to research eukaryotic genome. Chinese cabbage is one of the most important vegetable crops in cruciferous Brassica diploid species. In addition, the chromosome of Chinese cabbage is small, and the form nonhomologous chromosome is similar. Establishment of an effective method of chromosome recognition is important for karyotype analysis and cytogenetics research. Up to now, only 6 different chromosomes can be accurately distinguished from the total 10 different chromosomes based on FISH using rDNA as probes. Based on these, using the primer of Arabidopsis telomeric repeat sequence, the genome DNA of Chinese cabbage inbred line‘85-1’was amplified and the telomere repeat sequence was cloned and analyzed. Using the BAC clones anchored different chromosome in Chinese cabbage as probes, the FISH on the Chinese cabbage meteaphase chromosomes was conducted.According to above FISH results, the karyotype distinguished different chromosomes of Chinese cabbage was constructed. The main results are showed as follows:
1. Using Arabidopsis telomeric repeat sequence (TTTAGGG)3 as a primer, a DNA fragment with length of 344bp was amplified and cloned from Chinese cabbage. Sequence analysis showed that it is a telomeric repeat sequence (TAS), which is mapped to the proximal end of chromosome 2 of Chinese cabbage. In this TAS fragment, there is Arabidopsis telomeric repeat Sequence (TTTAGGG)3, it also contains telomeric repeat sequence of other creatures, such as silkworm.
2. Ten FISH signals of 5S rDNA probe were observed in Chinese cabbage, which were localized the long arms nearly centromere of chromosome 1, 2, 3, 5 and the end short arms of chromosome10. Ten FISH signals of 45S rDNA probe were also observed in Chinese cabbage, which were located on the long arms nearly the centromere of chromosome1~5.
3. Sixteen FISH signals of CentBr1 rDNA probe were observed in Chinese cabbage chromosomes, which were localized on the centromere of chromosome1,3,4, 6~10, 4 FISH signals of CentBr2 rDNA probe were observed in Chinese cabbage 10 chromosomes, which were localized on the long arms nearly centromere of chromosome 2 and the end of long arms of chromosome 5.
4. Except A1 linkage group, the BAC probes anchored the other nine linkage groups (A2 to A10) of Chinese cabbage displayed a pair of FISH signals in its corresponding chromosome. There into A2~A10 linkage groups were corresponding to chromosome 6, 2, 9, 5, 4, 7, 8, 1 and 10, respectively. A karyotype diagram based on FISH marker of rDNA, CentBr1, CentBr2 and chromosome specific BAC probes was established, which providing a reliablebasis basis for accurate identification of Chinese cabbage chromosomes.
引文
[1]Pinkel D, Straume T, et al. Cytogenetic analysisusing quantitative, high-sensitivity, fluorescence hybridization[J]. Proceedings of the National Academy of Sciences , 1986, 83(9): 2934-2938.
[2]杨壹羚,徐昕,陈虹.一种改进的重复多色荧光原位杂交技术[J].石河子大学学报, 2005, 23(5): 561-565.
[3]Speicher M, Carter N. The new cytogenetics: blurring the boundaries with molecular biology[J]. Nature Review Genetics, 2005, 6(10): 782-792.
[4]Raap A K . Adevance in fluorescence in situ hybridization[J]. Mutation Research, 1998, 400: 297-298.
[5]Hasterok R, Jenkins G, Langdon T. et al. Ribosomal DNA is an effective marker of Brassica chromosomes [J]. Theoretical and Applied Genetics, 2001, 103(4) : 486-490.
[6]Warwick S I, Sauder C. Phylogeny of tribe Brassiceae (Brassicaceae) based on chloroplast rest riction site polymorphisms and nuclear ribosomal internal transcribed spacer and chloroplast trnL intron sequences [J]. Canadian Journal of Botany, 2005, 83(5) : 467-483.
[7]奇文清,李愁学.植物染色体原位杂交技术的发展及应用闭[J].武汉植物学研究, 1996, 14(3): 269-278.
[8]Haaf T. Struchue analysis of a satellite DNA and centromere proteins using extended chromatin and chromosomes[J]. Human Molecular Genetics, 1994, 3: 697-709.
[9]Snowdon R J, Friedt W, Khler A, et al. Molecular cytogenetic localization and characterization of 5S and 25S rDNA loci for chromosome identification in oil seed rape (Brassicanapus L.) [J] . Annals of Botany, 2000, 86(1): 201-204.
[10]Koo D H, Plaha P, LimY P, et al. A high resolution karyotype of Brassica rapa ssp. pekinensis revealed by pachytene analysis and multicolour fluorescence in situ hybridisation[J]. Theoretical and Applied Genetics, 2004, 109(7): 1346-1352.
[11]Florijn R J. High resolution DNA fiber-FISH for genomic DNA mapping and colour bar-coding of large genes[J]. Human Molecular Genetics, 1995, 4: 831-836.
[12]Fukui K, Ohmido N, Khash G S. Variability in rDNA loci in the genus Oryza detected through fluorescence in situ hybridization[J]. Theoretical and Applied Genetics, 1994, 87: 893-899.
[13]Anamthawat-Jonsson K, Heslop-Harrison J P. Isolation and characterization of genome-specific DNA sequences in Triticeae species[J]. Molecular and General Genetics, 1993, 240:151-158.
[14]赵丽娟.端粒序列和rDNA在几种禾本科植物中的定位分析[D].武汉:武汉大学生命科学学院, 2005.
[15]Ning S B. Maize stress-related genes nacl and cld were localized on 10 L and 4 L respectively[J]. Ghromosome Research, 2000, (8): 273.
[16]Ried T, Baldini A, Rand Tc, et al. Simultaneously Visualization of seven different DNA probes by in situ hybridization using fluorescence and digital imaging microscopy[C]. Proceedings of NationalAcademy of Sciences of the U.S.A, 1992, 89: 1388-1392.
[17]Nederlof P M,Van De Flier , Vrolijk J, et al. Fluorescence. Ration measurements of double-labled probes for multiple in stitu hybridization by digital imaging microscopy[J]. Cytometry, 1992, 13:839-845.
[18]Li L J, Yang J L, Tong Q, et al. A Novel Approach to Prepare Extended DNA in Plant [J]. Cytometry Parta, 2005, 63(2): 114-117.
[19]Yang K, Qi H Y, Zhu L Q, et al. Localization of Genes on Extended DNA Fibers (EDFs) in Brassica oleraceaby High-Resolution FISH [J]. Acta Genetica Sinica, 2006, 33(3): 277-284.
[20]Dal-Hoe Koo, Prikshit Plaha, Yong Pyo Lim, et al. A high-resolution karyotype of Brassica rapa ssp. pekinensis revealed by pachytene analysis and multicolor fluorescence in situ hybridization[J]. Theoretical and Applied Genetics , 2004, 109: 1346–1352.
[21]Trask B J. Mapping of human chromosome by two-color fluorescence in situ hybridization of DNA sequences to interphase cell nuclei[J]. American Society and human Genetics ,1991, 48: 1-15.
[22]Van Den Engh, Sachs G, Trask, B. Estimaing genomic distance from DNA sequence location in cell nuclei by a random walk model[J]. Science, 1992, 257(4): 1410-1412.
[23]余舜武,张端品,宋运淳.基因组原位杂交的新进展及其在植物中的应用[J].武汉植物学研究, 2001, 19(3): 248-254.
[24]A K Io K, Vega J M, Han F P. Advances in plant chromosome identification and cytogenetic techniques[J]. Current Opintion in Plant Biology, 2005, 8: 148-154.
[25]King J, Armstead I P, Donnison I S. Physical and genetic mapping.In the grasses Loliumperenne and Festuca pratensis[J]. Genetics, 2002,161: 315-324.
[26]Khru Staleval L I, De-Melo P E, Van-Hu Sden A W. The integration of recombination and physicalmaps in a large-genomemonocot using haploid genome analysis in a trihybrid Allium population[ J]. Genetics, 2005, 169: 1673-1685.
[27]刘三宏,王坤波,宋国立,等.棉花GISH-NOR的初步探讨[J].科学通报, 2005, 50(5): 425-429.
[28]Han F, Fedak G, Benabdelmouna A. Characterization of six wheat Thinopyrum intermedium derivatives by GISH, RFLP and multicolor GISH[J]. Genome, 2003, 46: 490-495.
[29]Bennett M D. The development and use of genomic in situ hybridization (GISH) as a new tool in plant biosystematics[M] //BRANDHAM P E,BENNETTM D.Kew Chromosome Conference IV. Kew: Royal Botanic Gardens, 1995: 167-183.
[30]Wienberg J, Stanyon R, Nash W G, et al. Conservation of human vs. feline genome organization revealed by reciprocalchromosome painting[J]. Cytogenetic Cell Genetic, 1997, 77(3-4): 211-217.
[31]Kejnovsky E, Hobza R, Cermak T, et al. The role of repetitive DNA instructure and evolution of sex chromosomes in plants[J]. Heredity, 2009, 102(6): 533-541.
[32]Schubert I, Fransz P F, Fuchs J, et al. Chromosome painting in plants[J]. Methods in Cell Science, 2001, 23(1-3): 57-69.
[33]Lysak M A, Fransz P F, Ali H B, et al.Chromosome painting in Arabidopsis thaliana[J]. Plant Journal, 2001, 28(6): 689-697.
[34]Lysak M A, Pecinka A, Schubert I.Recent progress in chromosome painting of Arabidopsis and related species[J]. Chromosome Research, 2003, 11(3): 195-204.
[35]Jiang J M, Gill B S.Current status and the future of fluorescence in situ hybridization (FISH) in plant genome research[J]. Genome, 2006, 49(9): 1057-1068.
[36]Han Y H, Zhang Z H, Liu C X, et al. Centromererepositioning in cucurbit species: Implication of the genomic impact fromcentromere activation and inactivation[J]. Proceedings of the National Academy of Sciences, 2009, 106(35): 14937-14941.
[37]Dong F, Tek A L, Frasca A B L, et al. Development and characterization of potato-Solanum brevidens chromosomaladdition substitution lines[J]. Cytogenet Genome Research, 2005, 109(1-3): 368-372.
[38]Tang X M, Bao W D, Zhang W L, et al. Identification of chromosomes frommultiple rice genomes using a universal molecular cytogenetic markersystem[J]. Integrate Plant Biology, 2007, 49(6): 953-960.
[39]向素琼,汪卫星,梁国鲁. 45SrDNA在不同倍性沙田柚染色体上的荧光原位杂交分析[J].园艺学报, 2007, 34(1): 75-80.
[40]孙瑞芬,高安.植物染色体分带及其荧光原位杂交技术研究进展[J].生物技术通报, 2010, (10): 68-75.
[41]Vischi M, Jurman I, Bianchi G, et al. Karyotype of norway spruce by multicolor FISH[J]. Theoretical and Applied Genetics, 2003, 107(4): 591-597.
[42]李宗芸,宋运淳,李立家. Application of fiber-FISH technique in plants genome research[J]. Chinese Bulletin of Botany, 2002, 19(3): 359-364.
[43]Chen C C, Chen C M, Hsu F C, et al. The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences[J]. Theoretical and Applied Genetics, 2000, 101: 30-36.
[44]Li W, Zhang P, Fellers J P, et al . Sequence composition , organization , and evolution of the core Triticeae genome[J]. Plant Journal, 2004 , 40 (4) : 500-511.
[45]Bennetzen J L, Jianxin M A, Devos K M. Mechanisms of recentgenome size variation in flowering plants[J]. Annals of Botany, 2005, 95(1) :127-132.
[46]Zwick M S, Hanson R E, Mcknight T D, et al. A rapid procedure for the isolation of Cot 21 DNA from plants[J] .Genome, 1997, 40: 138-142.
[47]Flavell R B. Repetitive DNA and chromosome evolution in plants[J]. Horticultural Science, 1986, 312: 227-224.
[48]柴守诚,员海燕.高等植物DNA重复序列的主要类型和特点[J].西北植物学报, 1999, 19(3) :555-563.
[49]铁双贵,郑用琏.玉米基因组的简单重复序列遗传研究进展[J].生物工程进展, 2001, 21(5): 59-62.
[50]Morgante M, Hanafey M, Powell W. Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes[J]. Nature Genetic, 2002, 30: 196-200.
[51]Harrison G E, Heslop-Harrison J S. Centromeric repetitive DNA sequences in the genus Brassica[J]. Theoretical and Applied Genetics, 1995, 90(2): 157-165.
[52]Yang T J, Kim J S, Lim K B, et al. The Korea Brassica Genome Project: A glimpse of the Brassica genome based on comparative genome analysis with Arabidopsis[J]. Comparative and Functional Genomics, 2005, 6: 138-146.
[53]Lim K B, De Jong H, Yang T J, et al. Characterisation of rDNAs and tandem repeats in heterochromatin of Brassica rapa[J]. Molecular Cells, 2005a, 19(3): 436-444.
[54]吴伟,李洪杰,王晓鸣.栽培大豆端粒相关序列的克隆及定位[J].大豆科学, 2010, 29(3): 380-384.
[55]Zakian V A. Telomeres: beginning to understand the end [J]. Science, 1995, 270: 1601-1607.
[56]Mao L, Devos K M, Zhu L, et al. Cloning and genetic mapping of wheat-associated sequences[J]. Molecular and General Genetics, 1997, 254: 584-591.
[57]巩学千,龚继明,刘峰,等.筛选和定位含水稻端粒相关序列的BAC克隆[J].中国科学(C辑), 1998, 28: 437-443.
[58]Richards E J, Ausubel F M. Isolation of a higher eukaryotic telomere from Arabidopsis thaliana[J]. Cell, 1988, 53: 127-136.
[59]Ganal M W, Lapitan N L, Tanksley S D. Macrostructure of the tomato telomeres [J]. Plant Cell, 1991, 3: 87-94.
[60]Werner J E, Kota R S, Gill B S, et al. Distribution of telomeric repeats and their role in healing of broken chromosome ends in wheat[J]. Genome, 1992, 34: 844-848.
[61]Burr B, Burr F A, Matz E C, et al. Pinning down loose ends: mapping telomeres and factors affecting their length [J]. The Plant Cell, 1992, 4: 953-960.
[62]Wu T Y, Wang Y X, Wu R. Transcribed repetitive DNA sequences in telomeric regions of rice (Oryza sativa)[J]. Plant Molecular Biology, 1994, 26: 363-375.
[63]Fajkus J, Kovarik A, Kralovics R, et al. Organization of telomeric and subtelomeric chromatin in the higher plant Nicotiana tabacum[J]. Molecular and General Genetics, 1995, 247: 633-638.
[64]张德水,张志永,陈受宜.大豆染色体端粒DNA的Southern及荧光原位杂交(FISH)分析[J].高技术通讯, 1998, 12: 7- 10.
[65]Weiss H, Scherthan,H. Aloe spp. plants with vertebrate-like telomeric sequences[J]. Chromosome Research, 2002, 10:p. 155-164.
[66]李丹.杉木端粒相关序列和5S rDNA序列分析及染色体定位[D].南京:南京林业大学, 2010.
[67]Lapitan L V. Organization and evolution of higher plant nuclear genomes[J]. Genome, 1992, 35: 171-181.
[68]Inge U, Peter G. Nucleotide sequences of the 5.8S and 25S rRNA genes and of the internal transcribed spacers from Arabidopsis thaliana[J]. Nucleic Acids Research, 1990, 18: 4011.
[69]Badaeva E D, Friebe B, Gill B S. Genome differentiation in Aegilops umbellulata Distribulion of highly repetitive DNA sequences on chromosomes of diploid species [J]. Genome, 1996, 39: 293-306.
[70]Fransz P, Armstrong S, Alonso-Blanco C, et al. Cytogenetics for the model system Arabidopsis thaliana[J]. Plant Journal, 1998, 13: 867-876.
[71]Taketa S, Harrison G E, Heslop-Harrison J S. Comparative physical mapping of 5S and 18s-25S rDNA in nine wild Hordeum species and cytotypes[J]. Theoretical and Applied Genetics, 1999, 98: 1-9.
[72]Ansari H A, Ellison N W, Reader S M, et al. Molecular cytogenetic organization of 5S and 18S-26S rDNA loci in white clover (Trifolium repens L.) and related species[J]. Annals of Botany , 1999, 83: 199-206.
[73]Kim J S, Childs K L, Islam-Faridi M N, et al. Integrated karyotyping of sorghum by in situ hybridization of landed BACs[J]. Genome, 2002, 45 (2): 402-412.
[74]Wang K, Guo W Z, Zhang T Z. Development of one set of chromosome specifc microsatellite-containing BACs and their physical mapping in Gossypium hirsutum [J]. Theoretical and Applied Genetics, 2007, 115: 675–682.
[75]Yang T J, Kim J S, Lim K B, et al. The Korea Brassica Genome Project: A glimpse of the Brassica genome based on comparative genome analysis with Arabidopsis[J]. Comparative and Functional Genomics, 2005, 6: 138-146.
[76]轩淑欣.荧光原位杂交技术在大白菜染色体基因定位中的应用研究[D].保定:河北农业大学, 2006.
[77]Choi S R, Teakle G R, Plaha P, et al. The reference genetic linkage map for the multinatioonal Brassica rapa genome sequencing projet[J]. Theoretical and Applied Genetics, 2007, 115: 777-792.
[78]Nakayama S, Fujishita M, Sone T, et al. Additional locus of rDNA sequence specific to the chromosome of the liverwort, Marchantia polymorpha[J]. Chromosome Research, 2001, 9: 469-473.
[79]Koo D H, Hur Y, Jin D C, et al. Karyotype analysis of a Korean cucumber cultivar (Cucumis sativus L. cv. Winter Long) using C-banding and bicolor fluorescence in situ hybridization[J]. Molecules and Cells, 2002, 13: 413-418.
[80]Lim K.B, Yang J, Wang, Y, et al. 2007 Characterization of the centromere and peri-centromere retrotransposons in Brassica rapa and their distribution in related Brassica species[J]. The Plant Journal. 49: 173–183.
[81]巩学千,洪德军,陈受宜,等.水稻端粒相关序列的克隆及鉴定[J].中国科学(C辑), 1998, 28: 324-332.
[82]Fajkus J, Kovarik A, Kralovics R, et al. Organization of telomeric and suhtelomeric chromatic in the higher plant Nicotiaua tahacum[J]. Molecular and General Genetics, 1995,247 :633-638.
[83]Sykorova E ,Cartagena J, Horakova M, et al. Characterization of telomere-subtelomere junctions in Sileue latifolia[J]. Molecular Genetics Genomics, 2003,269:13-20.
[84]Kato A, Lamb .J C, Birchler J A. Chromosome painting using repetitive DNA sequences as probes for somatic chromosome identification in maize[J]. Proceedings of the National Academy of Sciences USA, 2004, 101: 13554-13559.
[85]Kulak S, Hasterok R, Maluszynska J. Karyotyping of Brassica amphidiploids using 5S and 25SrDNA as chromosome markers[J]. Hereditas, 2002, 136: 144-150.
[86]Lim K B, Hans De J, Yang T J, et al. Characterization of rDNAs and Tandem Repeats in the Heterochromatin of Brasscia rapa[J]. Molecules and Cells, 2005, 19(3): 436~444.
[87]Snowdon R J, Friedt W, K?hler A, et al. Molecular cytogenetic localization and characterization of 5S and 25S rDNA loci for chromosome identification in oilseed rape(Brassica napus L.)[J]. Annals of Botany, 2000, 86:201-204.
[88]Hasterok R, Jenkins G, Langdon T, et al. Ribosomal DNA is an effective marker of Brassica chromosomes[J]. Theoretical and Applied Genetics, 2001, 103: 486-490.