【作者】 李九欢；

【导师】 申书兴； 轩淑欣；

【作者基本信息】 河北农业大学 ， 蔬菜学， 2012， 硕士

【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。更多还原

【Abstract】 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.更多还原