白花甜菜9号染色体BAC芯片的构建及初步应用

【作者】 王志伟；

【导师】 郭德栋； 王桂芝；

【作者基本信息】 黑龙江大学 ， 生物化学及分子生物学， 2004， 硕士

【摘要】 甜菜无融合生殖单体附加系M14品系是通过二倍体栽培甜菜与四倍体野生白花甜菜杂交获得的异源三倍体再经过与栽培甜菜回交得到的后代，该材料经过鉴定为韭型二倍体孢子生殖的配子体无融合生殖材料，并认为无融合生殖基因定位在附加的白花甜菜染色体上。现已利用M14品系构建了BIBAC文库，并从中筛选出白花甜菜9号染色体BAC克隆2，377个，组成9号染色体BAC文库，该文库覆盖该染色体3.8倍。 本课题成功的发明了一种高效的BAC质粒提取方法，并建立了一种不同于常规芯片的BAC芯片构建平台，同时以白花甜菜9号染色体BAC2，377个克隆为基础制备了BAC芯片，经过阳性探针和随机引物探针检测，结果证明该芯片制备重复性好、背景噪声低、杂交信号清晰。同时将10个M14特异表达的EST定位在该BAC芯片上，证实为9号染色体特异表达。并利用M14和栽培甜菜的花期特异表达的mRNA对9号染色体BAC进行了初步表达分析。更多还原

【Abstract】 M14 beet is a monosomic addition line of Beta corolliflora Zoss which constitutes of the normal 18 B. vulgaris chromosomes plus Beta corolliflora chromosome 9. Through the investigation we found the transmission frequencies of M14 up to 96% in four generations. Our studies revealed that the reproduction mode of M14 is allium-odorum type diplospory, autonomous endospermgenesis but pollen stimulation is needed. We believed the gene(s) controlling apomixis may be located on the alien chromosome from Beta corolliflora Zoss.In order to clone the putative gene(s) controlling apomixes of M14, we constructed a plant-transformation-competent binary BAC library from the genomic DNA of M14, the library consists of 49,920 clones with an average insert size of 127kb, representing 7.5xhaploid genome equivalents and providing a grater than 99% probability of isolating single copy DNA sequences from this library. We used pBC1054 probe from the alien chromosome Beta corolliflora genome-specific dispersed repetitive DNA sequences to isolate BAC clones derived from the alien chromosome in the library. In result a total of 2.377 positive clones were obtained and rearrayed into a sublibary specific for Beta corolliflora chromosome 9, these clones represent 3.8xhaploid plus Beta corolliflora chromosome 9 equivalents.We invented a series new methods to isolated BAC plasmid and convalent DNA-glass chemistries attach to an unmodified glass surface. Make use of these technology we invented the BAC chip which 2,377 BAC DNA from Beta corolliflora chromosome 9 in the array. It can testified that the false-positive rate was very low through the positive probe and randomprimes detected. Our microarray system is fundamentally different from most current microarray technologies in that activated DNA is printed on nature glass surfaces, a strategy that invariably introduces hybridization backgrounds. Finally we make the simple express analysis between Ml4 beet and B.vulgaris through the BAC chip.更多还原