用于目标序列染色体定位的镜鲤BAC-FISH体系构建

为了构建用于镜鲤(Cyprinus carpio var. specularis)特定基因组序列染色体定位的实验体系, 在细菌人工染色体(Bacterial Artificial Chromosome, BAC)文库筛选池中对已知短序列基因组片段进行PCR扩增, 筛选出包含目标序列的BAC克隆, 提取BAC质粒进行缺刻平移标记制备探针, 开展荧光原位杂交(Fluorescence in situ hybridization, FISH)实验。通过对染色体片前处理、BAC质粒探针制备、C0t-1 DNA封闭基因组重复序列、预杂交、荧光染料选择、信号放大等一系列实验条件和方法的探索优化, 成功实现了目标序列在镜鲤有丝分裂中期染色体上的定位。定位对象既包括在染色体上有单一位点的序列, 如斑马鱼微卫星标记Z6884和Z4268, 也包括在染色体上有多个位点的重复序列, 如黄河鲤性别相关标记CCmf1。来自斑马鱼同一条染色体上的两个微卫星标记被分别定位于镜鲤不同染色体上, 为鲤鱼染色体数目加倍的进化假设提供了一项直接实验证据, 同时将现有遗传连锁图谱与染色体对应起来, 可作为染色体识别和细胞遗传学图谱构建的依据。黄河鲤性别相关重复序列被定位于不少于四条染色体上, 为性别决定相关基因的筛查提供了研究线索。这一BAC-FISH实验体系将成为鲤细胞遗传学图谱构建、基因组进化和比较基因组学研究中的重要研究工具。  

CONSTRUCTION OF A BAC-FISH EXPERIMENTAL SYSTEM FOR LOCALIZING SPECIFIC SEQUENCES ON MIRROR CARP CHROMOSOMES

An experimental system has been constructed for localizing specific genomic sequences on mirror carp (Cyprinus carpio var. specularis) chromosomes. In order to obtain individual BAC clones containing the target sequences, short genomic sequences were amplified by PCR in bacterial artificial chromosome (BAC) screening pools. BAC plasmid DNA was isolated and labeled by nick translation to prepare fluorescence in situ hybridization (FISH) probes. Via optimizing a series of protocols for chromosome slides pre-treatment, BAC plasmid probe preparation, repetitive sequences blocking by C0t-1 DNA, pre-hybridization, candidate fluorescence dyes evaluation, signal amplification and so on, the BAC-FISH system has been successfully established for mirror carp metaphase chromosomes, which is able to localize both single-copy markers and repetitive sequences. As supporting evidence to the genome duplication hypothesis of carp evolution, two microsatellite markers from zebrafish chromosome 17, namely Z6884 and Z4268, have been localized on two separate mirror carp chromosomes, with one marker on each chromosome. A male-specific marker CCmf1 from Yellow River carp has been localized on at least four chromosomes of mirror carp, suggesting that it might be a repetitive sequence useful for sex determining gene searching. The established mirror carp BAC-FISH experimental system would serve as a powerful tool for carp cytogenetic mapping, genome evolution and comparative genomics research.