摘要
显微分离出黑麦（ＳｅｃａｌｅｃｅｒｅａｌｅＬ．）１Ｒ染色体，用ＣｏｈｅｓｉｖｅａｄａｐｔｅｒｓｓｉｎｇｌｅｐｒｉｍｅｒＰＣＲ（ＣＡＳＰＰＣＲ）方法进行体外扩增，以ＤＩＧ１１ｄＵＴＰ标记扩增产物为探针，进行Ｓｏｕｔｈｅｒｎ分子杂交，结果表明扩增产物来自黑麦１Ｒ染色体。用１／１０体积的连接物转化Ｅ．ｃｏｌｉＤＨ５α，获得１００００多个重组菌落。经酶切分析，克隆子的插入片段为２５０～５００ｂｐ。
Two 1R chromosomes of Secale cereale L. were isolated from one metaphase cell by means of chromosome micro isolation, and the chromosomal DNA was amplified adopting the cohesive adapters single primer polymerase chain reaction (CASP PCR) technique. The CASP PCR products were labeled as probes. The results of Southern blot hybridization confirmed that the CASP PCR products derived from the chromosome 1R were homologous with the genomic DNA of S. cereale. The clones of PCR products were obtained with high efficiency. Over 10 000 recombinant clones were obtained from one tenth of the ligation mixture which was transferred into the competent E.coli DH5α. The size of the inserted fragments of clones ranged from 250 bp to 500 bp. This research has established the foundation for further selection of chromosome 1R markers.
基金
国家自然科学基金
关键词
黑麦
1R染色体
微切微克隆
Secale cereale, Chromosome 1R, Microdissection and microcloning, Cohesive adapters single primer polymerase chain reaction
