黑麦1R染色体的显微分离、体外扩增及扩增产物的鉴定

MICRODISSECTION AND AMPLIFICATION IN VITRO OF CHROMOSOME 1R IN RYE (Secale cereale L.) AND DETERMINATION OF THE AMPLIFIED PRODUCTS

借助Leitz显微操作器 ,在国产倒置显微镜下 ( 40 0× )用玻璃针对处于有丝分裂中期的黑麦根尖细胞中的 1R染色体成功地进行了分离。分离出来的 1R染色体转入 0 .5ml的Eppendorf管中 ,用蛋白酶K处理 ,把DNA释放出来 ;经Sau3A酶切 ,再与人工合成的Sau3A连接头连接 ;以连接头的一条链的核苷酸顺序片段为引物对DNA酶切片段进行了PCR扩增。琼脂糖凝胶电泳显示扩增产物的长度大约为 30 0～ 1 0 0 0bp。以生物素分别标记的黑麦总体DNA和小麦rDNA为探针进行斑点杂交 ,结果表明PCR扩增产物确实来源于黑麦的 1R染色体DNA。这个方法为构建黑麦 1R染色体亚基因组文库和筛选 1R染色体特异性探针奠定了基础。

The chromosome 1R of rye was microdissected and isolate d under reverse -microscope with a manipulator from the cells of root tip at mitotic metaphase. Single chromosome 1R was then transferred into 0.5 ml centrifuge tube. After the treatment with Protease K, the DNA of chromosome 1R was digested with restric tion enzyme Sau3A, then the Sau3A linker-adaptor was ligated to the ends of the DNA fragments, which was subsequently amplified by means of linker-adapter polym erase chain reaction (LA-PCR) with one of chains of the linker-adapter as primer . By dot blotting with biotinlatyled rDNA of wheat and genome DNA of rye as prob es, it was confirmed that the amplified products by LA-PCR are derived from the DNA of chromosome 1R in rye. This work would facilitated to construct the sub-ge nomic DNA library of chromosome 1R and to screen the specific probes of chromoso me 1R in rye.