摘要
建立了玉米单染色体的分离及体外扩增的方法。取95％乙醇固定后经果胶酶和纤维酶酶解的根尖制备染色体标本，用自制的微细玻璃针在倒置显微镜下挑取目的染色体。染色体DNA经Sau3A酶切后与人工合成的Sau3A连接接头连接，经两次PCR扩增获得足以用于构建单染色体DNA文库的扩增产物。片段大小为0．3～5kb，多数为0．5～3．5kb．与前人研究方法相比，所需底物量少（只需1条染色体），扩增片段大，为植物中小型染色体分离、体外扩增进而进行单染色体DNA文库构建奠定了基础。
A method of single chromosome isolation and amplification in Zea mays wasestablished. Root tips fixed by 95% ethonal were digested with an enzyme mixture ofcellulase and pectolyase, then used to prepare chromosome samples. The targetchromosome was microdissected by using a hand-made microglass needle under aninverted microscope. After chromosome DNA was digested by Sau3A, chromosomeDNA fragments were ligated to Sau3A linker-adaptor, then amplified by two roundPCR PCR preducts were enough to construct a library of specific chromosome. Thesize of DNA fragments in PCR products varied from 300 to 5 000bp with apredominance of fragments in the 500-3 500bp range. Compared with previous results,in this method, the template was less (one chromosome is enough) and the size ofPCR products was longer. The method may be suitable for single chromosomeisolation and amplification in other plants with small chromosomes like maize.
关键词
玉米
染色体
分离
DNA扩增
Zea mays, Chromosome microdissection, DNA amplification
