黄瓜SRAP-PCR反应体系的建立.pdfVIP

北基 学报 2008，1 7(3)：274 279 Acta Agriculturae Boreali—occidentalis 黄瓜SRAP—PCR反应体系的建立 柴丹丹，陈书霞，孟焕文，程智慧 ，李亚利 (西北农林科技大学园艺学院，陕西杨凌 7l21oo) 摘 要：为了建立黄瓜适宜的SRAP(相关序列多态性)反应体系，以长形白皮少刺黄瓜 13-2—2和圆形白皮黄 瓜 3为材料，分析了不同来源的Taq酶及其浓度、Mg 浓度、dNTP浓度、DNA模板浓度、引物浓度对扩增 结果的影响，并比较了琼脂糖凝胶和变性聚丙烯酰胺凝胶电泳检测扩增产物多态性的差异。结果表明，在 12．5 L反应体系中，Taq酶适用Takara酶，其最适用量为0．5U；DNA模板最适浓度为3O ng，Mg”最适浓 度为2．0 mmol／L，引物最适浓度为0．96 mol／L，dNTP最适浓度为 200~rnol／I 。6 的变性聚丙烯酰胺电 泳检测扩增产物效果比琼脂糖检测效果好。用不同黄瓜材料的基因组 DNA两次 SRAP—PCR扩增，6对引物 均能扩增出清晰且重复性好的谱带。因而建立的黄瓜SRAP反应体系稳定可靠。 关键词：黄瓜；SRAP；反应体系；琼脂糖凝胶；聚丙烯酰胺凝胶 中图分类号：$642．2 文献标识码：A 文章编号：1004—1389(2008)03—0274—06 Establishment of SRAP-PCR Reaction System on Cucumber CHAI Dan—dan，CHEN Shu—xia，MENG HHall—wen，CHENG Zhi-hui and LI Ya-1i (College of Horticulture．Northwest A＆ F University．Yangling Shaanxi 712100，China) Abstract：To establish appropriate cucumber SRAP(related polymorphism)reaction system，factors that affect the amplified result of SRAP such as Taq concentration grads，different Taq produced by different factories，Mg计 concentration grads，dNTP concentration grads，DNA template eoneentra— tion grads，primer concentration grads were optimized using cucumber as material，and the tWO meth— ods of analyzing PCR products including agarose gels and denaturing polyacylamide gels were corn— pared．It shows that in 12．5 uL system the proper concentration of Taq was 0．5U，the proper tern— plate concentration was 30 ng，the proper concentration of Mg was 2．0 mmol／L，the proper primer concentration was 0．96／~mol／L，and the proper dNTP concentration was 200／~mol／L，the Taq pro— duced by Takara was chosen from four factories，and the result of analyzing PCR products with 6 denaturing polyacylamide gels was better than agarose gels．In this tWO SRAP—PCR amplifications with different cucumber template，six