假臭草EST-SSRs标记的开发  

Development of Praxelis clematidea EST-SSRs

作　　者：黄敏[1] 陈振玺[1] 刘建福[1] 王奇志[1] 

机构地区：[1]华侨大学化工学院,福建厦门361021

出　　处：《热带农业科学》2014年第3期27-33,44,共8页Chinese Journal of Tropical Agriculture

基　　金：国家标本平台教学标本子平台(No.2005DKA21403-JK);华侨大学2012年实验教学改革与建设课题(No.66661220Y);福建省自然科学基金项目(No.2010J05076)

摘　　要：从NCBI的dbEST数据库中下载假臭草同族植物的EST序列,经EST序列处理及SSR位点查询,首次设计了27对假臭草EST-SSRs引物,以海南省文昌市假臭草DNA为模板,采用单因素和L16(45)正交试验对其EST-SSRs PCR反应体系进行优化。建立了假臭草EST-SSRs最佳20μLPCR反应体系:Mg2+浓度为2.75 mmol/L,模板DNA用量为35 ng,Tag DNA聚合酶为2.25 U,dNTPs浓度为0.3 mmol/L,引物浓度为0.6μmmol/L。并用14个不同来源的假臭草样本对其体系进行验证实验,结果均能获得稳定的、清晰明亮的扩增谱带。假臭草EST-SSRs标记的开发为深入研究其遗传多样性提供了基础。In this study, we download ESTs form dbEST database and first designed 27 primer pairs for Praxelis clematidea after EST pretreatment and SSR locus identification. The sample were collected from Wenchang City in Hainan province as DNA template, and the optimization and establishment of EST-derived SSR-PCR reaction system for Praxelis clematidea was carried out by single factor test and orthogonal experiment design of L16（45）. The result was indicated that the optimal EST-SSRs PCR reaction system （20μL） was contained 2.75 mmol/L Mg2＋, 35 ng DNA template, 2.25 U Tag DNA polymerase, 0.3 mmol/L dNTPs and 0.6 μmol/L primer. This system was tested by 14 Praxelis clematidea from different places, which stable and clear polymorphic bands were acquired. Accordingly, the development of EST-SSRs can be used for the genetic diversity research of Praxelis clematidea.

关 键 词：假臭草 引物设计 优化 

分 类 号：Q754[生物学—分子生物学] S451[农业科学—植物保护]