摘要
目的建立稳定性高、重现性好,适合地枫皮遗传差异分析的ISSR-PCR反应体系。方法利用正交试验设计,对地枫皮ISSR-PCR反应的5个因素(Mg2+、dNTPs、引物、Taq酶和模板DNA浓度)进行优化,PCR结果用SPSS统计软件分析,在此基础上对PCR反应过程中的退火温度和循环次数进行梯度检测。结果大部分因素的不同水平对PCR反应有显著影响,其中Taq酶影响最大;地枫皮ISSR-PCR最佳反应体系(20μL)为:Mg2+1.60 mmol/L、dNTP 0.22 mmol/L、引物0.90μmol/L、Taq酶0.50 U、模板DNA 70.00 ng;最佳退火温度和循环次数分别为51.8℃和40次;从62条引物中筛选出13条扩增稳定、多态性丰富的ISSR引物。结论建立的地枫皮ISSR-PCR反应体系,经过16份地枫皮样品检验,证明该体系稳定可靠,可用于地枫皮遗传差异分析。
Objective To establish a stable, reproducible, and suitable reaction system for ISSR analysis of genetic differences in Illicium difengpi. Methods The ISSR-PCR amplification system on/. difengpi in five factors （Mg^2＋, dNTPs, primers, Taq DNA polymerase, and DNA template） was optimized by orthogonal design, and the PCR result was analyzed by SPSS. Then based on the optimal ISSR-PCR amplification system, the annealing temperature and cycle times in PCR were proposed by gradient determenation. Results Most of the factors in different levels had the significant effects on the result of PCR, and the most remarkable factor was the quantity of Taq DNA polymerase. The optimized ISSR-PCR reaction system （20 μL） for/. difengpi was constructed of Mg^2＋ （1.60 mmol/L）, dNTP （0.22 mmol/L）, primer （0.90μmol/L）, Taq polymerase （0.50 U）, and DNA template （70.00 ng）. The optimized annealing temperature and cycle times were 51.8 ~C and 40 cycles, respectively. Thirteen ISSR primers with stable amplification and abundant polymorphism were selected from 62 ISSR primers. Conclusion The established and optimized ISSR reaction system is stable and credible according to the testing results of 16 samples of I. difengpi, and provides the basis for the genetic analysis of I. difengpi.
出处
《中草药》
CAS
CSCD
北大核心
2013年第5期610-615,共6页
Chinese Traditional and Herbal Drugs
基金
中国科学院"西部之光"人才培养计划项目[(2009)24]
广西科技创新能力与条件建设(0992028-10)
广西区专项基金[(2008)80]
广西科技攻关项目(10124008-1)
广西植物研究所基本业务费项目(09030)
关键词
地枫皮
ISSR—PCR
正交设计
最佳退火温度
最佳循环次数
遗传差异
Illicium difengpi K. I. B. et K. I. M.
ISSR-PCR
orthogonal design
optimized annealing temperature
optimized cycle times
genetic difference
