摘要
旨在建立稳定可靠的云生毛茛ISSR-PCR反应体系。采用正交试验设计方法,对影响云生毛茛ISSR-PCR扩增结果的Mg2+、d NTP、Taq DNA聚合酶、引物、模板DNA五个因素进行优化筛选,对反应程序进行优化,建立适用于云生毛茛的最佳反应体系和扩增程序,并对反应体系和扩增程序进行验证;在此基础上筛选多态性好的ISSR引物,采用梯度法筛选各个引物的最适退火温度。结果表明,云生毛茛20μL ISSR-PCR的最佳反应体系为:模板DNA 30 ng,Mg2+1.95 mmol/L,Taq DNA聚合酶0.04U/μL,d NTP 0.150 mmol/L,引物0.5μmol/L;最佳反应程序为:94℃预变性5 min;94℃变性20 s,49.6-60.6℃复性1 min,72℃延伸100 s,38个循环;72℃下延伸6 min。在优化的反应体系和反应程序条件下,从100条ISSR引物中筛选获得16条ISSR扩增引物,并确定了引物各自的最适退火温度。经过不同居群云生毛茛的验证,证明优化后体系扩增条带清晰且重复性好,可用于后续云生毛茛遗传多样性的研究。
This work is aimed to establish a stable ISSR-PCR system for Ranunculus nephelogenes var. nephelogenes. A L16 （ 45 ） orthogonal design was used to screen 5 main parameters （ Mg2＋, dNTP, Taq DNA polymerase, primer, and template DNA ） ; then the process of ISSR-PCR reaction was optimized, and consequently the optimal reaction system and amplifying procedure for R. nephelogenes vat. nephelogenes was established ; further, the optimized reaction system and amplifying procedure was verified. Results showed that the optimal concentrations in 20 ~L reaction mixture were template DNA 30 ng, Mg2~ 1.95 mmol/L, Taq DNA polymerase 0.04 U/~L, dNTP 0.150 mmol/L, and primer 0.5 ~tmol/L. The optimal reaction procedure was ： pre-denaturalization for 5 mins at 94℃, denaturalization for 20 s at 94℃, renaturation for 1 min at 49.6-60.6℃, followed by 38 cycles of extension for 100 s at 72℃, and final extension for 6 rain at 72℃. Under the above optimized reaction system and procedure, 16 amplified primers were screened from 100 ISSR primers, and the optimal annealing temperature for each primer was determined. Verification in R. nephelogenes var. nephelogenes of different populations with the optimized ISSR system confirmed that bands amplified were clear and steady, thus the established ISSR-PCR could favor further studies on the genetic diversity of R. nephelogenes var. nephelogenes.
出处
《生物技术通报》
CAS
CSCD
北大核心
2016年第9期65-71,共7页
Biotechnology Bulletin
基金
国家自然科学基金项目(31300269)
青海(应用)基础研究计划项目(2013-Z-756)
关键词
云生毛茛
ISSR-PCR
正交实验设计
体系优化
引物筛选
Ranunculu, nephelogenes var. nephelogenes
ISSR-PCR
orthogonal design
optimization of system
primer selection
