摘要
以藜芦为试验材料,通过L16(45)正交试验与单因素实验两种方法对藜芦ISSR-PCR反应体系中的5个主要因素(模板DNA,引物, Taq DNA聚合酶, dNTPs, Mg2+)进行筛选与优化,建立藜芦ISSR-PCR最佳反应体系;在此优化体系下,从100条引物中筛选适宜的ISSR引物,设置温度梯度检测各引物最佳退火温度。结果表明,藜芦20μL ISSR-PCR反应体系包括:0.30μmol/L引物、24 ng/20μL模板DNA、2 U/20μL Taq DNA聚合酶、0.20 mmol/L dNTP、1.00 mmol/L Mg2+;从100条引物中筛选出16条扩增稳定、多态性丰富的ISSR引物。利用14份藜芦样品验证该优化体系的稳定性,证明该体系稳定可靠,可为后续藜芦的遗传多样性分析研究提供帮助。
In this research,the ISSR-PCR amplification system in Veratrum nigrum was optimized by using the L16(45) orthogonal experimental design combined with signal factor analysis to screen 5 main factors(DNA template,primers,Taq DNA,dNTPs and Mg2+) of PCR reaction to establish the optimal ISSR-PCR reaction system of Veratrum nigrum.Based on this,suitable ISSR primers were screened out,and the optimal annealing temperature of selected primers were optimized by gradient method.The results showed that 20 μL ISSR-PCR reaction system contained 0.30 μmol/L primer,24 ng/20 μL template DNA,2 U/20 μL Taq DNA polymerase,0.20 mmol/L dNTP and 1.00 mmol/L Mg2+;16 amplified primers with stable amplification and rich polymorphism were selected from100 ISSR primers.Veratrum nigrum of 14 samples with the optimized ISSR system confirmed that the established ISSR-PCR was steady,thus the system provided the basis for the genetic analysis of Veratrum nigrum.
作者
唐雨晨
岳鑫
张屏
程杰
包保全
Tang Yuchen;Yue Xin;Zhang Ping;Cheng Jie;Bao Baoquan(College of Pharmacy,Inner Mongolia Medical University,Hohhot,010110)
出处
《分子植物育种》
CAS
CSCD
北大核心
2020年第15期5054-5062,共9页
Molecular Plant Breeding
基金
中国国家留学基金((2018)10006号)
国家自然科学基金地区项目(81460651)共同资助。
