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植物研究 ›› 2010, Vol. 30 ›› Issue (5): 588-593.doi: 10.7525/j.issn.1673-5102.2010.05.012

• 论文 • 上一篇    下一篇

华中五味子ISSR-PCR反应体系优化及引物筛选

罗成1;熊宇婷1;顾蔚1,2,3*;王喆之1,2,3   

  1. 1.陕西师范大学生命科学学院,西安 710062;2.陕西师范大学药用资源与天然药物化学教育部重点实验室,西安 710062;3.西北濒危药材资源开发国家工程实验室,西安 710062
  • 收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2010-09-20 发布日期:2010-09-20
  • 通讯作者: 顾蔚
  • 基金资助:
     

Optimization of ISSR-PCR Reaction System and Primers Screening in Schisandra sphenanthera

LUO Cheng;XIONG Yu-Ting;GU Wei;*;WANG Zhe-Zhi;   

  1. 1.College of Life Science,Shaanxi Normal University,Xi’an 710062;2.Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry,Xi’an 710062;3.National Engineering laboratory for Resource Development of Endangered Crude Drugs in Northwest of China,Shaanxi Normal University,Xi’an 710062
  • Received:1900-01-01 Revised:1900-01-01 Online:2010-09-20 Published:2010-09-20
  • Contact: GU Wei
  • Supported by:
     

摘要: 系统研究了华中五味子ISSRPCR反应体系中的主要影响因子,确立华中五味子ISSR-PCR最适反应体系,并筛选出12条有效引物。单因子实验结果显示,华中五味子ISSR-PCR反应体系中各主要成分的适宜浓度范围为,Mg2+ 1.50~3.50 mmol·L-1,dNTPs 0.10~0.35 mmol·L-1,引物0.25~0.60 μmol·L-1Taq酶0.50~1.50 U。4因子3水平正交实验确立了最适反应体系,即20 μL体系中包含2.50 mmol·L-1 Mg2+、0.20 mmol·L-1 dNTPs、0.25 μmol·L-1引物、1.50 U Taq酶、60 ng DNA模板、2.50 μL 10×PCR Buffer。本研究为华中五味子种质资源的评估及遗传多样性分析奠定基础。

关键词: 华中五味子, ISSR, 体系优化, 引物筛选

Abstract: The main influential factors of ISSR-PCR reaction system in Schisandra sphenanthera Rehd. et Wils. were systematically studied, optimization of ISSR-PCR system was established, and 12 effective ISSR primers were selected. Single-factor experimental results showed that the suitable concentration of composition of ISSR-PCR systen was 1.50~3.50 mmol·L-1 of Mg2+, 0.10~0.35 mmol·L-1 of dNTPs, 0.25~0.60 μmol·L-1 of primer and 0.50~1.50 U of Taq DNA polymerase, respectively. The optimal ISSR-PCR reaction system was defined by orthogonal experiment with four-factor of three-level, i.e. 20 μL reaction system contained 2.50 mmol·L-1 of Mg2+, 0.20 mmol·L-1 of dNTPs, 0.25 μmol·L-1 of primer, 1.50 U of Taq DNA polymerase, 60 ng of DNA template, 2.50 μL of 10×PCR Buffer. The present study could be used in the research for evaluation of germplasm resources and analysis of genetic diversity in S.sphenanthera Rehd. et Wils..

Key words: Schisandra sphenanthera Rehd. et Wils., ISSR, optimization of system, primers screening

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