茶树叶绿体DNA的PCR-RFLP反应体系优化

doi:10.7506/spkx1002-6630-201306016

食品科学 ›› 2013, Vol. 34 ›› Issue (6): 73-76.doi: 10.7506/spkx1002-6630-201306016

茶树叶绿体DNA的PCR-RFLP反应体系优化

陈盛相，齐桂年*，李欢

四川农业大学园艺学院茶学系，四川雅安 625014

收稿日期:2011-11-21 修回日期:2013-01-25 出版日期:2013-03-25 发布日期:2013-03-01
通讯作者: 齐桂年 E-mail:guinian5612@sina.com
基金资助:
四川省科技厅项目

Optimization of RFLP-PCR Reaction System for cpDNA in Tea Plants

CHEN Sheng-xiang，QI Gui-nian*，LI Huan

Department of Tea Science, College of Horticulture, Sichuan Agricultural University, Ya’an 625014, China

Received:2011-11-21 Revised:2013-01-25 Online:2013-03-25 Published:2013-03-01
Contact: QI guinian E-mail:guinian5612@sina.com

摘要/Abstract

摘要： 通过正交设计对影响茶树聚合酶链反应-限制性片段长度多态性(PCR-RFLP)反应体系的主要因素进行优化，快速确立适合茶树叶绿体DNA PCR-RFLP分析的扩增体系和酶切体系。结果表明：最佳PCR扩增体系为100ng模板DNA、200μmol/L dNTPs、1.5mmol/L MgCl2、50ng叶绿体引物、3U TaqDNA聚合酶、加ddH2O至25μL；最佳酶切体系为6μL扩增产物用量、2U限制性内切酶量、1×限制性内切酶buffer、加ddH2O至15μL，37℃酶切6h。利用优化的反应体系，对30个茶树品种叶绿体DNA进行PCR-RFLP扩增，可获得清晰扩增图谱和多态性酶切图谱。

关键词: 茶树, 叶绿体DNA, PCR-RFLP, 优化

Abstract: Optimal reaction system of PCR-restriction fragment length polymorphism (RFLP) for cpDNA in tea plants was investigated by orthogonal array design. Results showed that the optimal amplification system was 100 ng DNA template, 200 μmol/L dNTPs, 1.5 mmol/L MgCl2, 50 ng primer, 3U Taq DNA polymerase, and addition of ddH2O up to total volume of 25 μL. The optimal digestion system included 6 μL amplified product, 2 U endonuclease, 1 × endonuclease buffer in digestion solution, digestion time of 6 h and digestion temperature of 37 ℃, and addition of ddH2O up to total volume of 15 μL. Under the optimal reaction system, 30 camellia cultivars were analyzed and polymorphic digestion maps were obtained.

Key words: tea plant, chloroplast DNA, PCR-RFLP (restriction fragment length polymorphism), optimization

中图分类号:

S571.1

陈盛相，齐桂年*，李欢. 茶树叶绿体DNA的PCR-RFLP反应体系优化[J]. 食品科学, 2013, 34(6): 73-76.

CHEN Sheng-xiang，QI Gui-nian*，LI Huan. Optimization of RFLP-PCR Reaction System for cpDNA in Tea Plants[J]. FOOD SCIENCE, 2013, 34(6): 73-76.

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茶树叶绿体DNA的PCR-RFLP反应体系优化

Optimization of RFLP-PCR Reaction System for cpDNA in Tea Plants

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