沉香DNA的提取及其ITS2-PCR体系的优化

刘少烽; 陈晓东; 朱爽; 陈晓颖; 钟兆健; 章卫民; 高晓霞

doi:10.13422/j.cnki.syfjx.2015020007

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沉香DNA的提取及其ITS2-PCR体系的优化

工艺与制剂 | 更新时间：2024-01-04

- 沉香DNA的提取及其ITS2-PCR体系的优化
- Extraction of DNA from Aquilariae Lignum Resinatum and Optimization of Its ITS2-PCR System
- 中国实验方剂学杂志 2015年21卷第2期页码：7-11
- 作者机构：
- 作者简介：
- 基金信息：
  
  国家自然科学基金项目(81102418);广东省科技计划项目(2012A030100014);广东省科学院野外科学实验站基金项目(Sytz201204)
- DOI：10.13422/j.cnki.syfjx.2015020007
  中图分类号： R284.2
- 纸质出版日期：2015
- 稿件说明：
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刘少烽, 陈晓东, 朱爽, 等. 沉香DNA的提取及其ITS2-PCR体系的优化[J]. 中国实验方剂学杂志, 2015,21(2):7-11.

LIU Shao-feng, CHEN Xiao-dong, ZHU Shuang, et al. Extraction of DNA from Aquilariae Lignum Resinatum and Optimization of Its ITS2-PCR System[J]. Chinese journal of experimental traditional medical formulae, 2015, 21(2): 7-11.
刘少烽, 陈晓东, 朱爽, 等. 沉香DNA的提取及其ITS2-PCR体系的优化[J]. 中国实验方剂学杂志, 2015,21(2):7-11. DOI： 10.13422/j.cnki.syfjx.2015020007.

LIU Shao-feng, CHEN Xiao-dong, ZHU Shuang, et al. Extraction of DNA from Aquilariae Lignum Resinatum and Optimization of Its ITS2-PCR System[J]. Chinese journal of experimental traditional medical formulae, 2015, 21(2): 7-11. DOI： 10.13422/j.cnki.syfjx.2015020007.

摘要

目的: 建立沉香的总DNA提取方法及rDNA第二内转录间隔区序列(ITS2)聚合酶链式反应(PCR)扩增体系。方法: 分别采用改良的CTAB法、改良的CTAB法联合DNA提取试剂盒法及DNA提取试剂盒法提取沉香总DNA

利用通用引物ITS2P1/ITS2P2 扩增rDNA ITS2目的片段

通过正交试验优选沉香的PCR扩增条件并对目的片段进行测序。结果: 改良的CTAB法联合DNA提取试剂盒法提取总DNA的A260 nm/A280 nm 1.79

质量浓度350 mg ·L-1

PCR扩增目的片段产率最高。不同因素水平对沉香ITS2-PCR反应的影响顺序为模板DNA>引物>Taq DNA聚合酶>dNTP。最佳反应体系为20 μL体系

DNA模板40 ng

引物0.2 mmol ·L-1

dNTP 0.5 mmol ·L-1

Taq DNA聚合酶1.5 U。测序获得的目前片段长度230 bp

与GenBank中瑞香科沉香属白木香序列相似性100%。结论: CTAB法联合DNA试剂盒法可简单、快速获得高质量沉香总DNA

正交试验可快速获得ITS2目的片段

为沉香的分子鉴定和系统发育分析提供参考。

Abstract

Objective: To establish extraction method of total DNA from Aquilariae Lignum Resinatum and its rDNA ITS2 PCR amplified system. Method: Modified CTAB method

DNA extraction kit method

modified CTAB method combined with DNA extraction kit were employed to extract total DNA from Aquilariae Lignum Resinatum.Fragment of rDNA ITS2 barcode amplified with universal primers of ITS2P1/ITS2P2

PCR amplified conditions were optimized by orthogonal test

rDNA ITS2 barcode was characterized by sequencing and blasting in GenBank. Result: A260 nm/A280 nm of total DNA from Aquilariae Lignum Resinatum was 1.79 which extracted by modified CTAB method combined with DNA extraction kit and the concentration of total DNA was 350 mg · L-1

PCR amplified fragment was highest.Effects of different level factors about ITS2-PCR reaction from large to small successively was template DNA>primers>Taq DNA polymerase>dNTPs.Optimum PCR(20 μL)reaction system was template DNA of 40 ng

primers of 0.2 mmol · L-1

dNTP of 0.5 mmol · L-1 and Taq DNA polymerase of 1.5 U through the intuitive analysis.Both the length of two sample sequences were 230 bp

identities were 100% by comparing with Aquilariae Lignum Resinatum in GenBank. Conclusion: Total DNA from Aquilariae Lignum Resinatum with high quality can receive by modified CTAB method combined DNA kit method

ITS2 barcode can be quickly obtain by this optimized process

this study provides basis for molecular identification and phylogenetic analysis of Aquilariae Lignum Resinatum.

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