[1]丁信誉,邱帅,席梦利*,等.东方百合ISSR-PCR反应体系的正交优化[J].南京林业大学学报(自然科学版),2012,36(05):042-46.[doi:10.3969/j.jssn.1000-2006.2012.05.007]
 DING Xinyu,QIU Shuai,XI Mengli*,et al.Optimizing the ISSR-PCR reaction system for oriental lily based on orthogonal design[J].Journal of Nanjing Forestry University(Natural Science Edition),2012,36(05):042-46.[doi:10.3969/j.jssn.1000-2006.2012.05.007]
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东方百合ISSR-PCR反应体系的正交优化
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
36
期数:
2012年05期
页码:
042-46
栏目:
研究论文
出版日期:
2012-09-30

文章信息/Info

Title:
Optimizing the ISSR-PCR reaction system for oriental lily based on orthogonal design
作者:
丁信誉邱帅席梦利*施季
南京林业大学,林木遗传与生物技术省部共建教育部重点实验室,江苏南京210037
Author(s):
DING Xinyu QIU Shuai XI Mengli* SHI Jisen
Key Laboratory of Forest Genetics and Biotechnology Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
关键词:
东方百合ISSR-PCR正交设计
Keywords:
oriental lily ISSR-PCR orthogonal design
分类号:
Q948
DOI:
10.3969/j.jssn.1000-2006.2012.05.007
文献标志码:
A
摘要:
以东方百合‘Constanta’和‘Sorbonne’以及9份‘Constanta’ × ‘Sorbonne’的F1代为试材,采用改良的CTAB法提取百合嫩叶DNA,利用正交设计 L16(45)对东方百合ISSR-PCR 反应体系的Taq 酶、Mg2+、模板 DNA、dNTPs和引物5种因素在4个水平上进行优化试验,采用直观分析结合方差分析评价扩增结果。最终获得的最佳反应体系为:在20 μL 反应体系中,含Taq DNA 聚合酶 0.5 μmol/min、Mg2+ 1.75 mmol/mL、模板DNA 40~100 ng、dNTPs 0.15 mmol/mL、引物 0.6 μmol/L、10×PCR Buffer 2μL,不足部分以ddH2O 补足。在获得最佳反应体系的基础上采用单因素实验确定了引物最佳退火温度范围为55~45 ℃,最佳循环次数为30次以及延伸时间为1.5 min。应用该优化的反应体系,对‘Constanta’和‘Sorbonne’以及9份‘Constanta’ × ‘Sorbonne’的 F1代DNA进行ISSR-PCR扩增,结果显示优化的反应体系具有较高的扩增稳定性。
Abstract:
‘Constanta’ and ‘Sorbonne’ and 9 F1 hybrids of ‘Constanta’ × ‘Sorbonne’ were used as materials in this experiment, and a modified method of CTAB was applied to extract the genomic DNA from the young leaves. The orthogonal design L16(45)was used to optimize the ISSR-PCR system for oriental lily at four levels and five factors, namely, DNA polymerase, Mg2+, DNA template, dNTPs and primers. The results of PCR were evaluated by intuitive and variance analysis. Results showed that the best reaction system was 20 μL reaction solution, containing 0.5 μmol/min Taq DNA polymerase, 1.75 mmol/mL Mg2+ , 40-100 ng template DNA, 0.15 mmol/mL dNTPs, 0.6 μmol/L primers, 2 μL 10×PCR buffer and ddH2O. Using single factor experiment design, we found that 55-45 ℃ was the best touch down PCR annealing temperature, the best cycle times was 30 and the best extend time was 15 min. The stabilization of the optimized reaction system was tested by using ‘Constanta’ and ‘Sorbonne’ and 9 F1 hybrids of ‘Constanta’ × ‘Sorbonne’.

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备注/Memo

备注/Memo:
收稿日期:2011-08-04修回日期:2011-09-08
基金项目:国家自然科学基金项目(30972407);江苏高校优势学科建设工程资助项目(PAPD)
第一作者:丁信誉,硕士。*通信作者:席梦利,副教授。E-mail: ximenglinjfu@126.com。
引文格式:丁信誉,邱帅,席梦利,等. 东方百合ISSR-PCR反应体系的正交优化[J]. 南京林业大学学报:自然科学版,2012,36(5):42-46.
更新日期/Last Update: 2012-09-30