| 高GC含量青枯菌aac基因PCR扩增体系的建立与优化 |
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| 引用本文： | 张争,张杨,徐进,许景升,何礼远,冯洁.高GC含量青枯菌aac基因PCR扩增体系的建立与优化[J].植物保护,2008,34(2):90-93. |
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| 作者姓名： | 张争 张杨 徐进 许景升 何礼远 冯洁 |
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| 作者单位： | 中国农业科学院植物保护研究所 植物病虫害生物学国家重点试验室，北京100094 |
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| 摘 要： | 通过添加增效剂、正交试验设计优化PCR反应体系、联合采用多种PCR程序等措施，建立并优化了PCR扩增体系，成功地从高GC青枯菌基因组中扩增出了长度为2 434 bp且GC含量高达70.9%的aac基因。PCR反应体系为20 μL包括5%DMSO、2.5 mmol/L MgCl2、500 μmol/L dNTP、10 pmol/L引物、1.25 U Taq酶、50 ng模板DNA。首先采用热启动PCR：95 ℃5 min，保持80 ℃，加入Taq酶；然后采用二步PCR：5个循环包括变性95 ℃1 min，65 ℃1 min；最后采用降落PCR：30个循环为95 ℃1 min，78 ℃1 min，每个循环降低0.5 ℃，72 ℃3 min；补充10个循环为95 ℃ 1 min，63 ℃1 min，72 ℃3 min；72 ℃10 min。该试验体系的建立与优化为研究高GC含量生物的基因功能提供了方法。
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Establishment and optimization of the PCR system for amplification of aac gene from GC rich Ralstonia solanacearum |
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| Zhang Zheng,Zhang Yang,Xu Jin,Xu Jingsheng,He Liyuan,Feng Jie.Establishment and optimization of the PCR system for amplification of aac gene from GC rich Ralstonia solanacearum[J].Plant Protection,2008,34(2):90-93. |
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| Authors: | Zhang Zheng Zhang Yang Xu Jin Xu Jingsheng He Liyuan Feng Jie |
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| Institution: | State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, CAAS, Beijing 100094, China |
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| Abstract: | By introducing different additives and optimizing concentrations with orthogonal test method and combination of different PCR methods, the aac gene was obtained, which is 2 434 bp containing 70.9% of G+C. The PCR reactions were 20 μL, containing 5% DMSO, 2.5 mmol/L MgCl2, 500 μmol/L dNTPs, 10 pmol/L of each primer, 1.25 unit Taq polymerase and 50 ng genomic DNA. Firstly, Hot start PCR was performed by denaturing the DNA mixture at 95°C for 5 min, holding at 80℃ before adding Taq polymerase. Secondly, Two Step PCR was conducted as followed: 5 cycles at 95 ℃ for 1 min and 65 ℃ for 1 min. Thirdly, the touchdown PCR was carried out as followed: 95 ℃ for 1 min, 78 ℃ for 1 min with a decrease of 0.5 ℃ per cycle, and 72 ℃ for 3 min. Finally, 10 cycles of 95 ℃ for 1 min, 63 ℃ for 1 min, and 72 ℃ for 3 min were carried out, ended with a 10 min extension at 72 ℃. The strategy used in this study is very useful for investigation of the genes with high GC content. |
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