| 瘤胃甲烷生成菌PCR反应条件的优化 |
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| 引用本文： | 淡瑞芳,藏丽丽,龙瑞军,魏小红,张海涛.瘤胃甲烷生成菌PCR反应条件的优化[J].安徽农业科学,2009,35(19):8898-8900. |
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| 作者姓名： | 淡瑞芳 藏丽丽 龙瑞军 魏小红 张海涛 |
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| 作者单位： | 江苏畜牧兽医职业技术学院,江苏泰州,225300;甘肃农业大学生物技术工程学院,甘肃兰州,730070;兰州大学草地农业科技学院甘肃草原生态研究所,甘肃兰州,730020;兰州大学青藏高原生态系统管理国际中心,甘肃兰州,730020 |
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| 基金项目： | 国家自然科学基金重点项目 |
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| 摘 要： | 目的]优化适合于瘤胃甲烷生成菌的PCR反应体系。方法]以1．5周岁健康的藏系绵羯羊瘤胃内容物总DNA为模板，用特异性引物对甲烷生成菌16SrDNA保守序列进行PCR扩增，并通过改变PCR反应体系中的各参数，优化其PCR反应体系中的相关参数。结果]PCR最优程序为：94℃预变性3min；94℃变性45s，51℃退火458，72℃延伸90s，35个循环；72℃后延伸7min。优化后的PCR反应体系为：5．00山缓冲液，4．00μl d-NTP，2．00μl引物（前引物和后引物各1．00μl），4．00μl MgCl2，0．25μl TaqDNA聚合酶和1．00μl 1：100倍稀释的DNA模板，然后加水补足25．00μl。结论]该研究为瘤胃甲烷生成菌功能的研究奠定了基础。
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| 关 键 词： | 甲烷生成菌 16SrDNA PCR反应条件 优化 |
Optimization on the PCR Reaction Condition of Methane Producing Bacteria in Rumen |
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| Institution: | DAN Rui-fang et al (Jiangsu Vocational and Technical College of Animal Husbandry and Veterinary, Taizhou, Jiangsu 225300) |
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| Abstract: | Objective] The aim was to optimize PCR reaction system suitable for methane producing bacteria in rumen.Method] With the total DNA in rumen content of 1.5-year old healthy Tibetan sheep as template, the conservative sequence of methane producing bacteria 16S rDNA was amplified by PCR using specific primers.The related parameters in the PCR reaction system were optimized through changing each parameter in the PCR reaction system.Result] The optimum PCR program was as follows: pre-denaturing under 94 ℃ for 3 min, then denaturing under 94 ℃ for 45 s, annealing under 51 ℃ for 45 s and extending under 72 ℃ for 90 s.After 35 cycles, the sample was extended under 72 ℃ for 7 min.The optimized PCR reaction system was as follows: 5.00 μl buffer, 4.00 μl d-NTP, 2.00 μl primers (upstream primer and down primer were 1.00 μl each), 4.00 μl MgCl2, 0.25 μl Taq DNA polymerase, 1.00 μl DNA (1∶ 100), then adding ddH2O to 25.00 μl.Conclusion] The research laid the foundation for study on the function of methane producing bacteria in rumen. |
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| Keywords: | 16SrDNA Methane producing bacteria 16S rDNA PCR reaction condition Opitimization |
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