微生物发酵床垫料微生物总DNA提取方法的研究

福建农业学报 ›› 2011, Vol. ›› Issue (2): 153-158.

微生物发酵床垫料微生物总DNA提取方法的研究

刘云浩^1,2, 蓝江林², 刘波², 朱昌雄¹, 郭萍¹, 陈燕萍²

1. 中国农业科学院农业环境与可持续发展研究所, 北京100081;
2. 福建省农业科学院农业生物资源研究所, 福建福州350003

收稿日期:2011-03-03 修回日期:2011-03-24 出版日期:2011-04-15 发布日期:2011-04-15
通讯作者: 刘波(1957-),研究员,博士,博导,研究方向:微生物生物技术与农业生物药物(E-mail:liubofz@163.com);朱昌雄(1963-),研究员,博导,研究方向:微生物与环境修复(E-mail:zhucx120@163.com)
基金资助:
国家水体污染控制与治理科技重大专项(2008ZX07425-002);国家科技支撑计划项目(2008BAD96B07、2009BADC2B02);福建省发改委项目(闽发改投资[2008]762号);福建省科技计划——省属公益类科研院所基本科研专项(2010R1020-1)

Extraction Methods for Bedding Microbial DNA from Fermentation Bed

LIU Yun-hao^1,2, LAN Jiang-lin², LIU Bo², ZHU Chang-xiong¹, CHEN Yan-ping¹

1. Institute of Environment and Sustainable Development in Agriculture, The Chinese Academy of Agricultural Sciences, Beijing 100081, China;
2. Agricultural Biological Resource Research Institute, Fujian Academy of Agricultural Sciences, Fuzhou, Fujian 350001, China

Received:2011-03-03 Revised:2011-03-24 Online:2011-04-15 Published:2011-04-15

摘要/Abstract

摘要： 传统的平板分离培养法,不能全面反映垫料微生物的基因信息,所以获得高质量的垫料微生物总DNA对于研究垫料微生物的群落结构就显得尤为重要。本实验设计对比了6种关于养猪发酵床垫料微生物总DNA的提取方法,对6种方法提取的DNA的浓度和纯度进行比较评价,结果表明,试剂盒法中因为没有专门针对本实验垫料的总DNA提取试剂盒,所采用的粪便,土壤以及淤泥总DNA提取试剂盒都没有达到很好的效果;而在化学法当中,单纯的SDS法和CTAB法都不能有效除去腐殖酸和杂蛋白的污染,通过核酸蛋白分析仪测定的A₂₆₀/A₂₈₀的比值基本都在1.5以下,说明纯度很低,用16S rDNA通用引物进行PCR扩增也没有得到目的条带。在SDS-CTAB结合法中利用10%的PEG-8000进行沉淀不进行回收纯化所提取的DNA具有完整性好,纯度高的优点,浓度达到200 ng·μL^-1以上,通过核酸蛋白分析仪测定的A₂₆₀/A₂₈₀的比值达到1.8左右。用16S rDNA通用引物扩增得到比较亮的目的条带,因此SDS-CTAB结合法是一种高效、可靠的垫料微生物总DNA提取方法,有利于进行下游的分子生态学研究。

关键词: 垫料微生物, DNA提取, SDS-CTAB, 16S rDNA

Abstract: Traditional plate culture method can not fully reflect the microbial genetic information in microbiological fermentation bed,so obtaining high quality DNA is necessary to study microbial community structure in fermentation bed.In this research we designed and compared six extraction methods for bedding microbial DNA from fermentation bed,mainly in evaluating concentration and purification of extracted DNA.Results indicated that the kit methods have high pertinence,without specific bedding material microorganism DNA extraction kit,the use of excrement,soil and silt kit did not achieve good results.Simply applying SDS or CTAB methods could not effectively eliminate the pollution of humic acid and remove the complex proteins;so that,the ratio of A₂₆₀ to A₂₈₀ of the DNA was lower than 1.5 and no result obtained by PCR amplification.Comparatively,in SDS-CTAB method,using 10% PEG8000 in precipitation obtained the DNA with good integrity and high purity,the concentration of DNA was more than 200 ng·μL^-1,the ratio of A₂₆₀ to A₂₈₀ was about 1.8,and the PCR amplified targeted the DNA.Results indicated that the SDS-CTAB was an efficient and reliable method for DNA extraction from microbiological fermentation bed and it would be also conducive to the research of molecular ecology.

Key words: Bedding microorganism, DNA extraction, SDS-CTAB, 16S rDNA

中图分类号:

Q781

刘云浩, 蓝江林, 刘波, 朱昌雄, 郭萍, 陈燕萍. 微生物发酵床垫料微生物总DNA提取方法的研究[J]. 福建农业学报, 2011, (2): 153-158.

LIU Yun-hao, LAN Jiang-lin, LIU Bo, ZHU Chang-xiong, CHEN Yan-ping. Extraction Methods for Bedding Microbial DNA from Fermentation Bed[J]. Fujian Journal of Agricultural Sciences, 2011, (2): 153-158.

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