杨树人工林土壤细菌DNA的提取与扩增

J4 ›› 2013, Vol. 48 ›› Issue (05): 23-28.

杨树人工林土壤细菌DNA的提取与扩增

倪桂萍,王延平,王华田*,韩亚飞,桑亚林

山东农业大学林学院, 山东省森林培育重点实验室, 山东农业大学农业环境与生态重点实验室, 山东泰安 271018

收稿日期:2013-01-21 出版日期:2013-05-20 发布日期:2013-05-10
通讯作者: 王华田(1960- ),男,博士,教授,研究方向为工业人工林培育.Email: wanght@sdau.edu.cn
作者简介:倪桂萍(1986- ),女,硕士研究生,研究方向为工业人工林培育.Email: djngp@163.com
基金资助:
国家自然科学基金资助项目(31270670、31070550);山东省优秀中青年科学家奖励基金资助项目(BS2012NY006)

Extraction and amplification of bacterial DNA from soils of poplar plantation

NI Gui-ping, WANG Yan-ping, WANG Hua-tian*, HAN Ya-fei, SANG Ya-lin

Forest College of Shandong Agricultural University, Key Laboratory of Silviculture of Shandong Province,
AgroEcoenvironmental Laboratory of Shandong Agricultural University, Taian 271018, Shandong, China

Received:2013-01-21 Online:2013-05-20 Published:2013-05-10

摘要/Abstract

摘要：

土壤微生物多样性反映土壤生态系统健康程度,对土壤养分循环发挥决定作用,并在很大程度上影响林地生产力。为研究人工林长期经营对土壤微生物多样性的影响,本研究以杨树人工林为研究对象,取连作林地根际和非根际土壤,采用MOBIO PowerSoil DNA Isolation kit试剂盒提取土壤中微生物基因组DNA,并进行16S rDNA V3区扩增。结果表明:6种杨树人工林土壤中均能提取出微生物基因组DNA,基因组DNA片段大于2000bp,且DNA带型清晰完整,无明显降解,为后续土壤细菌的PCR扩增提供了良好模板;6种土壤基因组DNA在电泳图中的亮度不一,其中Ⅰ代林根际土壤(B3)微生物DNA条带最亮,Ⅲ代林非根际土壤(FB18)微生物DNA条带亮度最弱,经检测,B3样品DNA含量约为6.9ng·cm-3,而FB18仅有2.0ng·cm-3左右;选用27F／1492R和F338-GC／R518两对引物,采用巢式PCR策略,成功扩增出杨树人工林土壤细菌16S rDNA V3区DNA,片段大小为220bp左右。

关键词: 杨树人工林;土壤细菌DNA;巢式PCR

Abstract:

Soil microbial diversity reflects the degree of soil ecosystem health, plays an important role in soil nutrient cycling, and largely affects the forest land productivity. Using the rhizosphere and bulk soils from poplar plantations, extracting the genome DNA of total microorganism by MOBIO PowerSoil DNA Isolation Kit, the DNA extraction and amplification of 16S rDNA V3 area of microbes were studied in this paper to study the effect of plantation longterm management on soil microbial diversity. The results showed that this method can extract genome DNA of total microorganism from 6 kinds of soils from poplar plantation. The genomic DNA fragments was more than 2 000bp and the electrophoresis bands of DNA were clear, complete and without obvious degradation, and the genome DNA could be the favorable templates of 16S rDNA PCR amplification of soil bacteria. The brightness of 6 genomic DNA electrophoresis bands in electrophoresis figure is different, of which the DNA band of rhizosphere soil(B3)from first generation poplar plantation was the most bright, while the one of bulk soil(FB18)from third generation plantation was the most dark. According to the concentration detection of different DNA samples, the DNA concentration of B3 was about 69ng·ml-1, and that of FB18 was only 20ng·ml-1. Choosing 27F／1492R and F338GC／R518 primers, and using nested PCR strategy, this study successfully amplified the strip of 16S rDNA V3 area with about 220bp.

Key words: poplar plantation; genome DNA of soil bacteria; nested PCR

倪桂萍,王延平,王华田*,韩亚飞,桑亚林. 杨树人工林土壤细菌DNA的提取与扩增[J]. J4, 2013, 48(05): 23-28.

NI Gui-ping, WANG Yan-ping, WANG Hua-tian*, HAN Ya-fei, SANG Ya-lin. Extraction and amplification of bacterial DNA from soils of poplar plantation[J]. J4, 2013, 48(05): 23-28.

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