Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil
Method for RNA extraction and cDNA library construction from microbes in crop rhizosphere soil
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作物根际土壤微生物RNA提取及cDNA文库构建方法
DOI：
10.1007/s11274-013-1504-0
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发表时间：
2014-02
影响因子：
4.1
通讯作者：
Wenxiong Lin
中科院分区：
文献类型：
--
作者：
Changliang Ye;Likun Huang;Qingshui Wang;Wenxiong Lin
分析土壤根际转录组的技术对于揭示土壤生态系统中植物与微生物之间的相互作用和通讯至关重要。在根际土壤样品中加入不同体积的Al 2（SO 4）3沉淀腐殖物质，这些腐殖物质干扰RNA和DNA分析的大多数程序。腐殖物质沉淀后，用玻璃珠涡旋破碎土壤微生物细胞，然后用含有LiCl、SDS和EDTA的Tris-HCl缓冲液回收DNA和RNA。将粗提物沉淀并溶解在无RNA酶的水中，然后通过琼脂糖凝胶电泳分离。研究了Al_2（SO_4）_3处理水稻、烟草、甘蔗、紫茎泽兰、太子参根际土壤的最佳用量。从水稻土中提取的核酸粗提物用DNase I处理，然后用凝胶过滤柱纯化RNA。将纯化的RNA逆转录成单链cDNA，然后在扩增ds cDNA之前在每个末端与接头连接。将ds cDNA亚克隆用于随后的基因序列分析。我们进行qPCR以扩增16 S核糖体DNA并观察到高效扩增。这些结果表明，优化的提取方法可以从不同根际土壤微生物中分离和获得高质量的核酸，适用于基因组和后基因组分析。
Techniques to analyze the transcriptome of the soil rhizosphere are essential to reveal the interactions and communications between plants and microorganisms in the soil ecosystem. In this study, different volumes of Al2(SO4)3were added to rhizosphere soil samples to precipitate humic substances, which interfere with most procedures of RNA and DNA analyses. After humic substances were precipitated, cells of soil microorganisms were broken by vortexing with glass beads, and then DNA and RNA were recovered using Tris–HCl buffer with LiCl, SDS, and EDTA. The crude extract was precipitated and dissolved in RNAse-free water, and then separated by agarose gel electrophoresis. We determined the optimum volume of Al2(SO4)3for treating rhizosphere soil of rice, tobacco, sugarcane,Rehmannia glutinosa, andPseudostellaria heterophylla. The crude nucleic acids extract from rice soil was treated with DNase I and then RNA was purified using a gel filtration column. The purified RNA was reverse-transcribed into single-strand cDNA and then ligated with an adaptor at each end before amplifying ds cDNA. The ds cDNA was sub-cloned for subsequent gene sequence analysis. We conducted qPCR to amplify 16S ribosomal DNA and observed highly efficient amplification. These results show that the extraction method can be optimized to isolate and obtain high-quality nucleic acids from microbes in different rhizosphere soils, suitable for genomic and post-genomic analyses.
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DOI：
10.1007/3-540-29449-x_3
发表时间：
2006
期刊：
--
影响因子：
--
作者：
L. Bakken;Å. Frostegård
通讯作者：
L. Bakken;Å. Frostegård
DOI：
10.1007/3-540-29449-x_11
发表时间：
2006
期刊：
--
影响因子：
--
作者：
M. Krsek;W. Gaze;N. Z. Morris;E. Wellington
通讯作者：
M. Krsek;W. Gaze;N. Z. Morris;E. Wellington
影响因子：
4
作者：
Y. Wang;S. Morimoto;N. Ogawa;T. Oomori;T. Fujii
通讯作者：
Y. Wang;S. Morimoto;N. Ogawa;T. Oomori;T. Fujii
影响因子：
4.2
作者：
A. Schmalenberger;C. Tebbe;M. Kertesz;H. Drake;K. Küsel
通讯作者：
A. Schmalenberger;C. Tebbe;M. Kertesz;H. Drake;K. Küsel
影响因子：
9.7
作者：
Murase, A;Yoneda, M;Yonebayashi, K
通讯作者：
Yonebayashi, K