基于宏基因组测序的植物叶表微生物富集及DNA提取方法  

Microbial Enrichment on Leaf Surface and DNA Extraction Method Based on the Metagenomics Sequencing

作　　者：张雨函 范熠 李婷婷 庞爽 刘为 白可喻[2] 张西美 ZHANG Yu-han;FAN Yi;LI Ting-ting;PANG Shuang;LIU Wei;BAI Ke-yu;ZHANG Xi-mei(Institute of Environment and Sustainable Development in Agriculture,Chinese Academy of Agricultural Sciences,Beijing 100081;Alliance of Bioversity International and CIAT,Beijing Office,Institute of Agricultural Resources and Regional Planning,Chinese Academy of Agricultural Sciences,Beijing 100081)

机构地区：[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]中国农业科学院农业资源与农业区划研究所国际生物多样性中心东亚办事处,北京100081

出　　处：《生物技术通报》2022年第3期256-263,共8页Biotechnology Bulletin

基　　金：国家自然科学基金项目(32071547)。

摘　　要：获得高质量的微生物基因组DNA是进行复杂微生物群落宏基因组学研究的基础和难点。植物叶表是一个微生物多样性丰富的复杂生态系统,这些微生物群落可以调节叶片功能性状,影响植物的适应性。深入了解叶表微生物群落的基本结构和功能原理,有助于在促进植物生长和植物保护方面发挥重要的应用价值。由于叶表严苛的环境,导致富集叶表微生物难度较大,严重限制了高质量叶表微生物基因组DNA的提取。基于现有DNA提取方法,加入表面活性剂Silwet L-77进行前处理,同时循环利用洗脱液,加强叶表微生物的富集,以提高叶表微生物的获取量。结合商业试剂盒方法进行提取得到高纯度、高浓度的基因组DNA。经过质量控制和建库测序验证,DNA质量达到宏基组建库的要求。通过此方法可以提高叶表微生物分离和收集效率的方法,提高叶表微生物DNA提取成功率,为应用高通量测序技术研究叶表微生物组成和其他植物分子生物学研究提供参考。Obtaining high-quality total genomic DNA of microbial community is the basis and difficulty in the study of metagenomics of complex microbial communities.The plant leaf surface is a complex ecosystem with abundant microbial diversity,and the microbial community mediates leaf functional traits and affects plant adaptability.An in-depth understanding of the basic structure and functional principle of the microbial community on leaf surface will contribute to the applications of promoting plant growth and plant protection.Due to the harsh living environment of leaf surface,it is difficult to enrich microorganisms on leaf surface,which severely limits the extraction of high-quality genomic DNA of microorganisms on leaf surface.Based on the existing DNA extraction method,the surfactant Silwet L-77 was added for pretreatment,and the eluent was recycled,which increased the enrichment of microorganisms on leaf surface.The genomic DNA with high purity and concentration was extracted in combination with commercial kit methods.After quality control and library building sequencing as verification,DNA quality met the requirements for metagenomic library construction.This method may improve the enrichment and collection efficiency of microorganisms on leaf surface,increase the success rate of DNA extraction of microorganisms on leaf surface,and provide a reference for applying high-throughput sequencing technology to study the composition of microorganisms on surface and molecular biology research of plants.

关 键 词：叶表微生物 Silwet L-77 DNA提取 宏基因组测序 质量控制 

分 类 号：Q933[生物学—微生物学]