基于宏基因组测序的植物叶表微生物富集及DNA提取方法

doi:10.13560/j.cnki.biotech.bull.1985.2021-0550

摘要/Abstract

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。

关键词: 叶表微生物, Silwet L-77, DNA提取, 宏基因组测序, 质量控制

Abstract:

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.

Key words: microorganism on leaf surface, Silwet L-77, DNA extraction, metagenome sequencing, quality control

张雨函, 范熠, 李婷婷, 庞爽, 刘为, 白可喻, 张西美. 基于宏基因组测序的植物叶表微生物富集及DNA提取方法[J]. 生物技术通报, 2022, 38(3): 256-263.

ZHANG Yu-han, FAN Yi, LI Ting-ting, PANG Shuang, LIU Wei, BAI Ke-yu, ZHANG Xi-mei. Microbial Enrichment on Leaf Surface and DNA Extraction Method Based on the Metagenomics Sequencing[J]. Biotechnology Bulletin, 2022, 38(3): 256-263.

图/表 7

表1 无菌水和PBS缓冲液+Silwet L-77表面活性剂对叶表微生物基因组DNA的提取效果

Table 1 Extraction effect of sterile water and PBS buffer solution +Silwet L-77 surfactant on microbial genomic DNA from leaf surface

样品名称 Sample name	无菌水Sterile water			PBS+Silwet L-77
样品名称 Sample name	DNA浓度DNA concentration/（ng·µL^-1）	A₂₆₀/A₂₈₀	质量 Mass/µg	DNA浓度DNA concentration/（ng·µL^-1）	A₂₆₀/A₂₈₀	质量 Mass/ µg
LC	14.43±2.79	1.86	2.94	40.08±10.29	1.82	19.30
PD	13.61±7.52	1.91	1.44	74.25±5.77	1.96	19.85
AF	11.51±2.33	1.84	1.15	49.55±0.21	1.93	7.44
IL	19.51±8.13	1.89	1.95	64.42±13.03	1.93	34.97
TL	18.75±3.61	1.99	1.78	35.45±7.42	1.81	22.28
AR	18.14±3.87	1.97	1.81	44.93±31.67	1.96	30.31
All	15.99±4.95	1.93	1.63	51.44±18.10	1.86	22.36

表2 植物种类与不同富集方法对DNA浓度的影响

Table 2 Effects of plant species and different enrichment methods on DNA concentration

	Df	F value	Pr（>F）
富集方法Enrichment method	1	59.11	P<0.05
植物种类Plant species	5	1.71	0.21
富集方法:植物种类 Enrichment method:Plant species	5	1.96	0.16
残差Residual	12	NA	NA

图1 不同洗脱液提取不同植物种类叶表微生物的DNA浓度

Fig. 1 DNA concentration of leaf surface microorganism extracted from different plant species by different eluents

图2 六种植物叶表微生物DNA电泳图 1-6和7-12分别是羊草、叉分蓼、冷蒿、马蔺、披针叶黄华和野韭,1-6以PBS+ Silwet L-77为洗脱液,7-12以无菌水为洗脱液

Fig. 2 Microbial DNA electrophoresis patterns on the leaf surfaces of 6 plants 1-6 and 7-12 were L. chinensis（Trin.）Tzvel.,P. divaricatum L.,A. frigida Willd.,I. lactea Pall. var. chinensis（Fisch.）Koidz.,T. lanceolata R. Br. and A.ramosum L.. PBS+ Silwet L-77 as eluent for 1-6,and sterile water as eluent for 7-12,respectively

表3 宏基因组测序统计

Table 3 Metagenomics sequencing statistics

样品名称Sample name	Clean reads	Clean base/bp	Percent in raw reads/%	Percent in raw bases/%
LC	84554592	12743415315	99.08±0.12	98.89±0.26
PD	76636682	11558890595	98.97±0.04	98.86±0.09
AF	70985020	10682348648	98.70±0.10	98.36±0.14
IL	77764022	11727532109	99.11±0.08	98.97±0.11
TL	87048063	13081919500	98.93±0.08	98.47±0.24
AR	80930005	12202864242	98.82±0.69	98.67±0.74

图3 六种植物叶表微生物不同分类群的百分比

Fig. 3 Percentages of different taxa microorganism on the leaf surfaces of 6 plants

图4 六种植物叶表微生物COG功能注释

Fig. 4 Functional annotation of microorganism COG on the leaf surfaces of 6 plants

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