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

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

Abstract

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

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.

Figures/Tables 7

References 33

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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

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

	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

	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

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