Method for constructing mangrove forest soil large section metagenome library

A metagenomic library and mangrove technology, which is applied in the field of construction of a large fragment metagenomic library of mangrove soil, can solve the problems of inability to break through the size of soil eDNA fragments, unable to capture gene clusters encoding antibiotics, and insufficient to construct metagenomic libraries, etc. To achieve the effect of improving the possibility of successful construction of large fragment metagenomic libraries, reducing the risk of DNA molecule breakage, and reducing the possibility of degradation and loss

Inactive Publication Date: 2008-03-26
XIAMEN UNIV
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Problems solved by technology

However, so far, there are still special difficulties in using the in situ cleavage method to extract eDNA to construct a large-fragment DNA insertion library, and there are few reports on obtaining DNA that can satisfy the quality of the large-fragment library by direct cleavage (Patrick Robe, Renaud Nalin, Carmela Capellano , et al.Extraction of DNA from soil[J].European Journal of Soil Biology, 2003, 39(4):183~190), especially when the research object is a soil sample with high content of clay and organic matter, it is more difficult (ZhouJ, Bruns MA, and Tiedje JM. DNA recovery from soils of diverse composition [J]. Appl. Environ. Microbiol., 1996, 62(2): 316~322)
The specific performance is as follows: (1) Even if the mildest lysis method is used, the size of the obtained soil eDNA fragments still cannot exceed 100kb (1, Patrick Robe, Renaud Nalin, Carmela Capellano, et al. Extraction of DNA from soil [J]. European Journal of Soil Biology, 2003, 39 (4): 183~190; 2, Zhou J, Bruns MA, and Tiedje JM. DNA recovery from soils of diverse composition [J]. Appl. Environ. Microbiol., 1996, 62 (2 ): 316~322)
The DNA released by lysing microbial cells, especially the large fragments of DNA, is easily re-adsorbed to soil particles (clay) and is vulnerable to nuclease at

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  • Method for constructing mangrove forest soil large section metagenome library

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[0032] The following embodiments will further illustrate the present invention in conjunction with the accompanying drawings.

[0033] 1) Selected bacterial strain, cloning vector and culture medium: the bacterial strain is Escherichia coli EPI-100, and the cloning vector is pWEB TM , purchased from Epicentre Company, the culture medium is LB broth and LB agar, both added at a concentration of 200 μg mL -1 ampicillin, and the incubation temperature was 37°C.

[0034] 2) Collecting mangrove soil samples: mangrove soil was collected in Zhangjiangkou Mangrove National Nature Reserve, Fujian Province, in Dongxia Town, Yunxiao County, Fujian Province. The reserve is located at east longitude 117°24′07″~117°30 ′00″, latitude 23°53′45″~23°56′00″, altitude -6~8m, total area 2360hm 2 . Samples were taken in all four seasons of the year. The specific sampling plan is as follows: after low tide, set 9 sampling sites in the mangrove area (low, middle, and high tide zones, 3 sampling p...

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Abstract

The present invention relates to construction of macro genomic library, and is especially one fast and efficient process of constructing macro genomic library of mangrove forest edaphic community. The process includes the steps of selecting strain, cloning carrier and culture medium; extracting mangrove forest edaphic eDNA; processing mangrove forest edaphic eDNA; filling the ends of mangrove forest edaphic eDNA; connecting the end filling object to the carrier; outer packing; measuring the potency of the packed product; verifying the inserted segment of the cloning vector; and preserving positive cloning vector.

Description

technical field [0001] The invention relates to the construction of a metagenomic library, in particular to a method for constructing a mangrove soil large fragment metagenomic library. Background technique [0002] In the process of constructing environmental metagenomic library, the in situ lysis extraction method has the advantages of higher yield of environmental DNA (eDNA) than the indirect extraction method, and more truly represents the real information of the microbial community in the environment. At present, there are many reports of small fragments (insert fragments are 3-10kbp) environmental metagenomic libraries based on the in situ cleavage extraction method (1, Henne A, Schmitz RA, Bomeke M, et al.Screening of environmental DNA libraries for the presence of genes conferring lipolytic activity on Escherichia coli[J].Appl.Environ.Microbiol., 2000, 66(7):3113~3116; 2. Gabor EM, de Vries EJ and Janssen DB.Construction, characterization, and use of small -insert g...

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Application Information

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IPC IPC(8): C12N15/31
Inventor 蒋云霞郑天凌
Owner XIAMEN UNIV
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