Method for constructing mangrove forest soil large section metagenome library

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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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 attacks, resulting in a high yield of eDNA, but in fact the proportion of large fragments of DNA is low, which is not enough to construct metagenomic library

(2) The problem of co-extraction and co-purification of humic acid, fulvic acid and humic acid and other inhibitory substances in soil and eDNA is difficult to solve (1, Krsek M, Wellington EM. Comparison of different methods for the isolation and purification of total community DNA from soil[J].J Microbiol Methods., 1999, 39(1):1~16; 2. Tsai YL and Olson BH. Rapid method for separation of bacterial DNA from humic substances in sediments for polymerase chain reaction[J] .Appl.Environ.Microbiol., 1992,58(7):2292~2295; 3. Voget S, Leggewie C, Uesbeck A, et al.Prospecting for novel biocatalysts in a soil metagenome[J].Appl.Environ.Microbiol ., 2003, 69(10):6235~6242; 4. Daniel R.The soil metagenome-a rich resource for the discovery of novel natural products[J].Curr.Opin.Biotechnol., 2004,15(3):199 -204; 5. Pettit RK. Soil DNA libraries for anticancer drug discovery [J]. Cancer. Chemother. Pharmacol., 2004, 54(1): 1~6)

It is well known that small-fragment environmental genome libraries cannot capture complete gene clusters encoding antibiotics and other substances, and are of little value for new drug development

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