Method for constructing metagenomic Fosmid library of soil microorganisms in tropical rainforest

Abstract

The invention belongs to the field of microorganisms, and discloses a method for constructing a metagenomic Fosmid library of soil microorganisms in a tropical rainforest. The method comprises the following steps: washing by using cross-linked polyvinyl pyrrolidone (PVPP), removing impurities, and then adopting a lysozyme-protease K-SDS-CTAB lysis method to extract and purify deoxyribonucleic acid (DNA) in large fragments of soil microbial genomes; carrying out agarose gel low-voltage electrophoresis detection and pulsed electrophoresis detection; carrying out tail end repairing on the metagenomic DNA, cutting off a marker and a small sample, and using ligase to enable a recycled product to be ligated with pCC1FOS of a Fosmid carrier; carrying out a ligation reaction within 2h; packing a ligation product with a lambda packaging extract, carrying out transfection by using an EPI300-T1R strain, coating an LB flat plate, which contains 12.5mu g / ml of chloramphenicol, with a product of the transfection, and culturing overnight at the temperature of 37 DEG C. The method provided by the invention reflects that in the extreme environment of the tropical rainforest, the microorganisms and genetic resources thereof are extremely rich and abundant; the method is beneficial to the excavation and utilization of functional genes of the microorganisms.

Description

technical field [0001] The invention belongs to the field of microorganisms, and in particular relates to a method for constructing a macrogenome Fosmid library of tropical rainforest soil microorganisms. Background technique [0002] Fusarium wilt of melon is a devastating soil-borne disease of Cucurbitaceae, which is not easy to control with common chemical agents. The control of Fusarium wilt of melons mainly includes agricultural control (grafting and cultivating disease-resistant varieties, etc.), physical control, chemical control, biological control, etc. At present, most of them are controlled by chemical agents. However, due to the strong masking ability of pathogenic bacteria in soil-borne diseases, it is difficult for chemical fungicides to directly contact with them, and they cannot fundamentally kill pathogenic bacteria, and the application cost is high. A series of side effects have become increasingly prominent, and production and use are being gradually rest...

AI Technical Summary

Problems solved by technology

[0003] In summary, the problems in the prior art are: one, common chemical agents for melon wilt are not easy to prevent and cure

However, the success rate of discovering new antibiotic drugs based on microbial pure culture technology is getting smaller and smaller, and the phenomenon of repeated screening often occurs

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