Molecular identification method for sponge associated dominant bacterium constitution and host specific bacterium

Abstract

The process of identifying the co-epiphytic dominant bacteria composition and the specific host bacteria of sponge includes the following steps: preparing co-epiphytic microbe macro genome total DNA sample via grinding sponge, treating with lysozyme and proteinase K to release DNA, and phenol-chloroform treatment; PCR amplification with the sample as template and general bacterial primer to obtain 16S rDNA segment smaller than 500 bp; denaturing gradient gel electrophoresis to separate mixed segment and obtain 16S rDNA-DGGE gene fingerprint; recovering DNA bands, secondary PCR amplification, denaturing gradient gel electrophoresis, purification, cloning and sequencing to complete homology and system development analysis; analyzing bands in the DGGE fingerprint to complete the molecular identification of the co-epiphytic dominant bacteria composition; and finding out the differential bands in the DGGE fingerprint to complete the molecular identification of specific host bacteria.

Description

technical field [0001] The invention relates to a molecular identification method for the composition of the dominant symbiotic bacteria of the sponge and the specific bacteria of the host, and is specifically aimed at the in situ gene level identification of the symbiotic bacteria of the marine invertebrate sponge. It belongs to the technical field of marine microbial molecular ecology. Background technique [0002] Sponge has a unique cavity-like structure, relying on filtered seawater to absorb nutrients to survive, and a large number of symbiotic microorganisms gather in the body, generally accounting for more than 40% of the sponge's volume. Some of these microorganisms can be digested as food for the sponge, while others survive inside and on the surface of the sponge, and may participate in the chemical defense of the sponge or provide energy for the sponge. Therefore, revealing the composition information of these microorganisms is a prerequisite for the development...

AI Technical Summary

Problems solved by technology

16S rDNA library analysis is a classic molecular technique to reveal the microbial diversity composition of an environmental flora. Its advantage is that it can comprehensively reveal the overall composition information of the microbial flora. The disadvantage is that the operation is complicated and time-consuming, and it is not suitable for different samples and Comparison of microbial composition of the same sample at different times or spaces

Although techniques such as fluorescence in situ hybridization can reveal the distribution of microorganisms with known sequences, because the probe design needs to be based on known genetic information, it is limited to the disclosure of information on a few microorganisms

At present, there is no report on the method of using 16S rDNA-DGGE technology to reveal the structure and diversity of symbiotic microorganisms and host-specific bacteria in different sponges

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