Molecule detection method of spongioblast endosymbiotic actinomycetes
A molecular detection and actinomycete technology, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problem that actinomycetes are not paid enough attention, and the diversity information and diversity of sponge actinomycetes are rarely obtained. Distribution characteristics, lack of actinomycete species-specific primers, etc., to achieve accurate diversity information and improve sensitivity
Inactive Publication Date: 2013-02-06
SHANGHAI JIAOTONG UNIV
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However, in the research on the diversity of sponge symbiotic microorganisms, actinomycetes are often not paid enough attention. In modern molecular ecology research, there is a lack of application of actinomycete species-specific primers. Complete diversity information and distribution characteristics of actinomycetes spongiosa
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[0016] The technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments. The following examples are not intended to limit the present invention.
[0017] The flow process of the inventive method is as figure 1 As shown, first dissociate the sponge tissue and sort the cells to obtain the sponge cells, extract the total DNA of the symbiotic microorganisms in the cells, amplify the 16S rRNA gene fragments of actinomycetes by polymerase chain reaction, and construct a clone library combined with statistical Phylogenetic and phylogenetic analysis revealed the composition of symbiotic actinomycetes in sponge cells, and found specific actinomycetes in sponge cells.
[0018] The specific implementation steps are as follows:
[0019] 1. Spongy tissue dissociation and cell sorting:
[0020] (1) The following operations are performed under sterile conditions. Spongy tissue (1-2g) was thawed at 4°C and cut i...
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The invention relates to a molecule detection method of spongioblast endosymbiotic actinomycetes, comprising the following steps of: obtaining sponge single cells by combining a mechanical method and a chemical method and extracting the total DNA in the spongioblast endosymbiotic microorganisms; amplifying the segment of an actinomycetes 16SrRNA gene through a polymerase chain reaction by using an actinomycetes species specificity primer by taking the extracted total DNA as a template; purifying the amplified product to construct and clone a library, randomly selecting positive clone for sequencing until the library is saturated, and importing a sequencing result and the closest sequence into an ARB sequence analysis software for phylogenetic analysis to finish the molecule detection of spongioblast endosymbiotic actinomycetes. The invention establishes a set of relatively comprehensive and accurate molecule detection method of spongioblast endosymbiotic actinomycetes.
Description
technical field [0001] The invention relates to a molecular detection method for endosymbiotic actinomycetes in sponge cells, which aims at identifying the endosymbiotic actinomycetes in marine invertebrate sponge cells at the in situ gene level. The invention belongs to the technical field of marine molecular ecology. Background technique [0002] The sponge has a unique cavity-like structure and lives by filtering microorganisms in seawater. A large number of symbiotic microorganisms gather in the body, generally accounting for more than 40% of the sponge's volume. A lot of evidence shows that the medicinal natural products derived from sponges may be produced by their symbiotic microorganisms, and sponge symbiotic microorganisms have therefore become one of the focuses of marine drug research and development. Analyzing the diversity of these microorganisms and identifying host-specific microorganisms are the prerequisites for the development and utilization of sponge sym...
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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 刘放张风丽李志勇周康
Owner SHANGHAI JIAOTONG UNIV