A method for isolation and identification of cellulose-degrading bacteria in the intestinal tract of monochamus monochamus larvae

A technology of cellulose degradation and P. serrata, applied in the field of microorganisms, can solve the problems of rare reports on separation and identification research.

Active Publication Date: 2021-04-09
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the research on microbial degradation of cellulose has been relatively in-depth, but there are few reports on the isolation and identification of intestinal bacteria in Monochamus alternatus

Method used

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  • A method for isolation and identification of cellulose-degrading bacteria in the intestinal tract of monochamus monochamus larvae
  • A method for isolation and identification of cellulose-degrading bacteria in the intestinal tract of monochamus monochamus larvae
  • A method for isolation and identification of cellulose-degrading bacteria in the intestinal tract of monochamus monochamus larvae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] The larvae of Monochamus alternata were collected in Guantou Town, Lianjiang, Fujian Province (N 26.150°; E 119.593°). The insect-infested wood was sawed into a section of about 1m long and placed in a 1.5m long × 1.5m wide × 1.0m high growth chamber. In the insect cage, the insect cage is sealed with an iron sand net with a mesh diameter less than 2mm. The larvae of Monochamus alternatus were collected by peeling off the bark of Pinus massoniana and splitting the wood sections for experimental needs.

[0081] Isolation and identification method of flora structure

[0082] 1) placing Monochamus alternatus larvae in a sterile environment with a humidity of 90wt% and starvation treatment for 48 hours to obtain Monochamus alternatus larvae whose intestines are emptied of food residues;

[0083] 2) soaking the Monochamus alternatus larvae whose intestines have been emptied of food residues with alcohol with a volume concentration of 75% for 3 minutes, and rinsing with ster...

Embodiment 2

[0089] A single colony was picked from the No. 4 solid medium plate with a single colony obtained in Example 1 for morphological identification and physiological characteristic determination.

[0090] Observe and record the color, shape, texture and other characteristics of a single colony under an optical microscope. The identification of bacterial cell morphology refers to the method of "Bergey's Bacterial Identification Manual" to identify bacterial colony color, bacterium shape, size, Gram Negative and positive distinction, presence or absence of flagella, presence or absence of sterile membrane, and the number of bacterial colonies represented by the strain, etc. (Smibert, 1994).

Embodiment 3

[0092] A single colony was picked from the No. 4 solid medium plate with a single colony obtained in Example 1 for molecular identification.

[0093] According to the instructions of the bacterial DNA extraction kit (Omega, USA), the total DNA of each bacteria was extracted respectively, and then the bacterial 16S rDNA was amplified using the bacterial universal primers fD1 and rP1. For specific methods, see Huetal. (2014b). The 16S rDNA sequence of the sequencing data was uploaded to Genbank (http: / / www.ncbi.nlm.nih / gov / blast / ).

[0094] The 16S rDNA sequence gene of bacterial strains with cellulose-degrading activity in the intestinal tract of Monochamus monochamus larvae was submitted to the NCBI database with accession numbers: KX461909~KX461918.

[0095] In order to further determine the taxonomic status of the intestinal cellulose-degrading bacteria of each Monochamus alternata, all the sequences were first matched with the RDP II database sequences (Cole et al. Knowing...

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Abstract

The invention provides a method for separating and identifying cellulose-degrading bacteria in the intestinal tract of monochamus monochamus larvae, belonging to the technical field of microbes. Grind the hindgut of Monochamus alternatus larvae to obtain the original bacterial liquid, which is then serially diluted and spread on No. 4 medium with sodium carboxymethyl cellulose as the only carbon source for aerobic culture , Obtained single colony for morphological observation and molecular identification. According to the method provided by the present invention, 154 strains of bacteria with cellulase activity are isolated from the middle hindgut of Monochamus alternata larvae, belonging to 8 genera of Proteobacteria, Firmicutes and Bacteroidetes respectively, 10 strains. The separation and identification method provides a theoretical basis for revealing the interaction relationship between intestinal bacteria and Monochamus alternatus.

Description

technical field [0001] The invention belongs to the technical field of microbes, and in particular relates to a method for separating and identifying cellulose-degrading bacteria in the intestinal tract of monochamus monochamus larvae. Background technique [0002] Cellulose is the main nutrient in the food of tree borer pests and plays an important role in the normal growth and development of the pests (Peterson et al., 2015). A large number of studies have shown that there are many microorganisms growing in the intestinal tract of borer pests, and the number of them far exceeds the total number of insect cells (Six, 2013). These microorganisms can secrete cellulose-degrading enzymes, so that they can efficiently degrade, digest, absorb and utilize lignocellulose under the synergy of the insect's own digestive system and endosymbiotic microorganisms (Ning Na et al., 2015). It can be said that the gut microbiota of the borer pest is an indispensable synergistic factor for t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/02C12Q1/04
CPCC12N1/02C12Q1/04
Inventor 胡霞张飞萍李明吴松青梁光红王荣罗巧玉陈昊
Owner FUJIAN AGRI & FORESTRY UNIV
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