Pseudomonas plecoglossicida tonB gene silencing strain and application thereof

A Pseudomonas mutans, gene silencing technology, applied in the field of microorganisms

Active Publication Date: 2021-10-22
JIMEI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Related reports have confirmed that the TonB system is related to the virulence of strains, but about ton B Genes are less reported

Method used

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  • Pseudomonas plecoglossicida tonB gene silencing strain and application thereof
  • Pseudomonas plecoglossicida tonB gene silencing strain and application thereof
  • Pseudomonas plecoglossicida tonB gene silencing strain and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Pseudomonas mutans ton B A method for constructing a gene stable silencing strain, comprising the following steps:

[0030] Step 1: Using comparative transcriptomics technology to analyze the gene expression of Pseudomonas mutans in the spleen of grouper, it was found that ton B Gene-specific high expression will lock the target ton B Gene; ton B The gene sequence is shown in SEQ ID NO.1;

[0031] Step 2: For ton B Gene sequence using online shRNA design tool from Thermo-fisher Scientific , using the online shRNA design tool to design and synthesize 4 pairs of shRNA primers, which were respectively connected into pCM130 / tac to construct recombinant vectors, and then each recombinant vector was electroporated into Pseudomonas mutans competent cells, and Pseudomonas mutans was successfully constructed ton B Stable gene silencing strains; using qRT-PCR technology, the primers are (F: 5'-GTGGACACGCCGCCAAGCAA-3'; R: 5'- GCCAGGTGACGCAGCAAATCG-3') to test the silencing e...

Embodiment 2

[0047] Using artificial infection experiments, the wild strains of Pseudomonas mutans and ton B The virulence of gene stable and efficient silenced strains was compared.

[0048] ton B The gene stable and efficient silencing strain and the wild strain of Pseudomonas mutans came from our research group, were isolated and identified by Hu Jiao et al. Large yellow croaker ( Pseudosciaena crocea ) Pathogen isolation and identification of visceral white spot disease and its pathogenicity[J]. Ocean and Limnology, 2014, 45(02): 409-417. 2 HPO 4 0.36g, KH 2 PO 4 0.024g, H 2 O 1L, PH 7.0) were used to infect the three groups of grouper by intrathoracic injection, and the infection concentration of the strain was 5 10 4 cfu / tail, each fish was injected with 0.2 mL, 20 fish in each group, and then continued to be raised normally (under non-pathogenic laboratory conditions, water temperature 18 ± 2°C), and the survival status of each group of fish was recorded every day .

...

Embodiment 3

[0051] Using RNA-seq technology to ton B Transcriptome sequencing and comparative analysis of spleens from gene-silenced and wild-type Pseudomonas mutans infection ton B Effects of gene silencing on gene expression in Pseudomonas metamorphans and grouper.

[0052] The results of RNA-seq analysis showed that at the mRNA level, according to statistical criteria (|log2 fold change| ≥1, padj ≤0.05), a total of 375 differentially expressed genes were identified in the grouper on the third day of infection. In these differentially expressed genes, compared with infected wild-type strains, infection ton B -84 of the RNAi strain group were significantly down-regulated, and 291 were significantly up-regulated ( image 3 ). GO classification and KEGG pathway analysis to determine the biological functions of the differentially expressed genes of the host grouper. The results of KEGG enrichment analysis also showed that DEMs (Differential Expression mRNAs) are highly related to immuni...

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Abstract

The invention provides a Pseudomonas plecoglossicida tonB gene silencing strain and application of the Pseudomonas plecoglossicida tonB gene silencing strain. The bacterial strain is Pseudomonas plecoglossicida tonB-RNAi, the bacterial strain is preserved in the China Center for Type Culture Collection on December 18, 2020, and the preservation number is CCTCC (China Center for Type Culture Collection) NO: M 2020919. Compared with a wild strain, the strain for silencing the Pseudomonas plecoglossicida tonB gene has the advantages that the pathogenicity of the strain to the epinephelus coioides is remarkably reduced, and a spleen transcriptome of the epinephelus coioides can be remarkably changed, so that the strain can be used for researching the pathogenesis of the pseudomonas deformation, and particularly, the Pseudomonas plecoglossicida tonB gene silencing strain has unique advantages in studying the pathogenesis of pseudomonas deformation from the perspective of Epinephelus coioides infection and material transportation.

Description

technical field [0001] The invention belongs to the technical field of microorganisms, and more specifically relates to a strain of Pseudomonas mutans ton B Gene silencing strains and their applications. Background technique [0002] Pseudomonas mutans ( Pseudomonas plecoglossicida ) is the pathogenic bacterium of "visceral white spot disease" of mariculture fish such as large yellow croaker and oblique grouper, and the direct economic loss caused by it exceeds 100 million yuan every year. [0003] ton B The gene encodes the TonB protein in the TonB system (TonB-ExbB-ExbD). Early research showed that ton B Genes are ubiquitous in Gram-negative bacteria. The TonB system, consisting of ExbB-ExbD anchored on the cell membrane and the periplasmic protein TonB, provides energy to TonB-dependent outer membrane receptors (TBDTs) and supports the active transport of important nutrients, including iron, hemoglobin, vitamin B12 and carbohydrate. Iron plays an important but oft...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/113C12N15/78C12N15/31C12R1/38
CPCC07K14/21C12N15/113C12N15/78C12N2310/14
Inventor 赵玲敏胡伶飞鄢庆枇黄力行覃映雪
Owner JIMEI UNIV
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