Bacteriophage trp574 gene and application thereof

A phage and gene technology, applied in the phage trp574 gene and its application fields, to achieve the effect of great application prospects

Active Publication Date: 2020-12-08
SOUTH CHINA AGRI UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, non-pathogenic strains of R. solanacearum are mainly isolated and screened from plant tissues (Xiao Tian, ​​Xiao Chonggang, Zou Yang, et al. Preliminary study on the control effect of non-pathogenic strains of R. solanacearum on tobacco bacterial wilt [J].Plant Protection, 2008,34(002):79-82.), or through artificial selective cultivation, pathogenic strains are transformed into non-pathogenic strains, or obtained by ultraviolet mutagenesis (Chen Qinghe , Weng Qiyong, Hu Fangping. Control effect of non-pathogenic R. solanacearum strains on tomato bacterial wilt[J]. Knockout of disease-related virulence factors to obtain non-pathogenic R. solanacearum strains, and there is no report on introducing foreign genes into R. solanacearum strains to obtain non-pathogenic R. solanacearum strains

Method used

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  • Bacteriophage trp574 gene and application thereof
  • Bacteriophage trp574 gene and application thereof
  • Bacteriophage trp574 gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1 Bioinformatics analysis of trp574 gene (orf30)

[0050] In the early stage of the present invention, a transcription regulator orf30 was found by sequencing the genome of the lytic phage P574, and the gene protein sequence was compared using NCBI's Blastp; using the ProtParam tool (https: / / web.expasy.org / protparam / ) to analyze protein physicochemical properties; use SMART server (http: / / smart.embl-heidelberg.de / ) for protein domain analysis; use SWISS-MODEL (https: / / www.swissmodel.expasy.org / ) for Protein model construction.

[0051] NCBI's Blastp results show that the orf30 gene belongs to the XRE family, and the comparison results show that it has 69% homology with the transcription regulator of the Burkholderia phage Bcepmigl belonging to the XRE family. The four protein sequences with the highest scores in the download comparison results are, Use ClustalX software for multiple sequence alignment, and the alignment results are colored by Boxshade (https: / ...

Embodiment 2

[0052] Embodiment 2trp574 gene (orf30) cloning and recombinant plasmid construction

[0053] 1. Method

[0054] (1) Use the Universal Phage DNA Extraction Kit (Product No.: KG005-1) of Guangzhou Nuojing Biotechnology Co., Ltd. to extract phage DNA, and design the amplification primers orf30-F / orf30-R of the target gene orf30 according to the P574 genome information (such as shown in Table 2); with phage DNA as template and orf30-F / orf30-R as primers, Novozyme MasterMix amplifies the target gene orf30, the amplification system is Master Mix 25μL, Primer1 1μL, Primer2 1μL, template DNA 1μL (2 0 to 50 μL. The PCR amplification conditions were: 95°C for 3min; 95°C for 10s, 55°C for 30s, 72°C for 40s, 35 cycles; 72°C for 10min, 4°C, ∞. After the PCR products were electrophoresed, the target bands in the gel electrophoresis were recovered by gel cutting.

[0055] (2) The pBBR1MCS4 plasmid was extracted according to the instructions of the Axygen plasmid mini-extraction kit, an...

Embodiment 3

[0061] The preparation of embodiment 3 transfer trp574 gene (orf30) solanacearum Tb1546 and Tb15

[0062] 1. Preparation and electrotransformation of R. solanacearum competent cells

[0063] For the preparation of R. solanacearum Tb15 electroshock-competent and the electroshock transformation method, refer to the method of Lavie et al. (2002).

[0064] (1) Ralstonia solanacearum Tb1546 and Tb15 were inoculated in 10 mL NA medium for overnight culture, and the bacterial solution was placed on ice for 15 min, then packed into pre-cooled 1.5 mL centrifuge tubes, centrifuged at 6000 rpm at 4 °C for 5 min, and discarded. supernatant.

[0065](2) Add 1 mL of 10% glycerol to resuspend, centrifuge at 6000 rpm at 4°C for 5 min, and discard the supernatant.

[0066] (3) Repeat step (2) twice.

[0067] (4) Add 100 μL of 10% glycerol to resuspend to obtain Ralstonia solanacearum competence.

[0068] (5) Take 50 μL of prepared Tb1546 and Tb15 Ralstonia solanacearum competently in 0.1 c...

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Abstract

The invention discloses a bacteriophage trp574 gene and application thereof. A nucleotide sequence of the bacteriophage trp574 gene is as shown by SEQ ID NO:1, and a coded amino acid sequence of the bacteriophage trp574 gene is as shown by SEQ ID NO:2. The invention discloses a transcriptional regulation factor trp574 gene belonging to the XRE family for the first time, and researches of the invention shows that a ralstonia solanacearum strain into which the trp574 gene is transformed almost loses pathogenicity, and the trp574 gene has the comprehensive effects of remarkably interfering the growth, metabolism, pathogenicity and the like of ralstonia solanacearum, can be used for preparing ralstonia solanacearum strains without pathogenicity, and has a great application prospect in preventing and treating bacterial wilt.

Description

technical field [0001] The invention relates to the technical field of Ralstonia solanacearum control technology, and more specifically relates to a bacteriophage trp574 gene and its application. Background technique [0002] Ralstonia solanacearum (Ralstonia solanacearum for short) is a Gram-negative bacterium of the β-Proteobacteria subphylum that grows in soil. The pathogen usually invades the intercellular spaces of the cortex from the wounds of plant roots or stems or from the root caps of uninjured secondary roots. By destroying the intercellular glue layer, the cell walls are separated, deformed, and cavities are formed, thereby infecting The xylem parenchyma stimulates the small cells near the vessel to form the intrusion and migrate into the intrusion. Ralstonia solanacearum continued to be released into the duct after the invading body ruptured, and multiplied and spread rapidly in the duct, causing the plants to wilt and die. Known as crop bacterial wilt, this d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/34C12N15/11C12N1/21C07K14/01C12R1/01C12R1/19
CPCC07K14/005C12N2795/14122Y02A50/30
Inventor 刘琼光胡蓉花余成鹏钟敏
Owner SOUTH CHINA AGRI UNIV
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