A non-pathogenic R. solanacearum strain transfected with phage trp574 gene and its preparation method and application

A non-pathogenic, bacteriophage technology, applied in the field of bacterial wilt control, can solve the problems of pathogenic bacteria resistance environment, pollution, and the need to improve the control effect.

Active Publication Date: 2022-05-31
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The chemical control of bacterial wilt is more difficult, because there is no ideal chemical agent at present. The agricultural streptomycin that has been used for a long time has now withdrawn from the market. The application of preparations has a certain effect on delaying and slowing down the occurrence of bacterial wilt, but it cannot fundamentally solve the problem of bacterial wilt control, and the use of a large number of chemical agents will lead to drug resistance of pathogenic bacteria and environmental pollution. Therefore, bacterial wilt The biological control of blight has received extensive attention at home and abroad
[0003] Among them, utilizing non-pathogenic R. solanacearum strains to prevent and treat crop bacterial wilt is one of the key points of research in the biological control of R. solanacearum. In the prior art (Dong Chun, Zeng Xianming, Liu Qiongguang. Utilizing non-pathogenic R. Research on bacterial wilt control of tomato bacterial wilt [J]. Journal of South China Agricultural University, 1999, 020(004): 1-4.; Chen Qinghe, Weng Qiyong, Hu Fangping. Control effect of non-pathogenic bacterial wilt on tomato bacterial wilt [J]. Chinese Journal of Biological Control, 2004,20(1):42-44.) Although there are reports on the successful use of non-pathogenic R. solanacearum for the control of R. solanacearum, the control effect still needs to be improved. In the prior art, methods such as ultraviolet mutagenesis, plant tissue isolation, and knockout of pathogenic genes are mainly used to obtain non-pathogenic strains of R. solanacearum. Reports about R. solanacearum

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  • A non-pathogenic R. solanacearum strain transfected with phage trp574 gene and its preparation method and application
  • A non-pathogenic R. solanacearum strain transfected with phage trp574 gene and its preparation method and application
  • A non-pathogenic R. solanacearum strain transfected with phage trp574 gene and its preparation method and application

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Embodiment 1

[0035] Example 1 Preparation of bacterial wilt engineered bacteria transfected with bacteriophage trp574 gene

[0036] 1. Construction of recombinant plasmids

[0037] In the early stage, the inventors discovered a transcriptional regulator orf30 by sequencing the genome of the lytic phage P574, and further designed amplification primers for the target gene orf30 according to the P574 genome information. The purpose fragment of about 688bp was increased, and the purpose fragment was sequenced, and it was found that the orf30 sequence was 555bp, and its nucleotide sequence was as shown in SEQ ID NO:1, and its encoded amino acid sequence was as shown in SEQ ID NO:2; BALST alignment search found that orf30 only had 82% homology with Ralstonia phage GP4. The protein search results showed that orf30 has a conserved sequence of transcriptional regulator, and the highest amino acid sequence homology is less than 70%. Therefore, rf30 was named trp574 gene, where T stands for Transcri...

Embodiment 2

[0060] Embodiment 2 Tobacco bacterial wilt control pot experiment

[0061] 1. Method

[0062] R. solanacearum culture: activate Tb1546 stored at -80°C with TTC medium, cultivate at 30°C for 48h to obtain bacterial colonies of R. solanacearum, pick a single colony and cultivate in LB liquid medium for 24h for use. Similarly, the activated orf30-Tb1546 strain was cultured in LB liquid for 24-48 h, and set aside. Potted plant inoculation: The time was on June 26, 2019 in the Bacteria Research Room of South China Agricultural University. Each pot is filled with 15kg of soil, and 2 tobacco seedlings with 6 leaf ages are planted, and the number of each treated tobacco is 48, with a total of 24 pots. First inoculate the orf30-Tb1546 strain of engineering bacteria, and inoculate 200 mL of engineering bacteria solution per pot with a concentration of 2 × 10 6 CFU / mL. The next day, the concentration of 5 × 10 6 Ralstonia solanacearum Tb1546 of CFU / mL was sprayed on the rhizosphere...

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Abstract

The invention discloses a non-pathogenic R. solanacearum strain transfected with a phage trp574 gene, a preparation method and application thereof. The strain was deposited in the Guangdong Microbial Culture Collection Center on July 15, 2020, with the preservation number GDMCC No: 61049. In the present invention, the phage trp574 gene is introduced into R. solanacearum by means of molecular biology technology to obtain the transgenic R. solanacearum engineering bacteria. The engineering bacteria has lost its pathogenicity, but has strong colonization ability in rhizosphere soil and tobacco plants , and showed a control effect on tobacco bacterial wilt.

Description

technical field [0001] The invention relates to the technical field of prevention and control of R. solanacearum, more particularly, to a pathogenic R. solanacearum strain transformed with a phage trp574 gene and a preparation method and application thereof. Background technique [0002] Tobacco bacterial wilt disease caused by Ralstonia solanacearum (commonly known as Ralstonia solanacearum) is a devastating disease in tobacco production. Economic losses. R. solanacearum has a wide host range, prefers high temperature, and has complex populations, making it very difficult to control. Production is generally based on agricultural control, disease-resistant breeding and chemical control, but the effect is not very satisfactory. The use of disease-resistant varieties to control bacterial wilt is an economical and easy-to-operate measure. However, at present, my country still lacks high-quality, high-yield and high-resistant tobacco varieties. Moreover, due to environmental c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/15C12N15/33C12N15/80A01N63/30A01P3/00C12R1/645
CPCC07K14/005C12N15/80A01N63/30
Inventor 刘琼光胡蓉花余成鹏钟敏
Owner SOUTH CHINA AGRI UNIV
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