Fusarium oxyxporum f.sp. niveum type strain FON-GFP (Fusarium oxyxporum f.sp. niveum-green fluorescent protein) for expressing GFP as well as preparation method and application of strain

A FON-GFP, green fluorescent protein technology, applied in the field of genetic engineering, to achieve the effect of wide application field, clear disease resistance mechanism and simple operation

Inactive Publication Date: 2014-04-30
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

In contrast, the studies on rootstock grafting to improve watermelon wilt resistance mainly focus on the physiological and biochemical aspects of grafted plants, but there is no information about the infection characteristics of Fusarium oxysporum watermelon-specific bacteria in grafted rootstocks, especially grafted A report on the colonization antagonism of rootstocks to pathogenic bacteria

Method used

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  • Fusarium oxyxporum f.sp. niveum type strain FON-GFP (Fusarium oxyxporum f.sp. niveum-green fluorescent protein) for expressing GFP as well as preparation method and application of strain
  • Fusarium oxyxporum f.sp. niveum type strain FON-GFP (Fusarium oxyxporum f.sp. niveum-green fluorescent protein) for expressing GFP as well as preparation method and application of strain
  • Fusarium oxyxporum f.sp. niveum type strain FON-GFP (Fusarium oxyxporum f.sp. niveum-green fluorescent protein) for expressing GFP as well as preparation method and application of strain

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Embodiment 1

[0037] 1. Construction of fungal expression vector pBC-GFP

[0038] (1) Obtaining the fungal promoter trpC gene fragment

[0039] According to the trpC gene sequence on the pBARGPE1 plasmid, combined with the characteristics of the Gateway vector construction system, the trpC gene-specific primers trpcp-f and trpcp-r containing the attB site were designed, and the pBARGPE1 plasmid was used as a template to amplify the promoter trpC gene to obtain The 365bp trpC band (such as figure 1 : A), namely the trpC gene, the sequences of primer trpcp-f, primer trpcp-r and trpC gene are respectively as SEQ NO1, SEQ NO2, SEQ NO3;

[0040] (2) Using Gateway cloning technology, through two-step reactions of BP and LR, the trpC gene was replaced into the pMDC83 vector, and a 1436bp band was obtained by PCR amplification, which is the green fluorescent protein expression cassette "trpC+" initiated by the promoter trpC GFP+nos" (such as figure 1 : B), the plasmid containing this expressio...

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Abstract

The invention provides a Fusarium oxyxporum f.sp. niveum type strain FON-GFP (Fusarium oxyxporum f.sp. niveum-green fluorescent protein) for expressing GFP as well as a preparation method and application of the strain. A fungal expression vector with GFP is provided and contains a homomycin B resistance gene (i) hph ( / i) and a GFP gene (i) gfp ( / i), the vector is converted into a Fusarium oxyxporum f.sp. niveum type strain race1 by adopting an agrobacterium-mediated method, a transformant can express the GFP, the converted strain is infected by a grafting rootstock and a root of citrullus lanatus respectively, and the infection condition of Fusarium oxyxporum f.sp. niveum type bacteria in tissues of a plant root and hypocotyls can be accurately analyzed by monitoring fluorescent strains in real time. The Fusarium oxyxporum f.sp. niveum type strain disclosed by the invention has the characteristics of quickly, simply and conveniently indicating the infection way of the Fusarium oxyxporum f.sp. niveum type bacteria in real time, and the method provided by the invention is suitable for various filamentous fungi and can be widely applied to scientific research fields.

Description

technical field [0001] The invention belongs to the field of genetic engineering in biotechnology, and in particular relates to a Fusarium oxysporum watermelon-specific strain FON-GFP expressing green fluorescent protein and a preparation method and application thereof. Background technique [0002] Watermelon wilt is caused by Fusarium oxysporum Fusarium oxyxporum f.sp. niveum , FON) infection caused by soil-borne fungal diseases, is one of the diseases that seriously restrict the production of watermelon. According to the different resistance responses of watermelon varieties to pathogens, four physiological races of Fusarium oxysporum specialized in watermelon have been reported, namely 0, 1, 2 and 3. Green fluorescent protein (green fluorescent protein, GFP) is a widely used reporter gene, which has stable, intuitive, and easy-to-operate fluorescent properties. As a reporter gene, it has been successfully used in the molecular biology research of filamentous fungi. ...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/80A01G7/06C12R1/77
Inventor 张曼羊杏平徐锦华刘广姚协丰李苹芳
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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