Application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel

A technology of Trichoderma viridans and hygromycin, applied in the field of genetic engineering, can solve problems such as the influence of killing activity

Active Publication Date: 2016-03-02
SHANGHAI JIAO TONG UNIV
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  • Claims
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Problems solved by technology

[0002] At present, the global demand for agricultural and sideline products (quantity and quality) is increasing year by year. The traditional mode of crop cultivation needs to rely on highly efficient chemical pesticides to prevent pathogenic microorganisms from invading, so as to create stable and considerable economic benefits.

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  • Application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel
  • Application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel
  • Application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel

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Embodiment

[0055] The present invention utilizes the improved Agrobacterium tumefaciens-mediated transformation technology (ATMT) to knock out the carbon metabolism inhibitor CRE1 that antagonizes Trichoderma aeruginosa T. atroviride23. By comparing the growth traits of the mutant strain (Δcre1) and the wild type (widetype, WT), the regulation effect of cre1 gene on the growth and metabolism of Trichoderma aureides was ascertained. The study found that after the cre1 gene was silenced, the growth of Δcre1 mycelium was slower than that of WT, and sporulation lagged behind. However, the activities of cell wall degrading enzymes (chitinase, β-1,3-glucanase, cellulase, protease, amylase) secreted by a variety of Trichoderma regulated by it are increased, which in turn can improve the resistance of Trichoderma to multiple The effect of plant pathogens.

[0056] 1. Trichoderma dark green carbon metabolism inhibitor CRE1

[0057] Check out the published Trichoderma reesei CRE1 protein sequenc...

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Abstract

The invention discloses an application of improved ATMT (Agrobacterium Tumefaciens-Mediated Transformation) to construction of trichoderma atroviride T23 deltaCrel. The application is characterized in that the genomic DNA of trichoderma atroviride T23 is taken as a template, PCR (Polymerase Chain Reaction) amplification is carried out on the Crel gene sequence, and thus upstream and downstream flanking sequences of the Crel gene are obtained; then leading in respectively to knock out upstream and downstream of plasmid pC1300qh, so as to form recombinant plasmid pC1300qh-deltaCrel; converting escherichia coli competence of the recombinant plasmid pC1300qh-deltaCrel, performing verification, screening positive clone, extracting recombinant plasmid pC1300qh-deltaCrel, leading in agrobacterium tumefaciens AGL1, and carrying out improved ATMT. By using the improved ATMT technique, a trichoderma atroviride carbon metabolism inhibiting factor CRE1-knocked-out mutant strain is successfully constructed; the T23 deltaCrel bacterial strain has the functions of promoting secretion and activity of degrading enzyme of cell walls of trichoderma spp., and indirectly improving the function of trichoderma spp. to antagonize pathogenic bacteria.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the application of an improved Agrobacterium-mediated ATMT transformation method in the construction of Trichoderma dark green T23△Crel strain. Background technique [0002] At present, the global demand for agricultural and sideline products (quantity and quality) is increasing year by year. The traditional mode of crop cultivation needs to rely on highly efficient chemical pesticides to prevent pathogenic microorganisms from invading, so as to create stable and considerable economic benefits. The negative effects of use have become increasingly apparent. First of all, some pathogenic bacteria are resistant to low-dose chemical pesticides, and the killing activity is affected; more importantly, consumers are concerned about eating healthy, safe, and pesticide-free agricultural products and food. The degree of attention has been continuously increased. Therefore, the research ...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/885
Inventor 李雅乾周于聪余传金谢秋瑾陈捷
Owner SHANGHAI JIAO TONG UNIV
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