A kind of Trichoderma harzianum gene knockout method

A gene knockout and Trichoderma technology, applied in the biological field, can solve the problems of off-target, multiple false positives, and high off-target probability, and achieve the effect of promoting plant growth and shortening the experimental period.

Active Publication Date: 2018-11-16
NANJING AGRICULTURAL UNIVERSITY
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Problems solved by technology

During the transformation process, both HR and NHEJ are relatively active, but in filamentous fungi, the efficiency of NHEJ is much higher than that of HR, which directly leads to off-target gene knockout and more false positives
In the knockout of the Trichoderma harzianum gene, the homologous recombination efficiency is about 2% (Montero Barrientos et al., 2011), and the off-target probability is high. Currently, there are few cases of knockout of the Trichoderma harzianum gene in the world

Method used

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  • A kind of Trichoderma harzianum gene knockout method
  • A kind of Trichoderma harzianum gene knockout method
  • A kind of Trichoderma harzianum gene knockout method

Examples

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Embodiment 1

[0060] Example 1 Trichoderma harzianum Hydrophobic Protein Gene Knockout

[0061] 1. Trichoderma strains: Trichoderma harzianum SQR-T037 (CGMCC No.3308) and Trichoderma harzianum CBS 226.95 (Joint Genome Institute (JGI), CBS 226.95).

[0062] 2. Knockout vector construction and amplification of knockout fragments (2 days)

[0063] Transcriptomic analysis showed that Trichoderma SQR-T037 and CBS 226.95 antagonized the pathogenic fungus Fusarium oxysporum (Foc4), and the transcript levels of the hydrophobic protein hfb4 in SQR-T037 and hfb10 in CBS 226.95 were significantly increased. Mutants were constructed by gene knockout, and the function of hydrophobic proteins in Trichoderma antagonizing pathogenic fungi was analyzed.

[0064] Amplify the 5' and 3' homology arm primers of the target gene (hfb4 (SEQ ID NO.1) in SQR-T037 and hfb10 (SEQ ID NO.2) in CBS 226.95) respectively:

[0065] Using SQR-T037 genomic DNA as a template, primers hfb4_5F: gacggccagtgaattcttggagtagggatggg...

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Abstract

The invention relates to a trichoderma harzianum gene knockout method. The trichoderma harzianum gene knockout method comprises the following steps: (1) constructing a knockeout gene segment; (2) converting PEG-CaCl2 protoplast; and (3) screening a positive muton through bacterial colony PCR. A complete trichoderma harzianum gene knockout method is established, homologous assembly efficiency is 20%; and meanwhile, the bacterial colony PCR technology greatly shortens the period of obtaining positive converters, and efficiently knocks out a target gene.

Description

technical field [0001] The invention belongs to the field of biological technology and relates to a gene knockout method of Trichoderma harzianum. technical background [0002] The genus Trichoderma is an important soil habitant fungus, which belongs to the kingdom Fungi, Dikarycota, Deuteromycotina, Hyphospora, Polychospora, and from the family Stemporaceae. Its mycelial growth and spore germination can It adapts to a wide range of temperature, humidity and pH, and has strong saprophytic properties and fast reproduction speed, so it can quickly occupy a favorable space. At present, Trichoderma has been widely used in the control of various plant fungal diseases, and is an ideal biocontrol fungus and plant growth promoting fungus (Plant Growth Promoting Fungus, PGPF) (Shoresh et al. 2010). In the past five years, Trichoderma preparations in the global market have grown exponentially, and there are 38 officially registered Trichoderma preparations (Woo et al. 2014). Current...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/90C12N1/15C12R1/885
CPCC07K14/37C12N15/902
Inventor 沈其荣张建黄启为凌宁
Owner NANJING AGRICULTURAL UNIVERSITY
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