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.
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[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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