Fondcl1 gene deletion mutant of watermelon Fusarium wilt RNAi component and its construction method
A watermelon wilt pathogen and gene deletion technology, applied in the field of bioengineering, can solve problems such as environmental pollution, lack of long-term antigens in disease-resistant breeding, and ecological destruction
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Embodiment 1
[0037] Example 1: Obtaining of the FonDCL1 gene deletion mutant of Fusarium wilt of watermelon
[0038] 1. Construction of FonDCL1 gene deletion recombinant DNA fragment
[0039] The knockout of the watermelon wilt fungus FonDCL1 gene knockout adopts the principle of homologous replacement, and replaces the DNA fragment of the target gene FonDCL1 with the DNA fragment of the resistance gene hygromycin B (HPH). The FonDCL1 gene sequence is SEQ ID NO:2.
[0040] 1) Design of primers for recombinant DNA fragments with deletion of FonDCL1 gene
[0041] Using the 1.4Kb HPH gene with a promoter and a terminator as a template, the first round of PCR amplification, designed primers HYG-F / HYG-R for amplifying the 1.4Kb HYG gene fragment, used to amplify FonDCL1 Primers Fon1DCL1-LBCK / Fon1DCL1-HPH-LB-R for the upstream 2Kb gene fragment, primers Fon1DCL1-HPH-RB-F / Fon1DCL1-RBCK for amplifying the downstream 2Kb gene fragment of FonDCL1;
[0042] In the second round of PCR amplification...
Embodiment 2
[0060] Example 2: Functional Analysis of FonDCL1 Gene Deletion Mutants
[0061] 1. Determination of pathogenicity of FonDCL1 deletion mutants
[0062] 1) Take the FonDCL1 deletion mutant that has been cultured for 7 days, and use a 5mm hole punch to get 3 pieces of mycelium, add it to 200ml of PDB medium, shake it at 28°C and 150rpm for 3 days, filter it with 3 layers of lens paper, and collect the meristems The spore liquid was centrifuged at 5000rpm for 10min, and the concentration of the spore suspension was adjusted to 2×10 with sterile water. 6 pieces / ml.
[0063] 2) Take watermelon seeds, soak them in 0.1% sodium hypochlorite at 28°C for 6-12h, wrap the watermelon seeds in gauze and wash them for 3-5 times, evenly spread the watermelon seeds on a moist towel, and accelerate germination at 28°C for 2 days in the dark. Sow the germinated seeds in a nutrient bowl (15×15cm) containing nutrient soil such as coconut bran, 10 watermelon seeds per nutrient bowl, under 25°C, un...
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