Fusarium oxysporum f.sp.niveum RNAi component FonDCL1 gene deletion mutant and construction method thereof
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A watermelon wilt pathogen and gene deletion technology, applied in the field of bioengineering, can solve problems such as environmental pollution, ecological damage, and lack of long-term antigens in disease-resistant breeding
Active Publication Date: 2018-11-02
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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[0011] At present, people have adopted various measures to strengthen the prevention and treatment of Fusarium wilt of watermelon, such as grafting seedlings, disease-resistant breeding, chemical control, etc., but their effects have certain limitations, and also cause certain negative effects. , Quality has an important impact, the long cycle of disease-resistant breeding and the lack of antigens, the extensive use of pesticides in chemical control will bring negative effects such as environmental pollution, ecological damage and disease resistance
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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 take 3 pieces of mycelium, add them to 200ml of PDB medium, shake at 28°C and 150rpm for 3 days, filter 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 6-12 hours, wrap the watermelon seeds in gauze and wash them for 3-5 times, evenly spread the watermelon seeds on a wet 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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Abstract
The invention discloses a fusarium oxysporum f.sp.niveum RNAi component FonDCL1 gene deletion mutant and a construction method thereof. According to the fusarium oxysporum f.sp.niveum RNAi component FonDCL1 gene deletion mutant and the construction method thereof, according to a reported Dicer like 1 protein sequence, homologous alignment is conducted on a Fon genomic sequence to obtain a fusariumoxysporum f.sp.niveum FonDCL1 gene, by adopting a homologous gene substitution principle, by means of a Split-marker strategy, the target gene FonDCL1 is substituted for a hygromycin B (HPH) gene DNAfragment, and by constructing a homologous gene recombinant fragment, through genetic transformation of a wild strain Fon1, the FonDCL1 gene deletion mutant is obtained. The FonDCL1 mutant has the advantages that the virulence to watermelon seedlings is enhanced, and the FonDCL1 mutant is more sensitive to abiotic stress factors of monensin sodium salt, hydroxycarbamide, a fluorescent whitening agent CFW, Congo red (CR), hydrogen peroxide and the like, so that a research foundation is provided for functional assignment of small RNA generated in processes of self growth and development and watermelon infection of the Fon1.
Description
technical field [0001] The invention relates to a FonDCL1 gene deletion mutant of the RNAi component of Fusarium wilt of watermelon and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Fusarium wilt of watermelon is a watermelon vascular system disease caused by Fusarium oxysporum f.sp.niveum infection. The pathogen mainly invades through the root wound or the intercellular space at the top of the root hair. After growing in the cells, it enters the vascular bundle, decomposes and destroys the cells, accumulates pectin substances in the ducts, blocks the ducts, affects water transport, and causes plant wilting. [0003] Fusarium wilt of watermelon can occur from seedlings to adult plants, and the incidence is the most serious in the stage of melon setting and the later stage of melon expansion. Injured seedlings can cause rotten seeds before emergence and become ill after emergence. The cotyledons and true leav...
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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/15C12N15/55C12N9/16C12R1/77
CPCC12N9/16C12Y301/26003
Inventor 曾凡云彭军漆艳香丁兆健谢艺贤张欣
Owner ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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