Pathogenic fonagl3 gene of Fusarium wilt of watermelon, its deletion dna fragment, deletion mutant and application thereof

A technology of watermelon wilt pathogen and watermelon wilt, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of watermelon industry economic loss, lack of long-term antigens for disease-resistant breeding, environmental pollution, etc., and achieve good fusarium wilt The effect of prevention and control, low cost of prevention and protection, and environmental friendliness

Inactive Publication Date: 2020-09-25
河南省农业科学院园艺研究所 +1
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

In recent years, watermelon cultivation, especially the area of ​​protected land, has rapidly expanded, and the area of ​​heavy cropping and continuous cropping has increased. Watermelon wilt caused by high temperature and high humidity in protected land has become more and more common and serious, causing huge economic losses to the watermelon industry.
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: grafting seedlings has a negative impact on watermelon maturity. , 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 about negative effects such as environmental pollution, ecological damage, and disease resistance, etc.

Method used

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  • Pathogenic fonagl3 gene of Fusarium wilt of watermelon, its deletion dna fragment, deletion mutant and application thereof
  • Pathogenic fonagl3 gene of Fusarium wilt of watermelon, its deletion dna fragment, deletion mutant and application thereof
  • Pathogenic fonagl3 gene of Fusarium wilt of watermelon, its deletion dna fragment, deletion mutant and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment one: Fusarium oxysporum of watermelon FonAGL3 Obtaining of gene deletion mutants

[0050] 1. FonAGL3 Gene deletion DNA fragment construction

[0051] Fusarium wilt of watermelon FonAGL3 Gene knockout adopts the principle of homologous replacement, replacing the target gene with a DNA fragment of the resistance gene hygromycin B (HPH) gene FonAGL3 DNA fragments, replacement fragments specific sequence vector construction takes such as figure 1 , SEQID NO.4 ( FonAGL3 Genes and their upstream and downstream sequences, primers required for gene deletion mutations and their positions), SEQ ID NO.5 ( Partial sequence replacement of FonAGL3 gene and HPH gene ) and SEQ ID NO.6 (HPH gene). Wherein, the primer sequences used in each step in the figure are as follows:

[0052] P1 alg3UF: AGTTTCGTCATCGACAGGTTCC

[0053] P2 alg3UR:TTGACCTCCACTAGCTCCAGCCAAGCCTCTATGTAAGCCCGACCCTCTGGT

[0054] P3 alg3DF: ATAGAGTAGATGCCGACCGCGGGTTCGTACTTATATATCTGGTTCGCGT

[0055]...

Embodiment 2

[0074] Embodiment two: FonAGL3 Gene function analysis of deletion mutants

[0075] 1. FonAGL3 Deletion mutant pathogenicity assay

[0076] ① Take the cultured 10d FonAGL3 Deletion mutant PDA plate, add 20ml of sterile water to prepare a concentration of 1x10 5 Conidia / ml spore suspension for inoculation experiments;

[0077] ② Take watermelon seedlings with two leaves and one heart, remove the root matrix and rinse, then cut off part of the fibrous roots, and put them in FonAGL3 After dipping the roots in the conidia suspension of the deletion mutant for 30 minutes, they were replanted in the plug trays, and each strain was inoculated with 35 watermelon seedlings. Under the temperature of 25°C, they were managed routinely, and the incidence was observed. After the onset, the incidence was recorded and recorded. Calculate the disease index; watermelon seedling disease investigation is according to the following diseased plant grading standards:

[0078] Level 0: The plant...

Embodiment 3

[0101] Embodiment three: FonAGL 3 Application of gene deletion mutants in production

[0102] ① Take the cultured 10d FonAGL3 Gene deletion mutant A6325 strain PDA plate, add 20ml of sterile water, filter through sterile lens paper, collect conidia of Fusarium wilt, and prepare a concentration of 1x10 5 Conidia / ml spore suspension for inoculation experiments;

[0103] ② Soak 105 high-quality watermelon seeds in 2% sodium hypochlorite solution for 5 minutes, wash them with water, and sow 70 watermelon seeds directly in the seedling medium, and 35 watermelon seeds are taken FonAGL3 After soaking the seeds of the gene deletion mutant strain A6325, they were sown in a sterilized substrate, and cultivated at 25°C until the watermelon seedlings had two leaves and one heart;

[0104] ③ Watermelon seedlings with two leaves and one heart were taken, and the root matrix was removed and rinsed. Among them, 35 FonAGL3 Gene deletion mutant A6325 strain soaked watermelon seedlings and ...

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Abstract

The invention discloses a watermelon oxysporum pathogenic FonAGL3 gene as well as a deleted DNA fragment, a deletion mutant and application thereof, and aims to solve the technical problem of biological prevention and treatment on watermelon oxysporum. A pathogenic gene FonAGL3 derived from watermelon fusarium oxysporum is analyzed and screened by inserting watermelon oxysporum T-DNA into a mutant library, by utilizing the homologous gene replacement principle, DNA fragments of the target gene FonAGL3 are replaced by gene DNA fragments of a resistance gene hygromycin B (HPH), a FonAGL3 gene deletion mutant is obtained by establishing a gene deletion carrier and implementing genetic transformation of a wild strain FON-11-06, and the FonAGL3 mutant bacterium has a good wilt prevention and treatment effect and is environmental-friendly and low in prevention and treatment cost.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a pathogenic gene of watermelon Fusarium wilt FonAGL3 , its deletion DNA fragment, deletion mutant and application thereof. Background technique [0002] Fusarium wilt of watermelon is a disease of the vascular system of watermelon caused by Fusarium oxysporum infection of watermelon; the pathogen mainly invades through the root wound or the intercellular space at the top of the root hair, grows between and within the host main wall cells, enters the vascular bundle, decomposes and destroys cells, so that pectin substances accumulate in the ducts, block the ducts, affect water transport, and cause plant wilting. 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. Damaged seedlings can cause rotten seeds before emergence; after emergence, the cotyl...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/31C12N1/15C07K14/37A01N63/30A01P3/00C12R1/77
CPCA01N63/30C07K14/37
Inventor 尚泓泉徐小利梁慎赵卫星常高正李晓慧杨晓明程丹丹高宁宁康利允王彬王龙飞
Owner 河南省农业科学院园艺研究所
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