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Watermelon oxysporum pathogenic FonAGL3 gene as well as deleted DNA fragment, deletion mutant and application thereof

A watermelon fusarium wilt pathogen and gene deletion technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of watermelon industry economic losses, environmental pollution, long-term lack of antigens in disease-resistant breeding, etc., to achieve low cost of prevention and control, Environmentally friendly, good effect of Fusarium wilt control

Inactive Publication Date: 2017-10-27
河南省农业科学院园艺研究所 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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  • Watermelon oxysporum pathogenic FonAGL3 gene as well as deleted DNA fragment, deletion mutant and application thereof
  • Watermelon oxysporum pathogenic FonAGL3 gene as well as deleted DNA fragment, deletion mutant and application thereof
  • Watermelon oxysporum pathogenic FonAGL3 gene as well as deleted DNA fragment, deletion mutant and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Watermelon Fusarium oxysporum FonAGL3 Obtaining Gene Deletion Mutants

[0050] 1. FonAGL3 Gene deletion DNA fragment construction

[0051] Watermelon Fusarium wilt FonAGL3 Gene knockout adopts the principle of homologous replacement, and replaces the target gene with the DNA fragment of the resistance gene hygromycin B (HPH) gene FonAGL3 DNA fragment, replacement fragment specific sequence vector construction takes as figure 1 , SEQ ID NO.4 ( FonAGL3 Gene and its upstream and downstream sequences, primers required for gene deletion mutation and their positions), SEQ ID NO.5 ( FonAGL3 gene and HPH gene replace part of the sequence ) and SEQ ID NO. 6 (HPH gene). Among them, the primer sequences used in each step in the figure are as follows:

[0052] P1 alg3UF:AGTTTCGTCATCGACAGGTTCC

[0053] P2 alg3UR: TTGACCTCCACTAGCTCCAGCCAAGCCTCTATGTAAG CCCGACCTCT GGT

[0054] P3 alg3DF: ATAGAGTAGATGCCGACCGCGGGTTCGTACTTATATATCTGGTTCGCGT

[0055] P4 alg3DR: ATCGC...

Embodiment 2

[0074] Embodiment 2: FonAGL3 Deletion mutant gene function analysis

[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 the watermelon seedlings with two leaves and one heart, remove the root matrix and rinse them, cut off some fibrous roots, FonAGL3 Deletion mutant conidia suspensions were soaked in the root for 30 minutes, and then replanted in plug trays. Each strain was inoculated with 35 watermelon seedlings. At 25°C, they were routinely managed, and the incidence was observed. The disease index was calculated; the disease investigation of watermelon seedlings was based on the following classification standards for diseased plants:

[0078] Level 0: The plant is robust, true leaves and cotyledons are green;

[0079] Grade 1: 1 or both cotyledons turn slightly ...

Embodiment 3

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

[0102] ① Take the cultured 10d FonAGL3 The gene deletion mutant A6325 strain PDA plate was added with 20ml of sterile water, filtered with sterile lens paper, and the conidia of Fusarium wilt was collected and prepared to a concentration of 1×10 5 Conidia / ml spore suspension for inoculation experiments;

[0103] ② 105 high-quality watermelon seeds were soaked in 2% sodium hypochlorite solution for 5 minutes and washed with water. Among them, 70 watermelon seeds were directly sown in the seedling matrix, and 35 watermelon seeds were taken FonAGL3 After the gene deletion mutant A6325 strain was soaked, it was sown in a sterilized substrate, and cultivated at 25°C until the watermelon seedlings had two leaves and one heart;

[0104] ③ Take the watermelon seedlings with two leaves and one heart, remove the root matrix and rinse them. Among them, 35 FonAGL3 Gene deletion mutant A6325 strain...

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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 Fusarium wilt of watermelon FonAGL3 , its deletion DNA fragment, deletion mutant and its application. Background technique [0002] Watermelon fusarium wilt is a disease of the watermelon vascular system caused by the infection of Fusarium oxysporum. The pathogen mainly invades through the root wound or the intercellular space at the top of the root hair, and after growing between and within the cells of the host wall, it enters the vascular bundle and decomposes Cells accumulate pectin substances in the ducts, block the ducts, affect water transport, and cause plant wilting. Watermelon fusarium wilt can occur from seedlings to adult plants, and the disease is the most serious at the stage of melon setting and the late stage of melon expansion. Damaged seedlings can cause rotten seeds before emergence; after unearthed, the cotyledons and true leaves ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N1/15C07K14/37A01N63/04A01P3/00C12R1/77
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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