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RNAi component FonAgol gene deletion mutant of fusarium oxyspirum f.sp.niveum and construction method of RNAi component FonAgol gene deletion mutant

A watermelon fusarium wilt, gene deletion technology, applied in genetic engineering, plant genetic improvement, recombinant DNA technology, etc., can solve the problems of disease resistance breeding, lack of long periodic antigens, ecological damage, environmental pollution, etc.

Inactive Publication Date: 2018-10-30
ENVIRONMENT & PLANT PROTECTION INST CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0009] 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

Method used

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  • RNAi component FonAgol gene deletion mutant of fusarium oxyspirum f.sp.niveum and construction method of RNAi component FonAgol gene deletion mutant
  • RNAi component FonAgol gene deletion mutant of fusarium oxyspirum f.sp.niveum and construction method of RNAi component FonAgol gene deletion mutant
  • RNAi component FonAgol gene deletion mutant of fusarium oxyspirum f.sp.niveum and construction method of RNAi component FonAgol gene deletion mutant

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Embodiment 1

[0035] Example 1: Obtaining of the FonAgo1 gene deletion mutant of Fusarium wilt of watermelon

[0036] 1. Construction of FonAgo1 gene deletion recombinant DNA fragment

[0037] The knockout of the watermelon wilt fungus FonAgo1 gene knockout adopts the principle of homologous replacement, and replaces the DNA fragment of the target gene FonAgo1 with the DNA fragment of the resistance gene hygromycin B (HPH). The FonAgo1 gene sequence is SEQ ID NO:2.

[0038] 1) Design of primers for recombinant DNA fragments with deletion of FonAgo1 gene

[0039] 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 FonAgo1 Primers Fon1Ago1-LBCK / Fon1Ago1-HPH-LB-R for the upstream 2Kb gene fragment, primers Fon1Ago1-HPH-RB-F / Fon1Ago1-RBCK for amplifying the downstream 2Kb gene fragment of FonAgo1;

[0040] In the second round of PCR amplification...

Embodiment 2

[0057] Example 2: Functional Analysis of FonAgo1 Gene Deletion Mutants

[0058] 1. Determination of pathogenicity of FonAgo1 deletion mutants

[0059] 1) Take the FonAgo1 deletion mutant that has been cultured for 7 days, and use a 5mm hole punch to take 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.

[0060] 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...

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Abstract

The invention discloses an RNAi component FonAgol gene deletion mutant of fusarium oxyspirum f.sp.niveum and a construction method of the RNAi component FonAgol gene deletion mutant. The RNAi component FonAgol gene deletion mutant and the construction method thereof are characterized in that a reported Argonaute protein sequence is homologously compared with a Fon gemonic sequence to obtain a FonAgol gene of the fusarium oxyspirum f.sp.niveum; a target gene FonAgol is replaced with a DNA fragment of a hygromycin B (HPH) gene by adopting a homologous gene replacement principle and using a Split-marker strategy; a gene deletion mutant of the FonAgol is obtained by constructing a gene homologous recombination fragment and using genetic transformation of a wild strain Fon1. The FonAgol mutanthas the characteristics of higher sensitivity to pathogenicity weakening of watermelon seedlings, abiotic stress factors such as fluorescent brightener CFW, Congo red CR and hydroxycarbamide and the like, which provides a research foundation for growth and development of Fon1 and functional assignment of small RNA produced in the process of infection watermelons.

Description

technical field [0001] The invention relates to a FonAgo1 gene deletion mutant of RNAi component of Fusarium wilt of watermelon and a construction method thereof, belonging to the technical field of bioengineering. Background technique [0002] Watermelon wilt is a watermelon vascular system disease caused by the infection of Fusarium oxysporum f. sp.niveum. 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 leaves are d...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/37C12N1/15C12N15/80
CPCC07K14/37C12N15/80
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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