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Regulation and control system for enhancing disease resistance of tomato plants and regulation and control method and application thereof

A control system and disease resistance technology, applied in the field of plant genetic engineering, can solve problems affecting tomato yield and quality, leaf wilting, environmental and human health risks and impacts, and achieve enhanced regulation, enhanced disease resistance, and improved resistance. disease effects

Pending Publication Date: 2022-03-01
山东狮克现代农业投资有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In tomato, Pseudomonas syringae frequently infects the leaves and veins, usually starting from the lower mature leaves, causing the leaves to wither, seriously affecting the yield and quality of tomatoes
[0003] At present, the control of tomato diseases caused by Pseudomonas syringae is mostly chemical control, but chemical control of diseases will also bring potential risks and impacts on the environment and human health

Method used

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  • Regulation and control system for enhancing disease resistance of tomato plants and regulation and control method and application thereof
  • Regulation and control system for enhancing disease resistance of tomato plants and regulation and control method and application thereof
  • Regulation and control system for enhancing disease resistance of tomato plants and regulation and control method and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: Obtaining and phenotypic analysis of each transgenic strain

[0050] 1. Experimental method:

[0051] 1.1. Obtaining of WRKY75 transgenic strains

[0052]The specific sequence of WRKY75 coding region and the complete CDS sequence of WRKY75 (https: / / phytozome.jgi.doe.gov / pz / portal.html database, the number of WRKY75 is Solyc05g015850) were obtained by BLAST analysis, and the sequences were amplified and ligated into Plant RNAi interference vector and pSuper1300 overexpression vector. The above-mentioned vectors were transformed into Agrobacterium strain LBA4404 by freeze-thaw method, and then transformed into tomato by Agrobacterium-mediated leaf disk method to obtain the RNAi silencing strain of WRKY75 and its overexpression strain. The expression level of the obtained strains was detected by qRT-PCR.

[0053] 1.2 Phenotype observation and bacterial biomass determination of each strain of SlWRKY75

[0054] with 10mM MgCl 2 The Pseudomonas syringae cel...

Embodiment 2

[0061] experimental method:

[0062] 1.1 Yeast one-hybrid experiment

[0063] The full-length sequence of the WRKY75 coding region was constructed into the GAD vector; the four promoter sequences (P1-P4) of GH3.3 were connected into the pLacZi2u yeast expression vector. Then the constructed WRKY75-GAD+P1-LacZ, WRKY75-GAD+P2-LacZ, WRKY75-GAD+P3-LacZ and WRKY75-GAD+P4-LacZ were transformed into the yeast EGY48 strain respectively.

[0064] 1.2. Luciferase (LUC) activation experiment

[0065] The full-length sequence of the WRKY75 coding region was constructed into the pZP211-FLAG plant expression vector; the GH3.3 promoter sequence (P2 region) was connected into the pZP211-LUC plant expression vector. The constructed WRKY75-FLAG / -FLAG+P2pro-LUC were transformed into Agrobacterium GV3101 respectively, and then the transient transformation experiment of tobacco was carried out.

[0066] 1.3. Gel Migration Arrest Assay (EMSA)

[0067] The experiment was mainly carried out with ...

Embodiment 3

[0075] experimental method:

[0076] 1.1. Yeast two-hybrid experiment

[0077] The full-length sequence of WRKY75 coding region was constructed into AD vector; the full-length sequence of VQ10 coding region was connected into BD yeast expression vector. Then the constructed WRKY75-AD and VQ10-BD were transformed into yeast Y2H strain.

[0078] 1.2. Luciferase complementation experiment

[0079] The full-length sequence of WRKY75 coding region was constructed into pCambia-nLUC plant expression vector; the full-length sequence of VQ10 coding region was connected into pCambia-cLUC plant expression vector. The constructed WRKY75-nLUC and VQ10-nLUC were respectively transformed into Agrobacterium GV3101, and then the transient transformation experiment of tobacco was carried out.

[0080] Planting method: Tobacco (Niba benthamiana) is planted in the greenhouse, the sunshine time is 16 hours, the dark cultivation is 8 hours, the cultivation temperature is normal temperature 25 ℃,...

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Abstract

The invention discloses a regulation and control system for enhancing disease resistance of tomato plants and a regulation and control method and application thereof, and relates to the technical field of plant genetic engineering. The invention provides a regulation and control system for enhancing disease resistance of tomato plants. The regulation and control system comprises a transcription factor WRKY75 and auxin amino synthase GH3.3, the amino acid sequence of the transcription factor WRKY75 is SEQ ID NO. 1; the amino acid sequence of the auxin amino synthase GH3.3 is SEQ ID NO. 2. In the prevention and treatment of tomato diseases caused by pseudomonas syringae, a WRKY75-mediated auxin-related disease-resistant regulation and control path is found and confirmed in tomatoes for the first time by utilizing reverse genetics; the transcription factor WRKY75 can activate the expression of the encoding auxin amino synthase gene GH3.3 and reduce the level of active auxin (free state), thereby improving the disease resistance of tomato plants. In the process, the transcription cofactor VQ10 interacts with the transcription factor WRKY75, so that the regulation of the WRKY75 on GH3.3 is enhanced, and the disease resistance of tomato plants is enhanced.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a control system for enhancing the disease resistance of tomato plants, a control method and an application thereof. Background technique [0002] Pseudomonas syringae (Pseudomonas syringae) is an aerobic, saprophytic Gram-negative bacteria, and the incidence of plant diseases caused by it ranks first among the top ten bacterial plant diseases. In tomato, Pseudomonas syringae often occurs on the leaves and veins, usually from the lower mature leaves, causing the leaves to wither and seriously affecting the yield and quality of tomatoes. [0003] At present, the control of tomato diseases caused by Pseudomonas syringae is mostly chemical control, but chemical control of diseases will also bring potential risks and impacts on the environment and human health. Therefore, how to adopt environmentally friendly control measures to improve tomato disease resistance is...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N9/00C12N15/29C12N15/52C12N15/84A01H5/00A01H6/82
CPCC07K14/415C12N9/93C12N15/8205C12N15/8218C12N15/8281C12N15/8294C12Y603/02
Inventor 孟伦庄焜扬陈飞勇史庆华许思思李彦刘汉敬
Owner 山东狮克现代农业投资有限公司
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