Application of cucumber CsWRKY50 gene to strengthening downy mildew resistance of cucumber

A cucumber downy mildew and cucumber technology, applied in the field of crop genetic engineering, can solve the problems of pesticide pollution, yield decline, etc., and achieve the effects of safe application methods, improved resistance, and enhanced resistance

Active Publication Date: 2018-09-04
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0005] The purpose of the present invention is to provide the application of a cucumber CsWRKY50 gene in enhancing the resistance of cucumber downy mildew, so as to solve the

Method used

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  • Application of cucumber CsWRKY50 gene to strengthening downy mildew resistance of cucumber
  • Application of cucumber CsWRKY50 gene to strengthening downy mildew resistance of cucumber
  • Application of cucumber CsWRKY50 gene to strengthening downy mildew resistance of cucumber

Examples

Experimental program
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Effect test

Example Embodiment

[0020] Example 1 Cloning of Cucumber CsWRKY50 Gene and Construction of Overexpression Vector

[0021] (1) For the yellow light variety, Xintaimi thorn was selected as the test material. This variety has strong growth potential, with rooting melons on the 4th to 5th nodes, melon-shaped rod-shaped, well-proportioned, bluish green, dense thorns, with longitudinal edges, but not obvious. The cucumber seeds are sown in a solar greenhouse for ordinary cultivation and management. When the plants begin to set fruit, the young leaves are quickly frozen with liquid nitrogen, and the RNA of the young cucumber leaves is extracted by the Trizol (purchased from Life Technologies) extract. Use Thermo Fisher's reverse transcription kit for reverse transcription to obtain cDNA, and store at -20°C for later use.

[0022] (2) Design the amplification primers with restriction sites for cucumber CsWRKY50 gene, the primer sequence and the restriction sites carried are as follows (the underlined are the...

Example Embodiment

[0040] Example 2 Genetic transformation of cucumber CsWRKY50 gene and obtaining of transgenic plants

[0041] 1. Agrobacterium-mediated genetic transformation of cucumber genes

[0042] After infecting cucumber cotyledons with the Agrobacterium engineering strain LBA4404 containing the pBI121-35S::CsWRKY50 overexpression vector and screening the differentiation medium and rooting medium, the regenerated plants obtained were subjected to PCR detection with 35S upstream primers and gene downstream primers . The specific steps are as follows:

[0043] (1) Choose plump cucumber seeds and soak them in water for about 1 hour, such as figure 2 As shown in middle A, first sterilize with ethanol with a mass ratio of 75% on a sterile table for 30 seconds, then sterilize with NaClO with a mass ratio of 4.0% for 13 minutes, and finally rinse with sterilized ultrapure water for 3 to 4 times. Use high-temperature sterilized tweezers to sow the seeds on the MS medium plate and cultivate in the d...

Example Embodiment

[0061] Example 3 Cucumber Downy Mildew Resistance Test

[0062] (1) Acquisition of cucumber downy mildew pathogen:

[0063] The diseased leaves containing the downy mildew layer are collected from the greenhouse, rinse the mildew layer on the diseased spots with clean water, and then placed in an incubator, 20℃, relative humidity 90%, dark and moisturizing for 24 hours, when the gray mold layer grows, The diseased leaves are soaked in water, and the sporangia are brushed into a test tube filled with water with a writing brush to obtain an isolated substance. The isolate was inoculated on the leaves of the previously planted cucumber susceptible varieties. The leaves were placed in a large petri dish with a water-soaked filter paper at the bottom. The inoculum was sucked with a pipette and dropped on the back of the leaves, and placed at 22°C during the day and night In a light incubator at 18°C, leaves full of sporangia can be obtained from 4 to 6 days. Before inoculation, use a ...

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Abstract

The invention discloses application of a cucumber CsWRKY50 gene to strengthening the downy mildew resistance of the cucumber, the specific application is to overexpress the cucumber CsWRKY50 gene in acucumber plant, and the nucleotide sequence of the cucumber CsWRKY50 gene is shown as in SEQ ID:1. That overexpressing the CsWRKY50 in the cucumber can obviously improve the resistance of the cucumber to the downy mildew is found for the first time, the experiment proves that the cucumber CsWRKY50 gene is transferred into the cucumber plant as a target gene, the expression of the resistance geneis increased by changing the expression quantity of the cucumber CsWRKY50 gene in the cucumber plant, the resistance of the cucumber to the downy mildew is improved, the character is used in the breeding of the cucumber, the cucumber variety capable of resisting the downy mildew is bred by choosing the cucumber CsWRKY50 gene overexpressed plant through the gene detection method, the application method is safe and reliable, and the application has an excellent application prospect in the researches of the resistant variety of the cucumber.

Description

technical field [0001] The invention relates to the technical field of crop genetic engineering, in particular to the application of a cucumber CsWRKY50 gene in enhancing cucumber downy mildew resistance. Background technique [0002] Cucumber (Cucumis sativus L.) is a herbaceous plant of Cucurbitaceae, which is a vegetable crop widely cultivated in the world. Cucumber has many health effects and is deeply loved by people. At present, our country is mainly cultivated in greenhouses and greenhouses. Due to the special climatic conditions and continuous cropping in greenhouses and greenhouses, cucumbers are vulnerable to various diseases, among which downy mildew, powdery mildew, and gray mold are the most serious. The onset and spread of these diseases are fast, causing a series of problems such as a substantial decline in yield, poor quality, and premature plant aging. In order to solve this problem, people usually rely on spraying a large amount of chemical pesticides to p...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/29A01H5/00A01H6/34
CPCC07K14/415C12N15/8282
Inventor 任仲海栾倩倩刘梦雨刘盼静张振
Owner SHANDONG AGRICULTURAL UNIVERSITY
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