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Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita

A technology of root-knot nematode incognita and construction method, which is applied in the field of construction of transgenic plants, can solve problems such as deviation of phenotype and root system response, changes affecting root system, etc.

Pending Publication Date: 2022-04-12
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] The object of the present invention is to provide a method for constructing a transgenic plant in which the cucumber root system interacts with Meloidogyne incognita and its application. The method of the present invention can solve the problem that the change of the target gene in the aerial part caused by the whole plant transgenic system may affect the root system. Therefore, the phenotype and the response of the root system to nematode infection will be biased, and the screening and functional verification of root-related nematode resistance genes will be performed more accurately.

Method used

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  • Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita
  • Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita
  • Construction method and application of transgenic plant with interaction of cucumber root system and meloidogyne incognita

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preparation example Construction

[0035] In the present invention, the preparation method of the bacterial liquid preferably includes the following steps:

[0036] Inoculate the Agrobacterium rhizogenes transformed with the target gene carrier to be tested or knock out the target gene carrier in the resistant medium, and culture until monoclonal plaques grow, and collect the monoclonal plaques with sterile water to obtain Monoclonal plaque resuspension: mixing the monoclonal plaque resuspension with acetosyringone and incubating to obtain a bacterial solution.

[0037] In the present invention, the culture time is preferably 48-72 hours.

[0038] In the present invention, the resistant culture medium is water-based, preferably comprising components of the following concentrations: peptone 5-10g / L, yeast extract 3-5g / L, calcium chloride aqueous solution 10ml / L, agar 10~15g / L, streptomycin 50~80mg / L and kanamycin sulfate 50~80mg / L; the pH value of the resistant medium is 7.0~7.2; the calcium chloride in the cal...

Embodiment 1

[0051] Example 1 Transgenic root system of GUS gene cucumber

[0052] (1) Transformation of the target gene vector: the PBI121 expression vector carrying the GUS gene was transformed into Agrobacterium rhizogenes K599 competent cells by chemical transformation method, and after incubation at 28°C for 2 hours at a constant temperature shaker, the plates were plated with kanamycin sulfate and streptavidin Take the positive single clone in 5ml TY liquid medium + 50mg / L streptomycin + 50mg / L kanamycin sulfate at 28°C, 180rpm for overnight shaking, then keep the bacteria for preservation and transform the target gene carrier bacteria liquid.

[0053] TY liquid medium: 5g tryptone, 3g yeast extract, add water to 1L volume, after complete dissolution, sterilize at 121°C for 20min. Prepare 1M calcium chloride aqueous solution and sterilize at 121°C for 20 minutes. Add 10ml of sterile 1M calcium chloride aqueous solution to every 1L of sterilized TY liquid nutrient solution.

[0054...

Embodiment 2

[0061] Example 2 Obtaining transgenic roots of cucumber malate synthase gene CsMS induced by nematode infection

[0062] (1) Construction and transformation of the gene CRISPR knockout vector: two targets were obtained by analyzing and predicting the gene sequence of the CsMS gene, and the two targets were added to the CRISPR expression vector by four-primer amplification and restriction enzyme ligation Used to knock out the cucumber CsMS gene. Transform the expression vector into competent cells of Agrobacterium rhizogenes K599 by chemical transformation method, incubate at 28°C for 2 hours in a constant temperature shaker, plate kanamycin sulfate and streptomycin-resistant TY solid plates, and take positive single clones in 5ml TY +50mg / L Streptomycin+50mg / L Kanamycin Sulfate liquid culture medium was shaken overnight at 28°C and 180rpm, and then the bacterial solution transformed with the target gene carrier was preserved for bacterial preservation.

[0063] (2) Preparatio...

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Abstract

The invention provides a construction method and application of a transgenic plant with interaction of a cucumber root system and meloidogyne incognita, and belongs to the technical field of plant genetic engineering and plant protection. According to the method disclosed by the invention, in the period of relatively vigorous division and differentiation of cucumber seedlings with unexpanded cucumber cotyledons, a bacterial solution of agrobacterium rhizogenes transformed with a target gene vector to be inspected or knocked out of the target gene vector is inoculated, so that a hairy root system is generated, and an efficient transgenic root system is established and used for nematode infection and research on interaction between root systems. The method provided by the invention can solve the problem that the response of phenotypes and root systems to nematode infection is deviated due to the fact that the change of overground part target genes caused by a whole-plant transgenic system possibly affects the change of the root systems, and screening and functional verification of root system related nematode-resistant genes are more accurately carried out.

Description

technical field [0001] The invention relates to the fields of plant genetic engineering technology and plant protection technology, in particular to a method for constructing a transgenic plant in which cucumber roots interact with root-knot nematode incognita and its application. Background technique [0002] Cucumber is an annual vining plant in the genus Cucurbitaceae. Cucumber occupies an important position in the cultivation system of solar greenhouses in northern my country. However, due to the closed soil environment of solar greenhouses, and most of the greenhouses continue to grow the same species or crops of the same family all year round, it leads to the accumulation of soil pathogens and the occurrence of diseases. Plant parasitic nematodes are extremely harmful pathogens in solar greenhouses, while root-knot nematodes in northern solar greenhouses are mainly dominated by southern root-knot nematodes. [0003] Meloidogyne incognita has caused serious losses to ...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/06A01H6/34
Inventor 田永强张旭高丽红马斯李欣王星怡李诗卉梁美婷冀婷婷
Owner CHINA AGRI UNIV
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