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Oxathiapiprolin-resistance selection marker for genetic transformation of phytophthora sojae

A fluthiazopyridone and resistance-related technology, applied in the field of genetic engineering, can solve the problems of limited selection markers and difficult to achieve re-knockout, etc.

Active Publication Date: 2018-08-07
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the selection markers available in oomycetes are very limited at present, and the only reliable selection marker is the NPT Ⅱ gene, which encodes a protein encoded by the universal strong promoter ham34 of oomycetes, which can make oomycetes resistant to geneticin (G418) , so that transformants were screened out
However, when NPT II is used in the CRISPR / Cas9 gene knockout system, the transformant carrying the marker has G418 resistance and cannot be used again for complementation experiments; Re-knockout of other genes in knockout transformants is still difficult to achieve

Method used

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  • Oxathiapiprolin-resistance selection marker for genetic transformation of phytophthora sojae
  • Oxathiapiprolin-resistance selection marker for genetic transformation of phytophthora sojae
  • Oxathiapiprolin-resistance selection marker for genetic transformation of phytophthora sojae

Examples

Experimental program
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Effect test

Embodiment 1

[0073] Example 1. Obtaining of Fluthiapiprofen Resistance Gene and Its Recombinant Expression Vector

[0074] 1. Acquisition of Fluthiapiprofen Resistance Gene

[0075] The inventors of the present invention have found that when the oxysterol-binding protein 1 (ORP1, GENBANK Accession Number is XP_009522569.1) expressed by the Phytophthora sojae wild-type strain P6497 fluthiapiprofen resistance gene is from the 777th amino acid at the amino terminal ( When the asparagine) is missing, the resistance of the strain to fluthiapiprofen can be increased by more than 500 times. That is, the deletion mutant strain can increase the anti-fluthiapiprofen concentration by more than 500 times, and the mutant obtained by deleting the 777th amino acid (asparagine) of ORP1 after the wild type cannot grow the fluthiapiprofen concentration increased by 500 times can still grow.

[0076] Accordingly, the inventors identified a Phytophthora sojae fluthiazolidin resistance protein as a screening...

Embodiment 2

[0090] Example 2, screening marker PsORP1 ΔN777 Application in Genetic Transformation of Phytophthora soybean

[0091] In order to verify the application potential of the screening marker, the above-mentioned plasmid (pBS-PsORP1 ΔN777 ) into Phytophthora sojae protoplasts, to verify whether the selection marker of the present invention can be applied to the selection of transformants during genetic transformation of Phytophthora soybean, and the working efficiency of the selection marker when used for genetic transformation of Phytophthora soybean.

[0092] The specific operation process is as follows:

[0093] The wild-type strain of Phytophthora sojae was used as the experimental material, and protoplasts were obtained by referring to the method of Fang et al. 2 The method of mediated transfer into the protoplast with the vector (pBS-PsORP1 ΔN777 ), positive transformants were obtained after screening, and in the positive transformants, the expression cassette of the flut...

Embodiment 3

[0099] Embodiment 3, the analysis of biological character of transformant

[0100] Four strains of Phytophthora sojae positive transformants, the strain numbers are T777-1, T777-4, T777-5, T777-8, and one Phytophthora sojae wild type strain P6497.

[0101] V8 solid medium: 100ml V8 vegetable juice, centrifuge at 1500 rpm (5000g) for 10min, take the supernatant and dilute it with deionized water at a ratio of 1:9 (add 900ml deionized water to the supernatant of 100ml), add 1g of carbonic acid Calcium, 15g agar, autoclaved (121°C) for 20min.

[0102] (1) Sensitivity to fluthiapiprofen

[0103] Fluthiapiprofen was formulated with dimethyl sulfoxide (DMSO) into 10 4μg / ml stock solution. First prepare a 1000-fold concentrated drug solution, and then use V8 solid medium to dilute fluthiapiprofen according to a 1000-fold ratio to contain 0.001 μg / ml, 0.005 μg / ml, 0.01 μg / ml, 0.05 μg / ml, 0.1 V8 solid medium with a concentration gradient of μg / ml and 0.5 μg / ml. The specific method...

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Abstract

The invention discloses an oxathiapiprolin-resistance gene and application thereof as a selection marker for transformation of oomycetes. Protein of a sequence 2 in a coding sequence table of the oxathiapiprolin-resistance gene is mutant protein capable of leading the oomycetes to generate 500 times or more of resistance to oxathiapiprolin. The oxathiapiprolin-resistance coding gene is used for selection of transformant in transformation of phytophthora sojae. By development of the selection marker, the problem of single selection marker in transformation of the oomycetes is solved, and the strong guarantee is provided for working in the aspects of supplementing the genes and knocking out the genes for more times and the like in transformation of the oomycetes.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a novel screening marker for fluthiapiprofen resistance transformed by Phytophthora soybean. Background technique [0002] Oomycetes is a class of eukaryotic organisms similar to fungi. Researchers have discovered at least 1,800 species of oomycetes. Except for some saprophytic oomycetes, most oomycetes are pathogenic bacteria of plants, animals and other organisms. huge economic losses. Phytopathogenic oomycetes mainly include Downy mildews, Phytophthora, Pythium and Albugo. Compared with fungi, oomycetes have some unique physiological and biochemical characteristics, such as: the vegetative body is diploid; the main component of the cell wall is cellulose; most oomycetes are sterol auxotrophs; Produces lysine; hyphae have no or few septa; are not sensitive to DMI (sterol demethylation inhibitor) fungicides, but are sensitive to benzamide fungicides and carboxylic acid amide...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12N15/80C12N15/65C12N1/14C12R1/645
CPCC07K14/37C12N15/65C12N15/80
Inventor 苗建强刘西莉薛昭霖王为镇迟源冬林东刘鹏飞黄中乔
Owner NORTHWEST A & F UNIV
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