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Application of magnaporthe grisea gene MoHXT2 in regulation and control of botanical glycoside transport function

A technology of rice blast fungus and plant sugar, applied in the fields of application, plant products, genetic engineering, etc., can solve the problems of increased resistance of pathogenic bacteria, large environmental pollution, and increased cost of medication, and achieve reduced pathogenicity and lower hyphae melanin content Reduced effect

Active Publication Date: 2020-07-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Traditional chemically synthesized fungicides not only pollute the environment, but also tend to increase the resistance of pathogenic bacteria and increase the cost of medication

Method used

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  • Application of magnaporthe grisea gene MoHXT2 in regulation and control of botanical glycoside transport function
  • Application of magnaporthe grisea gene MoHXT2 in regulation and control of botanical glycoside transport function
  • Application of magnaporthe grisea gene MoHXT2 in regulation and control of botanical glycoside transport function

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0102] Example 1 Knockout and Identification of MoHXT2 Gene Mutants

[0103] (1) Amplify the target fragment

[0104] Inoculate the wild-type blast fungus WT into CM solid medium, place it in a 28°C incubator and grow in the dark for 5 days, collect the mycelia, then cut the mycelia into pieces and evenly distribute them into 50mL CM liquid medium, 28 ℃, 160rpm, after cultivating for 48 hours, collect mycelia, wash with sterile water and extract the genomic DNA of wild-type Magnaporthe grisea WT (DNA extraction is fungal genomic DNA extraction kit M5Fungal Genomic DNA Kit fungal genomic DNA extraction kit purchased from Beijing Jumei Biotechnology Co., Ltd.), stored at -20°C. Using it as a template, primers Flank1F / R and Flank2F / R were used to perform PCR amplification on the upstream and downstream homology arm sequences of the MoHXT2 gene (SEQ ID NO.1). The concentration of genomic DNA (gDNA) amplification was 5ng / μL, using The following reaction mixture: 2 μl 1× Taq buffe...

Embodiment 2

[0154] The growth situation of embodiment 2 Mohxt2 on different carbon source medium

[0155] (1) The mutant Mohxt2 obtained by screening in step (3) ⑤ of Example 1 (in this experiment, the knockout transformants of lane 2, lane 7 and lane 9 were selected and named in turn as ΔMohxt2-2, ΔMohxt2-7, ΔMohxt2-9 , and the wild-type WT strain as a control) were cultured on CM medium for 5 days, and the mycelium blocks were punched out with a 3 cm diameter hole punch along the edge of the colony, and transferred to different carbon sources (glucose, galactose, Mannose, fructose; The concentration is 2% (w / v); The monosaccharides used in the experiment were all purchased from Sigma) for growth observation on the CM solid medium, repeated three times. The difference in black accumulation was observed after culturing for 5 days. Differences between the wild-type WT strain and the mutant Mohxt2 on different carbon sources were observed on CM solid medium ( Figure 4 ). At the same tim...

Embodiment 3

[0187] Example 3 Mohxt2 pathogenicity analysis

[0188] The mycelia block of the mutant (Mohxt2-9) obtained by screening in Example 1 was inoculated on rice straw medium (RDC) (recipe is as follows: rice straw 100g, corn flour 40g, agar powder 15g; preparation method is: 100g rice straw Add the stem to 1L of sterile water and boil for 20 minutes, filter with gauze to obtain the filtrate, dissolve corn flour and agar, add water to 1L of high-temperature sterilization) to multiply, and after black light irradiation for 5 days, wash the spores with sterile water for microscopic examination ( Figure 8 ), inoculated on isolated rice leaves (rice CO39 (resistant variety), Rice Institute of Guangdong Academy of Agricultural Sciences), and took pictures for observation and pathogenicity analysis after infecting for 96 hours. Compared with the wild-type strain WT, the pathogenicity of ΔMohxt2 in the resistant variety CO39 was significantly reduced ( Figure 9 ).

[0189]In order to ...

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Abstract

The invention discloses an application of a magnaporthe grisea gene MoHXT2 in regulation and control of a botanical glycoside transport function. It is found in the invention that a transport proteinencoded by MoHXT2 accepts a plurality of carbohydrates as substrates; namely, the MoHXT2 gene is a hexose transport protein of magnaporthe grisea; after MoHXT2 is knocked out by utilizing a CRISPR / Cas9 gene knockout system, the melanin content of mutant hyphae is found to be obviously reduced, so pathogenicity is greatly reduced; and phlorizin can generate a certain inhibition effect on hexose transportation of MoHXT2, so the growth of magnaporthe grisea is inhibited. Therefore, the MoHXT2 gene can be used for regulating and controlling the botanical glycoside transport function, the pathogenicity of magnaporthe grisea and the growth and development of magnaporthe grisea, can also be used as a drug target of a novel pesticide, and provides a more reliable direction for synthesizing a targeted bactericide.

Description

technical field [0001] The invention belongs to the field of fungal genetic engineering, and particularly relates to the application of a rice blast fungus gene MoHXT2 in regulating the plant sugar transport function. Background technique [0002] Magnaporthe grisea is a complex, heterothallic ascomycete that can infect more than 50 species of grasses including rice, wheat crops, and corn. Upon exposure to rice leaves under high humidity conditions, conidia of Magnaporthe oryzae rapidly germinate and produce specialized infestation structures called appressoria. Episporium aggregates glycerol-like solutes through melanization to generate a high level of turgor pressure to help blast fungus invade rice cells [1] . After entering the rice cells, the blast fungus begins to form thick spherical primary infection hyphae, which last for 3 to 5 days before showing symptoms. The specific process of rice blast fungus infecting rice is as follows: 1) overwintering hyphae germinate ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/31C12N15/66A01H5/00A01H6/46
CPCC12N15/8213C12N15/66C07K14/37C12N15/8245
Inventor 林菲罗晓徐汉虹
Owner SOUTH CHINA AGRI UNIV
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