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Agrobacterium-mediated pythium oligadrum gene transformation method

A genetic transformation method and technology mediated by Agrobacterium, applied in the field of genetic transformation of Pythium oligandrum mediated by Agrobacterium, can solve the problems of reducing the transformation rate, complicated process, inability to transform, etc., to restore growth vitality and facilitate picking , The effect of simplifying the operation process

Active Publication Date: 2017-01-04
周剑平 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Refer to the transformation method of other filamentous fungi, the transformation cannot be carried out smoothly
[0005] In the traditional Agrobacterium-mediated genetic transformation method, the Agrobacterium is mixed with the infected object, and then the transfected bacteria or fungi are co-cultivated and selectively cultured using nitrocellulose membrane or filter paper and other auxiliary solid culture methods. A series of operation steps of laying film on the base plate, peeling off the film and then laying the film are complicated, easy to pollute, easy to reduce the conversion rate, and have a huge impact on the final results of the experiment. This method needs to be improved urgently

Method used

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  • Agrobacterium-mediated pythium oligadrum gene transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Agrobacterium-mediated genetic transformation experiment using Pythium oligandrum mycelial spore suspension

[0034] (1) Inoculate Pythium oligandrum hyphae on PDA plate medium, culture at 26°C for 6 days, wash the plate with 2-4mL induction medium IM, wash off the Pythium oligandrum spores and part of the mycelium on the plate, Make a concentration of 1×10 7 spore mycelia suspension per mL.

[0035] (2) Pick a single clone from the Agrobacterium engineering strain resistance plate containing the target gene carrier, and put it in 5ml LB (50mg / L rifampicin Rif + 50mg / L kanamycin Kan) liquid medium at 28°C , shake culture at 200rmp for 20h; pipette 2mL of the above bacterial solution into 20mL LB (50mg / L Rif + 50mg / L Kan + 300uM acetosyringone) liquid medium and culture at 28℃, 240rmp until OD 600 2.5; 4°C, centrifuge at 5000rmp for 10min, collect the bacteria, resuspend and dilute the bacteria with IM osmosis medium (PDB+300 uM acetosyringone, pH 5.0) to mak...

Embodiment 2

[0042]Example 2 Experiment on the influence of different co-cultivation time on genetic transformation.

[0043] With reference to the method described in Example 1, Agrobacterium bacterium liquid and Pythium oligandrum mycelial spore suspension are co-cultivated for different times, and the optimal co-cultivation time is determined according to the PCR detection results, and the experimental results are shown in the table below:

[0044]

[0045] It can be seen from the experimental results that a good transformation effect can be obtained when the Agrobacterium bacterial liquid is co-cultivated with Pythium oligandrum mycelia and spore suspension for 48-96 hours.

Embodiment 3

[0046] Example 3 Experiment on the influence of recovery culture on genetic transformation.

[0047] With reference to the method described in Example 1, whether Agrobacterium bacterium liquid and Pythium oligandrum hyphae and spore suspension are co-cultivated and whether they experience recovery and recovery time, the experimental results are shown in the table below:

[0048]

[0049] It can be seen from the experimental results that the recovery culture stage can well promote the recovery of growth vigor of the infected Pythium oligandrum and increase the transformation effect, and the recovery culture period can be 24-48 hours.

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Abstract

The invention provides an agrobacterium-mediated pythium oligadrum gene transformation method. The agrobacterium-mediated pythium oligadrum gene transformation method comprises the following steps that 1, a pythium oligadrumsuspension is prepared; 2, an agrobacterium solution containing a target gene carrier is prepared; 3, the agrobacterium solution infects the pythium oligadrum suspension; 4, culture is recovered; 5 converter screening is performed. The agrobacterium-mediated pythium oligadrum gene transformation method adopts a liquid oscillation and co-culture mode to improve the transformation rate, the transformation rate is improved in a liquid culture recovery mode, the operating process is simplified, the pollution rate is reduced, and resistant single colonies can be conveniently selected for purified culture and identification. The agrobacterium-mediated pythium oligadrum gene transformation method is simple, convenient to operate, efficient and stable, and a research way is provided for transformation by utilizing functional genes and their properties of biological control fungi.

Description

technical field [0001] The invention relates to the technical field of microbial genetic engineering, in particular to a method for genetic transformation of Pythium oligandrum mediated by Agrobacterium. Background technique [0002] Pythium oligandrum belongs to the genus Pythium of the family Pythium Oomycota. It is a soil-dwelling saprophytic fungus. It can colonize the root circle of a variety of important crops. It is non-toxic to plants and animals and is environmentally safe. The hyphae of Pythium oligandrum can parasitize the pathogenic bacteria, interfere with the metabolic activities of the host, consume nutrients in the cells, and cause the death of the pathogens. At the same time, they can also synthesize tryptamine, tryptophan, indole acetic acid and other growth active substances to promote Plant physiological metabolism, nutrient uptake and growth and development. Pythium oligandrum not only has a strong parasitic or antagonistic effect on most pathogenic fun...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12R1/645
CPCC12N15/80
Inventor 王沛雅周剑平杨晖祝英彭轶楠巩晓芳王治业
Owner 周剑平
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