Genetic transformation method of PEG/LiAc mediated plasmodiophora brassicae woronin

A genetic transformation method, the technology of Plasmodium brassicae, applied in the biological field, can solve the problems of long transformation time and no green fluorescence observed, and achieve the effect of increasing the fluorescence intensity

Inactive Publication Date: 2015-04-01
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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Problems solved by technology

[0003] Studying the growth, development and pathogenic mechanism of P. brassicae by means of molecular biology can provide new ideas for the breeding of disease-resistant varieties and the development of new drug targets, because the reproduction mode, biology of P. brassicae The characteristics are similar to fungi, so it is usually regarded as a class of inferior fungi ("Ainsworth & Bisby's Dictionary of the Fungi", Hawksworth et al, International Mycological Institute, 1995), since Mishra and Tatum first reported fungi in 1973 Since the genetic transformation of Neurospora crassa, many fungi have established efficient genetic transformation systems. However, compared with other pathogenic fungi, the genetic transformation research of Plasmodium brassicae started relatively late until 2013, Feng (Genetic transformation of the obligate parasite Plasmodiophora br

Method used

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  • Genetic transformation method of PEG/LiAc mediated plasmodiophora brassicae woronin
  • Genetic transformation method of PEG/LiAc mediated plasmodiophora brassicae woronin
  • Genetic transformation method of PEG/LiAc mediated plasmodiophora brassicae woronin

Examples

Experimental program
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Effect test

Embodiment 1

[0048] 1. Preparation of cabbage root exudate: plant cabbage seedlings in Hoagland nutrient solution, and cultivate in alternating light and dark at 24°C / 18°C. After 7 days, collect root exudate, and filter and sterilize root exudate with a 0.22um bacterial filter to obtain cabbage Root exudate, kept at 4°C for later use.

[0049] 2. Collection of dormant spore liquid of Plasmodium brassicae:

[0050] (a) Take the roots of cabbage clubroot stored at -20°C, and observe the diseased tissues under the scanning electron microscope, as shown in figure 1 shown;

[0051] Add ddH after chopping the diseased root 2 Stir into a homogenate in a juicer, filter through 8 layers of gauze, centrifuge the filtrate at 6000rpm for 5min, discard the supernatant, take the precipitate and suspend it with 50% sucrose solution, centrifuge at 500rpm for 5min, take the supernatant and the upper gray layer;

[0052] (b) Transfer the supernatant and the upper gray layer obtained in step a into a ne...

Embodiment 2

[0062] Example 2 PCR detection of Plasmodium brassicae transformants

[0063] 1. Extraction of transformant genomic DNA:

[0064] (a) Collect the diseased roots of wild-type HSP2 and Plasmodium brassicae transformants obtained in Example 1, wash them under tap water, rot at room temperature for 5 days, chop them up, take an appropriate amount in a 2ml centrifuge tube, and add a small amount of quartz sand;

[0065] (b) Add 500 μl of 2% CTAB and 500 μl of a mixed solution prepared from phenol, chloroform, and isoamyl alcohol at a volume ratio of 25:24:1 to the centrifuge tube described in step a, shake at room temperature at 200 rpm for 1 hour, and centrifuge at 12,000 rpm for 10 minutes. Add 2 times the volume of absolute ethanol to the supernatant, place it at -20°C for 2 hours, and centrifuge at 12,000 rpm for 10 minutes; discard the supernatant and take the precipitate;

[0066] (c) Take the precipitate obtained in step b, add 70% ethanol to wash, centrifuge at 12000rpm fo...

Embodiment 3

[0075] Example 3 Fluorescent microscope observation of Plasmodium brassicae transformants

[0076] Extract its spores from the diseased root whose GFP fragment was detected by PCR in Example 2 and make a temporary glass slide, observe with a Nikon fluorescence microscope and find that the successfully transformed spores can emit stronger green fluorescence, and the transformation efficiency reaches 1000 transformants / mg DNA ( Measured in unit mass of DNA). After the transformant was subcultured for 3 generations through bleomycin resistance selection, strong green fluorescence could still be observed, indicating that it had high genetic stability.

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Abstract

The invention discloses a genetic transformation method of PEG/LiAc mediated plasmodiophora brassicae woronin. The genetic transformation method comprises the following specific steps: (a) preparing root secretion; (b) collecting hypnospore liquid; (c) preparing competent hypnospore liquid; (d) carrying out genetic transformation on plasmodiophora brassicae woronin; (e) carrying out resistance screening on bleomycin, repeatedly inoculating and screening for three generations, thus obtaining stable genetic transformation spores. The genetic transformation method disclosed by the invention realizes the genetic transformation of PEG/LiAc mediated plasmodiophora brassicae woronin and obtains stably genetic transformation strain capable of expressing green fluorescent proteins with a high intensity, and the transformation efficiency reaches 1000 transformants/mg of DNA (by DAN in a unit mass); in addition, a carrier used for the transformation in the genetic transformation method disclosed by the invention has enhanced green fluorescent protein genes and strong promoter RP27 in fungus, thus effectively increasing the fluorescent intensity of the transformation strain.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a PEG / LiAc-mediated genetic transformation method of Plasmodium brassicae. Background technique [0002] Clubroot caused by the infection of Plasmodiophora brassicae Woronin is an important soil-borne disease worldwide. According to statistics, the global annual output loss of cruciferous crops due to clubroot accounts for about 10% of the total production. 10%-15% (The occurrence and economic impact of Plasmodiophora brassicae and clubroot disease, Dixon et al, Journal of Plant Growth Regulation, 2009, 28(3):194-202), in my country, cruciferous clubroot The perennial damage area accounts for more than 1 / 3 of the total planting area, and in severe cases, the field loss can reach more than 60%, which seriously affects the yield and quality of Cruciferous crops (Research Progress on Cruciferous Clubroot, Wang Jing et al., Plant Protection, 2011, 37(6):153-158). Because brassica c...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N1/15C12R1/645
Inventor 杜艳刘邮洲乔俊卿
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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