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Cytospora chrysosperma protoplast preparation method and prepared protoplast conversion method

A technology of rotting pathogens and protoplasts, applied in the field of molecular plant pathology, can solve problems such as inapplicable Ascospora aureus

Inactive Publication Date: 2016-10-05
BEIJING FORESTRY UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although this method has been successful in genetic transformation studies of various pathogenic bacteria such as Cytospora mali, Cryphonectria parasitica, and Verticillium dahliae, but due to differences in pathogenic bacteria, the original Some enzymatic hydrolysis systems are not suitable for the Ascospora aureus studied in the present invention

Method used

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  • Cytospora chrysosperma protoplast preparation method and prepared protoplast conversion method
  • Cytospora chrysosperma protoplast preparation method and prepared protoplast conversion method
  • Cytospora chrysosperma protoplast preparation method and prepared protoplast conversion method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0078] Example 1 Reagent preparation

[0079] 1. PDA solid medium

[0080] Potatoes 200g, D-glucose (D-glucose) 20g, agar 15g, sterile water to a volume of 1L, placed at 120°C, sterilized for 30min.

[0081] 2. Osmotic fluid (protoplast buffer)

[0082] Add KCl to a concentration of 10 mM NaH 2 PO 4 In the solution, stir and dissolve evenly to make a mixed solution in which the molar concentration of KCl in the solution is 1.2M; then dropwise add Na with a molar concentration of 1M 2 HPO 4 Solution, the pH value of the mixed solution is adjusted to 5.0-7.0, preferably 5.5-6.0, and made into a stabilized seepage solution for subsequent use.

[0083] 2. Enzyme hydrolysis solution (cell wall lysate)

[0084] Add Driselase Enzyme (collapsing enzyme) and Lysing Enzyme (lysing enzyme) into the steady seepage solution, stir and dissolve evenly, and prepare protoplast enzymatic solution, wherein, the concentration of Driselase Enzyme in the enzymatic solution is 15-25mg / mL, pref...

Embodiment 2

[0100] The preparation of the protoplast of embodiment 2 poplar rot fungus

[0101] 1. Preparation of conidia suspension

[0102] Pick activated poplar rot fungus (Cytospora chrysosperma (Pers.) Fr., China Forestry Microorganism Culture Collection and Management Center, preservation number CFCC 89981) and inoculate it in Cytospora chrysosperma medium (PDA solid medium), cultivated in the dark at 25°C for 15-60 days, after it produced conidia horns, picked the conidia horns, and made poplar rot fungus (i.e. golden ascocysts) with sterile water Bacteria) conidia suspension, wherein the conidia concentration of Ascosporium aureus in poplar rot pathogen conidia suspension is 10 7 -10 8 conidia / mL.

[0103] The Ascosporium aureus conidia concentration of preparation in the embodiment of the present invention is 5 * 10 7 conidia / mL.

[0104] 2. Mycelia culture and collection

[0105] 2-1) Put 1mL poplar rot fungus conidia suspension and 100mL YEPD liquid culture into a sterili...

Embodiment 3

[0113] Example 3 Genetic transformation of poplar rot fungus protoplasts

[0114] 1. Preparation of protoplast solution

[0115] 1-1) Place the protoplasts of poplar rot fungus (i.e. Ascospora aureus) prepared in Example 2 in a centrifuge tube, then add an appropriate amount of STC buffer, and mix with the tip of a 5mL pipette Evenly, the poplar rot fungus protoplast is suspended in the STC buffer solution to make the poplar rot fungus (i.e. Ascosporium aureus) protoplast mother liquor, for subsequent use;

[0116] 1-2) Use a hemocytometer to count the number of protoplasts in the protoplast mother liquor, add STC buffer solution to dilute the protoplast mother liquor according to the counting results, and obtain a protoplast concentration of 10 7 -10 8 protoplasts / mL of protoplast solution.

[0117] In the embodiment of the present invention, a hemocytometer is used to count the number of protoplasts in the protoplast mother liquor, and the counting results show that the c...

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Abstract

The invention provides cytospora chrysosperma protoplast preparation and genetic transformation methods, and belongs to the field of molecular plant pathology. The cytospora chrysosperma protoplast preparation method comprises the following steps: inoculating cytospora chrysosperma into a YEPD liquid culture medium for mycelium culture, and performing enzymolysis treatment on the cytospora chrysosperma mycelia, thereby obtaining protoplast. In addition, a PEG mediated method is adopted for genetic transformation on the prepared protoplast, and thus a mutant can be obtained. The method provided by the invention is high in protoplast preparation efficiency, low in cost and simple to operate, the number of protoplast after enzymolysis can be as high as 10<8>-10<9> / mL, the transformation efficiency is high, the protoplast is good in activity, the transformation efficiency can be improved, and the research time can be greatly shortened. By adopting the method, a good base is made for molecular biology study on cytospora chrysosperma in the future, and furthermore potential utilization values can be made for prevention and treatment and histopathologic study on cytospora chrysosperma.

Description

technical field [0001] The invention belongs to the field of molecular plant pathology, specifically relates to a preparation method of protoplast of poplar rot pathogen, and also provides a PEG-mediated genetic transformation method of poplar rot pathogen. Background technique [0002] Poplar is the main tree species for afforestation and afforestation on the plains in the area north of the Yangtze River in my country. Especially in recent years, the area of ​​poplar afforestation in my country has continued to expand, and it has become the country with the largest area of ​​poplar plantation in the world. Poplar is widely used in timber forests and shelter forests because of its rich germplasm resources, fast growth, short rotation period, easy sexual hybridization and asexual reproduction. But at the same time, various branch diseases have become the key factors that threaten the survival rate of poplar. Among them, the poplar rot fungus caused by Cytospora chrysosperma ...

Claims

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Application Information

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IPC IPC(8): C12N1/14C12N15/87C12R1/645
CPCC12N1/14C12N15/87
Inventor 梁英梅刘玲玲王永林田呈明
Owner BEIJING FORESTRY UNIVERSITY
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