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Method for rapidly extracting DNA of single sporangium of phytophythora infestans

A technology of potato late blight and sporangia, which is applied in the biological field, can solve the problems of increased pollution, cumbersome, difficult to master, etc., and achieves the effect of reducing loss and efficient extraction

Inactive Publication Date: 2016-05-11
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main reason is that Fusarium, Rhizoctonia, Pythium and other miscellaneous bacteria mixed with Phytophthora in disease spots grow faster. During the separation and purification process, Phytophthora infestans will grow first, inhibiting the growth of Phytophthora infestans or its hyphae cover Phytophthora infestans so that no pure strain can be obtained
Secondly, conventional single spore isolation methods, such as plate streaking method and suspension coating method, all need to transfer the dispersed single sporangia or spores from the water agar to the medium. The separation process is complicated / cumbersome and difficult to master. , the separation efficiency is low, and the probability of pollution is easily increased during operation. The separation efficiency is low
Therefore, it is difficult for these two methods to provide DNA of a large number of P. infestans strains for population genetic research in a short period of time.

Method used

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  • Method for rapidly extracting DNA of single sporangium of phytophythora infestans
  • Method for rapidly extracting DNA of single sporangium of phytophythora infestans
  • Method for rapidly extracting DNA of single sporangium of phytophythora infestans

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] 1. Isolation of individual sporangia

[0019] Use a sterile inoculation needle to pick a mycelium mass of Phytophthora infestans with a diameter of about 4mm from the edge of the lesion on the diseased leaf, put it in a 25mL centrifuge tube, add 7mL of sterile water and mix well, then filter with sterilized absorbent cotton to collect the spores The capsule suspension is placed in a 2ml centrifuge tube, and the next step should be carried out immediately to avoid the release of zoospores.

[0020] Adjust the concentration of the sporangia suspension to 1 / 2 μL, use a capillary glass tube with a diameter of 6 mm to self-absorb for 8 seconds, draw 2 μL of the suspension, observe it under a microscope, and transfer it to a 0.5 mL centrifuge tube after confirming that it contains a sporangia. Place in a refrigerator at 4°C for 2 hours to fully release the zoospores in the sporangia.

[0021] 2. Extraction of DNA from individual sporangia

[0022] Improve the CTAB method ...

Embodiment 2

[0029] 1) Isolation of single sporangia

[0030] a. Use a sterile inoculation needle to pick a mycelium mass of Phytophthora infestans with a diameter of about 4mm in a 25mL centrifuge tube at the edge of the diseased leaf lesion, add 10mL of sterile water and mix well, then use sterilized absorbent cotton to filter, Collect the sporangia suspension in a 2ml centrifuge tube and proceed to the next step immediately to avoid the release of zoospores.

[0031] b. Adjust the concentration of the sporangia suspension to 1 / 2 μL, use a capillary glass tube with a diameter of 6 mm to self-absorb for 7 seconds, draw 2 μL of the suspension, observe under a microscope, and transfer it to a 0.5 mL centrifuge tube after confirming that it contains a sporangia. Place in a refrigerator at 4°C for 2 hours to fully release the zoospores in the sporangia.

[0032] 2) Extraction of single sporangia DNA

[0033] Improve the CTAB method to extract the DNA of a single sporangia, the main steps ar...

Embodiment 3

[0039] 1) Isolation of single sporangia

[0040] a. Use a sterile inoculation needle to pick a mycelial mass of P. infestans with a diameter of about 4mm in a 25mL triangular flask at the edge of the diseased leaf lesion, add 5mL of sterile water and mix well, then filter with sterilized absorbent cotton, Collect the sporangia suspension in a 2ml centrifuge tube and proceed to the next step immediately to avoid the release of zoospores.

[0041] b. Adjust the concentration of the sporangia suspension to 1 / 2 μL, use a capillary glass tube with a diameter of 6 mm to self-absorb for 6 seconds, draw 2 μL of the suspension, observe under a microscope, and transfer it to a 0.5 mL centrifuge tube after confirming that it contains a sporangia. Place in a refrigerator at 4°C for 2 hours to fully release the zoospores in the sporangia.

[0042] 2) Extraction of single sporangia DNA

[0043] Improve the CTAB method to extract the DNA of a single sporangia, the main steps are as follows...

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Abstract

The invention provides a method for rapidly extracting DNA of a single sporangium of phytophythora infestans. The method comprises the steps that the single sporangium is obtained through the capillarity of a capillary glass tube, and then the DNA of the single sporangium is directly extracted through an improved CTAB method. Impurities such as protein and polysaccharide in the single sporangium are few, therefore, phenol-chloroform-isoamyl alcohol does not need to be utilized for removing the impurities, and losing of the DNA in the cumbersome operation process is greatly reduced. Pure DNA of the phytophythora infestans can be efficiently extracted within a short time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for rapidly extracting single sporangia DNA of potato infestans. Background technique [0002] Phytophthora infestans is one of the devastating diseases of potato, causing huge economic losses every year. In the laboratory, the success rate of obtaining P. infestans by single hyphae isolation method is low, generally around 30-50%. The main reason is that Fusarium, Rhizoctonia, Pythium and other miscellaneous bacteria mixed with Phytophthora in disease spots grow faster. During the separation and purification process, Phytophthora infestans will grow first, inhibiting the growth of Phytophthora infestans Or its hyphae will cover P. infestans so that pure strains cannot be obtained. Secondly, conventional single spore isolation methods, such as plate streaking method and suspension coating method, all need to transfer the dispersed single sporangia or spores fro...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003C12Q2547/101C12Q2565/125
Inventor 段国华杨丽娜詹家绥沈林林习京
Owner FUJIAN AGRI & FORESTRY UNIV
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