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Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades

A wheat stripe rust and leaf technology, applied in the field of plant protection, can solve the problems of cumbersome operation steps, time-consuming and laborious, cumbersome extraction of genomic DNA, etc.

Inactive Publication Date: 2012-08-15
YUNNAN AGRICULTURAL UNIVERSITY
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Problems solved by technology

These methods all have cumbersome operation steps, time-consuming and labor-intensive, a large amount of bacterial cells, and low DNA acquisition rate.
For the obligate parasitic fungus that cannot be artificially cultivated——Wheat stripe rust, when extracting the single-spore pile DNA, it is necessary to obtain enough wheat stripe rust through the isolation and inoculation of the single-spore pile, the propagation and cultivation of stripe rust, etc. spores, and because the cell wall of uredia spores is thicker, it is cumbersome and difficult to extract their genomic DNA compared with ordinary fungi
So far, although there are many reports on the exploration of DNA extraction methods for wheat stripe rust, there is still a lot of demand for wheat stripe rust urediospores in these reports

Method used

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  • Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades
  • Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades
  • Method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades

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Embodiment Construction

[0009] The present invention will be described in detail below in conjunction with specific embodiments.

[0010] A method for efficiently and rapidly extracting wheat stripe rust DNA directly from susceptible wheat leaves:

[0011] Dissolve 20 g of chelex-100 in 100 mL of sterilized double distilled water to prepare a 20% chelex-100 suspension for use. After mixing thoroughly, pipette 20 μl of the prepared chelex-100 suspension into a 100 μl PCR tube, pipette 5 μl of the Chelex-100 supernatant on the leaves of wheat stripe rust uredospores, and drip Chelex- 100 supernatant on the lesions and repeatedly smeared the leaves several times, washed the rust spores, sucked back the Chelex-100 supernatant containing the rust spores on the leaves and added it to the tank containing 20 μl 20% chelex-100 suspension. In the PCR tube, use a pipette to repeatedly absorb and mix well, put it in a boiling water bath for 2 minutes, then place it on a vortex mixer and shake and mix for 10 sec...

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Abstract

The invention discloses a method for quickly and efficiently extracting deoxyribonucleic acid (DNA) of wheat stripe rust directly from infected wheat leaf blades. The method is characterized in that 20mu l of prepared 20% chelex-100 suspension is drawn and injected into a 100mu l polymerase chain reaction (PCR) tube; 5mu l of chelex-100 supernate is drawn and dripped on the wheat leaf blades with wheat stripe rust uredispore and the spots of the leaf blades on which the chelex-100 supernate is dripped are smeared repeatedly to wash off the aecidiospore; the chelex-100 supernate containing the aecidiospore on the leaf blades is drawn back and added into the PCR tube containing 20mu l of 20% chelex-100 suspension; the mixture is drawn repeatedly and mixed evenly by a liquid-moving device; the mixture is put in boiling water bath for water bath for 2 minutes and then put in a vortex mixer to be oscillated and mixed evenly for 10 seconds; the mixture is subjected to water bath for 5 minutes and preserved at the temperature of minus 20 DEG C for later use; and the supernate can be used as templates of PCR. The invention provides an efficient and rapid method for extracting DNA of wheat stripe rust which is used as PCR amplification templates directly from infected wheat leaf blades.

Description

technical field [0001] The invention relates to a method for efficiently and rapidly extracting wheat stripe rust DNA directly from susceptible wheat leaves, belonging to the field of plant protection. Background technique [0002] Wheat stripe rust caused by Puccinia striiformis f.sp.tritici is one of the major diseases in wheat producing areas in the world. With the development of molecular biology, more and more researchers have rapidly increased their explorations at the level of proteins and DNA molecules related to wheat stripe rust, and the content of PCR-based research has gradually been enriched. The premise of PCR amplification is to extract relatively complete genomic DNA. The methods for extracting fungal DNA at home and abroad mainly include: CTAB method, SDS extraction method, urea extraction method, benzyl chloride extraction method and other chemical methods to break the cell wall, and then use organic solvents to extract DNA. These methods all have cumbers...

Claims

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Application Information

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IPC IPC(8): C12N15/10
Inventor 刘林李成云杨静朱有勇李进斌兰茗清武静雯
Owner YUNNAN AGRICULTURAL UNIVERSITY
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