Method for extracting mushroom genome

An extraction method and genome technology, applied in the field of molecular biology, can solve the problems of low DNA extraction rate, time-consuming, time-consuming and labor-intensive, and achieve the effects of reducing mechanical shear damage, wide application range, and avoiding cross-contamination.

Inactive Publication Date: 2009-05-20
INST OF SOIL & FERTILIZER SHANDONG ACAD OF AGRI SCI
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Problems solved by technology

[0003] At present, commonly used methods such as cesium chloride centrifugation, CTAB method, and kit method have been applied to the genome extraction of bacteria, animals, and plants. However, there are no commercial extraction reagents for genomes of higher edible fungi such as shiitake mushrooms. There are few reports on the corresponding extraction methods: in 1992, Lu Zuozhou proposed a method for extracting DNA from edible fungi, but this method required the preparation of protoplasts, which was complicated and time-consuming, and the extraction rate of DNA was low and the protein content was high; In 1994, Zhu Heng and others used the benzyl chloride method to extract the DNA of edible fungi. The DNA content extracted by this method was high and the protein content was low, but the polysaccharide content was more, and the extracted DNA needed further purification; in 1999, Wang Chunhui et al. The CTAB method was used to extract the DNA of edible fungi, and it was improved, and good results were obtained; in 2000, Jiang Yuji et al. used the CTAB method to conduct a more detailed experiment on straw mushrooms; in 2005, Shang Xiaodong et al. used LETS Improved method extracts the genome of shiitake mushrooms
However, in general, the reported methods for extracting the genomes of higher edible fungi such as shiitake mushrooms are very cumbersome. Whether it is using enzyme treatment, freeze-thaw method or liquid nitrogen grinding method, it takes hours or even a long time to prepare before extraction. It is time-consuming and labor-intensive, and the cost is high, and the liquid nitrogen grinding process is easy to cause cross-contamination between samples
Especially for the rapid extraction and detection of mushroom genomes with many varieties and large sample quantities, molecular identification, and gene operations such as PCR with low requirements, it is very uneconomical and practical

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Experimental program
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Effect test

Embodiment 1

[0032] The mushroom hyphae of three different varieties (Guxiang 66, Biyang No. 3, and Shenxiang No. 10) were picked and inoculated on PDA plates with cellophane on the surface of the culture medium, and cultured at 25°C for 10 days. After the plate was full, the mycelia were collected separately, and the extracted genomic DNA and EcoRI digestion results were shown in the appendix Figure 1 , two (L1 is Guxiang 66; L3 is Biyang No. 3; L5 is Shenxiang No. 10), the yield of extracted DNA is about 1.1 mg / g mycelia, and the spectrophotometric detection of A 260 / A 280 About 1.7 or so. Above-mentioned three shiitake mushroom varieties all adopt following steps as follows:

[0033] 1. Bacteria culture: Pick an appropriate amount of shiitake mushroom mycelium and inoculate it on a PDA plate with cellophane on the surface of the medium, culture at 25°C for 12 days, and collect the mycelium; PDA is a potato medium, and the preparation method is: peeled potatoes and thin slices 200g...

Embodiment 2

[0044] Pick mushroom hyphae of 3 different varieties (Guxiang 66, Biyang No. 3, Shenxiang No. 10) and inoculate them into conical flasks containing 10 mL of PDA liquid medium, shaker speed 200 rpm, and 25 °C. After culturing for 10 days, when the mycelium balls grew uniformly, the mycelia were collected by suction filtration respectively, and the genomic DNA and EcoRI digestion results were shown in the appendix. Figure 1 , two (L2 is Guxiang 66; L4 is Biyang 3; L6 is Shenxiang 10). The yield of DNA extracted per gram of mycelia is slightly higher than that of solid culture, about 1.4 mg, and the spectrophotometric detection of A 260 / A 280 About 1.8 or so. Above-mentioned three shiitake mushroom varieties all adopt following steps as follows:

[0045] 1. Bacteria culture: Pick an appropriate amount of shiitake mushroom mycelia and inoculate it in a shaker flask equipped with PDA liquid medium, culture it at 25°C for 10 days, and collect the mycelium; the preparation meth...

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Abstract

The invention relates to a method for quickly extracting the genome of lentinus edodes, pertaining to the technical field of molecular biology. The method for extracting the genome of the lentinus edodes comprises the following steps sequentially: cultivation of thalli, washing of the thalli, cellular wall breaking of the thalli, extraction of a genome, removal of RNA, removal of protein and deposition, washing and dissolution of genome DNA; and is characterized in that the purpose of simply and quickly extracting the lentinus-edodes genome DNA with stable effect is achieved by adopting the special steps of acid cleaning of glass beads and cellular wall breaking by oscillation.

Description

technical field [0001] The invention relates to a rapid extraction method of shiitake mushroom genome, which belongs to the technical field of molecular biology. Background technique [0002] Lentinus edodes is a class of edible fungi with important economic value. As the world's largest production and trade country of shiitake mushrooms and the birthplace of cultivated shiitake mushrooms, in recent years, more and more methods of molecular biology have been applied to the research on population genetics, phylogenetic evolution, and strain identification of shiitake mushrooms. Due to the variety of edible fungi such as shiitake mushrooms, if you want to carry out research based on genomics-related cloning of unknown genes, construction of genomic libraries, and molecular marker-assisted breeding, the number of experimental samples to be processed is large. Whether the method of extracting genome DNA from shiitake mushrooms is simple and fast , high efficiency and low cost h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/04C12N15/11C12N15/10
Inventor 姚强宫志远任海霞任鹏飞李瑾曲玲
Owner INST OF SOIL & FERTILIZER SHANDONG ACAD OF AGRI SCI
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