阅读说明：本技术 中草药微生物培养方法及dna提取方法 (Chinese herbal medicine microorganism culture method and DNA extraction method ) 是由 黄容琴 卢晨阳 张雅萍 于 2020-06-02 设计创作，主要内容包括：本发明涉及一种中草药微生物培养方法及DNA提取方法,该方法结合结构生物学对DNA高级结构的预测分析,运用计算机模型对各个细菌DNA的功能区序列进行设计和优化密码子,从而得到能够高效表达出目标细菌功能的基因序列,再将上述目标序列连接到合适的表达载体上,然后利用氯化铯密度梯度超速离心纯化,从样本中提取微生物DNA,从而以基因组的多样性反映物种的多样性。(The invention relates to a Chinese herbal medicine microorganism culture method and a DNA extraction method, the method combines the prediction analysis of structural biology on the higher structure of DNA, a computer model is used for designing and optimizing codons for functional region sequences of various bacterial DNAs so as to obtain a gene sequence capable of efficiently expressing the functions of target bacteria, then the target sequence is connected to a proper expression vector, and then cesium chloride density gradient ultracentrifugation purification is utilized to extract microorganism DNA from a sample, so that the diversity of species is reflected by the diversity of genome.)

1. A method for extracting Chinese herbal medicine microorganism DNA is characterized by comprising the following steps:

s1, analyzing the microorganism DNA functional region in the Chinese herbal medicine by using a computer model to obtain a target gene sequence capable of efficiently expressing the function of the target microorganism;

s2, extracting microorganisms in the Chinese herbal medicines by a glass bead shaking method, and then cracking the microorganisms in situ by combining a liquid nitrogen freeze thawing method, a plum method and a sds method;

s3, subjecting the crude DNA obtained in the step S2 to cesium chloride density gradient ultracentrifugation purification to obtain supernatant, adding potassium acetate for precipitation, and adding the obtained precipitate into TE solution to obtain Chinese herbal medicine microorganism DNA solution;

s4, diluting the DNA solution of the traditional Chinese medicine microorganism, and carrying out partial enzyme digestion on the DNA of the target microorganism to obtain the target gene sequence and amplification.

2. The method for extracting DNA from Chinese herbal medicine microorganisms as claimed in claim 1, wherein the step S2 specifically comprises:

weighing Chinese herbal medicines in a centrifuge tube containing a solution of glass beads, shaking and centrifuging to obtain microorganisms in the Chinese herbal medicines; and then repeatedly freezing and thawing in liquid nitrogen, adding cell lysis solution, DNA extracting solution, lysozyme and SDS solution to ensure that the microorganism is subjected to cell lysis to release DNA, and finally centrifuging to obtain the crude DNA.

3. The method for extracting DNA from Chinese herbal medicine microorganisms as claimed in claim 2, wherein the step S3 specifically comprises:

and dissolving the crude DNA in TE, adding cesium chloride, performing density gradient ultracentrifugation after the crude DNA is completely dissolved, then adding isopropanol for purification, adding potassium acetate for centrifugal precipitation, and dissolving the precipitate in TE to obtain the Chinese herbal medicine microorganism DNA solution.

4. The method for extracting Chinese herbal medicine microorganism DNA as claimed in claim 1, wherein in step S4, the specific steps of partial enzyme digestion comprise:

diluting the Chinese herbal medicine microorganism DNA solution, adding enzyme digestion buffer solution to obtain Chinese herbal medicine microorganism DNA diluent, adding restriction enzyme into the Chinese herbal medicine microorganism DNA diluent according to the analysis result of a computer model, uniformly mixing and culturing, and inactivating the restriction enzyme.

5. The method for extracting DNA from Chinese herbal medicine microorganisms according to claim 1, wherein in step S4, the specific steps of amplification comprise:

and adding primers into the Chinese herbal medicine microorganism DNA solution according to the analysis result of a computer model to perform PCR amplification.

6. A Chinese herbal medicine microorganism culture method is characterized by comprising the following steps:

the method for extracting DNA from Chinese herbal medicine microorganisms according to any one of claims 1 to 5, wherein the target sequence is linked to a carrier and then transferred into the Chinese herbal medicine microorganisms, and then the Chinese herbal medicine microorganisms are cultured in a bacterial flora solution.

7. The method for cultivating Chinese herbal medicine microorganisms of claim 6, wherein the bacterial flora solution is prepared by the following method:

mixing cell sap of wormwood, dandelion and houttuynia cordata, adding camphor wood powder, bamboo charcoal powder and distilled water, shaking up and mixing, and then centrifuging to remove impurities, and storing the obtained bacterial flora solution at-80 to-50 ℃.

8. The method of claim 6, wherein the amplified DNA product is separated by DGGE, the obtained DGGE film is stained and scanned to obtain an electron map, and the signal intensity profile of the resulting band is analyzed by software.

9. The method of claim 8, wherein the Chinese herbal medicine microorganism is subjected to colony PCR amplification after transferring the carrier, and the amplified colony product is simultaneously subjected to DGGE separation and staining together with the DNA product, and the characteristics of the microorganism corresponding to the band are known by comparing the bands on the electron spectrum.

Technical Field

The invention relates to a Chinese herbal medicine microorganism culture method and a DNA extraction method, belonging to the field of microbial engineering.

Background

The deodorizing method for refuse landfill is to use automatic spraying system and spraying truck to eliminate the bad smell from refuse. Spraying two deodorizers, namely plant liquid and biological bacteria, by covering. At present, most of the plant liquid is used. When landfill summer, produce the filtration liquid easily and leak, filtration liquid is harmful again to the environment, so generally all can carry out recovery processing, but filtration liquid can distribute very smelly gas. Conventional deodorants do not solve these problems.

The microorganism deodorization can quickly solve the problem of leakage of the percolate, and the product is compounded with a plurality of strains, so the product has obvious removal effect on the high compound malodorous substance gas of the percolate. However, in the conventional microbial extraction technology, the microbial community diversity and structural analysis are performed by isolating and culturing microorganisms and then analyzing the microorganisms by general biochemical properties and specific phenotypes. The traditional extraction technology has the following limitations:

limited isolation of the microorganism from the solid medium;

pure culture methods and principles are mostly introduced from medical microorganisms, and some methods are not suitable for specific microorganism research, for example, some microorganisms are in poor nutrition, while laboratory beef extract peptone rich in nutrition is used for measuring the total number of live bacteria in an environment, a large amount of poor nutrition microorganisms are not suitable for growth, and the measurement result has large error;

most microorganisms are at present uncultured;

therefore, the information of the living characteristics and the ecological functions of the microorganisms in a specific ecological system cannot be obtained by simple extraction and plate culture counting, so that a great amount of microorganism resources with great application value are buried.

Disclosure of Invention

The invention aims to provide a Chinese herbal medicine microorganism culture method and a DNA extraction method, which can extract DNA from Chinese herbal medicine microorganisms, so that the diversity of species is reflected by the diversity of genome, and the obtained product contains up to 122 microorganisms, and is more favorable for absorbing and decomposing harmful gases under the combined action of various microorganisms.

In order to achieve the purpose, the invention provides the following technical scheme: a method for extracting Chinese herbal medicine microorganism DNA comprises the following steps:

s1, analyzing the microorganism DNA functional region in the Chinese herbal medicine by using a computer model to obtain a target gene sequence capable of efficiently expressing the function of the target microorganism;

s2, extracting microorganisms in the Chinese herbal medicines by a glass bead shaking method, and then cracking the microorganisms in situ by combining a liquid nitrogen freeze thawing method, a plum method and a sds method;

s3, subjecting the crude DNA obtained in the step S2 to cesium chloride density gradient ultracentrifugation purification to obtain supernatant, adding potassium acetate for precipitation, and adding the obtained precipitate into TE solution to obtain Chinese herbal medicine microorganism DNA solution;

s4, diluting the DNA solution of the traditional Chinese medicine microorganism, and carrying out partial enzyme digestion on the DNA of the target microorganism to obtain the target gene sequence and amplification.

Further, step S2 specifically includes:

weighing Chinese herbal medicines in a centrifuge tube containing a solution of glass beads, shaking and centrifuging to obtain microorganisms in the Chinese herbal medicines; and then repeatedly freezing and thawing in liquid nitrogen, adding cell lysis solution, DNA extracting solution, lysozyme and SDS solution to ensure that the microorganism is subjected to cell lysis to release DNA, and finally centrifuging to obtain the crude DNA.

Further, step S3 specifically includes:

and dissolving the crude DNA in TE, adding cesium chloride, performing density gradient ultracentrifugation after the crude DNA is completely dissolved, then adding isopropanol for purification, adding potassium acetate for centrifugal precipitation, and dissolving the precipitate in TE to obtain the Chinese herbal medicine microorganism DNA solution.

Further, in step S4, the specific steps of the partial enzyme digestion include:

diluting the Chinese herbal medicine microorganism DNA solution, adding enzyme digestion buffer solution to obtain Chinese herbal medicine microorganism DNA diluent, adding restriction enzyme into the Chinese herbal medicine microorganism DNA diluent according to the analysis result of a computer model, uniformly mixing and culturing, and inactivating the restriction enzyme.

Further, in step S4, the specific steps of amplifying include:

and adding primers into the Chinese herbal medicine microorganism DNA solution according to the analysis result of a computer model to perform PCR amplification.

The invention also provides a Chinese herbal medicine microorganism culture method, which comprises the following steps:

the DNA extraction method of the Chinese herbal medicine microorganisms is carried out, the target sequences are connected to a carrier and then transferred into the Chinese herbal medicine microorganisms, and then the Chinese herbal medicine microorganisms are cultured in a bacterial flora solution.

Further, the bacterial flora solution is prepared by the following method:

mixing cell sap of wormwood, dandelion and houttuynia cordata, adding camphor wood powder, bamboo charcoal powder and distilled water, shaking up and mixing, and then centrifuging to remove impurities, and storing the obtained bacterial flora solution at-80 to-50 ℃.

Further, the amplified DNA product is separated by DGGE, the obtained DGGE film is dyed and scanned to obtain an electronic map, and the signal intensity distribution map of the produced band is analyzed by software.

Further, colony PCR amplification is carried out on the Chinese herbal medicine microorganisms after the Chinese herbal medicine microorganisms are transferred into the carrier, DGGE separation and staining are carried out on the amplified colony products and the DNA products at the same time, and the characteristics of the microorganisms corresponding to the bands are obtained by comparing the bands on the electronic map

Compared with the prior art, the invention has the beneficial effects that: the Chinese herbal medicine microorganism culture method and the DNA extraction method can extract DNA from Chinese herbal medicine microorganisms, so that the diversity of species is reflected by the diversity of genome, and the obtained product contains up to 122 microorganisms, and is more favorable for absorbing and decomposing harmful gas under the combined action of various microorganisms. Meanwhile, the microorganisms can generate inorganic acid, and simultaneously form an acidic environment which is not beneficial to the life of the putrefactive microorganisms, and fundamentally degrade and decompose substances which generate malodorous gases. According to different components of odor, the product is more targeted by screening, domesticating and culturing specific microorganisms by the Chinese herbal medicine microorganism culture method and the DNA extraction method.

The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a detailed description of the preferred embodiments of the present invention.

Detailed Description

The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.