阅读说明：本技术 一种山药dna提取的新方法 (novel method for extracting Chinese yam DNA ) 是由 薛刚 于 2018-05-31 设计创作，主要内容包括：本发明公开了一种山药DNA提取的新方法,包括：S1鲜山药去皮、洗净,在液氮中磨成山药粉；S2将山药粉在2-8倍重量的水中,水热处理1-2h获得混合浆液,处理温度为40-50℃,混合浆液经离心获得沉淀物；取沉淀物5-10g,加入15-20mL 65℃预热的裂解缓冲液和300-400μL的β-巯基乙醇,混合均匀,于60-70℃下保温40-50min;加入15-20mL体积比为25:24:1的酚-氯仿-异戊醇溶液,混合后,室温下离心分离获得上清液；S4向上清液中加入等体积的异丙醇,出现絮状沉淀,离心分离获得DNA沉淀；S5 DNA洗涤：用体积浓度为75%的乙醇洗涤DNA沉淀,达到了提高山药DNA纯度的效果。(The invention discloses a novel method for extracting Chinese yam DNA, which comprises the following steps: s1 peeling fresh rhizoma Dioscoreae, cleaning, and grinding into rhizoma Dioscoreae powder in liquid nitrogen; s2, putting the yam powder into water with the weight 2-8 times of that of the yam powder, carrying out hydro-thermal treatment for 1-2h to obtain mixed slurry, wherein the treatment temperature is 40-50 ℃, and the mixed slurry is centrifuged to obtain a precipitate; taking 5-10g of precipitate, adding 15-20mL of lysis buffer preheated at 65 ℃ and 400 mu L of beta-mercaptoethanol at 300-70 ℃, uniformly mixing, preserving the heat at 60-70 ℃ for 40-50min, adding 15-20mL of phenol-chloroform-isoamylol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant; s4, adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate; s5DNA wash: the DNA sediment is washed by ethanol with the volume concentration of 75 percent, and the effect of improving the purity of the yam DNA is achieved.)

1. A novel method for extracting Chinese yam DNA is characterized in that: the method comprises the following steps:

S1 liquid nitrogen grinding: peeling fresh rhizoma Dioscoreae, cleaning, and grinding into rhizoma Dioscoreae powder in liquid nitrogen;

S2 hydrothermal treatment: hydrothermally treating the yam powder in water of which the weight is 2-8 times that of the yam powder for 1-2 hours to obtain mixed slurry, wherein the treatment temperature is 40-50 ℃, and centrifuging the mixed slurry to obtain a precipitate;

s3DNA extraction: taking 5-10g of precipitate, adding 15-20mL of lysis buffer preheated at 65 ℃ and 400 mu L of beta-mercaptoethanol at 300-70 ℃, uniformly mixing, preserving the heat at 60-70 ℃ for 40-50min, adding 15-20mL of phenol-chloroform-isoamylol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;

S4DNA isolation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate;

S5DNA wash: washing the DNA precipitate with 75% ethanol by volume concentration to obtain the yam DNA.

2. The method for extracting Chinese yam DNA according to claim 1, which is characterized in that: in step S2, amylase is added, and 1-2 units of amylase with activity is added to every 1g of yam flour.

3. The method for extracting Chinese yam DNA according to claim 1, which is characterized in that: in step S2, the ultrasonic treatment is assisted, and the ultrasonic power is 100-.

4. The method for extracting Chinese yam DNA according to claim 3, which is characterized in that: in step S2, ultrasonic wave interval treatment is carried out for multiple times, each ultrasonic wave treatment time is 5-8min, and the interval between two adjacent ultrasonic wave treatments is 20-40 min.

5. the method for extracting Chinese yam DNA according to claim 4, wherein the method comprises the following steps: in step S2, the ultrasonic power is 200W, the ultrasonic treatment time is 6min each time, and the interval between two adjacent ultrasonic treatments is 30 min.

6. The method for extracting Chinese yam DNA according to claim 1, which is characterized in that: in step S3, 8g of precipitate is taken, 16mL of lysis buffer preheated at 65 ℃ and 320 μ L of beta-mercaptoethanol are added, the mixture is uniformly mixed, the temperature is kept at 65 ℃ for 45min, 16mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1 is added, and after mixing, the supernatant is obtained by centrifugal separation at room temperature.

7. a yam DNA prepared by the method of any one of claims 1-6.

Technical Field

the invention relates to the technical field of plant DNA extraction, in particular to a novel method for extracting yam DNA.

Background

The yam is an underground root tuber of the dioscoreaceae, is tender in meat quality, contains rich nutritional health-care substances, can be made into health-care food, and has a medicinal value for conditioning diseases.

Chinese yam germplasm resources are rich, and in actual production, the classification of Chinese yam is usually based on the shape of tubers or isozyme is used as identification basis, however, the two methods have limitations.

the molecular markers are utilized to reveal the difference and the correlation among the yam varieties from the DNA molecular level, more accurate identification can be made on the genetic relationship of the yam varieties, and a basis is provided for variety classification, quality evaluation, new variety breeding and core germplasm construction of yam germplasm resources on the molecular level.

the content of secondary metabolites such as polysaccharide in the Chinese yam is high, so that the extraction process of the DNA of the Chinese yam is influenced, a large amount of the DNA of the Chinese yam is easy to remain in the DNA extract of the Chinese yam, the purity of the DNA of the Chinese yam is not high, and the subsequent use of the DNA of the Chinese yam is further influenced.

disclosure of Invention

Aiming at the defects of the prior art, the invention provides a novel method for extracting yam DNA, and the purpose of improving the extraction purity of yam DNA is achieved.

The technical purpose of the invention is realized by the following technical scheme:

A novel method for extracting yam DNA comprises the following steps:

s1 liquid nitrogen grinding: peeling fresh rhizoma Dioscoreae, cleaning, and grinding into rhizoma Dioscoreae powder in liquid nitrogen;

S2 hydrothermal treatment: hydrothermally treating the yam powder in water of which the weight is 2-8 times that of the yam powder for 1-2 hours to obtain mixed slurry, wherein the treatment temperature is 40-50 ℃, and centrifuging the mixed slurry to obtain a precipitate;

S3, DNA extraction: taking 5-10g of precipitate, adding 15-20mL of 65 ℃ preheated lysis buffer and 300-400 mu L of beta-mercaptoethanol, uniformly mixing, and preserving heat at 60-70 ℃ for 40-50min; adding 15-20mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and performing centrifugal separation at room temperature to obtain supernatant;

S4DNA separation: adding isopropanol with the same volume into the supernatant to generate flocculent precipitate, and performing centrifugal separation to obtain DNA precipitate; s5DNA washing: washing the DNA precipitate with 75% ethanol by volume concentration to obtain the yam DNA.

By adopting the scheme, before DNA extraction, hydrothermal treatment is carried out in advance, reasonable hydrothermal treatment parameters are set, secondary metabolites such as a large amount of polysaccharide in the Chinese yam are decomposed and dissolved in water, most of Chinese yam DNA still exists in the precipitate, the precipitate containing a large amount of Chinese yam DNA is obtained through centrifugal separation, then, DNA extraction is carried out, the secondary metabolites such as Chinese yam polysaccharide in the Chinese yam DNA can be greatly reduced, the extraction purity of the Chinese yam DNA is improved, the analysis of the Chinese yam DNA is facilitated, and bases are provided for variety classification, quality evaluation, new variety breeding, core germplasm construction and the like of Chinese yam germplasm resources.

the lysis buffer solution for DNA extraction is 3% CTAB, 100mmol/L Tris-HCl, pH8.0, 20mmol/L EDTA, pH8.0, 1.4mol/L NaCl and 2% beta-mercaptoethanol, Tris-HCl (Ph8.0) provides a buffer environment to prevent nucleic acid from being damaged, EDTA chelates Mg ²⁺ or Mn ²⁺ ions to inhibit DNase activity, NaCl provides a high-salt environment to fully dissolve DNA and exists in a liquid phase, and beta-mercaptoethanol is an antioxidant to effectively prevent phenol from being oxidized into quinone and avoid browning.

Preferably, amylase is added, and 1-2 activity units of amylase are added to every 1g of yam flour.

By adopting the scheme, the amylase has a decomposition effect on starch in the Chinese yam, is dissolved in water, and is separated from the Chinese yam DNA in the precipitate through centrifugal separation, so that impurities in the precipitate are further reduced, and the extraction purity of the Chinese yam DNA is improved.

preferably, in step S2, the ultrasonic treatment is assisted, and the ultrasonic power is 100-.

By adopting the scheme, through ultrasonic treatment, ultrasonic waves are propagated in water to generate a special 'cavitation effect', countless micro air pockets with the internal pressure reaching thousands of atmospheric pressures are continuously generated, the micro air pockets are continuously 'blasted' to generate microcosmic powerful shock waves, and the shock waves continuously act on the Chinese yam tissues to further erode the internal structure of the Chinese yam tissues, promote escape of secondary metabolites such as polysaccharide and the like, and improve the removal effect of the polysaccharide.

Preferably, in step S2, the ultrasonic treatment is performed at intervals, each ultrasonic treatment time is 5-8min, and the interval between two adjacent ultrasonic treatments is 20-40 min.

Preferably, in step S2, the ultrasonic power is 200W, each ultrasonic treatment time is 6min, and the interval between two adjacent ultrasonic treatments is 30 min.

by adopting the scheme, the ultrasonic treatment time is too long, so that excessive fragments of the yam tissue generated in the ultrasonic treatment process are easily caused, the escape channels of secondary metabolites such as polysaccharide are blocked, and the escape of the secondary metabolites such as polysaccharide is influenced. The ultrasonic treatment time and the interval time are limited, sufficient flowing time is provided for yam tissue fragments, escape channels of secondary metabolites such as polysaccharide and the like are prevented from being blocked, and the removal effect of the polysaccharide is further improved. In addition, the ultrasonic wave interval treatment can also reduce energy consumption, and then reduce cost.

preferably, in step S3, the precipitate 8g is taken, 16mL of lysis buffer preheated at 65 ℃ and 320 μ L of beta-mercaptoethanol are added, mixed uniformly, and the temperature is kept at 65 ℃ for 45min; adding 16mL of phenol-chloroform-isoamyl alcohol solution with the volume ratio of 25:24:1, mixing, and centrifuging at room temperature to obtain supernatant.

Object two of the present invention: provides the yam DNA prepared by the method.

In conclusion, the invention has the following beneficial effects:

1. before DNA extraction, a large amount of secondary metabolites such as polysaccharide in the Chinese yam are removed through hydrothermal treatment in advance, and then DNA extraction is carried out on precipitates containing a large amount of Chinese yam DNA, so that the secondary metabolites such as Chinese yam polysaccharide in the Chinese yam DNA can be greatly reduced, and the extraction purity of the Chinese yam DNA is improved;

2. Amylase has decomposition effect on starch in the Chinese yam, is dissolved in water, and is separated from Chinese yam DNA in the precipitate through centrifugal separation, so that impurities in the precipitate are further reduced, and the extraction purity of the Chinese yam DNA is improved;

3. ultrasonic auxiliary treatment is carried out in the hydrothermal process, escape of secondary metabolites such as polysaccharide is promoted, and the removal effect of polysaccharide is improved; 4. the ultrasonic treatment time and the interval time are limited, sufficient flowing time is provided for yam tissue fragments, escape channels of secondary metabolites such as polysaccharide and the like are prevented from being blocked, and the removal effect of the polysaccharide is further improved. In addition, the ultrasonic wave interval treatment can also reduce energy consumption, and then reduce cost.

Detailed Description

The present invention will be described in further detail below.