CN105861495A - Method for extracting genome DNA from taxus chinensis medicinal material - Google Patents
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Abstract
The invention discloses a method for extracting genome DNA from a taxus chinensis medicinal material. A plant genome DNA extraction kit is improved, and phenol-chloroform-isoamyl alcohol (25:24:1) is added based on an original kit method, and extraction is performed twice to remove protein relatively well; and a DNA precipitator is precooled at -20 DEG C instead to realize a relatively good effect of precipitating DNA. ITS2 sequence amplification by virtue of DNA extracted from the taxus chinensis medicinal material through an improved kit is successful completely. The method disclosed by the invention is suitable for obtaining genome DNA from taxus chinensis processed into medicinal material decoction pieces or powder for follow-up conventional molecularly-biological operation and lays a foundation for realizing traditional Chinese medicine identification by using a DNA biological identification technology.
Description
Technical field
The present invention relates to TCD identification technical field, more particularly it relates to the extracting genome DNA technical field of Chinese medicine.The inventive method is suitable for from Ramulus et folium taxi cuspidatae medical material extracting genomic DNA.
Background technology
Taxaceae (Taxaceae) Taxus (Taxus) plant is aiphyllium or shrub, has 11 kinds in the whole world, is distributed in temperate zone, the Northern Hemisphere, cool temperature zone and the torrid zone, alpine region, subtropical zone, wherein there be 4 kind of 1 mutation in China, is distributed in southwest, northeast, Central China, south China and East China.Chinese yew genus plants is Relict Plant in the Tertiary Period, for rare tree species in imminent danger.
Ramulus et folium taxi cuspidatae, commonly referred to as " Ramulus et folium taxi cuspidatae " among the people, containing multiple medicinal ingredient.Medicinal function about Chinese yew genus plants is recorded in Compendium of Material Medica the earliest, can be used for treating the disease such as cholera, typhoid fever." book on Chinese herbal medicine pushes away old " also had a relevant record to its medicinal function: " Ramulus et folium taxi cuspidatae can be used as medicine, and diuresis, stimulates the menstrual flow, controls nephropathy, easily vomits with skin, and woody part and leaf are not told ".Among the people and Chinese Traditional Medicine also has long historical records for the utilization of Chinese yew genus plants, in order to removing food stagnancy, moisturize, diuresis, stimulate the menstrual flow, antiinflammatory, and multiple intestinal parasitical diseases can be treated.Up-to-date pharmaceutical research finds, Chinese yew genus plants has antibacterial holds back poison, antiinflammatory toxin expelling effect, upper respiratory tract infection is had therapeutical effect, diabetes, nephropathy, hypertension and hyperlipidemia etc. are had significant curative effect.
Paclitaxel is a kind of Fourth Ring two note amides compound that American scientist M.C.Wani in 1963 and Monre E.Wall extract isolated from the bark of Ramulus et folium taxi cuspidatae of the Pacific Ocean, it is that find up to now a kind of can promote the cohesion of cancerous cell micro-pipe the medicine of irreversible depolymerization, it is possible to effectively stop the propagation of cancerous cell.
In Chinese yew genus plants the most of the same race, content of taxol is different, and its drug effect is also the most different.Average content such as China Ramulus et folium taxi cuspidatae, T. yunnanensis, Xizang Taxus chinensis, Ramulus et folium taxi cuspidatae, Taxus mairei and each position of Taxus media trees is respectively 0.032%, 0.050%, 0.034%, 0.043%, 0.044% and 0.046%.Therefore, the discriminating of Ramulus et folium taxi cuspidatae medical material utilizes for conservation of plant resources and Chinese crude drug safe and reasonable and all has great importance.
Although can easily Ramulus et folium taxi cuspidatae be identified by plant morphology, but, Ramulus et folium taxi cuspidatae medical material in the market is mostly decoction pieces or powder, after being processed into decoction pieces or powder, is difficult with conventional TCD identification means and differentiates these crude drug sources are in which kind of Chinese yew genus plants.
Along with the development of molecular biology, method for identifying molecules is the most more and more applied in the discriminating of Chinese crude drug.DNA bar code technology, as a kind of hot topic, the Molecular Identification technology in forward position, is widely used in medicinal plants is identified.In the common bar code of plant, ITS2 sequence can well distinguish different affinity species.This Biological assay based on DNA often accuracy is high, reproducible, has more science, be suitable for the needs of actual application for traditional method.
The premise of DNA bar code Molecular Identification is to obtain the preferable DNA of quality.Conventional DNA extraction method, such as CTAB method, mainly it is applied to the extraction of plant cell dna, is generally based on what plant leaf blade was carried out.For Ramulus et folium taxi cuspidatae medical material, the greatest problem faced is how to extract to obtain DNA from the Chinese medicinal material of this deep processing.
On the one hand, due to Chinese medicine its DNA heavy damage in preparation process, and losing in a large number, therefore in finished product, contained DNA content is few, and fragment is extremely short, and how being enriched with these only a small amount of DNA fragmentations is the challenge that must face；On the other hand, in Ramulus et folium taxi cuspidatae medical material, topmost composition is various albumen, DNA extraction and purification work are necessarily caused severe jamming by the existence of these albumen, and how overcoming these protein existed in a large number is another challenge that must face during DNA extraction on the impact of DNA extraction.
Additionally, extract the DNA obtained also should be able to reach the requirement of some conventional molecular biological including PCR operation, in order to follow-up qualification.It appeared that, the simple DNA extraction method using the most any routine, also being able to from Ramulus et folium taxi cuspidatae medical material extract and obtain a small amount of DNA sample, but but hardly result in the DNA sample that disclosure satisfy that subsequent detection requires, it is extremely difficult for continuing to apply to follow-up molecular Biological Detection.Therefore, the extracting method of DNA in a kind of Ramulus et folium taxi cuspidatae medical material that can be applied to follow-up molecular biology manipulations to be set up, be necessary on the basis of existing, these conventional methods are carried out some necessity, adjust and improve targetedly.
Summary of the invention
It is an object of the invention to provide a kind of method extracting genomic DNA from Ramulus et folium taxi cuspidatae medical material, the inventive method is suitable for obtaining genomic DNA from the Ramulus et folium taxi cuspidatae being processed into pharmaceutical decocting piece or powder and operates for follow-up conventional molecular biological, provides basis for utilizing DNA Biology identification technology to realize TCD identification.
The method extracting genomic DNA from Ramulus et folium taxi cuspidatae medical material of the present invention specifically follows the steps below:
1) Ramulus et folium taxi cuspidatae medicinal powder is processed by GP1 buffer that preheat with 65 DEG C, containing 0.1wt% mercaptoethanol；
2) with volume ratio as phenol: chloroform: isoamyl alcohol=25: the above-mentioned Ramulus et folium taxi cuspidatae medical material processed is extracted by the extracting solution of 24: 1, isolated the first supernatant；
3) adding volume ratio in described first supernatant is phenol: chloroform=25: the extracting solution of 24 extracts, isolated the second supernatant；
4) in described second supernatant, chloroform, isolated the 3rd supernatant are added；
5) in described 3rd supernatant, add buffer GP2, mixing, proceed in adsorption column CB3 ,-20 DEG C of refrigerators are placed 5min, take out centrifugal, remove waste liquid；
6) clean adsorption column CB3 with the rinsing liquid GD of pre-cooling and the rinsing liquid PW of pre-cooling successively, dry rinsing liquid remaining in adsorption column CB3；
7) being eluted from adsorption column CB3 by genomic DNA with elution buffer TE, room temperature places 5min, and centrifugal solution of collecting obtains the genomic DNA of described Ramulus et folium taxi cuspidatae medical material.
Specifically, described step 1) the process time be 30min～1h.
Further, in the method for the invention, can be by step 2) extraction process be repeated 2 times.
Further, described step 6) in, clean 2 adsorption column CB3 with the rinsing liquid PW of pre-cooling.
For increasing the yield of genomic DNA, it is also possible to by step 7) to collect the solution that obtains and add in adsorption column CB3, room temperature places 4 DEG C of centrifugal 2min of 2min, 12000rpm.
And then, it is template that method described above extracts the Ramulus et folium taxi cuspidatae medical material STb gene obtained, ITS2F and ITS3R primer is used to carry out pcr amplification reaction, by direct for PCR primer two-way order-checking, sequence assembly, contrast, compare with the Chinese yew NJ tree set up, it is possible to identify which kind of Chinese yew genus plants Ramulus et folium taxi cuspidatae medical material belongs to.
Test shows, with plant genome DNA extract test kit (Tiangen Biotech Co.,
China) DNA in the Chinese yew genus plants leaf sample of silica dehydrator is extracted, with being dried DNA cloning ITS2 sequence 100% success that blade extracts.But when extracting the DNA in Ramulus et folium taxi cuspidatae medical material with same test kit, the effect of extracting directly is unsatisfactory.Therefore, plant genome DNA extraction test kit is improved by the present invention.Firstly, since various secondary metabolites and protein are more in Ramulus et folium taxi cuspidatae medical material, therefore on the basis of original reagent cassette method, add phenol chloroform-isoamyl alcohol (25: 24: 1) extract 2 times, can preferably remove protein.Secondly, although the DNA precipitant in original reagent box is containing a certain amount of isopropanol, but the scarce capacity of its precipitation DNA, change the precipitant of-20 DEG C of pre-coolings after test into, it is possible to reach to precipitate the better effects of DNA.The present invention, after above-mentioned improvement, by the DNA cloning ITS2 sequence extracted from Ramulus et folium taxi cuspidatae medical material, also achieves 100% success.
Accompanying drawing explanation
Fig. 1 is to use original reagent box extraction Chinese yew genus plants DNA cloning ITS2 sequence and use original reagent box and improvement test kit to extract the PCR amplification electrophoresis detection figure of commercially available Ramulus et folium taxi cuspidatae medical material DNA cloning ITS2 sequence respectively.
Wherein: MDY be Taxus media (Taxus x
media)；HJ be gracefully sub-red arrow (Taxus
x media var. Hongjian)；LX be gracefully sub-green star (Taxus x media var. Lvxing)；ZK be Man Diyazi section (Taxus x
media var. Zike)；DB be Ramulus et folium taxi cuspidatae (Taxus cuspidata)；NF be Taxus mairei (Taxus chinensis var. Mairei)；1 is Ramulus et folium taxi cuspidatae；2 is Ramulus et folium taxi cuspidatae；3 is Ramulus et folium taxi cuspidatae board taxus chinensis powder；M is DNA Marker.
The left side is the PCR amplification electrophoresis detection figure that original reagent box extracts Chinese yew genus plants and commercially available Ramulus et folium taxi cuspidatae medical material DNA cloning ITS2 sequence；The PCR that Chinese yew genus plants DNA and the improvement test kit commercially available Ramulus et folium taxi cuspidatae medical material DNA cloning ITS2 sequence of extraction are extracted for original reagent box in the right expands electrophoresis detection figure.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment that the inventive method, step or condition are made or replacement, belong to the scope of the present invention.
If not specializing, the conventional means that the technological means used by embodiment is well known to those skilled in the art.
Embodiment 1: use ITS2 sequence to differentiate commercially available Ramulus et folium taxi cuspidatae medical material.
1, sample source: 3 parts of Ramulus et folium taxi cuspidatae medical materials of test are collected from commodity Ramulus et folium taxi cuspidatae, Ramulus et folium taxi cuspidatae board taxus chinensis powder and the commodity Ramulus et folium taxi cuspidatae that medical material market, respectively different company produce.
2, DNA extraction: by each for above-mentioned sample 100mg that about takes, adds liquid nitrogen and is fully ground into powder, utilizes the plant genome DNA of improvement to extract test kit and extracts sample DNA.
1) ground medicinal powder is added to 2mL centrifuge tube.
2) add 1000 L 65 DEG C preheating buffer GP1 and (in the GP1 of preheating, before experiment, add mercaptoethanol, make its final concentration of 0.1%), after the most reverse mixing, centrifuge tube being placed on 1h in 65 DEG C of water-baths, during water-bath, every 10min is reverse once with biased sample.
3) material temperature bathed takes out to be put at room temperature, after being cooled to room temperature, adds 700 L and consists of phenol: chloroform: the extracting solution of isoamyl alcohol (25: 24: 1), fully mixes, and 12000rpm room temperature is centrifuged 5min, collects supernatant.
4) by centrifugal sediment repetitive operation step 3), collect supernatant, merge twice supernatant.
5) being proceeded to by supernatant carefully in new centrifuge tube, add 700 L and consist of phenol: the extracting solution of chloroform (25: 24), fully mix, 12000rpm room temperature is centrifuged 5min, collects supernatant.
6) proceeding to supernatant carefully, in new centrifuge tube, add 700 L extracting solution chloroforms, fully mix, 12000rpm room temperature is centrifuged 5min, collects supernatant.
7) proceed to supernatant carefully, in new centrifuge tube, add 700 L buffer GP2, fully mix.
8) liquid of mixing is proceeded in adsorption column CB3, be placed in 5min in-20 DEG C of refrigerators, take out 4 DEG C of centrifugal 1min of 12000rpm, outwell waste liquid.(adsorption column volume 700 about L, graded adds).
9) in adsorption column CB3, add 4 DEG C of centrifugal 1min of rinsing liquid GD, 12000rpm of 500 μ L pre-coolings, outwell waste liquid, adsorption column CB3 is put in collecting pipe.
10) in adsorption column CB3, add 4 DEG C of centrifugal 1min of rinsing liquid PW, 12000rpm of 600 μ L pre-coolings, outwell waste liquid, adsorption column CB3 is put in collecting pipe.
11) repetitive operation step 10).
12) adsorption column CB3 is put back in collecting pipe, 4 DEG C of centrifugal 2min of 12000rpm, after outwelling waste liquid, uncap adsorption column CB3 and are placed in room temperature placement several minutes, thoroughly to dry rinsing liquid remaining in adsorption column.
13) adsorption column CB3 is proceeded in clean centrifuge tube, to the middle part of adsorbed film unsettled dropping 50 μ L elution buffer TE, room temperature places 4 DEG C of centrifugal 2min of 5min, 12000rpm, solution is collected in centrifuge tube, obtain the genomic DNA of Ramulus et folium taxi cuspidatae medical material.
14) for increasing the yield of genomic DNA, can add in adsorption column CB3 by the solution that collection obtains, room temperature places 4 DEG C of centrifugal 2min of 2min, 12000rpm, is collected in centrifuge tube by solution.
3, PCR amplification.
Pcr amplification primer thing:
ITS2F:5'-ATGCGATACTTGGTGTGAAT-3'；
ITS3R:5'-GACGCTTCTCCAGACTACAAT-3'.
PCR reaction system: be calculated as with 20 μ L: ddH2O, 8 μ L；2 × Taq PCR Mix, 10 μ L；ITS2F/3R primer, 0.5 μ L；DNA profiling, 1 μ L.
PCR reaction condition: 94 DEG C of 5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 45s, 40cycles；72℃10min.
PCR result as it is shown in figure 1, in Fig. 13, rightmost side swimming lane be use improvement test kit extract commercially available Ramulus et folium taxi cuspidatae medical material DNA carry out ITS2 sequence PCR amplification electrophoresis detection figure, display DNA cloning success, can check order.
4, order-checking: application ABI 3730 sequenator (Applied
Biosystems Co., USA) pcr amplification product after purification carried out direct two-way order-checking.
5, the splicing of sequence, comparison, process.
Use sequence assembly software CodonCode Aligener V3.0 (CodonCode., USA) that order-checking peak figure is corrected, remove low quality sequence and guiding region, carry out Multiple Sequence Alignment and desk checking.Use based on hidden Markov model (hidden Markov
Model, HMMer) annotation method remove all sequences two ends 5.8S and 28S section, and then obtain ITS2 intervening sequence.K2P (Kimura 2-parameter) Genetic Distance Analysis in using MEGA (Molecular Evolutionary Genetics Analysis) 5.1 softwares to plant between carrying out Multiple Sequence Alignment sequence and carrying out planting, based on K2P distance method, by NJ method to comparison sequence construct system clustering tree, Bootstrap (repeating for 1000 times) is utilized to check the supporting rate of each branch.
From GenBank, download Chinese yew genus plants ITS2 sequence data, use bioinformatics software CodonCode Aligner and MEGA5.1 to be analyzed, set up the NJ tree of Chinese yew genus plants.
By the analysis to NJ tree, it can be seen that Ramulus et folium taxi cuspidatae medical material divides on same with corresponding Chinese yew genus plants, commodity Ramulus et folium taxi cuspidatae, Ramulus et folium taxi cuspidatae board taxus chinensis powder and commodity Ramulus et folium taxi cuspidatae are nearest with the sibship of all kinds, Taxus mairei and the Ramulus et folium taxi cuspidatae of Taxus media respectively, thus show that commodity Taxus is in Taxus media, Ramulus et folium taxi cuspidatae board taxus chinensis powder belongs to Taxus mairei, commodity Ramulus et folium taxi cuspidatae belongs to Ramulus et folium taxi cuspidatae, has well identified which kind of Chinese yew genus plants Ramulus et folium taxi cuspidatae medical material belongs to.
Comparative example 1: use ITS2 sequence to differentiate Chinese yew genus plants.
1, sample source: the Chinese yew genus plants of employing include Taxus media (Taxus x media), the red arrow of Man Diya (Taxus x media var. hongjian), the green star of Man Diya (Taxus x media var. lvxing), Man Diyazi section (Taxus x media var. zike), Ramulus et folium taxi cuspidatae (Taxus cuspidata), Taxus mairei (Taxus
chinensis var.mairei), it is collected from Agricultural University Of Shanxi's Medicinal Plant Germplasm Resources garden.
2, DNA extraction: use 70% ethanol Chinese yew genus plants blade surface, uses silica gel to be dried.Take the appropriate blade being dried, add liquid nitrogen and be fully ground into powder, extract test kit (Tiangen Biotech Co., China) with plant genome DNA and extract the genomic DNA in Chinese yew genus plants blade.
1) ground blade powder is added to 2mL centrifuge tube.
2) add 700 L 65 DEG C preheating buffer GP1 and (in the GP1 of preheating, before experiment, add mercaptoethanol, make its final concentration of 0.1%), after the most reverse mixing, centrifuge tube is placed on 20min in 65 DEG C of water-baths, reverse no less than once with biased sample during water-bath.
3) material temperature bathed takes out to be put at room temperature, after being cooled to room temperature, adds 700 L and consists of phenol: the extracting solution of chloroform (25: 24), fully mix, and 12000rpm room temperature is centrifuged 5min, collects supernatant.
4) proceeding to supernatant carefully, in new centrifuge tube, add 700 L extracting solution chloroforms, fully mix, 12000rpm room temperature is centrifuged 5min, collects supernatant.
5) proceed to supernatant carefully, in new centrifuge tube, add 700 L buffer GP2, fully mix.
6) liquid of mixing is proceeded in adsorption column CB3,4 DEG C of centrifugal 1min of 12000rpm, outwell waste liquid.(adsorption column volume 700 about L, graded adds).
7) in adsorption column CB3, add 500 μ L rinsing liquid GD, 4 DEG C of centrifugal 1min of 12000rpm, outwell waste liquid, adsorption column CB3 is put in collecting pipe.
8) in adsorption column CB3, add 600 μ L rinsing liquid PW, 4 DEG C of centrifugal 1min of 12000rpm, outwell waste liquid, adsorption column CB3 is put in collecting pipe.
9) repetitive operation step 8).
10) adsorption column CB3 is put back in collecting pipe, 4 DEG C of centrifugal 2min of 12000rpm, after outwelling waste liquid, uncap adsorption column CB3 and are placed in room temperature placement several minutes, thoroughly to dry rinsing liquid remaining in adsorption column.
11) adsorption column CB3 is proceeded in clean centrifuge tube, to the middle part of adsorbed film unsettled dropping 50 μ L elution buffer TE, room temperature places 4 DEG C of centrifugal 2min of 5min, 12000rpm, solution is collected in centrifuge tube, obtain the genomic DNA in Chinese yew genus plants blade.
12) for increasing the yield of genomic DNA, can add in adsorption column CB3 by the solution that collection obtains, room temperature places 4 DEG C of centrifugal 2min of 2min, 12000rpm, is collected in centrifuge tube by solution.
3, PCR amplification.
Carry out according to embodiment 1 method.
PCR result, as it is shown in figure 1, each 6 swimming lanes of Fig. 1 middle left and right both sides letter are the electrophoresis detection figure using original reagent box extraction Chinese yew genus plants DNA to carry out the amplification of ITS2 sequence PCR, all show DNA cloning success, can check order.
4, order-checking: carry out according to embodiment 1 method.
5, the splicing of sequence, comparison, process.
Carry out according to embodiment 1 method.
By the analysis to NJ tree, it can be seen that Chinese yew genus plants the most of the same race, all in different branches, can significantly distinguish.
Comparative example 2: use ITS2 sequence to differentiate commercially available Ramulus et folium taxi cuspidatae medical material.
3 parts of Ramulus et folium taxi cuspidatae medical materials of Example 1, extract the genomic DNA of Ramulus et folium taxi cuspidatae medical material according to the DNA extraction step of comparative example 1.PCR amplification is carried out according to embodiment 1 method, result is as shown in Figure 1, in Fig. 1 left side 3 be 1,2,3 sequence number swimming lane be use original reagent box extract commercially available Ramulus et folium taxi cuspidatae medical material DNA carry out ITS2 sequence PCR amplification electrophoresis detection figure, display DNA cloning unsuccessful, it is impossible to carry out lower pacing sequence.
Claims (5)
1. the method extracting genomic DNA from Ramulus et folium taxi cuspidatae medical material, described method follows the steps below:
1) Ramulus et folium taxi cuspidatae medicinal powder is processed by GP1 buffer that preheat with 65 DEG C, containing 0.1wt% mercaptoethanol；
2) with volume ratio as phenol: chloroform: isoamyl alcohol=25: the above-mentioned Ramulus et folium taxi cuspidatae medical material processed is extracted by the extracting solution of 24: 1, isolated the first supernatant；
3) adding volume ratio in described first supernatant is phenol: chloroform=25: the extracting solution of 24 extracts, isolated the second supernatant；
4) in described second supernatant, chloroform, isolated the 3rd supernatant are added；
5) in described 3rd supernatant, add buffer GP2, mixing, proceed in adsorption column CB3 ,-20 DEG C of refrigerators are placed 5min, take out centrifugal, remove waste liquid；
6) clean adsorption column CB3 with the rinsing liquid GD of pre-cooling and the rinsing liquid PW of pre-cooling successively, dry rinsing liquid remaining in adsorption column CB3；
7) being eluted from adsorption column CB3 by genomic DNA with elution buffer TE, room temperature places 5min, and centrifugal solution of collecting obtains the genomic DNA of described Ramulus et folium taxi cuspidatae medical material.
Method the most according to claim 1, is characterized in that described step 1) the process time be 30min～1h.
Method the most according to claim 1, is characterized in that described step 2) extraction process be repeated 2 times.
Method the most according to claim 1, is characterized in that described step 6) in, clean 2 adsorption column CB3 with the rinsing liquid PW of pre-cooling.
Method the most according to claim 1, is characterized in that step 7) to collect the solution that obtains and add in adsorption column CB3, room temperature places 4 DEG C of centrifugal 2min of 2min, 12000rpm.
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| CN110972933A (en) * | 2019-12-26 | 2020-04-10 | 山西农业大学 | Tetraploid taxus media cultivation method |
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2016
- 2016-06-20 CN CN201610441493.1A patent/CN105861495A/en active Pending
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