CN103074326A - Changbai mount rhododendron chrysanthum pall genome DNA extraction method - Google Patents
Changbai mount rhododendron chrysanthum pall genome DNA extraction method Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and discloses an alpine plant Changbai mount rhododendron chrysanthum pall genome DNA extraction method. According to the method, a CTAB method and a SDS method are combined at the first time to extract and purify Changbai mountain rhododendron chrysanthum pall genome DNA; in the extraction method, a PVP use amount in a CTAB extraction buffer solution is 4% and is increased by 2% compared to a PVP use amount in the conventional method, a 2 beta-mercaptoethanol use amount is 4.3% and is increased by about 1 time compared with a 2-mercaptoethanol use amount in the conventional method, and a 65 DEG C warm bath time is prolonged to 4 h from 1 h of the conventional method; and the SDS method is combined to purify the extracted genome so as to successfully extract the high purity and good quality Changbai mountain rhododendron chrysanthum pall genome DNA. With the present invention, disadvantages of difficult extraction, low purity of the extracted genome and easy degradation of the existing alpine plant are overcome, and important significances are provided for subsequent molecular level researches and alpine plant genome researches.
Description
Technical field
The present invention relates to a kind of method of alpine plant extracting genome DNA, belong to gene engineering technology field, relate to specifically the method for a kind of Changbai Mountain Rhododendron aureum extracting genome DNA.
Background technology
Because the alpine plant envrionment conditions is abominable, so alpine plant is considered to the most extreme biology in land usually, enriches valuable genetic resources but alpine plant has also.Since 1896, as focus and the difficult point in the organic evolution research, high mountain and arctic plants are to adaptation mechanism and always extremely investigators' the concern of strategy in habitat.The extractive technique of alpine plant genomic dna is one of basic fundamental of carrying out molecular biology of plants research, the height of DNA productive rate, and the quality of quality directly has influence on the success or failure of subsequent experimental.The extracting method of inquiring into the alpine plant genomic dna is a very significant job, for guaranteeing excellent germplasm to be provided aspect the breeding of farm crop tolerance to environmental stress and to select index, provide genes involved for agriculture as the foundation of the economy research and genetically engineered.This special genetic resources is applied in views and admires land plant; to protect and rationally utilize effective combination; go away sustainable development road with natural harmony development, this not only is conducive to the stable of the growing of this zone vegetation, the ecosystem, more is conducive to the development of regional economy.
The Extraction Methods of Genome of alpine plant has multiple, and in recent years, people are devoted to study a kind of efficiently extracting method fast always, but because the specificity of species, it not is to make things convenient for so that the genome of alpine plant extracts.The envrionment temperature of alpine plant existence is low, radiation is strong, in such environment, plant-growth is extremely slow, product consumption reduces, keep out the cold and the physiological drought aspect for improving it, make the accumulation of part sugar and lipid material and be stored in the leaf tissue cell, so that the extraction difficulty of alpine plant genomic dna strengthens.Wang Zhuowei waits and find effectively Polysaccharide removing of PVP in to mulberry leaf DNA extraction method research process.The brave grade under study for action of Luo Zhi carried out some improvement to this method: the concentration that 1. improves CTAB; 2. for containing the more plant of Polyphenols, increase in the CTAB Extraction buffer-content of mercaptoethanol, add simultaneously PVP and make it be combined the formation mixture with polyphenols; 3. increase the amount of less salt precipitation buffering liquid; 4. increase the concentration of salt in the high level salt solution.Separate from the medicinal plants such as ginseng, Radix Panacis Quinquefolii, pseudo-ginseng with Innovative method and to have obtained highly purified DNA; Porebski waits in precipitation when slightly carrying DNA, and the concentration of sodium-chlor in the Extraction buffer is increased to 2.5 molL
-1So that polysaccharide can be more effectively removed in DNA preferential precipitation under high salt buffer condition.Yu Zhixiong to Hylocereus undatus total DNA extraction method comparative studies verified that it is effective that the CTAB method is extracted the Hylocereus undatus genome, and should beautiful comparison to two kinds of different Sugarcane Genome DNA Extracting Methods, proved that simultaneously SDS and CTAB method can both extract genomic dna and two kinds of methods are improved.
The Changbai Mountain cuckoo is a kind of representative alpine plant, at height above sea level 1700 m-2500 m height above sea level bands distribution is arranged all, its physiological and biochemical index is comparatively special, the scheme of extracting about the Changbai Mountain cuckoo genome of mentioning in this research also has multiple, but different methods differs widely to the genome extraction effects of different plants.For the research of the extracting genome DNA aspect of cuckoo still seldom.Zhao Xihua waits and adopts respectively CTAB method, alkaline lysis, SDS method to extract the cuckoo genomic dna take Genus Rhododendron Subgenus Hymenanthes Rhododendron jinggangshanicum Tam leaf as material, and DNA purity and productive rate that three kinds of methods are extracted have difference, and wherein the CTAB method is better.Zhuan Defeng, Cao Hounan adopts the CTAB method (1. to get the ground fresh blade in the 0.2 g left and right sides and place 1.5 mL centrifuge tubes, add 800 ul through the CTAB of 65 ℃ of preheatings (2% CTAB; 100 mmolL
-1Tris-HCl, pH8.0; 20 mmolL
-1EDTA; 1.4 molL
-1NaC1; 2% PVP) Extraction buffer adds 2% β-mercaptoethanol before grinding; Fully behind the mixing, 65 ℃ of temperature are bathed 1h in water-bath; Deng) extract the genomic dna of several kinds Changbai Mountain cuckoo, the DNA purity of gained is high, quality is good, but Rhododendron aureum and spore leaf cuckoo genome fail to extract.This experiment with the SDS method extract Plant Genome (Wei Qun. molecular biology experiment instructs [M]. Beijing: Higher Education Publishing House, 2007.) method: " get 0.2g fresh plant blade, in liquid nitrogen, be ground into powder; Be transferred in the 2 mL centrifuge tubes, add 800 ul cell extracts (pH 8.0,100 mmolL
-1TrisHCl, 5 mmolL
-1EDTA, 500 mmolL
-1NaCl, 1.25% SDS, 100 ul beta-mercaptoethanols), abundant mixing; 65
oC water bath heat preservation 20 minutes; From water-bath, take out centrifuge tube, add 250 ul, 5 molL
-1KCl solution, mixing, ice bath 20 minutes; Deng " extract the genome of Changbai Mountain cuckoo, failing extracts its genomic dna.
Summary of the invention
For above-mentioned deficiency, the object of the invention is to overcome prior art in the cuckoo genome DNA extracting method of Changbai Mountain, the genome extraction is difficult, purity is low, the deficiency of easy degraded, a kind of method of new Changbai Mountain cuckoo extracting genome DNA is provided, thereby obtains the Changbai Mountain cuckoo genomic dna that purity is high, stability is strong.
The present invention realizes by following technical scheme: the method adopts the CTAB method to combine with the SDS method, namely at first adopts the CTAB method to extract the Rhododendron aureum genome, and then the Rhododendron aureum genomic dna that adopts SDS method purifying to extract.
The concrete steps of the method are as follows:
(1), the CTAB method is extracted the Rhododendron aureum genomic dna:
In extraction vessel, add the CTAB Extraction buffer, be preheated to 65 ℃; The fresh blade of Rhododendron aureum is added before grinding-mercaptoethanol, and add-on is the 4.3%V/V of CTAB Extraction buffer volume, joins rapidly behind liquid nitrogen grinding gelation powdery in the CTAB Extraction buffer of above-mentioned preheating, fully mixing; Bathed 4 hours in 65 ℃ of temperature; Then extract according to a conventional method genomic dna;
(2), the genomic dna that extracts in conjunction with SDS method purifying:
Add 1/10 volume (SDS volume: 2% the SDS of genomic dna liquor capacity=1:10), ice bath 10 minutes in the Rhododendron aureum genomic dna solution that extracts to above-mentioned CTAB method; Add 1/10 volume, 5 molL
-1KAc, ice bath 30 minutes; 12,000 rpm centrifugal 10 minutes under 18 ℃, get supernatant liquor; The SDS that adds 1/20 volume 2%, ice bath 10 minutes; Add 1/20 volume, 5 molL
-1KAc, ice bath 30 minutes; Centrifugal, get supernatant liquor; The extracting of phenol chloroform is once got supernatant liquor; Alcohol precipitation, washing precipitation, dissolution precipitation is the Rhododendron aureum genomic dna of purifying.
The concrete steps that described CTAB method is extracted the Rhododendron aureum genomic dna are as follows:
Get 2.0 ml centrifuge tubes, add the CTAB Extraction buffer of 700 ul, 65 ℃ of preheatings; Get tender Rhododendron aureum material 0.3 g of fresh children, add 30 ul-mercaptoethanol before grinding, grind to form the agar powder in the liquid nitrogen and rapidly this agar is changed in the above-mentioned centrifuge tube over to abundant mixing; 65 ℃ of temperature were bathed 4 hours in the water-bath; Then extract according to a conventional method Rhododendron aureum genomic dna crude product;
The described genomic concrete steps of extracting in conjunction with SDS method purifying are as follows:
The SDS that adds 1/10 volume 2% in the Rhododendron aureum genomic dna solution that extracts to above-mentioned CTAB method, ice bath 10 minutes; Add 1/10 volume, 5 molL
-1KAc, ice bath 30 minutes; 12,000 rpm centrifugal 10 minutes under 18 ℃, get supernatant liquor; The SDS that adds 1/20 volume 2%, ice bath 10 minutes; Add 1/20 volume, 5 molL
-1KAc, ice bath 30 minutes; Centrifugal, get supernatant liquor; The extracting of phenol chloroform is once got supernatant liquor; Alcohol precipitation, washing precipitation, dissolution precipitation is the Rhododendron aureum genomic dna of purifying.
In the method for above Rhododendron aureum extracting genome DNA, described CTAB Extraction buffer is by 2% CTAB, 100 mmolL
-1Tris-HCl pH 8.0,20mmolL
-1EDTA, 1.4 molL
-1NaCl, 4% PVP forms; Described phenol chloroform is that phenol and chloroform are formulated by the 1:1 volume ratio.
The present invention has following useful effect:
1, in the Rhododendron aureum Extraction Methods of Genome of the present invention, taked first the CTAB method to be combined with the SDS method, namely the CTAB method is extracted after the genome, the genome that extracts in conjunction with SDS method purifying.
2, method of the present invention has been carried out creative improvement to existing CTAB method and SDS method, has outstanding substantive distinguishing features and significant progressive, is embodied in:
Step (1) CTAB method is extracted in the Rhododendron aureum genomic dna process: conventional CTAB method ratio: the PVP consumption in the CTAB Extraction buffer has increased by 2%, namely increases to 4% by 2%; Before grinding-and the conventional CTAB method of mercaptoethanol consumption, increased about 1 times, namely increase to 4.3% by 2%; Incubative time extended to 4 hours in 1 hour by the CTAB method of routine.
In the genome process that step (2) is extracted in conjunction with SDS method purifying: the SDS dosage is respectively the SDS of 0.2%(1/10 volume 2%) with the SDS of 0.1%(1/20 volume 2%), and after extracting, genome adds separately, and in the conventional SDS method, the dosage of SDS is 1.25%, and is blended in the cell extract before genome extracts and adds; Purification step is also used respectively 0.5 molL
-1With 0.25 molL
-1KAc replaced 1.56 molL in the conventional SDS method
-1KCl.
Because the combination and improvement and usefulness of CTAB method and SDS method have overcome the difficulty that the alpine plant genome separates with secondary metabolite difficulties such as other polysaccharide, Polyphenols.Extraction has obtained being difficult for ordinary method the Rhododendron aureum genomic dna of extraction.
3, method of the present invention is compared with the method that the other plant genome extracts, after testing, the Rhododendron aureum genomic dna that the present invention obtains, not only extraction efficiency is high, and the purity of genomic dna is high, is not easy degraded, for solid foundation has been established in follow-up molecular biological research, and operation is simple and feasible, practical.
Description of drawings
Fig. 1 is the photo figure in kind of the Changbai Mountain Rhododendron aureum about height above sea level 2000 m.
Fig. 2 is polar region, Changbai Mountain cuckoo genome dna electrophoresis detected result figure, wherein 1-is the Changbai Mountain cuckoo genomic dna of test kit rapid extraction, 2-is the Changbai Mountain cuckoo genomic dna that the SDS method is extracted, and 3,4-is the Changbai Mountain Rhododendron aureum genomic dna that the CTAB-SDS method is extracted.
Embodiment
The invention will be further described below in conjunction with case study on implementation:
One, the CTAB-SDS method is extracted Changbai Mountain cuckoo genomic dna
(1), the improved CTAB method method is extracted plant genome DNA
1, get the fresh blade of Changbai Mountain Rhododendron aureum about 0.4 g, liquid nitrogen grinding is powdered, places 2 ml centrifuge tubes, adds in advance 700 ul in the pipe through 65
oThe CTAB Extraction buffer of C preheating (2% CTAB, 100 mmolL
-1Tris-HCl pH 8.0,20mmolL
-1EDTA; 1.4 molL
-1NaCl, 4% PVP), add 30 ul beta-mercaptoethanols before grinding.
2, rapidly fully behind the mixing, in water-bath 65
oThe C temperature was bathed 4 hours, shook up gently every 30 minutes.
3, the phenol that adds equal-volume (730 ul): chloroform (1:1) mixed solution, put upside down the mixing mixture, 18
o12000 rpm centrifugal 10 minutes under the C; Repeat once.
4, get supernatant liquor, add the dehydrated alcohol of 3 times of volumes, put upside down mixing ,-20
oC alcohol precipitation 1-2 hour.
5,18
o12000 rpm centrifugal 30 minutes under the C, abandon supernatant liquor.
6, add 1 ml, 70% ethanol, washing and precipitating, 18 ℃ were descended 12000 rpm centrifugal 6 minutes; Repeat 2 times.
7, abandon supernatant, air-dry precipitation.
8, add an amount of 103 ul (ddH
2O: the Pancreatic RNase premixed liquid) (100 ul:3 ul) dissolution precipitation, 37
oC water-bath 1 hour.
9, add isopyknic phenol: chloroform (1:1) mixed solution, put upside down the mixing mixture, 18
o12000 rpm centrifugal 10 minutes under the C.
10, get supernatant liquor, add 1/10 volume, 3 molL
-1NaAc (pH 5.2), the dehydrated alcohol of 3 times of volumes ,-20
oC left standstill 1-2 hour.
11,18
o12000 rpm centrifugal 30 minutes under the C, abandon supernatant liquor.
12, add 1 ml, 70% ethanol, washing and precipitating, 18 ℃ of 12000 rpm are centrifugal 6 minutes; Repeat 2 times.
13, abandon supernatant, air-dry precipitation.
14, add an amount of (30-80 ul) (ddH
2O: the Pancreatic RNase premixed liquid) dissolution precipitation.
(2), in conjunction with SDS method purifying plant genome DNA
15, carry adding 1/10 volume 2% SDS in the genomic dna solution, ice bath 10 minutes in above-mentioned institute.
16, add 1/10 volume KAc(5mol/L), ice bath 30 minutes.
17,18
o12000 rpm centrifugal 10 minutes under the C, get supernatant liquor.
18, repeat above-mentioned steps 15-18, but 1/10 changes 1/20 into.
19, add 1/10 volume, 3 molL
-1NaAc adds 3 times of volume dehydrated alcohols ,-20 again
o C alcohol precipitation 2 hours takes out when the adularescent flocks occurs.
20,18
o12000 rpm centrifugal 30 minutes under the C, abandon supernatant.
21, add 1 ml, 70% ethanol, washing and precipitating, 18 ℃ were descended 12000 rpm centrifugal 6 minutes; Repeat 2 times.
22, abandon supernatant, air-dry precipitation adds 20ul ddH
2The O dissolution precipitation.
23, short-term is with being stored in 4
oC, prolonged preservation is stored in-20
oC.
Three, agarose gel electrophoresis detects (see figure 2)
Sepharose with 1.2% carries out electrophoresis detection, and electrophoretic buffer is 1 * TAE, and sample-loading buffer is 10 * LoadingBuffer, and molecular weight standard Marker is DL2, and 000, record point sample order and point sample amount (sample: 5ul, Marker:DL2,000).Under ultraviolet lamp 312nm, observe electrophoresis result with ultraviolet gel imaging instrument, take a picture, preserve.
Claims (4)
1. the method for a Changbai Mountain Rhododendron aureum extracting genome DNA is characterized in that: the method adopts the CTAB method to combine with the SDS method, namely at first adopts the CTAB method to extract the Rhododendron aureum genomic dna, and then the genomic dna that extracts of employing SDS method purifying.
2. the method for a Changbai Mountain Rhododendron aureum extracting genome DNA, it is characterized in that: the concrete steps of the method are as follows:
(1), the CTAB method is extracted the Rhododendron aureum genomic dna:
Add the CTAB Extraction buffer in the extraction vessel and be preheated to 65 ℃; The fresh blade of Rhododendron aureum is added before grinding-mercaptoethanol, and add-on is the 4.3%V/V of CTAB Extraction buffer volume, joins rapidly behind liquid nitrogen grinding gelation powdery in the CTAB Extraction buffer of above-mentioned preheating, fully mixing; Bathed 4 hours in 65 ℃ of temperature; Then extract according to a conventional method the Rhododendron aureum genomic dna;
(2), the genomic dna that extracts in conjunction with SDS method purifying:
The SDS that adds 1/10 volume 2% in the above-mentioned Rhododendron aureum genomic dna solution that extracts, ice bath 10 minutes; Add 1/10 volume, 5 molL
-1KAc, ice bath 30 minutes; 12,000 rpm centrifugal 10 minutes under 18 ℃, get supernatant liquor; The SDS that adds 1/20 volume 2%, ice bath 10 minutes; Add 1/20 volume, 5 molL
-1KAc, ice bath 30 minutes; Centrifugal, get supernatant liquor; The extracting of phenol chloroform is once got supernatant liquor; Alcohol precipitation, washing precipitation, dissolution precipitation is the Rhododendron aureum genomic dna of purifying.
3. the method for a kind of Changbai Mountain according to claim 2 Rhododendron aureum extracting genome DNA, it is characterized in that: the concrete steps of the method are as follows:
(1), the CTAB method is extracted the Rhododendron aureum genomic dna:
Get 2.0 ml centrifuge tubes, add the CTAB Extraction buffer of 700 ul, 65 ℃ of preheatings; Get tender Rhododendron aureum material 0.3 g of fresh children, add 30 ul-mercaptoethanol before grinding, grind to form the agar powder in the liquid nitrogen and rapidly this agar is changed in the above-mentioned centrifuge tube over to abundant mixing; 65 ℃ of temperature were bathed 4 hours in the water-bath; Then extract according to a conventional method the Rhododendron aureum genomic dna;
(2), the genome that extracts in conjunction with SDS method purifying:
The SDS that adds 1/10 volume 2% in the above-mentioned Rhododendron aureum genomic dna solution that extracts, ice bath 10 minutes; Add 1/10 volume, 5 molL
-1KAc, ice bath 30 minutes; 12,000 rpm centrifugal 10 minutes under 18 ℃, get supernatant liquor; The SDS that adds 1/20 volume 2%, ice bath 10 minutes; Add 1/20 volume, 5 molL
-1KAc, ice bath 30 minutes; Centrifugal, get supernatant liquor; The extracting of phenol chloroform is once got supernatant liquor; Alcohol precipitation, washing precipitation, dissolution precipitation is the Rhododendron aureum genomic dna of purifying.
4. according to claim 2 or the method for 3 described Rhododendron aureum extracting genome DNA, it is characterized in that: described CTAB Extraction buffer is by 2% CTAB, 100 mmolL
-1Tris-HCl pH 8.0,20mmolL
-1EDTA, 1.4 molL
-1NaCl, 4% PVP forms; Described phenol chloroform is that phenol and chloroform are formulated by the 1:1 volume ratio.
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Cited By (1)
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| CN104342433A (en) * | 2014-09-19 | 2015-02-11 | 中国农业科学院烟草研究所 | Extracting solution for extracting genome DNA (Deoxyribose Nucleic Acid), application of extracting solution and method for rapidly and efficiently extracting genome DNA of tobaccos by utilizing extracting solution |
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| CN102154308A (en) * | 2010-12-03 | 2011-08-17 | 中国海洋大学 | Preparation method of sea tangle sporophyte DNA |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102154308A (en) * | 2010-12-03 | 2011-08-17 | 中国海洋大学 | Preparation method of sea tangle sporophyte DNA |
Non-Patent Citations (2)
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| CHEN NIU 等: "A safe inexpensive method to isolate high quality plant and fungal DNA in an open laboratory environment", 《AFRICAN JOURNAL OF BIOTECHNOLOGY》, vol. 7, no. 16, 18 August 2008 (2008-08-18), pages 2818 - 2822 * |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104342433A (en) * | 2014-09-19 | 2015-02-11 | 中国农业科学院烟草研究所 | Extracting solution for extracting genome DNA (Deoxyribose Nucleic Acid), application of extracting solution and method for rapidly and efficiently extracting genome DNA of tobaccos by utilizing extracting solution |
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