CN105586398A - Method for identifying aloe vera species with ITS sequence - Google Patents
Method for identifying aloe vera species with ITS sequence Download PDFInfo
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- CN105586398A CN105586398A CN201510498425.4A CN201510498425A CN105586398A CN 105586398 A CN105586398 A CN 105586398A CN 201510498425 A CN201510498425 A CN 201510498425A CN 105586398 A CN105586398 A CN 105586398A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
Abstract
The invention discloses a method for identifying aloe vera species with an ITS sequence. The method includes the following steps that firstly, mature leaves of healthy and fresh aloe samples are collected, and total DNA is extracted; or aloe gel total DNA is extracted from aloe food; secondly, the DNA extracted in the first step serves as a template, primers shown by SEQ ID NO:2 and 3 are used for performing a PCR amplification reaction, and the ITS sequence is amplified; thirdly, PCR products are subjected to direct sequencing; fourthly, a sequencing result is subjected to sequence alignment and clustering analysis through MEGA4.1 software, a system development tree is drawn, and the aloe vera species are identified through a sequence alignment and clustering analysis result. The ITS sequence comes from the rDNA ITS sequence of aloe vera and is a DNA sequence located between 18SrRNA and 26SrRNA, and the nucleotide sequence of the ITS sequence is as shown in SEQ ID NO:1. The method is easy to operate, and the purpose of conveniently, fast, objectively and accurately distinguishing aloe vera from other aloe species can be achieved.
Description
Technical field
The present invention relates to a kind of method of identifying aloe kind, particularly one is utilized ITS Sequence Identification CuracaoThe method of aloe kind, belongs to the authenticate technology field of plant germplasm resource.
Background technology
Aloe (Aloe) is a kind of long green fleshiness herbaceous plant, is under the jurisdiction of perennial Monocotyledonae(Monoctyledoneae) Asparagales (Asparagales) Du Weicao section (Asphodelaceae). This section withIn front classification, divide most of genus in Liliaceae (Liliaceae), is listed in subfamily. Aloe (AloeL.)Be the foundation of Sweden taxology great master Lin Nai in 1754, be published in his classical works " plant belongs to will " the 5thVersion. " Chinese Plants will " 14 volumes (Liliaceae) of publishing for 1980, are under the jurisdiction of Aloe in the Liliaceae of broad sense.Liliaceae has 230 genus, and 3500 kinds have more than 300 to plant in this genus.
Having many uses of aloe, its active ingredient has special health care and antineoplastic action to human body, at presentBe widely used in health products, cosmetics, food, view and admire with pharmaceutical industries on. Aloe (AloeL.) plant speciesClass is various, makes a variation various, and germ plasm resource is extremely abundant, and traditional aloe kind classification Main Basis is morphology, thinBorn of the same parents learn. Classify according to morphology more directly perceived. But because aloe and relative genus intermolecular hybrid can be educated, constantly occurThe generic cross kind that some are new; And in Aloe, xenogenesis of the same name and different name phenomenon of the same race have generation more, cause not of the same raceOr the affiliation of subspecies is more chaotic, morphology is difficult to reflect hereditary difference and the affiliation between planting sometimes.And about the research data of affiliation between its kind very few. Confirm to be used at present the Curacao that only has of food in ChinaThis kind of aloe, therefore has ten from molecular level to the research of the classification of aloe kind, qualification and definite affiliationDivide important meaning.
DNA bar code is to utilize the genetic fragment of standard species to be carried out to the technology of Rapid identification, successfully usesIn research fields such as living species classification and qualification, Ecological Investigation and biodiversity assessments, for plant genetic manySample detects and provides more directly, more accurate method, the i.e. sequence variation of direct-detection DNA itself. Due toIssuable variety of issue while having avoided inferring genotype according to phenotype, DNA analysis method becomes at presentTill the most effective genetic analysis method, ITS sequence is moderately repetitive sequence, be distributed widely in genome andBe synchronous evolution, the degree of its base sequence homology determines the affiliation distance between biology. It is deposited simultaneouslyBe in nucleus, avoided other DNA bar code genes to be present in chloroplaset, and contained in aloe gelMeasure extremely low problem. Therefore patent of the present invention is applied to DNA bar codes technique to differentiate aloe kind, by expandingIncrease ITS sequence and it is analyzed, inquiring into the affiliation between them from molecular level, having set up at moleculeThe aloe true and false and whether be the method for aloe barbadensis Miller in horizontal detection food.
Summary of the invention
Main purpose of the present invention is the aloe raw material true and false and the variety problem for current existence, provides a kind of and differentiatesThe aloe material true and false and whether be nucleotide sequence and the method for aloe barbadensis Miller, analyzes reed from DNA levelThe hereditary feature of luxuriant growth germplasm resource, for qualification aloe barbadensis Miller provides effective molecular labeling.
The technical solution adopted for the present invention to solve the technical problems is:
A method of utilizing ITS Sequence Identification aloe barbadensis Miller kind, is characterized in that, comprises the following steps:
(1) collect the mature leaf of healthy fresh aloe sample, extract total DNA; Or carry from aloe foodGet the total DNA of aloe gel;
(2), taking the middle DNA extracting of step (1) as template, use primer shown in SEQIDNO:2 and 3 to enterPerforming PCR amplified reaction, amplification ITS sequence;
(3) PCR product carries out direct Sequencing;
(4) utilize MEGA4.1 software to carry out sequence alignment and cluster analysis to sequencing result, drawing system is grownTree, according to sequence alignment and cluster analysis result qualification aloe barbadensis Miller kind.
Wherein, described ITS sequence derives from the rDNAITS sequence of aloe barbadensis Miller, be positioned at 18S andDNA sequence dna between 26SrRNA, its nucleotide sequence is as shown in SEQIDNO:1.
In the present invention, preferably, collect the mature leaf of healthy fresh aloe sample, carry out cutting process postpositionIn softness and the good paper bag of gas permeability, and be placed in the drying box that silica gel is housed and be dried; Or collect healthyThe mature leaf of fresh aloe sample, carries out putting into batch cultur ware after cutting process, seals with preservative film,-20 DEG C to be refrigerated to aloe leaf freezing completely, puts into subsequently freeze dryer freeze drying 24h, after be placed on silica gel be housedDrying box in be dried.
In the present invention, preferred, described silica gel is that pulverous diameter is the white silica gel and indigo plant of 20～30AThe mixture of look discolour silica gel.
In the present invention, preferred, the reaction body of described pcr amplification reaction is counted with 25 μ L:
In the present invention, preferred, it is characterized in that, the reaction condition of described pcr amplification reaction is: 94 DEG CDenaturation 4min, 94 DEG C of sex change 40s, 52 DEG C of renaturation 40s, 72 DEG C are extended 2min, 30 circulations, last 72 DEG CExtend 10min, 4 DEG C of preservations.
The present invention also provides the application of a kind of ITS sequence in qualification aloe barbadensis Miller kind, described ITS orderRow derive from the rDNAITS sequence of aloe barbadensis Miller, are the DNA sequence dnas between 18S and 26SrRNA,Its nucleotide sequence is as shown in SEQIDNO:1.
Compared with prior art, beneficial effect of the present invention is embodied in:
The rDNAITS sequence that derives from aloe barbadensis Miller provided by the invention, can be used as contrast and differentiates Curacao reedLuxuriant growth raw material; By analyze the hereditary feature of aloe barbadensis Miller on DNA level, for distinguishing aloe barbadensis Miller, woodVertical aloe, opening general aloe etc. effective molecular labeling is provided, is kind authenticity and the sieve of aloe barbadensis MillerSelect improved seeds etc. to lay a good foundation.
Meanwhile, the method for utilizing above-mentioned ITS sequence to differentiate aloe barbadensis Miller kind provided by the invention, simple to operateYi Hang, can reach easily and fast, objective, the object of accurately distinguishing aloe barbadensis Miller and other aloe kinds.
Brief description of the drawings
Fig. 1 is 4 kind aloe genome dna electrophoresis collection of illustrative plates; Wherein, M is DNAMarker, and 1 is storehouseDrag-line aloe samples, 2 for opening general aloe samples, and 3 is aloe sample, and 4 is aloe paste sample;
Fig. 2 is the pcr amplification product electrophoresis pattern of 4 aloe samples material DNA; Wherein, M is DNAMarker, 1 is aloe barbadensis Miller sample, and 2 for opening general aloe samples, and 3 is aloe sample, and 4 is aloe pasteSample;
Fig. 3 is different cultivars aloe material ITS Sequence Cluster Analysis result figure;
Fig. 4 is different place of production aloe barbadensis Miller material ITS Sequence Cluster Analysis result figure.
Detailed description of the invention
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be along with descriptionAnd it is more clear. But these embodiment are only exemplary, scope of the present invention are not formed to any restriction. ThisThose skilled in the art it should be understood that can be to the technology of the present invention side under without departing from the spirit and scope of the present inventionThe details of case and form are modified or are replaced, but these amendments and replacement all fall within the scope of protection of the present invention.
Embodiment 1
The present embodiment has been measured the rDNA ITS sequence from the Aloe resources of 3 different cultivars, aloe productKind comprises aloe barbadensis Miller, opens general aloe and aloe, and the method for employing comprises the steps:
(1) sample collection and processing
Adopt following two kinds of methods to process sample:
1. gather then raw ripe, healthy fresh aloe blade as the material that extracts genomic DNA,Carry out cutting process and be placed on softness and the good paper bag of gas permeability, and be placed in the drying box that silica gel is housed and doDry. Ventilative paper bag can separate DNA of plants material when dry with silica gel, even if like this plant leaf blade because ofAfter dry becoming fragile, also can not mix with silica gel, and cause the cross pollution of sample room. Desiccant silica gel is powderThe white silica gel (diameter is 20～30A) of shape and the mixture of discolour silica gel (blueness). Or
2. choose healthy fresh plant DNA material, cutting process becomes small pieces, puts into batch cultur ware,Seal with preservative film ,-20 DEG C to be refrigerated to aloe leaf freezing completely. Put into subsequently freeze dryer freeze drying 24h.To be placed in equally afterwards drying box and carry out kept dry. This kind of method first carried out freeze drying by aloe gel, then entersRow silica dehydrator.
(2) genome DNA is extracted
Adopt CTAB method to carry out the extraction of aloe genomic DNA, extracting method is as follows: get by above-mentioned stepsThe aloe leaf or the gel drying sample that obtain, each material is got 3 strains, carries out extracting genome DNA after mixing.
1. by 2 × CTAB extract at least 20min of preheating in 70 DEG C of water-baths, take 0.2g aloe leaf, putEnter in sterilized mortar, the fast liquid nitrogen of pouring into, is not evaporated completely full-time being clayed into power at it, and proceed to 2mL fromIn core barrel.
2. add rapidly 2 × CTAB extract, the 800 μ L after preheating, put upside down and mix, 70 DEG C of water-baths60-90min, shakes centrifuge tube therebetween frequently.
3. take out centrifuge tube, after it is cooled to room temperature, add isopyknic chloroform/isoamyl alcohol (24:1), carefully topThe about 10min of heterogeneous example showing an absence of inverse disconnection between the middle term and the major term core barrel, oscillator concussion 2min, leaves standstill 5min on ice and makes solution layering (be divided into four from top to bottomLayer: green chloroform uvea, green cell debris layer, white polysaccharide layer, colourless DNA water), 12000rpmCentrifugal 5min.
4. get supernatant, add isopyknic phenol/chloroform/isoamyl alcohol (25:24:1), carefully put upside down centrifuge tube and make solutionLayering, the centrifugal 10min of 12000rpm.
5. get supernatant, add isopyknic chloroform/isoamyl alcohol (24:1), carefully put upside down centrifuge tube and make solution layering,The centrifugal 10min of 12000rpm.
6. get supernatant, add 3 μ LRNaseA, 30-60min is placed in 37 DEG C of water-baths.
7. take out centrifuge tube, add 1/10 volume sodium acetate, isopropyl alcohol (or 2 volume-20 DEG C of 2/3 volume-20 DEG C precoolingThe ethanol of precooling), mix 4 DEG C of precipitation 12h.
8. the centrifugal 10min of 10000rpm, abandons supernatant. With 70% alcohol washing precipitation 2 times, when pressure-vaccum solution, moveLight and slow, remove alcohol, back-off 5min on aseptic filter paper, then this DNA is dried.
9. add the aseptic ultra-pure water of 80 μ L to dissolve above-mentioned DNA precipitation, play gently centrifuge tube bottom, mix, putPut-20 DEG C of Refrigerator stores.
(3) detection of aloe genomic DNA quality
The quality that detects the aloe genomic DNA of carrying with 0.8% agarose gel electrophoresis, step is as follows:
1. the preparation of Ago-Gel
Take 0.48g agarose and be placed in conical flask, add 40mL1 × TAE, boil with microwave-oven-heating to secondaryRise, shake up gently (avoiding producing bubble). Treat that room temperature is cooled to 80 DEG C of left and right, select suitable gel slab, first inhaleTake a morsel glue by gel slab edge edge sealing, in order to avoid leak glue. Treat that agarose room temperature is cooled to 60 DEG C of left and right, by glueSlowly pour into, then insert comb, thick about 5mm, room temperature is placed 30-40min, after glue solidifies completely, lightGently extract comb and whether sample for reference hole is complete. Then gel piece is put into Horizontal electrophoresis tank, add electrophoresis slowRush liquid 1 × TAE, liquid level exceeds gel face 1-2mm.
2. point sample
Each DNA sample is drawn 8 μ L, then adds 2 μ L6 × LoadingBuffer (or bromjophenol blue), fullyMix rear point sample. Select 200bpDNAMarker, draw 5 μ L point samples. Attention: the point that does not use edgeSample hole, because the electric field at edge is unstable, can affect electrophoretic effects.
3. electrophoresis
Adopt voltage stabilizing electrophoresis, voltage 110V, electrophoresis 70min left and right.
4. dyeing
After electrophoresis finishes, gel piece is put into EB (ethidium bromide) the solution 10min that dyes.
5. rinsing
Rinsing 5min.
6. observe
Alpha gel imaging system is taken pictures, and result as shown in Figure 1. The genomic DNA extracting is one and becomes clearBand, its molecular weight is more than 100kb, without degraded, and concentration reaches subsequent experimental requirement.
(4) pcr amplification
Analyze and experimental study by lot of documents, choose karyogene ITS, be positioned between 18S and 26SrRNADNA sequencing fragment, as testing goal sequence, is designed the pcr amplification primer of aloe ITS in the present invention, justDivide to primer primerF (shown in SEQIDNO:2) and reverse primer primerR (shown in SEQIDNO:3)Not as follows:
PrimerF：5’-AGAAGTCGTAACAAGGTTTCCGTAGG-3’
PrimerR：5’-TCCTCCGCTTATTGATATGCTTAA-3’
The total DNA extracting taking above-mentioned steps (2) and (3) respectively, as template, carries out pcr amplification reaction:
The PCR reaction system of ITS is to contain in every 25 μ L:
Pcr amplification reaction program is: 94 DEG C of denaturation 4min, 94 DEG C of sex change 40s, 52 DEG C of renaturation 40s, 72 DEG CExtend 2min, 30 circulations, last 72 DEG C are extended 10min, 4 DEG C of preservations.
(5) PCR product detects and purifying
The detection of PCR product:
PCR product detects by 1.2% agarose gel electrophoresis the candidate sequence that whether successfully increased, and getsThe product of 8 μ L, and 6 × LoadingBuffer of 2 μ L mix, using the DNAMarker of 5 μ L asMolecular labeling, sample enters before glue, and Current Control is at 50mA, and sample enters after glue, and Current Control is at 100mA, electricityPressure-controlled, at 120V, in the time that sample runs to full glue length 2/3, stops electrophoresis, and the time is approximately 60min, at purpleUnder outer gel imaging analysis system, detect, by AlphalmagerHp software editing, and take pictures, by withMarker compares, and obtain a band clearly, and fragment length size is also correct, and result is as Fig. 2 instituteShow
Adopt the plain agar sugar gel DNA of Tian Gen biochemical technology Co., Ltd to reclaim kit. Concrete steps are as follows:
Before using, first in rinsing liquid PW, add absolute ethyl alcohol.
1. column equilibration step: (adsorption column is put into collecting pipe) adds 500 μ L equilibrium liquids in adsorption column CA2BL, (～13,400 × g) centrifugal 1min, outwells the waste liquid in collecting pipe to 12,000rpm, and adsorption column is put back to againIn collecting pipe.
2. single target DNA band being cut to (excising redundance) from Ago-Gel as far as possible puts into dryIn clean centrifuge tube, take weight.
3. to add in blob of viscose isoploid amass solution PN (if gel is heavily 0.1g, its volume can be considered 100 μ L,Add 100 μ LPN solution), 50 DEG C of water-baths are placed, and constantly leniently spin upside down centrifuge tube therebetween, with reallyProtecting blob of viscose fully dissolves. Continue place a few minutes or add again some sol solutionses, until blob of viscose dissolves completely.
4. previous step gained solution is added in an adsorption column CA2 (adsorption column is put into collecting pipe), room temperature is putPut 2min, (～13,400 × g) centrifugal 30-60sec, outwells the waste liquid in collecting pipe, by adsorption column to 12,000rpmCA2 puts into collecting pipe.
5. to add in adsorption column CA2 600 μ L rinsing liquid PW (before use, please first check whether added anhydrousEthanol), (～13,400 × g) centrifugal 30-60sec, outwells the waste liquid in collecting pipe, by adsorption column CA2 to 12,000rpmPut into collecting pipe. Add rear standing 2-5min centrifugal again.
6. repeat step 5.
7. adsorption column CA2 is put back in collecting pipe, (～13,400 × g) centrifugal 2min, eliminates 12,000rpm as far as possibleRinsing liquid. Adsorption column CA2 is placed in to room temperature and places several minutes, dry up hill and dale, to prevent residual rinsing liquid shadowRing next step experiment.
8. adsorption column CA2 is put in a clean centrifuge tube, to the appropriate wash-out of the unsettled dropping in adsorbed film centre positionBuffer solution EB,
Room temperature is placed 2min. 12,000rpm (～13,400 × g) centrifugal 2min collects DNA solution.
(6) PCR product sequencing
Amplified production is by two-way order-checking to guarantee the accuracy of data, and purifying and order-checking are all in the biological skill of Shanghai English fine horseArt Co., Ltd (InvitrogenCo., Ltd.) completes.
(7) DNA sequence data analysis
According to rDNAITS sequencing result, utilize MEGA4.1 software, drawing system is grown tree, cluster resultAs shown in Figure 3, can obviously find out aloe barbadensis Miller and other aloe kind (opening general aloe and aloe) by Fig. 3Distinguish in different branches, utilize ITS sequence well aloe barbadensis Miller and other aloe kind (to be openedGeneral aloe and aloe district) make a distinction.
The rDNAITS sequence (shown in SEQIDNO:1) of gained aloe barbadensis Miller with open general aloe and the reed of standing motionlessThe rDNAITS sequence of luxuriant growth contrasts and cluster analysis, distinguishes different kinds according to sequence alignment and cluster resultMatter resource, reaches the object of differentiating the aloe barbadensis Miller kind true and false.
Embodiment 2
According to method described in embodiment 1, respectively to 9 places of production (Huizhou, Zhanjiang, Xi'an, Chaoyang, Wanning,Yuanjiang, Xiong County, Weihai and Dalian) the rDNAITS sequence of aloe barbadensis Miller kind increases, sequencing,Sequence alignment and cluster analysis, result as shown in Figure 4. The aloe barbadensis Miller ITS sequence in the different places of production does not almost haveDifference.
Embodiment 3
Buy from the market aloe paste product, the total DNA of aloe gel is wherein extracted, adopt day root biochemistryThe deep-processed food DNA of Technology Co., Ltd. extracts kit, and concrete extracting method is as follows:
(1) take aloe gel 10:1 0mg in the food of grinding, add 500 μ L buffer solution GMO1 and 20 μ LProteinaseK (20mg/mL), vortex vibration 1min.
Hatch 1h for (2) 56 DEG C. Hatching every 15min in process vibrates once.
(3) add 200 μ L buffer solution GMO2, fully mix, vortex vibration 1min. Room temperature leaves standstill 10min.
(～13,400 × g) centrifugal 5min, is transferred to supernatant in new centrifuge tube (4) 12,000rpm.
(5) to the isopropyl alcohol that adds 0.7 times of volume in supernatant, fully mix. 12,000rpm (～13,400 × g)Centrifugal 3min, abandons supernatant, retains precipitation.
(6) add 700 μ L70% ethanol, vortex vibration 5sec, 12,000rpm (～13,400 × g) centrifugal 2min,Abandon supernatant.
(7) inversion of uncapping, room temperature 5-10min, thoroughly dries remaining ethanol.
(8) add 20-50 μ L elution buffer TE, vortex vibration 1min, finally obtains DNA solution.
According to method described in embodiment 1, to the total DNAITS sequence of extracted aloe paste increase, orderRow are measured, sequence alignment, and result shows that it is in full accord with the ITS sequence that comes from Curacao blade. VisibleThe specific fragment that invention is developed is suitable as the bar code sequence of differentiating aloe kind and aloe product.
Claims (6)
1. a method of utilizing ITS Sequence Identification aloe barbadensis Miller kind, is characterized in that, comprises following stepRapid:
(1) collect the mature leaf of healthy fresh aloe sample, extract total DNA; Or carry from aloe foodGet the total DNA of aloe gel;
(2), taking the middle DNA extracting of step (1) as template, use primer shown in SEQIDNO:2 and 3 to enterPerforming PCR amplified reaction, amplification ITS sequence;
(3) PCR product carries out direct Sequencing;
(4) utilize MEGA4.1 software to carry out sequence alignment and cluster analysis to sequencing result, drawing system is grownTree, according to sequence alignment and cluster analysis result qualification aloe barbadensis Miller kind.
Wherein, described ITS sequence derives from the rDNAITS sequence of aloe barbadensis Miller, be positioned at 18S andDNA sequence dna between 26SrRNA, its nucleotide sequence is as shown in SEQIDNO:1.
2. method according to claim 1, is characterized in that, collects the maturation of healthy fresh aloe sampleBlade, carries out cutting process and is placed on softness and the good paper bag of gas permeability, and be placed in the drying box that silica gel is housedBe dried; Or collect the mature leaf of healthy fresh aloe sample, carry out putting into disposable training after cutting processSupport in ware, seal with preservative film ,-20 DEG C to be refrigerated to aloe leaf freezing completely, puts into subsequently freeze dryer freezing dryDry 24h, after be placed in the drying box that silica gel is housed and be dried.
3. method according to claim 2, is characterized in that, described silica gel is that pulverous diameter isThe white silica gel of 20～30A and the mixture of blue variable colour silica gel.
4. method according to claim 1, is characterized in that, the reaction body of described pcr amplification reactionCount with 25 μ L:
5. method according to claim 1, is characterized in that, the reaction bar of described pcr amplification reactionPart is: 94 DEG C of denaturation 4min, and 94 DEG C of sex change 40s, 52 DEG C of renaturation 40s, 72 DEG C are extended 2min, 30 circulations,Last 72 DEG C are extended 10min, 4 DEG C of preservations.
6. the application of ITS sequence in qualification aloe barbadensis Miller kind, described ITS sequence derives from storehouseThe rDNAITS sequence of drag-line aloe, is the DNA sequence dna between 18S and 26SrRNA, its nucleotidesSequence is as shown in SEQIDNO:1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106755431A (en) * | 2016-12-27 | 2017-05-31 | 中国环境科学研究院 | A kind of method for identifying cynomorium songaricum |
| WO2018101781A1 (en) * | 2016-12-02 | 2018-06-07 | 김권희 | Species identifying marker and primer set based on complete sequencing of chloroplast genome and nuclear ribosomal dna sequence of aloe vera, aloe saponaria, and aloe saengjang, and use thereof |
| CN114134249A (en) * | 2021-09-29 | 2022-03-04 | 丽水市中医院 | Multiplex PCR primer combination and multiplex PCR kit for rapidly detecting aflatoxin toxigenic bacteria and detection method |
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| CN102191318A (en) * | 2011-03-04 | 2011-09-21 | 广州中医药大学 | Application of nucleotide sequence of rDNA (recombinant deoxyribonucleic acid) ITS (internal transcribed spacer)-D3 region in establishment of DNA (deoxyribonucleic acid) bar code identification system for medicinal plants |
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| Title |
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| STUART P. ADAMS等: "RIBOSOMAL DNA EVOLUTION AND PHYLOGENY IN ALOE (ASPHODELACEAE)", 《AMERICAN JOURNAL OF BOTANY》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018101781A1 (en) * | 2016-12-02 | 2018-06-07 | 김권희 | Species identifying marker and primer set based on complete sequencing of chloroplast genome and nuclear ribosomal dna sequence of aloe vera, aloe saponaria, and aloe saengjang, and use thereof |
| CN106755431A (en) * | 2016-12-27 | 2017-05-31 | 中国环境科学研究院 | A kind of method for identifying cynomorium songaricum |
| CN114134249A (en) * | 2021-09-29 | 2022-03-04 | 丽水市中医院 | Multiplex PCR primer combination and multiplex PCR kit for rapidly detecting aflatoxin toxigenic bacteria and detection method |
| CN114134249B (en) * | 2021-09-29 | 2024-02-23 | 丽水市中医院 | Multiplex PCR primer combination and multiplex PCR kit for rapidly detecting aflatoxin-producing bacteria and detection method |
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Application publication date: 20160518 |