CN103849687A - Molecular biological identification method of fritillaria taipaiensis - Google Patents
Molecular biological identification method of fritillaria taipaiensis Download PDFInfo
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Abstract
The invention discloses a molecular biological identification method of fritillaria taipaiensis. The method comprises the following steps: extracting chloroplast genome DNA (Desoxvribose Nucleic Acid) of a medicinal material to be identified; performing sequencing and sequence splicing to obtain a complete chloroplast genome sequence; comparing the sequence with the chloroplast genome sequence of fritillaria taipaiensis to realize rapid and accurate identification of fritillaria taipaiensis.
Description
Technical field
The invention belongs to identification and assessment of Chinese medicines technical field, specifically disclose a kind of molecular biological variety identification method of Bulbus Fritillariae taipaiensis.
Background technology
Bulbus Fritillariae taipaiensis, claim again too shellfish, sharp shellfish, Qin shellfish, for the dry bulb of Liliaceae Liliaceae Fritillaria Fritillaria L. plant Bulbus Fritillariae taipaiensis F.taipaiensis P.Y.Li, cure mainly the few phlegm of lung-heat type cough, dry cough, sputum streaked with blood is coughed, coughed up to deficiency of Yin labor, is that 2010 editions Pharmacopoeias of the People's Republic of China are included kind.Bulbus Fritillariae taipaiensis and the Wa Bu bulb of fritillary are all used as medicine as Unibract Fritillary Bulb in pharmacopeia, in addition Unibract Fritillary Bulb, Bulbus Fritillariae cirrhosae, the Gansu bulb of fritillary and the dark violet bulb of fritillary, Unibract Fritillary Bulb Original plant reaches 6, but its profile is very similar, only there is small variation, traditional classical taxonomy Main Basis flower feature is identified, but because floral organ characteristic difference is little and transitional type is more, the method for qualitative non-quantitation is difficult to objectively distinguish.Other common pharmacopeia of the source plant and Fritillaria of being used as medicine with Unibract Fritillary Bulb carry kind same be difficult to distinguish, as Ussuriensis Fritillary Bulb, Thunberg Fritillary Bulb and Fritillaria pallidiflora Schrenk etc.The evaluation science of bio-barcode to species, fast, effectively, is easy to formation standard, significant to the discriminating of Fritillaria medicinal plant.
Chloroplast gene gains public acceptance as the status of DNA Barcoding standard sequence, but find in the work of DNA barcode identification research, that although the method has is simple to operate, repeatable strong, be applicable to the plurality of advantages such as multiple taxonomic categories, but DNA Barcode Length is many between 300-700bp, sometimes the variation information that comprises sequence is inadequate, distinguish and identify Part Medicinal Plant (as Fritillaria) plant in and between kind, have difficulties when sample; Simultaneously the high variability of Plant Genome causes also not finding so far generally acknowledged general DNA bar code sequence in plant, proves that theoretically some conventional DNA barcodes exist limitation in versatility above.Chloroplast gene group extensively exists in plant, and length reaches 120-160kb, compared with DNA barcode, has better versatility and stronger resolving power.But due to sequencing template extract also there is certain difficulty, in the time there is no the sequence of nearly edge species to sequencing data splice assembling remain complexity.Therefore, in prior art, think that the evaluation that adopts chloroplast gene group to carry out species is not also a good selection at present.The present invention is by the acquisition to Bulbus Fritillariae taipaiensis chloroplast gene group and belong to the analysis of nearly edge species together, proposes first to utilize the full genome of chloroplast(id) to identify fast and accurately Bulbus Fritillariae taipaiensis.
Summary of the invention
The technical problem to be solved in the present invention is the Molecular Identification mark using chloroplast gene group complete sequence as Bulbus Fritillariae taipaiensis, and Bulbus Fritillariae taipaiensis is identified fast and accurately.
For solving the problems of the technologies described above, the present invention adopts following technical proposals:
A kind of molecular biological variety identification method of Bulbus Fritillariae taipaiensis, the method comprises the chloroplast genomic dna the purifying that extract medicinal material to be identified, check order and sequence assembly, obtain complete chloroplast gene group sequence, the chloroplast gene group sequence of this sequence and Bulbus Fritillariae taipaiensis is compared, obtain Analysis and Identification result, wherein, the NCBI sequence number of Bulbus Fritillariae taipaiensis chloroplast gene group sequence is KC543997.
In aforesaid method, the chloroplast genomic dna of described extraction medicinal material to be identified the concrete grammar of purifying are:
(1) get fresh young leaflet tablet, 4 ℃ of dark place reason 24h, clean, and defoliation arteries and veins, tears to bits, and the buffer A that adds 4 ℃ of precoolings is placed in homogenizer, and then fully homogenate filter, and collects filtrate, waste;
(2) by filtrate, in 4 ℃, 1500g, centrifugal 15min, abandons supernatant;
(3) by damping fluid 1 washing and precipitating;
(4) by the lavation buffer solution precipitation that suspends, in a new centrifuge tube, add 30% sucrose solution, precipitation suspension is covered to the surface of 30% sucrose solution, in 4 ℃, 2500g, centrifugal 15min, precipitation chloroplast(id);
(5) precipitation is resuspended in lavation buffer solution, in 4 ℃, and the centrifugal 5min of 100g, supernatant is transferred to new pipe and at the centrifugal 10min of 380g;
(6) precipitation is resuspended in lavation buffer solution, this solution is covered in to the top of saccharose gradient, in 4 ℃, and the centrifugal 70min of 20000g; Wherein, saccharose gradient is made up of the sucrose solution of 7mL30% and 16.5～18mL52%;
(7) remove upper strata, the middle phase of the sucrose solution from 52% and 30%, collects chloroplast DNA in centrifuge tube, then adds lavation buffer solution, in 4 ℃, and the centrifugal 15min precipitation of 1500g chloroplast DNA;
(8) by damping fluid 2 washing and precipitating, in 4 ℃, 1500g, centrifugal 15min, get precipitation, adding damping fluid 2 and concentration is the Proteinase K of 10mg/mL, and room temperature leaves standstill 2min, adds the lysate of 1/5 volume, put upside down and mix, be then incubated at least 1h, cooled on ice at 50 ℃;
(9) transfer in sterilizing centrifuge tube, add isopyknic phenol: chloroform: primary isoamyl alcohol, put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature; Wherein, phenol: chloroform: the volume ratio of primary isoamyl alcohol is 25:24:1;
(10) supernatant is transferred in new centrifuge tube, adds the 5molL of 2/5 volume
-1naCl, the CTAB/NaCl that is preheated to 65 ℃ of 2/5 volume, and 68 ℃ heating 15 minutes, add isopyknic chloroform: primary isoamyl alcohol, put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature, gets supernatant; Wherein, chloroform: the volume ratio of primary isoamyl alcohol is 24:1;
(11) repeat (10)
(12) add isopyknic chloroform: primary isoamyl alcohol, put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature; Wherein, chloroform: the volume ratio of primary isoamyl alcohol is 24:1;
(13) get supernatant, add the 3molL of 0.1 times of volume
-1the pre-cold isopropanol of NaAc and 0.8 times of volume or add the 3molL of 0.1 times of volume
-1the dehydrated alcohol of NaAc and 2 times of volumes, places at least 20min precipitation chloroplast DNA for-20 ℃;
(14) 4 ℃, the centrifugal 20min of 13500g, cleans the centrifugal 10min of twice, 13500g by precipitation by 70% ethanol room temperature, air-dry, resuspended with the TE that contains RNA enzyme, and 37 ℃ of insulation 1h, obtain chloroplast DNA;
Wherein, described buffer A contains 50mM Tris, 25mM EDTA, 1.25M NaCl, 0.25M xitix, 1%PVP40,0.1%BSA, 1mM DTT, pH3.6;
Described damping fluid 1 contains 1.25M NaCl, the 50mM Tris-HCl of pH8.0, the 7mMEDTA of pH8.0,5%PVP40,0.1%BSA, 1mM DTT;
Described lavation buffer solution contains 0.35M sorbyl alcohol, the 50mM Tris-HCl of pH8.0, the 25mM EDTA of pH8.0;
The 10mM Tris-HCl that described damping fluid 2 contains pH8.0, the 5mM EDTA of pH8.0;
Described lysate contains 20% sarcosyl, the 50mM Tris-HCl of pH8.0, the 25mM EDTA of pH8.0.
Further, chloroplast genomic dna order-checking with the concrete grammar of sequence assembly is: adopt s-generation high throughput sequencing technologies, complete order-checking and the sequence assembly of chloroplast gene group by Roche454GS FLX high-flux sequence instrument, obtain chloroplast(id) whole genome sequence.Sometimes, on spliced chloroplast(id) whole genome sequence, may leave a small amount of space, according to the primers on both sides, space, carry out pcr amplification, and with Sanger method order-checking, obtain complete chloroplast gene group sequence after filling space.
The application is by comparing the sequence of the full gene of Bulbus Fritillariae taipaiensis Chloroplast in the chloroplast gene group complete sequence of medicinal material to be identified and GenBank, identify more accurately medicinal material, if its gene order is consistent with it, be that homology reaches more than 98%, this sample is exactly Bulbus Fritillariae taipaiensis, otherwise is not just this plant.
The application also carries out genome annotation (adopting online annotating software DOGMA (http://dogma.ccbb.utexas.edu/)) to Bulbus Fritillariae taipaiensis chloroplast(id) complete sequence, and has drawn the full physical map of genome of chloroplast(id) (adopting online graph drawing instrument (http://wolfe.gen.tcd.ie/GenomeVx/)).
Beneficial effect of the present invention is as follows:
(1) compare with total DNA extraction method, chloroplast DNA Study on Extraction Method is less, and there is no at present cheapness, efficient commercially available reagent box.Being optimized extraction flow process for species often needs a couple of days or tens of days, has hindered the propelling of scientific research project.The method of application documents and materials is extracted chloroplast DNA often needs 30-50 hour, and considerably beyond the extraction time of conventional DNA1-2 hour, and output was unstable.The extracting method of chloroplast gene group provided by the invention has been improved cpDNA and has been extracted flow process, and Bulbus Fritillariae taipaiensis cpDNA extraction efficiency is 206.0 μ g/100g, and OD260/OD280 is 1.94.
(2) the present invention has proposed first chloroplast gene group and has been more suitable for the Molecular Identification mark as Bulbus Fritillariae taipaiensis and other Fritillaria Linn species.By the full genom sequence of testing sample chloroplast(id) is measured, can differentiate Bulbus Fritillariae taipaiensis at molecular level, can also effectively distinguish Bulbus Fritillariae taipaiensis and belong to kindred plant together.For the monoid that is difficult in traditional taxonomy distinguish, adopt the method for chloroplast gene group sequential analysis can cast aside plesiomorphic illusion, provide new classification foundation from gene level.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
Fig. 1 is the full physical map of genome of Bulbus Fritillariae taipaiensis chloroplast(id).
Embodiment
In order to be illustrated more clearly in the present invention, below in conjunction with preferred embodiments and drawings, the present invention is described further.It will be appreciated by those skilled in the art that specifically described content is illustrative and nonrestrictive below, should not limit the scope of the invention with this.
Experiment reagent and laboratory apparatus
(1) buffer A (pH=3.6): 50mM Tris, 25mM EDTA, 1.25M NaCl, 0.25M xitix (VC), 1%PVP40(kollidon), 0.1%BSA, 1mM DTT;
(2) damping fluid 1:1.25M NaCl, 50mM Tris-HCl(pH8.0), 7mM EDTA(pH8.0) and, 5%PVP40,0.1%BSA, 1mM DTT;
(3) damping fluid 2:10mM Tris-HCl(pH8.0), 5mM EDTA(pH8.0);
(4) lavation buffer solution: 0.35M sorbyl alcohol, 50mM Tris-HCl(pH8.0), 25mMEDTA(pH8.0);
(5) 52% sucrose: 52% sucrose is dissolved in the Tris-HCl(pH8.0 of 50mM), 25mMEDTA(pH8.0);
(6) 30% sucrose: 30% sucrose is dissolved in the Tris-HCl(pH8.0 of 50mM), 25mMEDTA(pH8.0);
(7) lysate: 20% sarcosyl, 50mM Tris-HCl(pH8.0), 25mMEDTA(pH8.0);
(8) CTAB/NaCl solution: 10%CTAB, 0.7M NaCl.
(9) agarose: Biowest Agrose(Spain).
(10) the TE:200uL TE that contains RNA enzyme adds the RNAase of 1uL50mg/mL
Other reagent consumptive material: 10ml syringe peace scalp acupuncture, Virahol, SYNTHETIC OPTICAL WHITNER, dehydrated alcohol, 70% ethanol, above reagent component is all purchased from Beijing Bi Chenglan biotechnology limited liability company.
It takes root in fabric texture DNA extraction test kit: catalog number (Cat.No.) DP305, TIANGEN Biotech (Beijing) Co., Ltd. (China).
PCR material:
code DRR001A, precious biotechnology (Dalian) company limited (China).
Degreasing cotton gauze: non-sterilizing, Cao County, Shandong Province Hua Lu sanitary material company limited (China); Miracloth:cat#475855,
merck(Germany);
All size centrifuge tube: Haimen City three and glass experiment instrument factory of Xinhua.
Laboratory apparatus
Low-temperature and high-speed whizzer: SIGMA Laboratory Centrifuges3K30(Germany);
Ice-making machine: Sanyo (Japan);
The freezing large capacity whizzer of desk type high speed (horizontal rotor): Eppendorf Centrifuge5810R(Germany);
High-speed homogenization machine:
(Germany);
Ultra-high speed refrigerated centrifuge: Beckman CO Μ LTER Optima
tMthe L-80XP Μ Ltracentrifuge(U.S.);
Gel imaging instrument: the Biorad(U.S.);
Electric-heated thermostatic water bath: DK-S24 type, the gloomy reliable Instrument Ltd. (China) that tests in Shanghai;
Electronic balance: METTLER TOLEDO PL203, Mettler-Toledo Instrument (Shanghai) Co., Ltd. (China);
Electrophoresis apparatus: DYY-8C type, Beijing Liuyi Instrument Factory (China);
Micro-spectrophotometer: the Thermo Scientific NanoDrop2000(U.S.);
PCR instrument:
the PCR System9700(U.S.);
The Applied Biosystems2720Thermal Cycler(U.S.);
Biohazard Safety Equipment: Su Jing safe and sound (China);
Vortex instrument:
mS3(Germany);
High-pressure sterilizing pot: Sanyo (Japan).
The acquisition of embodiment 1 Bulbus Fritillariae taipaiensis chloroplast gene group
(1) Bulbus Fritillariae taipaiensis chloroplast genomic dna extraction and purification
(the fresh blade of Bulbus Fritillariae taipaiensis picks up from Hong Chi dam, Wen Feng shop, Wuxi County, Chongqing City (latitude: 31.4 ° to get the fresh young leaflet tablet of Bulbus Fritillariae taipaiensis, longitude: 109.6 °), be accredited as Bulbus Fritillariae taipaiensis Fritillaria.taipaiensis P.Y.Li through China Medical Sciences Academy Medical Plants Institute,), sucrose density gradient centrifugation separation and Extraction the Purification of Chloroplast genome (cpDNA) of taking improvement, step is as follows:
1. get fresh young leaflet tablet 100g, 4 ℃ of dark place reason 24h, clean, and defoliation arteries and veins is torn into the big or small fragment in 1cm2 left and right, and the buffer A that adds 500mL4 ℃ of precooling is placed in homogenizer, makes the basic submergence of blade, fully homogenate.Then use 4 layers of filtered through gauze 1 time, 4 layers of Miracloth filter-cloth filtering 1 time, collects filtrate, waste.
2. installed to filtrate dividing in 50ml centrifuge tube, 1500g at 4 ℃, 15min is centrifugal, abandons supernatant.
3. in precipitation, add 20mL damping fluid 1, by the glass stick precipitation that fully suspends, the centrifugal 15min of 1500g at 4 ℃, abandons supernatant; The resuspended precipitation of 20mL damping fluid 1 once, centrifugal, with thorough washing and precipitating.
4. respectively by the 20mL lavation buffer solution each pipe precipitation that suspends.In a new 50mL centrifuge tube, add the sucrose solution of 20mL30%, the precipitation suspension of 20mL is carefully covered to the surface of sucrose solution, in 4 ℃, 2500g horizontal centrifugal 15min, precipitation chloroplast(id).Precipitation after centrifugal is merged, be again resuspended in 20mL lavation buffer solution, and repeat carefully to cover on the sucrose solution of 20mL30% 2500g horizontal centrifugal 10min.To precipitate merging, be resuspended in 20mL lavation buffer solution, again carefully cover on the sucrose solution of 20mL30%, 2500g horizontal centrifugal 5min.
5. last throw out is resuspended in 40mL lavation buffer solution, and 4 ℃, the centrifugal 5min of 100g.Supernatant is transferred to new pipe and at the centrifugal 10min of 380g.If used a large amount of parent materials, the third round of carrying out a 850g is centrifugal.If still do not have centrifugally to get off, can suitably strengthen revolution and extend centrifugation time.
6. precipitation is resuspended in to 5mL lavation buffer solution, this chloroplast(id) solution is carefully covered in to the top of saccharose gradient, and (saccharose gradient is made up of the sucrose solution of 7mL30% and 16.5～18mL52%, configuration in advance, place at least one hour for 4 ℃), go up immediately horizontal rotor whizzer, the centrifugal 1h10min of 20000g at 4 ℃.
7. remove upper strata, the middle phase of the sucrose solution from 52% and 30%, collects chloroplast DNA in the centrifuge tube of 50mL, is then diluted to 45mL with lavation buffer solution, gets 1.2mL4 ℃ and saves backup.45mL pipe is at 4 ℃, and the centrifugal 15min of 1500g precipitates chloroplast DNA.
8. be deposited in resuspended twice (1500g in the damping fluid 2 of 10mL, 15min, 4 ℃ are centrifugal), get precipitation, add 2mL damping fluid 2 and 200uL Proteinase K (10mg/mL), room temperature leaves standstill 2min, add the lysate of 1/5 volume (440uL), slightly put upside down pipe and mix for several times, be then incubated at least general 2～3h of 1h(at 50 ℃), middle gentle concussion mixes, cooled on ice.
9. transfer in 2mL sterilizing centrifuge tube, add isopyknic phenol: chloroform: primary isoamyl alcohol (25:24:1), put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature.
10. supernatant is transferred in new 2mL centrifuge tube, adds the 5molL of 2/5 volume
-1naCl, the CTAB/NaCl that is preheated to 65 ℃ of 2/5 volume, and 68 ℃ heating 15 minutes.Add isopyknic chloroform: primary isoamyl alcohol (24:1), put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature, gets supernatant.
get supernatant, add isopyknic chloroform: primary isoamyl alcohol (24:1), put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature.
CpDNA through improvement extracts flow process, and Bulbus Fritillariae taipaiensis cpDNA extraction efficiency is 206.0 μ g/100g, and 260/280 is 1.94.
(2) order-checking and sequence assembly
Bulbus Fritillariae taipaiensis cpDNA sample after purified adopts s-generation high-flux sequence platform Roche454GS FLX Titanium to check order.First double-stranded DNA is interrupted to the small segment into 800bp, add specificity adapter at fragment two ends, making it sex change is that strand is connected on magnetic bead, through Emulsion PCR, sample cpDNA is increased, and is enriched to PicoTiterPlate plate, upper machine order-checking.Concrete grammar can be referring to Margulies M, Egholm M, Altman WE, et al.Genome sequencing in microfabricated high-density picolitre reactors[J] .Nature, the record in 2005,437:376 380.
(3) filling in space in chloroplast(id) sequence
The data that order-checking produces are removed after inferior quality sequence, and adopting 454 sequenators to carry Newbler software, to carry out de novo assembled.Order-checking obtains 41296 CCS sequences (14.5Mbp), approximately 76 times of coverages altogether to Bulbus Fritillariae taipaiensis chloroplast gene group to adopt 454FLX order-checking platform.After splicing, obtain 45 contig, that the longest is 42,767bp.The wherein coverage of 20 contig higher (>=20 is doubly), overall length 126.398bp.These contig are further spliced, and determine its position relationship, finally obtain complete Bulbus Fritillariae taipaiensis chloroplast gene group sequence.
(4) genome sequence annotation and submission
Adopt online annotating software DOGMA (http://dogma.ccbb.utexas.edu/) to carry out genome annotation to Bulbus Fritillariae taipaiensis chloroplast(id) complete sequence.According to the encoding egg white gene scope of initiator codon and the preliminary annotation of termination codon subsequence manual setting DOGMA, the Seqin software that the result of annotation is specified with NCBI website is submitted Bulbus Fritillariae taipaiensis cpDNA sequence data and annotation information to, generates the sqn file of Bulbus Fritillariae taipaiensis sequence.The Genbank formatted file of Bulbus Fritillariae taipaiensis sequence is drawn to the full physical map of genome of Bulbus Fritillariae taipaiensis chloroplast(id) (Fig. 1) with GenomeVx online graph drawing instrument (http://wolfe.gen.tcd.ie/GenomeVx/).
Bulbus Fritillariae taipaiensis chloroplast DNA (chloroplast DNA, cpDNA) is ring shaped molecule structure, and its size is 151693bp (NCBI sequence number KC543997).The long 26335bp in a pair of inverted repeat district (IRs), large single copy area (LSC) and little single copy area (SSC) are respectively 81471bp, 17552bp.Bulbus Fritillariae taipaiensis cpDNA 132 genes of encoding, comprise 86 encoding egg white genes, 8 rRNA genes and 38 tRNA genes.Protein-coding region accounts for whole genomic 44.88%, and rRNA accounts for 5.97%, tRNA and accounts for 6.61%, and intergenic region and intron account for 42.54%.Wherein 18 genes (6 tRNA and 12 protein coding genes) comprise intron.The GC content of Bulbus Fritillariae taipaiensis chloroplast gene group is 42.49%, QiIR district GC content (42.49%) is higher than LSC (34.82%) and SSC (30.36%), and the high GC content in IR district is because IR district comprises 4 rRNA gene: rrn16, rrn23, rrn4.5, rrn5 (GC content 55.07%).
The feasibility that embodiment 2 carries out Bulbus Fritillariae taipaiensis evaluation based on chloroplast gene group complete sequence
(1) Bulbus Fritillariae taipaiensis based on single-gene sequence and Unibract Fritillary Bulb bio-barcode are identified
The genomic dna that extracts medicinal material according to method well known in the prior art, utilizes chloroplast(id) trnh-psbA sequence to carry out species evaluation.The primer that the amplification of trnh-psbA district is used is:
Upstream primer: 5 '-GTTATGCATGACGTAATGCTC-3 '
Downstream primer: 5 '-CGCGCATGGTGATTCACAATCC-3 '.
Pcr amplification condition: 94 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ are extended 1.5min, 30 circulations, last 72 ℃ are extended 7min.
After order-checking, Unibract Fritillary Bulb trnH-psbA sequence:
ACAATTTCCCTCTAGACCTAGCTGCTATTGAAGTTCCATCTACAAATGGATAAGACTTACATTCTGTCTTAGTGTATACGAATCCTTGATGGAGCAATATCTTTGTATCTTGCTAAAGATATTGCTCCATCAAGGATTTTTTTTTATTGAACGTGTCACAGGGGATTACTCCTTTATTACATTACATTTTTAAAATTGGCATTCTATGCCCAATATATCGATCTAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAATAAAAAAGCTAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGT
Bulbus Fritillariae taipaiensis trnH-psbA sequence:
ACAATTTCCCTCTAGACCTAGCTGCTATTGAAGTTCCATCTACAAATGGATAAGACTTACATTCTGTCTTAGTGTATACGAATCCTTGATGGAGCAATATCTTTGTATCTTGCTAAAGATATTGCTCCATCAAGGATTTTTTTTTATTGAACGTGTCACAGGGGATTACTCCTTTATTACATTACATTTTTAAAATTGGCATTCTATGCCCAATATATCGATCTAAGTATGAAGGTAAGAATAAATACAATAATGATGAATGGAAAAATAAAAAAGCTAAAATCCTTTAGCTAGATAAGGGGCGGATGTAGCCAAGTGGATCAAGGCAGT
From the current result to the research of trnH-psbA sequence, this sequence is when to Unibract Fritillary Bulb and Bulbus Fritillariae taipaiensis analysis, sequence similarity degree is 100%, points out our two species sibship in the time evolving nearer, adopts single-gene to carry out the larger difficulty of Molecular Identification existence to Bulbus Fritillariae taipaiensis.
(2) based on chloroplast gene group complete sequence, Bulbus Fritillariae taipaiensis is identified
A high-flux sequence and data are extracted
According to high-flux sequence and the annotation of Bulbus Fritillariae taipaiensis in embodiment 1, obtain 86 albumen coded sequences.Based on these 86 protein gene sequences, obtain the sequence of 86 total genes of 14 samples of other nearly edge species (table 1).
B data processing
The total gene of 14 samples is carried out respectively to sequence alignment, composition sequence matrix, carry out the Genetic Distance Analysis in kind between wilcoxon two sample test kind, application paup software also adopts blast and the method for distance is carried out different category level (genus, species, population) comparative analysis.For the identification capacity of checking chloroplast gene group sequence to the nearly edge species of Fritillaria, adopt the optimum single-gene combination of approval in the world to contrast with genome sequence.
C interpretation of result
On the basis of chloroplast(id) whole genome sequence, extract the total protein coding gene sequence of all samples and carry out Bulbus Fritillariae taipaiensis and the nucleotide sequence variance analysis that belongs to nearly edge species together, with the hereditary difference object as a comparison obtaining, carry out species and identify (the results are shown in Table 2 and table 3).
Between planting, plant inner analysis, genetic distance using chloroplast gene group as identifying mark is 2.15 times that international barcode tissue is recommended sequence matK+rbcL, the average difference between species of chloroplast gene group is 1.46 times of minimum difference between species, matK+rbcL and mean distance (1.05 times) about the same.Genomic level, average difference between species is 16.2 times of intraspecies variation, and matK+rbcL only has 4 times, and therefore chloroplast gene group is more suitable for as Molecular Identification mark.
From different category level, no matter chloroplast gene group is blast or distance statistical method, determination rates is 100%, matK+rbcL is 78.6, for further verifying the evaluation effect of chloroplast gene group, on the basis of matK+rbcL, we have increased the non-coding region psbA-trnH with larger variant sites generally acknowledging, analytical results shows that two kinds of average determination rates of statistical method are 85.8, increase not remarkable, the conclusion of the international barcode tissue of Zhe Yu is consistent, and polygenic combination can not obviously improve the identification capacity of DNA barcode.Grade under kind, the evaluation advantage of chloroplast gene group is more obvious, and the determination rates of two kinds of methods is respectively 92.9% and 100%, and average specific Matk+rbcL and Matk+rbcL+psbA-trnH exceed respectively 50.05% and 28.6%.Therefore, chloroplast gene group is suitable as the identifying mark of the nearly edge species of Fritillaria, can be used as the molecular bar code of Bulbus Fritillariae taipaiensis and other Fritillaria Linn species.
Table 1 experiment material
| Title | Latin name |
| Bulbus Fritillariae taipaiensis | Fritillaria.taibaiensis |
| Ussuriensis Fritillary Bulb 1 | Fritillaria?ussurieusis |
| Ussuriensis Fritillary Bulb 2 | Fritillaria?ussurieusis |
| Thunberg Fritillary Bulb 1 | F.thunbergii |
| Thunberg Fritillary Bulb 2 | F.thunbergii |
| Thunberg Fritillary Bulb 3 | F.thunbergii |
| The dense sweet bulb of fritillary 1 | F.millea |
| The dark violet bulb of fritillary 1 | F.unibracteata |
| The dark violet bulb of fritillary 2 | F.unibracteata |
| Watt cloth bulb of fritillary 1 | F.wabuensis |
| Watt cloth bulb of fritillary 2 | F.wabuensis |
| The scrolling bulb of fritillary | F.cirrhosa |
| Fritillaria pallidiflora Schrenk 1 | F.pallidiflora |
| Fritillaria pallidiflora Schrenk 2 | F.pallidiflora |
Table 2 chloroplast gene group sequence is planted interior Genetic distance comparison to Fritillaria different plant species
| ? | Genome sequence | matK+rbcL |
| Average sample difference between species | 0.0131±0.0275 | 0.0061±0.006862 |
| Minimum difference between species | 0.0089±0.0315 | 0.00582±0.01048 |
| Average intraspecies variation | 0.00081±0.000729 | 0.001513±0.001423 |
| Maximum intraspecies variation | 0.001719±0.002342 | 0.001643±0.001847 |
Table 3 adopts different identifying marks and different authentication method to identify the nearly edge species of Fritillaria
Obviously; the above embodiment of the present invention is only for example of the present invention is clearly described; and be not the restriction to embodiments of the present invention; for those of ordinary skill in the field; can also make other changes in different forms on the basis of the above description; here cannot give all embodiments exhaustively, everyly belong to apparent variation or the still row in protection scope of the present invention of variation that technical scheme of the present invention extends out.
Claims (5)
1. the molecular biological variety identification method of a Bulbus Fritillariae taipaiensis, it is characterized in that, the method comprises the chloroplast genomic dna the purifying that extract medicinal material to be identified, check order and sequence assembly, obtain complete chloroplast gene group sequence, the chloroplast gene group sequence of this sequence and Bulbus Fritillariae taipaiensis is compared, obtain Analysis and Identification result.
2. the molecular biological variety identification method of Bulbus Fritillariae taipaiensis according to claim 1, is characterized in that, the NCBI sequence number of Bulbus Fritillariae taipaiensis chloroplast gene group sequence is KC543997.
3. the molecular biological variety identification method of Bulbus Fritillariae taipaiensis according to claim 1 and 2, is characterized in that, extracts the chloroplast genomic dna of medicinal material to be identified and the concrete grammar of purifying is:
(1) get fresh young leaflet tablet, 4 ℃ of dark place reason 24h, clean, and defoliation arteries and veins, tears to bits, and the buffer A that adds 4 ℃ of precoolings is placed in homogenizer, and then fully homogenate filter, and collects filtrate, waste;
(2) by filtrate, in 4 ℃, 1500g, centrifugal 15min, abandons supernatant;
(3) by damping fluid 1 washing and precipitating;
(4) by the lavation buffer solution precipitation that suspends, in a new centrifuge tube, add 30% sucrose solution, precipitation suspension is covered to the surface of 30% sucrose solution, in 4 ℃, 2500g, centrifugal 15min, precipitation chloroplast(id);
(5) precipitation is resuspended in lavation buffer solution, in 4 ℃, and the centrifugal 5min of 100g, supernatant is transferred to new pipe and at the centrifugal 10min of 380g;
(6) precipitation is resuspended in lavation buffer solution, this solution is covered in to the top of saccharose gradient, in 4 ℃, and the centrifugal 70min of 20000g; Wherein, saccharose gradient is made up of the sucrose solution of 7mL30% and 16.5～18mL52%;
(7) remove upper strata, the middle phase of the sucrose solution from 52% and 30%, collects chloroplast DNA in centrifuge tube, then adds lavation buffer solution, in 4 ℃, and the centrifugal 15min precipitation of 1500g chloroplast DNA;
(8) by damping fluid 2 washing and precipitating, in 4 ℃, 1500g, centrifugal 15min, get precipitation, adding damping fluid 2 and concentration is the Proteinase K of 10mg/mL, and room temperature leaves standstill 2min, adds the lysate of 1/5 volume, put upside down and mix, be then incubated at least 1h, cooled on ice at 50 ℃;
(9) transfer in sterilizing centrifuge tube, add isopyknic phenol: chloroform: primary isoamyl alcohol, put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature; Wherein, phenol: chloroform: the volume ratio of primary isoamyl alcohol is 25:24:1;
(10) supernatant is transferred in new centrifuge tube, adds the 5molL of 2/5 volume
-1naCl, the CTAB/NaCl that is preheated to 65 ℃ of 2/5 volume, and 68 ℃ heating 15 minutes, add isopyknic chloroform: primary isoamyl alcohol, put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature, gets supernatant; Wherein, chloroform: the volume ratio of primary isoamyl alcohol is 24:1;
(11) repeat (10)
(12) add isopyknic chloroform: primary isoamyl alcohol, put upside down and mix, leave standstill 5min, the centrifugal 20min of 2600g room temperature; Wherein, chloroform: the volume ratio of primary isoamyl alcohol is 24:1;
(13) get supernatant, add the 3molL of 0.1 times of volume
-1the pre-cold isopropanol of NaAc and 0.8 times of volume or add the 3molL of 0.1 times of volume
-1the dehydrated alcohol of NaAc and 2 times of volumes, places at least 20min precipitation chloroplast DNA for-20 ℃;
(14) 4 ℃, the centrifugal 20min of 13500g, cleans the centrifugal 10min of twice, 13500g by precipitation by 70% ethanol room temperature, air-dry, resuspended with the TE that contains RNA enzyme, and 37 ℃ of insulation 1h, obtain chloroplast DNA;
Wherein, described buffer A contains 50mM Tris, 25mM EDTA, 1.25M NaCl, 0.25M xitix, 1%PVP40,0.1%BSA, 1mM DTT, pH3.6;
Described damping fluid 1 contains 1.25M NaCl, the 50mM Tris-HCl of pH8.0, the 7mMEDTA of pH8.0,5%PVP40,0.1%BSA, 1mM DTT;
Described lavation buffer solution contains 0.35M sorbyl alcohol, the 50mM Tris-HCl of pH8.0, the 25mM EDTA of pH8.0;
The 10mM Tris-HCl that described damping fluid 2 contains pH8.0, the 5mM EDTA of pH8.0;
Described lysate contains 20% sarcosyl, the 50mM Tris-HCl of pH8.0, the 25mM EDTA of pH8.0.
4. the molecular biological variety identification method of Bulbus Fritillariae taipaiensis according to claim 1, is characterized in that, chloroplast genomic dna order-checking is to adopt s-generation high throughput sequencing technologies with sequence assembly.
5. the molecular biological variety identification method of Bulbus Fritillariae taipaiensis according to claim 3, is characterized in that, described s-generation high throughput sequencing technologies is to complete by Roche454GS FLX high-flux sequence instrument.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104862300A (en) * | 2015-01-23 | 2015-08-26 | 王恒 | Application of highly pure capsicum genome DNA extraction technology |
| CN105734153A (en) * | 2016-04-20 | 2016-07-06 | 上海派森诺生物科技股份有限公司 | Method of using genes for species identification of plants |
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| CN109411014A (en) * | 2018-10-09 | 2019-03-01 | 中国科学院昆明植物研究所 | A kind of cyclic method of plant chloroplast full-length genome assembling based on the sequencing of two generations |
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| CN111575273A (en) * | 2020-04-08 | 2020-08-25 | 康美华大基因技术有限公司 | Traditional Chinese medicine fritillary DNA extraction reagent and extraction method |
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-
2014
- 2014-02-25 CN CN201410064966.1A patent/CN103849687A/en active Pending
Non-Patent Citations (4)
| Title |
|---|
| LI,Y ET AL: "KC543997", 《GENBANK》 * |
| 林小涵等: "药用植物叶绿体基因组研究", 《世界科学技术-中医药现代化》 * |
| 段宝忠等: "太白贝母资源学研究概况", 《中国现代中药》 * |
| 胡志刚: "菊科药用植物DNA条形码及叶绿体基因组研究", 《中国博士学位论文全文数据库农业科技辑》 * |
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| CN105734153A (en) * | 2016-04-20 | 2016-07-06 | 上海派森诺生物科技股份有限公司 | Method of using genes for species identification of plants |
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