CN102703444B - Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker - Google Patents
Radix polygonati officinalis microsatellite DNA (deoxyribonucleic acid) molecular marker Download PDFInfo
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Abstract
The invention discloses a radix polygonati officinalis microsatellite DNA molecular marker, comprising the steps of constructing a radix polygonati officinalis microsatellite (CT) n enrichment library; screening and sequencing microsatellite sequence positive clone; obtaining 55 clones containing the microsatellite repeated sequence; and screening to obtain 9 high polymorphic microsatellite molecular markers Po1, Po2, Po3, Po4, Po5, Po6, Po7, Po8 and Po9. The radix polygonati officinalis microsatellite DNA molecular marker can be used for researching genetic diversity of the radix polygonati officinalis, variation and evolutionary relationship between polygonatum plants and population, has good repeatability and is a reliable and efficient molecular marker.
Description
Technical field
The present invention relates to molecular marking technique, be specifically related to a kind of radix polygonati officinalis micro-satellite molecule genetic marker.
Background technology
Little satellite (Microsatellite) be called again short polyphone repeat (Short Tandem Repeats, STRs), simple repeated sequence (Simple Sequence Repeat, SSRs), SSLP (SSLP).Refer to that a class is dispersed throughout (being called core sequence, being generally 1～6bps) the simple series connection of forming for repeating unit and repeating by several nucleotide pairs in the biological gene group.Its multiplicity is alterable height between the different genotype of same species, but the two ends of this section repetition are the single-copy sequences of guarding relatively, design primer accordingly, can analyze the variation of core sequence multiplicity by round pcr, between different genotype, detect polymorphic.According to the formation of repeating unit, the microsatellite DNA sequence is divided into 3 types: single type (pure), compound (compound) and discontinuous form (interrupted).For example:
Single type: ATATATATATATATATATATATAT
Compound: ATATATCACACACACACACAC
Discontinuous form: ATATATCA ATATATCA ATATATA
As a kind of molecule marker, microsatellite DNA has following characteristics: be distributed widely in the Eukaryotic genome, in per 6 kb length 1 microsatellite locus just arranged according to estimates; The multiplicity of core sequence is height variability, polymorphism information capacity height: be codominance, follow the heredity of broad-mouthed receptacle for holding liquid Dare rule between individuality; Select neutrality etc.
Based on above characteristics, microsatellite DNA is widely used in that structure, the assignment of genes gene mapping, cultivar identification and the germplasm of plant genetic collection of illustrative plates are preserved, the research of analysis, assistant breeding, structure finger printing, genetic diversity and the aspects such as spore and sibship of quantitative character gene.
Radix polygonati officinalis has another name called the tail ginseng, beautiful ginseng, and the small bell dish, ground tube and sweet grass are under the jurisdiction of Liliaceae (Liliaceae L.) Polygonatum (Polygonatum Mill.), are per nnial herb, and happiness is cloudy gives birth to, and is distributed widely in area, temperate zone, Eurasia.
The radix polygonati officinalis subterraneous stem can be used as medicine, and its medicinal history has exceeded more than 2,000 year in China.Traditional Chinese medicine thinks that radix polygonati officinalis property is sweet, is slightly cold, attaches to the lung and stomach meridians, can Yin nourishing and lung moistening, reinforcing stomach reg fluid is used for scorching impairment of yin, the disease of thermal burn stomach-Yin.Modern study proof radix polygonati officinalis contains polysaccharide and multiple extract, is delaying senility, and strengthening immunity suppresses tumour, and aspects such as promotion lymphocyte transformation have certain effect.
Since among the people dig excessively serious, in addition the natural crude drugs cycle long, the radix polygonati officinalis wild resource is exhaustion day by day.In fact, radix polygonati officinalis medicine source is comparatively complicated, and little satellite helps to identify the radix polygonati officinalis strain, and distinguishes the pseudo-product of radix polygonati officinalis.Simultaneously, radix polygonati officinalis a series of problems occurred in the cultivation domestication process, and is low as breeding coefficient, even utilize microsatellite marker to the research of radix polygonati officinalis molecular breeding seldom.
Summary of the invention
The technical problem that the present invention solves: the microsatellite DNA molecule marker of separating clone radix polygonati officinalis, set up radix polygonati officinalis satellite DNA technical system and carry out the radix polygonati officinalis genetic diversity with these molecule markers, the research of Polygonatum platymiscium genetic affinity, and carry out the Polygonatum Idioplasm identification.
The technical scheme of technical solution problem of the present invention: radix polygonati officinalis microsatellite DNA molecule marker, comprise the structure of the little satellite of radix polygonati officinalis (CT) n enriched library, the screening of microsatellite sequence positive colony and order-checking obtain 55 clones that contain little satellite tumor-necrosis factor glycoproteins.Obtain 9 microsatellite molecular marker Po1, Po2, Po3, Po4, Po5, Po6, Po7, Po8, Po9 that polymorphism is high; Po1 is 360 Nucleotide, and Po2 is 471 Nucleotide, and Po3 is 447 Nucleotide, and Po4 is 408 Nucleotide, and Po5 is 463 Nucleotide, and Po6 is 497 Nucleotide, and Po7 is 424 Nucleotide, and Po8 is 509 Nucleotide, and Po9 is 342 Nucleotide.
Description of drawings
Fig. 1: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po1 site dyes PAGE figure.
Fig. 2: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po2 site dyes PAGE figure.
Fig. 3: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po3 site dyes PAGE figure.
Fig. 4: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po4 site dyes PAGE figure.
Fig. 5: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po5 site dyes PAGE figure.
Fig. 6: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po6 site dyes PAGE figure.
Fig. 7: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po7 site dyes PAGE figure.
Fig. 8: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po8 site dyes PAGE figure.
Fig. 9: the silver of the DNA of 24 radix polygonati officinalis of primer amplified in Po9 site dyes PAGE figure.
Embodiment
Do detailed explanation below in conjunction with the present invention of embodiment.
Embodiment 1:
The structure of radix polygonati officinalis microsatellite DNA enriched library
1) genomic extraction and enzyme are cut:
The CTAB method of introducing with Doyle J (Doyle J. DNA protocols for plants-CTAB total DNA isolation [A]. In Hewitt GM, Johnston A (eds.), Molecular Techniques in Taxonomy [M]. Berlin:Springer, 1991,283-293) extract genome.Cut genome with restriction enzyme Sau 3AI enzyme.Behind 2% the agarose gel electrophoresis, under UV-light, downcut the dna fragmentation of 300-900 bp.Reclaiming test kit with glue reclaims.
2) add joint:
The product that glue is reclaimed behind the purifying adds joint:
oligo A(5’-GGCCAGAGACCCCAAGCTTCG-3’)
oligo B (5’-PO
4-GATCCGAAGCTTGGGGTCTCTGGCC-3’),
Under T4 DNA Ligase effect, spend the night in 16 ℃ of water-baths connections.
3) the PCR detection tabs connects:
Being primer with oligo A, is that template is carried out pcr amplification to connect product, (50 μ l reaction systems are as follows: 25 μ l GoTaq Green Master Mix, and 5 μ l primer oligo A, 5 μ l connect product, add the water benefit to 50 μ l.The PCR response procedures: 94 ℃ of pre-sex change 5 minutes, 94 ℃ of sex change 50 seconds, 56 ℃ of annealing one minute, 72 ℃ were extended 2 minutes, sex change, annealing, three step cycle of extension 25 times, last 72 ℃ were fully extended 10 minutes.After agarose gel electrophoresis 2% detected, the PCR product carried out purifying with purification kit.
4) enrichment with magnetic bead:
Dna fragmentation sex change behind the purifying is formed after the strand and the little satellite probe hybridization that contains vitamin H, form " little satellite probe comprises the genomic dna strand of little satellite " complex body.Hybridization solution is added the magnetic bead of having handled well, and room temperature left standstill 30 minutes.Centrifuge tube is placed on the magnetic force frame, inhales and remove supernatant, use 6 * SSC successively, 2 * SSC, 1 * SSC washes twice under 60 ℃, use at last under 0.1 * SSC room temperature and wash once.Add the TE damping fluid, 95 ℃ of sex change are 15 minutes on the PCR instrument, collect supernatant.Repeat twice.
5) PCR enrichment:
Be template with the enrichment with magnetic bead product, oligo A is that primer carries out pcr amplification, and 25 μ l reaction systems are as follows, 12.5 μ l GoTaq Green Master Mix, and 1.5 μ l primer oligo A, the DNA after the 2.5 μ l enrichments adds water and mends to 25 μ l.The PCR response procedures is as follows: 94 ℃ of pre-sex change 3 minutes, and 94 ℃ of sex change 35 seconds, 60 ℃ of annealing property 35 seconds, 72 ℃ were extended 1 minute, sex change, annealing, three step cycle of extension 25 times, last 72 ℃ were fully extended 12 minutes.The PCR product carries out purifying with purification kit after 2% agarose gel electrophoresis detection.
6) preparation of competent cell:
The single bacterium colony of E.coli DH-5 α is inoculated in the LB substratum that does not contain Amp of 5 ml on the picking flat board, and 37 ℃ of shaking culture are spent the night.Get 1 ml nutrient solution in the LB of 50 ml liquid nutrient medium, 37 ℃ of shaking culture, 2～4 h are to logarithmic phase.Survey OD(0.2～0.6).The nutrient solution branch is filled to (every pipe 1.5 ml bacterium liquid) in the EP pipe, water-bath 10～15 min.4 ℃ centrifugal, centrifugal again after 4000 rpm, the 10 min(cooling earlier) abandon supernatant.Abandon three pipe and pipes behind the supernatant, ice bath 30 min(first pipe adds 0.1 mol/L, and 1 ml is the CaCl2 of precooling in the ice in advance, mixing, sucking-off to the second pipe is so with three pipes and a pipe).Take out the back gradient centrifugation, 4 ℃, 4000 rpm, 5～8 min.Abandon supernatant.Add 200 μ l precooling glycerine (15%)/CaCl
2(0.1 M) mixing ,-80 ℃ of preservations.
7) connect conversion:
The purified product of getting after the PCR enrichment connects into TA cloning vector (pMD19-T Simple Vector), and 16 ℃ of water-baths connect 2-3 hour.Take out frozen pipe, 10 min on ice recover; The connection product of 10 μ l is added in the 200 μ l competent cells mixing, ice bath 30 min; 42 ℃ of water-bath heat-shocked 90 s; Ice bath 2 min add 600 μ l LB substratum, 37 ℃ of shaking culture 1.5 h; Get 70 μ l nutrient solutions, splash on the LB/Amp/X-gal/IPTG flat board and coating even (X-Gal needs lucifuge); 37 ℃ of forwards of flat board are placed cultivation 0.5h, in 37 ℃ of baking ovens, be inverted overnight incubation then.Through the cultivation of 12 h, treat that the bacterium colony on the flat board is of moderate size, take out flat board and in 4 ℃ of placements locus coeruleus is fully manifested.With white mono-clonal bacterium colony on the toothpick picking flat board of the bacterium of going out, place 96 deep-well plates (deep-well plates has added the LB/Amp substratum that contains the Amp resistance of 400 μ l in advance).After the single bacterium colony picking of white finishes, with non-setting adhesive orifice plate is built the back in 37 ℃, 225rpm shaking culture 4 h.
8) screening of positive colony:
The bacterium liquid that transforms gained with connection is template, utilizes primer oligo A and primer (CT)
12Carry out pcr amplification.15 μ l PCR reaction systems comprise: rTaq enzyme 0.375 U, 10 * Buffer, 1.5 μ l, dNTP 0.2 μ M, MgCl
21.5 μ M, and primer 0.4 μ M (oligo A, (CT)
12), water, template.Response procedures: 94 ℃ of pre-sex change 3 minutes, 94 ℃ of sex change 50 seconds, 56 ℃ of annealing 35 seconds, 72 ℃ were extended 1 minute, sex change, annealing, three step cycle of extension 30 times.Last 72 ℃ were fully extended 12 minutes.1.5% agarose gel electrophoresis detects.
9) positive colony order-checking and design of primers:
Choose the amplification fragment length and contain the bacterium liquid of positive colony at 300-900 bp, send the order-checking of the biological (Shanghai) Co., Ltd. of living worker, obtain 51 sequences that contain microsatellite DNA altogether.Utilize software Primer Premier 5.0 sequences according to the microsatellite DNA two ends to design primer.
10) screening has the primer of polymorphism:
Extract the genome of radix polygonati officinalis with above-mentioned CTAB method.With primer (table 1) the amplification gene group that designs.Response procedures: 94 ℃ of pre-sex change 4 minutes, 94 ℃ of sex change 45 seconds, corresponding annealing temperature 30 seconds, 72 ℃ were extended 35 seconds, sex change, annealing, three step cycle of extension 35 times.Last 72 ℃ were fully extended 8 minutes.1.5% agarose gel electrophoresis detects.The PCR product detects with the PAGE electrophoresis.Obtain 9 microsatellite markers that polymorphism is arranged altogether.Po1, Po2, Po3, Po4, Po5, Po6, Po7, Po8, Po9; Po1 is 360 Nucleotide, and Po2 is 471 Nucleotide, and Po3 is 447 Nucleotide, and Po4 is 408 Nucleotide, and Po5 is 463 Nucleotide, and Po6 is 497 Nucleotide, and Po7 is 424 Nucleotide, and Po8 is 509 Nucleotide, and Po9 is 342 Nucleotide.
11) interpretation of result:
Use Cervus3.0 computed in software expectation heterozygosity and observe heterozygosity.Use the value of Genepop3.4 computed in software Hardy-Weinberg and linkage disequilibrium, describe the feature of 9 radix polygonati officinalis microsatellite DNA polymorphic sites with this.Can be found out by accompanying drawing 1-12, the diversity that the length of 9 microsatellite sequences of the present invention all occurs in 21 radix polygonati officinalis for examination, this shows, these 9 microsatellite markers of the present invention can be used for the genetic diversity of research radix polygonati officinalis, between the Polygonatum plant species and the variation between population and evolutionary relationship, having very high repeatability, is a kind of reliable and effective molecule marker.
Table 1 primer property list:
F represents upstream primer in the table 1, and R represents. downstream primer.
Claims (1)
1. radix polygonati officinalis microsatellite DNA molecule marker, it is characterized in that: the label of described microsatellite marker is: Po1;
Its nucleotides sequence is classified as:
Po1：
gatcacatgc cgatagttcc tcggtcacct agcatattta gtgtcagtct tctagctggc 60
gaataaaaac actaagtcga aatgaactaa ggaaagtgct cttctaactg tgagagagag 120
agagagagag agagagagag agagagagag agatgttggt ccatccctca tggtgtactg 180
tggatttcat tttgcgctct ctagctaatt ggattttagt cagatttttc tagttagttt 240
agcaccactc ataggtagag ctgtaaaatg aactgagttt tggagtgttt gtgttcgttc 300
aataatttac acgagtttaa atttagctga ttctatatga acccaaacaa atactcaatt 360。
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