CN105907845A - Molecular identification method for Fuding Dabai commercial tea - Google Patents

Molecular identification method for Fuding Dabai commercial tea Download PDF

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CN105907845A
CN105907845A CN201610232109.7A CN201610232109A CN105907845A CN 105907845 A CN105907845 A CN 105907845A CN 201610232109 A CN201610232109 A CN 201610232109A CN 105907845 A CN105907845 A CN 105907845A
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fuding
dabai
tea
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buffer
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CN105907845B (en
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房婉萍
周琳
俞滢
李庆会
朱旭君
王玉花
叶乃兴
李磊
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Nanjing Agricultural University
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    • 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
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    • 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
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Abstract

Belonging to the technical field of molecular markers, the invention discloses a primer group for molecular identification of Fuding Dabai commercial tea, and also discloses a molecular identification method of Fuding Dabai commercial tea. According to the method, a to-be-identified sample is subjected to PCR reaction, sequencing is carried out on an amplification, then comparison with a Fuding Dabai variety bar code is conducted, when the similarity of the amplification product's SNP sequence and a difference base sequence shown as the Fuding Dabai variety bar code reaches more than 99.5%, the to-be-identified sample is Fuding Dabai variety. The method provided by the invention overcomes the technical problem that the authenticity of Fuding Dabai tea and the different Camellia sinensis varieties belonging to camellia are difficultly identified by sensory evaluation, physical and chemical analysis or other methods, and can effectively, simply and rapidly identify the variety of Fuding Dabai commercial tea accurately.

Description

A kind of method for identifying molecules of Fuding DABAI commodity tea
Technical field
The invention belongs to molecular marking technique field, be specifically related to the method for identifying molecules of a kind of Fuding DABAI commodity tea.
Background technology
Camellia sinensis [Camellia sinensis (L.) O.Kuntze] is a kind of important industrial crops, is to flow most 21 century One of healthy beverage plant of row.China is the original producton location of Camellia sinensis, produces tea with a long history.In recent years, Chinese tea industry is quickly sent out Exhibition, industry size constantly expands, and tea place cultivated area, yield, export volume and the amount of money hit new peak repeatly.Meanwhile, twentieth century nine The ten's started excavation in all parts of the country, and recovered historical famous tea and formulate new well-known tea and develop simultaneously, and Famous High-quality Tea quantity is continuously increased, quality by Year improves, and market is opened up comprehensively.
Ramulus et Folium Mussaendae Pubescentis primarily now producing region has two: Fuding and having stable political situation.Ramulus et Folium Mussaendae Pubescentis is to pluck dish tea fresh leaves to make the earliest, the most just uses Fuding white tea (China's tea 1), Fuding big milli tea (China's tea 2) make Ramulus et Folium Mussaendae Pubescentis.Fuding white tea is to originate in Fuding Mount Taimu Fuding white tea and Fuding big milli tea and the general designation of Ramulus et Folium Mussaendae Pubescentis series products made.Standard of plucking according to Folium Camelliae sinensis is different and processes The difference of processing technology, mainly divides Silver-needle Pekoe, white peony, tribute eyebrow longevity eyebrow and new technology Ramulus et Folium Mussaendae Pubescentis etc. four kinds.
Fuding white tea has another name called raw white tea, is called for short good fortune big.Clone, dungarunga type, middle period class, early non-hibernating eggs, diploid.Produce Ground and distribution: nod Bai Liu village, town in original Fuding City, existing more than 100 year cultivation history, it is mainly distributed on tea district, northeast, Fujian.20 After the sixties in century, there is large area the provinces and regions such as Fujian and Zhejiang, Hunan, Guizhou, Sichuan, Jiangxi, Guangxi, Hubei, Anhui, Jiangsu Cultivation.Within 1985, national Crop breed audit committee regards as country's kind, numbering GS13001 1985.Feature: Bud-leaf fertility and to hold tender property strong, yield is high, every 667 square metres up to more than 200 kilograms.Spring tea two leaves and a bud dry sample is containing about ammonia Base acid 4.3%, tea polyphenols 16.2%, catechin total amount 11.4%, caffeine 4.4%.Suitable black tea processed, green tea, Ramulus et Folium Mussaendae Pubescentis, quality Excellent.The Fuding white tea height of tree 1.5 2 meters, fabric width 1.6 2 meters, tree vigo(u)r half is opened a business, for dungarunga type.Branch is closeer, and internode is still Long.Bark Lycoperdon polymorphum Vitt.Elliptic leaf shape, tip is tapering and the most sagging, and base portion is the most blunt, and leaf margin is slightly upwardly.The biggest 12 × 5.4 centimetres, Length-width ratio average out to 2.2.Leaf yellow skin is green, tool gloss.Lateral vein is obvious, and 7 11 is right.Sawtooth is relatively neat, obvious, and 27 38 is right.Leaf Meat is thick, the softest.Two leaves and a bud length 5.1 centimetres, hundred-bud weight 23 grams.Flower pattern is relatively big, and stamen is less than gynoecium, full-bloom stage late October To mid-November, flower amount is many, and fruiting rate is high, and Fructus Camelliae sinensis is big and full.Germination period stopped growing in early March, mid-November.Growth Whole year phase reaches 8 months.Growth potential is vigorous, strong stress resistance, drought-enduring also resists cold, though at subzero 34 DEG C or the lowest do not endure cold.Numerous Power of growing is strong, and press strip, rooting are easy, and survival rate is up to more than 95%.Yield is higher than local dish tea.Gather and process acupuncture needle pure white with bud The many characteristics the most of stout and strong, fine hair.
Fuding big milli tea is called for short big milli, clone, dungarunga type, great Ye class, early non-hibernating eggs, diploid.The place of production and distribution: former Produce Fuding City to nod Wang Jiayang village, town, existing a century cultivation history.It is mainly distributed on fujian tea district.After 20 century 70s, Jiangsu, There is commerial growing the provinces and regions such as Zhejiang, Sichuan, Jiangxi, Hubei, Anhui.Within 1985, national Crop breed audit committee is assert For country's kind, numbering GS13002 1985.Feature: bud-leaf fertility and to hold tender property relatively strong, yield is high, every 667 squares Meter Ke Da 200～300 kilograms.Spring tea two leaves and a bud dry sample is containing about aminoacid 3.5%, tea polyphenols 25.7%, catechin total amount 18.4%, caffeine 4.3%.Suitable black tea processed, green tea, Ramulus et Folium Mussaendae Pubescentis.
Fuding white tea and the difference of Fuding big milli tea.In shape, Fuding white tea: blade ovalize, leaf color is green, Blade face is swelled, glossy, and leaf margin is put down, and blade is put down, and the blunt point of blade tip, leaf-teeth are sharp closeer, and the food value of leaf is thicker soft.Fuding big milli tea: leaf is ellipse Circle or nearly oblong, leaf color is green, rich gloss, and blade face is swelled, leaf margin microwave, and blade is rolled over slightly within, and blade tip is tapering, leaf-teeth sharp shallow, Closeer, food value of leaf thickness is crisp.Fuding white tea: bud-leaf yellow green, fine hair spy is many, a bud SANYE hundred-bud weight 63 grams.Fuding big milli tea: bud Stout and strong, fine hair is intensive, a bud SANYE hundred-bud weight 104 grams.In yield, learnt by comparing above, the bud-leaf of Fuding white tea Weight one bud SANYE hundred-bud weight 63 grams, Fuding big milli tea one bud SANYE hundred-bud weight 104 grams, two tea per mu yield difference have more than 1/3.At mouth In sense, due to two kinds of tea theanine and polyphenol content difference, so the shape of two kinds of tea, perfume (or spice), color, taste are slightly distinguished, but Totally it is more or less the same.
At present, Fuding DABAI because of its be worth in tea market high, effect is many and has reserve value and inevitable Ground has suffered counterfeit, it is therefore necessary to differentiate the quality true and false of Famous High-quality Tea, the interests of the fan that samples tea with protection, maintains The fair and just property in Famous High-quality Tea market.Sense organ differentiates and Physico-chemical tests is the most frequently used tea leaf quality discrimination method, but sense organ Method of discrimination is affected by the personal experience of teacher of the sampling tea, psychology and physiologic factor etc., often differentiates accuracy and the repeatability of result Difference；The method of Physico-chemical tests is only limited to the analysis of the internal specific components of some Folium Camelliae sinensis, and analysis process is many, and cost is high.
In recent years, molecular biology and biotechnology develop rapidly, and Genetic Markers have also been obtained fast on this basis Hail exhibition.Molecular marker as the morphology labelling that continues, cytological marker, the new Genetic Markers of latter of biochemical marker, Though development time is short but have broad application prospects and huge application potential, at genetic map construction, population genetic variations Significant with in the research of diversity analysis, material evolution and sibship.At present, molecular marking technique grinds in Camellia sinensis Extensively apply in studying carefully, obtain more progress, and research is also updated.More molecular marking technique is applied in Camellia sinensis For restriction fragment length polymorphism (RFLP), RAPD DNA marker (RAPD), simple sequence repeats (SSR), amplified fragment length polymorphism (AFLP), single nucleotide polymorphism (SNP) etc..Although RFLP, RAPD, SSR, AFLP exist Molecular marking technique has respective advantage, but there is also that experimental implementation is loaded down with trivial details, detection cycle length, high in cost of production shortcoming. Single nucleotide polymorphism (SNP) as modal one can hereditary variation, be widely present in animal-plant gene group.SNPs refers to In genomic level, the DNA sequence polymorphism that single nucleotide diversity is formed, for the representative of third generation molecular marker.SNP includes Two kinds of forms, the conversion of base or the transversion of base, have widely distributed, enormous amount, hereditary stability strong, gene coding region SNP may directly affect the advantage such as structure or gene expression dose of product albumen matter, and be also easy to the most quickly inspection Survey and automated analysis, be research complex inheritance character and the ideal mark of genome evolution.
Commodity Biluochun tea can be identified by the method through us, and the qualification of Fuding white tea also has important meaning Justice.And relative to Biluochun tea, the processing technique of Ramulus et Folium Mussaendae Pubescentis is only dry in the sun, is dried, without any parch, due to processing work Sequence is few, remains the various compositions in Folium Camelliae sinensis the most largely, and after extracting DNA, wherein impurity is more, have impact on SNP's Identify.At present there is assorted situation in tea market, therefore for the qualification of each commodity tea, only carry out single bud-leaf Extraction can not represent the overall condition of these commodity.In the case of having relatively Multi-example and needing duplicate detection, we utilize Method below carries out the DNA extraction of commodity tea, it is possible to the extraction DNA of efficiently and accurately, simultaneously can be with the carrying out of efficiently and accurately after Continuous experiment.
Summary of the invention
Goal of the invention: first purpose of the present invention there is provided a kind of Molecular Identification for Fuding DABAI commodity tea Primer sets.
Second object of the present invention there is provided the method for identifying molecules of a kind of Fuding DABAI commodity tea.
The present invention utilizes molecular marking technique to overcome sense organ differentiation and the deficiency of Physico-chemical tests method, utilizes multiple SNP Difference site, is finally reached the purpose detection that the Fuding white tea quality true and false quickly differentiates.
Technical scheme: in order to solve above-mentioned technical problem, the invention provides a kind of dividing for Fuding DABAI commodity tea The primer sets that son is identified, described primer sets includes 45 pairs of primer sequences, and described primer sequence is as shown in SEQ ID NO:1～90.
Present invention also offers the method for identifying molecules of a kind of Fuding DABAI commodity tea, comprise the following steps:
1) 45 SNP site of Fuding white tea leaf sample are determined, its SNP sequence such as SEQ ID NO:91～135 institute Show., 45 the corresponding gene orders found by blast in NCBI；S in the SNP site of the present invention represents: (C/G), R:(A/G), M:(A/C), W:(A/T) and, Y:(C/T), K:(G/T)；
2) design of primer；According to 45 gene orders found, according to the method for homologous clone, find kindred plant, The comparison of line order of going forward side by side row, finds the segment area that homology is higher, designs the upstream and downstream primer of these 45 genetic fragments；Described Primer sequence 45 pairs of primer sequences described above；
3) extracting genome DNA；
4) PCR amplification and detection；
5) with Fuding DABAI kind bar code comparison, the base sequence of Fuding DABAI kind bar code after amplified production being checked order Row (accession number on NCBI that 1～45 gene order is corresponding sees table 1), the SNP sequence of amplified production and Fuding DABAI product The similarity planting the distinguishing base sequence shown in bar code reaches more than 99.5%, then this sample to be identified is Fuding DABAI product Kind.
(1～45 represent 1～45 gene order to table 1.)
Wherein, the design length 18-25bp of above-mentioned primer, product size be 200-600bp, Tm value be 50 DEG C-60 DEG C, GC Content is between 40%-60%.
Wherein, said gene group DNA extraction uses QIAGEN 96plate kit method to extract genomic DNA from blade, Its main operational steps is:
For identifying sample on a small quantity, its extracting genome DNA operating procedure is as follows:
1) put into after 50mg blade being weighed in 2mL centrifuge tube；
2) in 2mL centrifuge tube, tungsten carbide pearl and quartz sand are added；
3) centrifuge tube is put in automatization's sample grinder by sample grinding fully；
4) in each 2mL centrifuge tube, add 400 μ l AP1 buffer and 4 μ l RNase A, build rear vortex abundant；
5) by mixture water-bath 20min at 65 DEG C, abundant every 5min vortex；
6) ice chest is prepared；
7), after water-bath terminates, 130 μ LAP2 buffer are added, after vortex is abundant, ice bath 5min；
8) 14000rpm is centrifuged 5min；
9) careful for the supernatant (about 500 μ l) being centrifuged in previous step sucking-off is placed in QIA shredder Mini Spin In Colum (purple centrifugal column), 14000rpm is centrifuged 2min；
10) (avoid being drawn onto lower floor to another new centrifuge tube by the filtrate (about 400 μ l) obtained in previous step slowly sucking-off Precipitation)；
11) in centrifuge tube, add the AP3/E buffer of filtrate (400 μ l) 1.5 times of volumes (600 μ l), and mixing is played in suction Uniformly；
12) mixed liquor in upper step (include formed precipitation) is added in Dneasy Mini Spin Colum (white from Stem), >=8000rpm is centrifuged 1min, abandons waste liquid；Remaining mixed liquor is added centrifugal column, again extracts；
13) AE is preheated in 65 DEG C of water-baths；
14) taking-up of white centrifugal column is put on 2mL collecting pipe；
15) adding the AW buffer of 500 μ l in centrifugal column, 8000rpm is centrifuged 1min
16) abandon waste liquid, centrifugal column is put back to collecting pipe, then in centrifugal column, add the AW buffer of 500 μ l, 14000rpm Centrifugal 2min；
17) DneasyMiniSpinColum (white centrifugal column) is positioned on 1.5 or 2mL centrifuge tubes, adds 50 μ l The AW of 65 DEG C of preheatings, room temperature places 20min；
18) 8000rpm is centrifuged 1min；
19) if need repeat step 17,18；
20) the DNA marker details that will extract, deposit in-20 DEG C of refrigerators standby.
For identifying sample in a large number, its extracting genome DNA operating procedure is as follows:
1) put into after 50mg blade being weighed in 96 hole sample collection tubes；
2) in collecting pipe, add tungsten carbide pearl and quartz sand, and cover collecting pipe with flexible glue；
3) 65 DEG C preheating AP1 buffer, by 90mL preheat AP1 buffer and 225 μ l RNase A (100mg/ml), 225 μ l Reagent DX mix homogeneously (mixed liquor is lysate), are put in water-bath in 65 DEG C；
4) 2 piece of 96 hole sample collection tube is put in automatization's sample grinder by sample grinding fully；
5) in each collecting pipe, add 400 μ l lysates with the volley of rifle fire, after covering new flexible glue lid, rock mixing up and down；
6) centrifugal, turn up 3000rpm can stop；
7) add 130 μ LAP2 buffer, after vortex is abundant, 96 orifice plates is put in-20 DEG C of refrigerators and stands 10min；
8) 96 orifice plate 6000rpm are centrifuged 5min；
9) throw away lid, be transferred to each collection 400 μ l supernatant in 96 new hole collecting pipes (need to guarantee new during operation Micro-new collector in the right direction)；
10) add 1.5 volume AP3/E buffer in each collecting pipe, cover new flexible glue lid；
11) the most acutely shaking 15s, centrifugal, rotating speed reaches centrifugal during 3000rpm；
12) two DNeasy 96Plates are placed in above S-Blocks, carry out labelling (to avoid sample mix)；
13) remove from miniature collecting pipe and throw away lid, carefully each sample 1ml being transferred to DNeasy 96Plates；
14) DNeasy 96Plate is sealed with AirPore Tape Sheet, centrifugal 4min under 6000rpm；
15) remove gummed paper, each sample adds 500 μ l buffer A W；
16) sealing Sheet DNeasy 96Plate with AirPore Tape, under 6000rpm, centrifugal 15min makes DNeasy film is dried；
17) remove AirPore Tape, each sample adds the AE buffer of 50 μ l 65 DEG C preheating, at 15-25 DEG C Ambient temperatare puts 20min, centrifugal 2min under 6000rpm；
18) the 18th step is repeated；
19) the DNA marker details that will extract, deposit in-20 DEG C of refrigerators standby.
Wherein, above-mentioned pcr amplification reaction program is: 94 DEG C of denaturations 10min；Subsequently into circulation, each circulation 94 DEG C Degeneration 30sec, 52～55 DEG C of annealing 30sec, 72 DEG C extend 90sec, totally 35 thermal cycles, finally extend 72 DEG C and extend 10min； Amplified production is taken out after 4 DEG C of coolings.
Beneficial effect: the present invention can be cost-effective, and does not limit for sample size, overcomes directly use single Site carries out confirming problem the most accurately, and the present invention is more accurate by using 45 SNP site to determine.The method is the easiest OK, reliable and stable, only need very small amount sample just can complete, there is good practical value.Fuding DABAI, can be similar with other Folium Camelliae sinensis as succedaneum, the inventive method can identify easily Fuding DABAI, can be special with from form by Fuding DABAI easily Levy other Folium Camelliae sinensis easily obscured with Fuding DABAI to differentiate.Therefore, the inventive method purchase and Folium Camelliae sinensis to protecting Fuding DABAI The quality of production has highly important using value.
Detailed description of the invention
For making the object, technical solutions and advantages of the present invention clearer, below in conjunction with specific embodiment, to this Bright further description.
Embodiment 1:
1, the choosing and the determination of 45 SNP site of material
Fuding white tea leaf sample is Fuding, different regions DABAI sample.In test, Fuding white tea sample is respectively at 2015 The 6-7 month in year is purchased from Fujian, Zhejiang, Hunan, Hubei, Jiangxi, Jiangsu and Deng Chanchaming district, Anhui.45 SNP positions of Fuding DABAI The gene order of point, according to these 45 SNP site, the corresponding gene found by blast in NCBI.
Table 2 is for examination Fuding, Fujian Province DABAI tea tree breed (being) source and original producton location
Table 3 is with reference to tea tree breed (being) source and original producton location
Table 4 commodity Fuding white tea is originated
2, the exploitation of EST-SNP
(http://www.ncbi.nlm.nih.gov/) Camellia sinensis est sequence is obtained in NCBI GenBank.At NCBI Obtaining 47789 est sequences in data base, these sequences derive from the cDNA of Different Tea Varieties, different tissues and organ Library.
(1) ' noise ' sequence is removed: the est sequence directly obtained from data base usually contains some low-quality small pieces , the most also there is the sequence of polyA/T " tail " with a small amount of carrier sequence and end, remove these sequences in section；(2) EST number According to cluster and splicing: utilize Phrap software cluster and splice；(3) screening: utilize AutoSNP software to screen, row Except the interference of paralog gene, and solve the false positive issue that order-checking mistake causes.Fuding is obtained by said method screening The gene order of 45 SNP site of DABAI.
3, the design of primer and amplification
Use Primer5.0 software at 45 gene order both sides design primers corresponding to candidate SNP, design of primers former Be then: length 18-25bp, product size be 200-600bp, Tm value be 50 DEG C-60 DEG C, G/C content is between 40%-60%.Altogether Devise 45 groups of primer sequences.
4, (using DNeasy Plant Mini Kit test kit, this test kit is at Shang Haijia for extracting genome DNA Close bio tech ltd to buy)
Using following methods to extract genomic DNA from blade, its main operational steps is:
1) put into after 50mg blade being weighed in 96 hole sample collection tubes；
2) in collecting pipe, add tungsten carbide pearl and quartz sand, and cover collecting pipe with flexible glue；
3) 65 DEG C preheating AP1 buffer, by 90mL preheat AP1 buffer and 225 μ l RNase A (100mg/ml), 225 μ l Reagent DX mix homogeneously (mixed liquor is lysate), are put in water-bath in 65 DEG C；
4) 2 piece of 96 hole sample collection tube is put in automatization's sample grinder by sample grinding fully；
5) in each collecting pipe, add 400 μ l lysates with the volley of rifle fire, after covering new flexible glue lid, rock mixing up and down；
6) centrifugal, turn up 3000rpm can stop；
7) add 130 μ LAP2 buffer, after vortex is abundant, 96 orifice plates is put in-20 DEG C of refrigerators and stands 10min；
8) 96 orifice plate 6000rpm are centrifuged 5min；
9) throw away lid, be transferred to each collection 400 μ l supernatant in 96 new hole collecting pipes (need to guarantee new during operation Micro-new collector in the right direction)；
10) add 1.5 volume AP3/E buffer in each collecting pipe, cover new flexible glue lid；
11) the most acutely shaking 15s, centrifugal, rotating speed reaches centrifugal during 3000rpm；
12) two DNeasy 96Plates are placed in above S-Blocks, carry out labelling (to avoid sample mix)；
13) remove from miniature collecting pipe and throw away lid, carefully each sample 1ml being transferred to DNeasy 96Plates；
14) DNeasy 96Plate is sealed with AirPore Tape Sheet, centrifugal 4min under 6000rpm；
15) remove gummed paper, each sample adds 500 μ l buffer A W；
16) sealing Sheet DNeasy 96Plate with AirPore Tape, under 6000rpm, centrifugal 15min makes DNeasy film is dried；
17) remove AirPore Tape, each sample adds the AE buffer of 50 μ l65 DEG C preheating, at 15-25 DEG C Ambient temperatare puts 20min, centrifugal 2min under 6000rpm；
18) the 18th step is repeated；
19) the DNA marker details that will extract, deposit in-20 DEG C of refrigerators standby.
5, PCR amplification and detection
PCR reaction is carried out in Bio-Rad DNA Engine Dyad PCR instrument, and reaction system and response procedures are as follows: 45 pairs of above-mentioned primers are carried out following PCR reaction respectively, respectively obtains 45 PCR primer.
Response procedures is:
The 45 groups of PCR primer obtained respectively through 1.0% agarose gel electrophoresis detect, purpose band use AxyPrep DNA Gel Extraction Kit (Axygen Corp.) reclaims.Draw 5 μ L and reclaim product, coagulate with 1.0% agarose Gel electrophoresis detects the presence of purpose fragment, if having, saves backup at recovery product is placed in-20 DEG C.
6, the preparation of E. coli competent DH5 α and conversion
1), the preparation of E. coli competent DH5 α
(1) take E. coli competent (TaKaRa, Japan) and dissolve on ice, solution is all added the LB liquid to 5mL In body culture medium, in constant-temperature table, at 37 DEG C, 220rpm shaken cultivation is overnight；
(2) 5mL bacterium solution is fully transferred in the 150mL conical flask of the LB fluid medium containing 100mL, shakes in constant temperature 220rpm shaken cultivation 2～3h at 37 DEG C in Chuan, being 0.4～0.5 to OD600, (antibacterial number is less than 108Individual/mL)；
(3) bacterium solution is transferred in the 50mL centrifuge tube of sterilizing, places 10min on ice；
At (4) 4 DEG C, 4 000rpm are centrifuged 10min, pour out culture fluid, are placed in superclean bench by centrifuge tube and are inverted about 1min, so that culture fluid flows to end, precipitates at the bottom of recovery tube；
(5) CaCl of cold 20mmol/L is added210mL suspends precipitation, places 30min on ice；
At (6) 4 DEG C, 4 000rpm are centrifuged 10min, recovery tube ins and outs bacterium；
(7) CaCl of cold 20mmol/L is added2(being previously added 15% glycerol) 2mL suspends precipitation；
(8) competent cell, every part of 100 μ L-150 μ L are encapsulated with cryopreservation tube；
(9) by after competent cell liquid nitrogen flash freezer, preserve stand-by at-70 DEG C.
2) competence converts, checks order and comparison
(1) target fragment (about 1500bp) recovery obtained is attached with pMDl8-T Vector carrier, connector It is as follows:
(2) after above-mentioned reactant liquor being connected 3h at 16 DEG C, for the conversion of E. coli competent DH5 α:
(3) competent cell is taken out to thawed on ice, then 5 μ L is connected liquid and adds to competent cell, mixing；
(4) after ice bath reaches 30min, in 42 DEG C of water-bath heat shock 90s, then 5min is placed rapidly on ice；
(5), after adding the LB fluid medium 1mL of sterilizing, after sealing with sealed membrane, it is placed in constant-temperature table at 37 DEG C 220rpm shaken cultivation 1h；
(6), after cultivating 1h, 4 DEG C of 12 000rpm is centrifuged 1min.
(7) with pipettor Aspirate supernatant 1mL, remaining solution is inhaled and plays mixing, draw bacterium solution L-shaped spreading rod uniform Coat on LB (containing 0.1%Amp) solid medium；
(8) culture dish is inverted in constant incubator 37 DEG C of dark culturing to there being bacterium colony to grow (about 8～12h).
(9) it is previously added the liquid LB medium of 0.1%Amp to 3mL with the single bacterium colony on the toothpick picking flat board of sterilizing In, 220rpm shaken cultivation 8～12h at 37 DEG C；
(10) above-mentioned bacterium solution PCR is reacted (reaction system is 10 μ L, and response procedures is constant) and agarose gel electrophoresis Verify whether to convert successfully.If purpose band can be detected, then explanation escherichia coli convert successfully.Will be able to detect that purpose band Bacterium solution deliver to Nanjing Jin Sirui biotech firm and check order.Sequencing result DNAMAN is analyzed.
7, data process&analysis
By amplified production sequencing result and Fuding DABAI sequence alignment, the base sequence such as sequence table of described Fuding DABAI Middle base sequence, when the similarity of the base sequence shown in the sequence of amplified production is higher, and 45 SNP site base phases With, this sample to be identified is Fuding DABAI；Otherwise, it not Fuding DABAI.
The above is only the preferred embodiment of the present invention, it should be pointed out that: for the ordinary skill people of the art For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should It is considered as protection scope of the present invention.

Claims (6)

1. the primer sets for the Molecular Identification of Fuding DABAI commodity tea, it is characterised in that described primer sets includes that 45 is right Primer sequence, described primer sequence is as shown in SEQ ID NO:1～90.
2. the method for identifying molecules of a Fuding DABAI commodity tea, it is characterised in that comprise the following steps:
1) determining 45 SNP site of Fuding white tea leaf sample, its SNP sequence is as shown in SEQ ID NO:91～135., 45 the corresponding gene orders found by blast in NCBI；
2) design of primer；According to 45 gene orders found, according to the method for homologous clone, find kindred plant, go forward side by side The comparison of line order row, finds the segment area that homology is higher, designs the upstream and downstream primer of these 45 genetic fragments；Described primer Sequence 45 pairs of primer sequences as claimed in claim 1；
3) extracting genome DNA；
4) PCR amplification and detection；
5) with Fuding DABAI kind bar code comparison after amplified production being checked order, the base sequence of Fuding DABAI kind bar code, The similarity of the SNP sequence of amplified production and the distinguishing base sequence shown in the DABAI kind bar code of Fuding reach 99.5% with On, then this sample to be identified is Fuding DABAI kind.
The method for identifying molecules of a kind of Fuding the most according to claim 2 DABAI commodity tea, it is characterised in that described primer Design length 18-25bp, product size be 200-600bp, Tm value be 50 DEG C-60 DEG C, G/C content is between 40%-60%.
The method for identifying molecules of a kind of Fuding the most according to claim 2 DABAI commodity tea, it is characterised in that described gene Group DNA extraction uses following methods to extract genomic DNA from blade, and for identifying sample on a small quantity, its extracting genome DNA is grasped Make step as follows:
1) put into after 50mg blade being weighed in 2mL centrifuge tube；
2) in 2mL centrifuge tube, tungsten carbide pearl and quartz sand are added；
3) centrifuge tube is put in automatization's sample grinder by sample grinding fully；
4) in each 2mL centrifuge tube, add 400 μ l AP1 buffer and 4 μ l RNase A, build rear vortex abundant；
5) by mixture water-bath 20min at 65 DEG C, abundant every 5min vortex；
6) ice chest is prepared；
7), after water-bath terminates, 130 μ LAP2 buffer are added, after vortex is abundant, ice bath 5min；
8) 14000rpm is centrifuged 5min；
9) careful for the supernatant being centrifuged in previous step sucking-off is placed in purple centrifugal column QIA shredder Mini Spin In Colum, 14000rpm is centrifuged 2min；
10) by the slow sucking-off of filtrate that obtains in previous step to another new centrifuge tube；
11) in centrifuge tube, add the AP3/E buffer of 1.5 times of volumes of filtrate, and mix homogeneously is beaten in suction；
12) being added in white centrifugal column Dneasy Mini Spin Colum by mixed liquor in upper step, >=8000rpm is centrifuged 1min, abandons waste liquid；Remaining mixed liquor is added centrifugal column, again extracts；
13) AE is preheated in 65 DEG C of water-baths；
14) taking-up of white centrifugal column is put on 2mL collecting pipe；
15) adding the AW buffer of 500 μ l in centrifugal column, 8000rpm is centrifuged 1min
16) abandoning waste liquid, centrifugal column puts back to collecting pipe, then add the AW buffer of 500 μ l in centrifugal column, 14000rpm is centrifuged 2min；
17) white centrifugal column DneasyMiniSpinColum is positioned on 1.5 or 2mL centrifuge tubes, adds 50 μ l 65 DEG C pre- The AW of heat, room temperature places 20min；
18) 8000rpm is centrifuged 1min；
19) repeat step 17,18；
20) the DNA marker details that will extract, deposit in-20 DEG C of refrigerators standby.
The method for identifying molecules of a kind of Fuding the most according to claim 2 DABAI commodity tea, it is characterised in that described gene Group DNA extraction uses following methods to extract genomic DNA from blade, and for identifying sample in a large number, its extracting genome DNA is grasped Make step as follows:
1) put into after 50mg blade being weighed in 96 hole sample collection tubes；
2) in collecting pipe, add tungsten carbide pearl and quartz sand, and cover collecting pipe with flexible glue；
3) 65 DEG C of preheating AP1 buffer, the AP1 buffer that 90mL is preheated and 225 μ l RNase A, 225 μ l Reagent DX Mix homogeneously, is put in water-bath in 65 DEG C；
4) 2 piece of 96 hole sample collection tube is put in automatization's sample grinder by sample grinding fully；
5) in each collecting pipe, add 400 μ l lysates with the volley of rifle fire, after covering new flexible glue lid, rock mixing up and down；
6) centrifugal, turn up 3000rpm can stop；
7) add 130 μ LAP2 buffer, after vortex is abundant, 96 orifice plates is put in-20 DEG C of refrigerators and stands 10min；
8) 96 orifice plate 6000rpm are centrifuged 5min；
9) throw away lid, each collection 400 μ l supernatant is transferred in 96 new hole collecting pipes；
10) add 1.5 volume AP3/E buffer in each collecting pipe, cover new flexible glue lid；
11) the most acutely shaking 15s, centrifugal, rotating speed reaches centrifugal during 3000rpm；
12) two DNeasy 96Plates are placed in above S-Blocks, carry out labelling；
13) remove from collecting pipe and throw away lid, carefully each sample 1ml being transferred to DNeasy 96Plates；
14) DNeasy 96Plate is sealed with AirPore Tape Sheet, centrifugal 4min under 6000rpm；
15) remove gummed paper, each sample adds 500 μ l buffer A W；
16) sealing Sheet DNeasy 96Plate with AirPore Tape, under 6000rpm, centrifugal 15min makes DNeasy film It is dried；
17) remove AirPore Tape, each sample adds the AE buffer of 50 μ l 65 DEG C preheating, 15-25 DEG C of room temperature Lower placement 20min, centrifugal 2min under 6000rpm；
18) the 18th step is repeated；
19) the DNA marker details that will extract, deposit in-20 DEG C of refrigerators standby.
The method for identifying molecules of a kind of Fuding the most according to claim 2 DABAI commodity tea, it is characterised in that described PCR Amplified reaction program is: 94 DEG C of denaturations 10min；Subsequently into circulation, 94 DEG C of degeneration 30sec of each circulation, 52～55 DEG C are moved back Fire 30sec, 72 DEG C extend 90sec, totally 35 thermal cycles, finally extend 72 DEG C and extend 10min；Take out amplification after 4 DEG C of coolings to produce Thing.
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CN106480224A (en) * 2016-12-28 2017-03-08 中国农业科学院茶叶研究所 The molecular marker combination of Rapid identification difference albino tea tree breed, method and application
CN114350847A (en) * 2022-02-11 2022-04-15 四川农业大学 SNP (Single nucleotide polymorphism) site for identifying early tea trees and application thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106480224A (en) * 2016-12-28 2017-03-08 中国农业科学院茶叶研究所 The molecular marker combination of Rapid identification difference albino tea tree breed, method and application
CN106480224B (en) * 2016-12-28 2019-09-24 中国农业科学院茶叶研究所 Molecular labeling combination, method and the application of Rapid identification difference albino tea tree breed
CN114350847A (en) * 2022-02-11 2022-04-15 四川农业大学 SNP (Single nucleotide polymorphism) site for identifying early tea trees and application thereof
CN114350847B (en) * 2022-02-11 2023-09-22 四川农业大学 SNP locus for identifying early-maturing tea trees and application thereof

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