CN106755308A - Screen the '-hydroxylase gene functional label of flavonoids 3 ', 5 ' and its application, application process of dihydroxy catechin tea tree high - Google Patents
Screen the '-hydroxylase gene functional label of flavonoids 3 ', 5 ' and its application, application process of dihydroxy catechin tea tree high Download PDFInfo
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Abstract
The invention belongs to biological technical field, and in particular to a kind of the '-hydroxylase gene functional label of flavonoids 3 ', 5 ' and its application, application process of screening dihydroxy catechin tea tree high.The invention discloses the functional label for identifying dihydroxy catechin tea tree plant high, functional label in the present invention is applied into molecular marker assisted selection, the tea tree vegetable material with dihydroxy catechin high is allowed rapid screening out, so as to accelerate the incubation paces of high-quality tea tree breed.The present invention using molecular marker assisted selection dihydroxy catechin tea tree breed high to having important theory significance and economic worth.
Description
Technical field
The invention belongs to biological technical field, and in particular to a kind of flavonoids 3 ' of screening dihydroxy catechin tea tree high,
5 '-'-hydroxylase gene functional label and its application, application process.
Background technology
Catechin（catechins）It is tea tree teas flavones（flavonoids）Bulk composition, account for green tea dry weight
8-26% (Chen ＆ Zhou, 2005), into tea nearly all characteristic such as flavour, soup look and fragrance etc. all directly or indirectly with
Catechin is relevant（Wang et al., 2000）.Catechin has anti-oxidant, anti-mutagenesis, anticancer, anti-cardiovascular disease, ultraviolet
Treatment of beta radiation protective effect, anti-diabetic, anti-inflammation, fat-reducing and Parkinson's etc. is acted on（Xia Tao and Koryo duckweed,
2009）, belong to flavan-3-alcohol class chemical combination.Catechin is the derivative that flavylium ion is muttered, according to the hydroxyl of catechin B rings
On number, C rings on 2,3 isomers, C rings 3 whether connect gallic acid etc. and can be divided into several component.According to
The hydroxy number of catechin B rings, catechin can be largely classified into the double hydroxyls of B rings and trihydroxy catechin, wherein epicatechin
(EC) and L-Epicatechin gallate (ECG) belongs to B ring dihydroxy catechins, and epigallocatechin (EGC) and table
Nutgall catechin gallic acid ester (EGCG) belongs to B ring trihydroxy catechins.3 ', 5 '-hydroxylase of flavonoids
(flavonoid 3 ', 5 '-hydroxylase, F3 ' 5 ' H) is the important enzyme in the synthesis of tea tree flavan-3-alcohol.F3′5′H
Belong to CYP75A subfamilies, can respectively be catalyzed flavones, flavanones, flavanonol and flavonols and be converted into 3 ', 4 ', 5 ' three
Hydroxylation product, is unique enzyme system of the hydroxylating of catalysis B- rings 5 ' in the Cytochrome P450 family being currently known.
During fermentation of black tea, the formation of each theaflavin molecule needs 1 dihydroxy catechin and 1 trihydroxy
Theine, therefore the fresh leaf of the dihydroxy catechin containing isoconcentration and trihydroxy catechin is red most beneficial for high-quality is formed
Tea.But patent applicant has carried out system identification at 2010 and 2011 to 403 parts of Resources of Tea Plant catechin compositions, find with
The main product black tea country such as Kenya compares, and China is no lack of high-catechin resource, but EGCG account for catechin total amount ratio it is very high, put down
It is 59.3-61.3%（Jin et al., 2014）, far above Kenya（All kinds are all below 32%）（Owuor &
Obanda, 2007）, and dihydroxy catechins ECG and EC contents are relatively low.Therefore, the improvement of China's tea tree breed from now on, especially
It is the breed breeding of black tea, it is necessary to screen and utilize the Resources of Tea Plant of dihydroxy catechin high, and then cultivates the black tea of high-quality
Kind.
So far, biochemical measurement method is mostly taken in domestic and international tea catechin content identification.The method needs certain
Quantity tea leaf, the plant of identification could identify after needing to be long to 3-4, expend overlong time, inefficiency.Additionally, mirror
Be colonized the catechin content of strain by planting environment and the tea tree age of tree influenceed it is very big, it is necessary to for many years, multiple spot identification can just accomplish accurate
Evaluate.With developing rapidly for Protocols in Molecular Biology, molecular marker assisted selection (MAS) is widely used in molecular breeding
In, because it is not influenceed by environment and breeding generation, Seedling selection and prediction can be carried out, substantially reduce Tea Breeding year
Limit.Functional label is the mark that a class is based on the exploitation of gene particular sequence, is isolated with target gene, substantially increases selection
Accuracy.
The content of the invention
For the problem that prior art is present, a kind of screening dihydroxy catechin high is provided it is an object of the invention to design
3 ', 5 '-'-hydroxylase gene of flavonoids functional label of tea tree and its application, the technical scheme of application process.
A kind of flavonoids 3 ', 5 '-'-hydroxylase gene functional label of described screening dihydroxy catechin tea tree high, it is special
Levy be the functional label upstream primer sequence as shown in SEQ ID No.1, anti-sense primer is as shown in SEQ ID No.2.
A kind of 3 ', 5 '-'-hydroxylase gene of flavonoids functional label of described screening dihydroxy catechin tea tree high is in sieve
Choosing is with the application in dihydroxy catechin tea tree high, it is characterised in that the upstream primer sequence of the functional label such as SEQ ID
Shown in No.1, anti-sense primer is as shown in SEQ ID No.2.
A kind of 3 ', 5 '-'-hydroxylase gene of flavonoids functional label of described screening dihydroxy catechin tea tree high is in sieve
Choosing is with the application process in dihydroxy catechin tea tree high, it is characterised in that comprise the following steps：
Using functional label to the bp to the bp of downstream 871 of the H gene initiation codon ATG of F3 ' 5 ' downstreams 676 in each tea plant material
DNA fragmentation enter performing PCR amplification, digestion and agarose gel analysis, if the fragment for being shown as 196bp after amplified production digestion
The tea tree is defined as low dihydroxy catechin tea tree, and the tea tree determines if the fragment of 176bp is shown as after amplified production digestion
It is dihydroxy catechin tea tree high.
Described application process, it is characterised in that the system and response procedures of described PCR amplifications be：PCR reaction systems：
32 µL ddH2The mM MgCl of O, 2 μ L DNA, 5 μ 10 × PCR of L Buffer, 4 μ L 252, 5 μ L 2 Mm dNTP, 1 μ L
KOD-Plus-Neo enzymes, 10 μM of each 3 μ L of upstream and downstream primer；PCR amplification programs are：94 DEG C of 2 min, then carry out with
Lower circulation, 94 DEG C of 15 sec, 58 DEG C of 25 sec, 68 DEG C of 5 sec, totally 35 circulations；Last 68 DEG C of extensions 2min；Described
Digestion and Ago-Gel condition are：PCR primer 10 μ L, restriction endonuclease EcoRI 10U, the μ L of inscribe enzyme buffer liquid 2, use ddH2O is mended
Together to 20 μ L digestion systems；The endonuclease reaction time is 1 hour, and temperature is 37 DEG C, agarose gel electrophoresis point of the amplified production 3%
From.
Compared with prior art, the advantage of the invention is that：Functional label in the present invention is applied into molecular labeling
Assisted Selection, allows rapid screening out the tea tree vegetable material with dihydroxy catechin high, so as to accelerate high-quality black tea tea tree product
The incubation paces planted.The present invention using molecular marker assisted selection dihydroxy catechin tea tree breed high to having important theory
Meaning and economic worth.
Brief description of the drawings
Fig. 1 is Dragon Well tea 43 and phoenix giant tea tree 3 ', 5 '-'-hydroxylase gene of flavonoidsF3′5′HComparison spectrogram；
In Fig. 1：LJ43 is Dragon Well tea 43, and FFDCS is phoenix giant tea tree.
Fig. 2 is the comparison spectrogram of Dragon Well tea 43 and phoenix giant tea tree digestion band difference；
In Fig. 2：M is Marker；The PCR primer that 1 and 2 DNA for being respectively Dragon Well tea 43 are expanded with primer pair 1 and 2 is through restriction endonucleaseEcoRBand before and after I digestions, the PCR primer that 3 and 4 DNA for being respectively phoenix giant tea tree are expanded with primer pair 1 and 2 is through inscribe
EnzymeEcoRBand before and after I digestions.
Fig. 3 is to 28 parts of genotype call results of Resources of Tea Plant SNP848 with the H of functional label dCAPS-F3 ' 5 '；
In Fig. 3：M is Marker；1-10 is followed successively by Fuding white tea, vine tea, white stone tooth tea, icicles, the big white tea in Fuan, Ning Zhouhou
No. 4 in leaf kind, Jiang Hua Sweet tea, big sun tea, water Gu tea and wood, stripe size is 196bp, the base of SNP848 after PCR primer digestion
Because type is AA；11-20 is followed successively by that Lechang youth field herb mixtures tea, newborn source willow cheat No. 1, Taishan white clouds tea, Ying Hong 1, the Five Ridges is red, Lechang is sharp
Leaf white hair, the red bud kind in Luoding, tin tea 10, daye tea colony and Red River Lang Di tea, stripe size is after PCR primer digestion
The genotype of 196bp and 176bp, SNP848 is AG；21-28 is followed successively by phoenix giant tea tree, master of great learning and integrity No. 5, town river flowing from Guizhou Province through Hunan into Dongting Lake 2, Changning 4
Number, Jing Gu Lao Cang colonies, Malipo 8, the general stamen of cloud tea and Yun County 1, stripe size is 176bp after PCR primer digestion,
SNP848 genotype is AG.
Specific embodiment
The present invention will be further described in detail with reference to the accompanying drawings and detailed description.
Reagent used in following embodiments, unless otherwise specified, can be commercially available from routine biochemistry reagent shop.With
Quantitative test in lower embodiment, is respectively provided with three repetitions and tests, results averaged.
Embodiment 1
F3′5′HThe exploitation of the discovery, primer pair and the H of functional label dCAPS-F3 ' 5 ' of allelic differences sequence
1. material to be tested
Selection Dragon Well tea 43 and phoenix giant tea tree are research material.
2. catechin content is determined
The two leaves and a bud of tea tree spring young sprout is plucked, sample is consolidated in time using 120 DEG C of heated-air drying 5min, 75 DEG C are dried to the dry low temperature of foot
Preserve.Ground with machinery when to be determined, cross Cord blood after 40 mesh sieves, it is standby.Weigh 0.2g samples（It is accurate to 0.0001g）,
It is placed in 10mL centrifuge tubes.Add 70 DEG C of 70% methanol solution 5mL of preheating, 70 DEG C of water-baths after mixing.Water-bath 10min, Zhong Jian Oscillating
It is even 2-3 times.3,500r/min centrifugation 10min, shift supernatant.Repeat step 2-4, the supernatant that merging is collected twice, constant volume
To 10mL.Pipette 2ml extract solutions to be placed in volumetric flask, use stabilizing solution（5% 10mg/mL EDTA solution, 5% 10mg/mL resist
Bad hematic acid solution, 10% acetonitrile）10ml is settled to, with 0.45 μm of membrane filtration after mixing.Using high pressure lipuid chromatography (HPLC)
（HPLC）Detected, qualitative and quantitative analysis is carried out to alkaloid and catechin with external standard method.Liquid chromatogram measuring condition：
Mm × 250 mm of C12 chromatographic columns 4.6 (4 μm, Guangzhou Féraud door)；Mobile phase A is 0.5% formic acid, and Mobile phase B is acetonitrile, stream
Fast 1mL/min, 30 DEG C of column temperature, Detection wavelength 280nm, the μ L of sample size 10, gradient elution：Mobile phase B is in 16min by 6.5%
Linear gradient changes to 16%, and 16min to 20min changes to 25% by 16% linear gradient, keeps 5min, returns to initial shape
State, releveling 5min.Measurement result shows that the trihydroxy catechin content of phoenix giant tea tree is suitable with Dragon Well tea 43, but dihydroxy
Catechin content is but 2.4 times of Dragon Well tea 43.
12 parts of differences of tea plant material spring tea catechin content of table（mg/g）
| Title material | EGC | EC | EGCG | ECG | Trihydroxy catechin（EGC+EGCG） | Dihydroxy catechin（EC+ECG） | Catechin total amount |
| Dragon Well tea 43 | 8.2 | 5.4 | 69.2 | 32.5 | 77.4 | 37.9 | 115.3 |
| Phoenix giant tea tree | 16.3 | 22.7 | 65.9 | 68.3 | 82.2 | 91.0 | 173.2 |
3. the extraction of genomic DNA
The fresh tender tips of 1g are taken, liquid feeding nitrogen is ground to powdered.0.2g powder is inserted into 1.5mL centrifuge tubes, plus 700 μ L CTAB are carried
Liquid is taken, water-bath 1h at 65 DEG C, even once per 20min Oscillating after fully mixing.Add isometric chloroform/isoamyl alcohol（24：1）, mix
After stand 2min.14,000g centrifugations 10min, takes supernatant at room temperature.Add the isopropanol of isometric precooling, -20 DEG C of standing 1h.
14,000g centrifugation 10min, abandon supernatant.Add 300 μ L high level salt solutions, 65 DEG C incubate 30min to precipitating dissolving.At room temperature 10,
000g is centrifuged 10min, takes supernatant.Add the NaAc of 1/10 volume precooling（pH 5.2）, the isopropanol of 2/3 volume precooling, fully
Mixing, -20 DEG C of placement 30min.14,000g centrifugation 5min, abandon supernatant.70% ethanol washing precipitation 1 time, absolute ethanol washing one
It is secondary.It is placed on and 30min is dried up on superclean bench, is dissolved in 200 μ L aqua sterilisas, -20 DEG C of preservations.
4.PCR is sequenced and sequencing analysis
Design special primer, in each tea plant material of amplificationF3′5′HThe bp of initiation codon ATG upstreams 54 is to the bp's of downstream 891
DNA fragmentation, primer is designed by software Primer5.0, and sequence is as follows：
Sense primer（As shown in SEQ ID No.3）：5 '-ACCAAAACACTCAACCAGGT-3 ',
Anti-sense primer（As shown in SEQ ID No.4）：5 '-TGCCTTGATGTTGGTCGTGT-3 '；
PCR reaction systems：32 µL ddH2The mM MgCl of O, 2 μ L DNA, 5 μ 10 × PCR of L Buffer, 4 μ L 252, 5 μ
The Mm dNTP of L 2,1 μ L KOD-Plus-Neo enzymes, 10 μM of each 3 μ L of forward and reverse primer.PCR amplification programs are：94℃ 2
Min, then carries out following circulation, 94 DEG C of 15 sec, 53 25 sec, 68 DEG C of 30 sec, totally 35 circulations；Last 68 DEG C
Extend 7min.Pcr amplification product carries out electrophoresis on 1.2% Ago-Gel, and observation and gel extraction, are connected under uviol lamp
Carrier, conversion, the sequencing of bacterium solution PCR screening positive clones.Analysis finds there are 3 mononucleotides in two parts of storeroom First Exons
Mutation（SNP）, wherein only SNP848 is nonsynonymous mutation（Fig. 1）.
5. the exploitation of the H of functional label dCAPS-F3 ' 5 '
Design special primer, in each tea plant material of amplificationF3′5′HThe bp of gene start codon ATG downstreams 676 is to downstream 871
The DNA fragmentation of bp, primer is by using dCAPS Finder 2.0（http://helix.wustl.edu/dcaps/
dcaps.html）Design.The mispairing of 1bp is introduced at the end of anti-sense primer 3 ', sequence is as follows：
Sense primer（As shown in SEQ ID No.1）：5 '-GATTTCATACCATCGATTGCGT-3 ',
Anti-sense primer（As shown in SEQ ID No.2）：5 '-TGAGCTTCTCTTCACCAGGAATT-3 '；
Enter performing PCR amplification, digestion and agarose gel analysis respectively using described primer sequence.
PCR reaction systems：32 µL ddH2The mM MgCl of O, 2 μ L DNA, 5 μ 10 × PCR of L Buffer, 4 μ L 252,
The Mm dNTP of 5 μ L 2,1 μ L KOD-Plus-Neo enzymes, 10 μM of each 3 μ L of forward and reverse primer.
PCR amplification programs are：94 DEG C of 2 min, then carries out following circulation, 94 DEG C of 15 sec, 58 DEG C of 25 sec, 68
DEG C 5 sec, totally 35 circulations；Last 68 DEG C of extensions 2min.
Digestion system, time, temperature and agarose gel electrophoresis are：The μ L of PCR primer 10, restriction endonucleaseEcoRI 10U, it is interior
The μ L of enzyme cutting buffer solution 2, use ddH2O polishings are to 20 μ L；The endonuclease reaction time is 1 hour, and temperature is 37 DEG C, and amplified production is 3%
Agarose gel electrophoresis are separated.
Analysis finds that still stripe size is still 196bp after the PCR primer digestion of Dragon Well tea 43, shows the genotype of SNP848
It is AA, and 1 band of 176bp is generated after phoenix giant tea tree digestion, the genotype for showing SNP848 is GG（Fig. 2）.Utilize
The molecular labeling can identify Resources of Tea PlantF3′5′HThe genotype of gene SNP 848, and can be the tea containing dihydroxy catechin high
Tree material is screened.
Embodiment 2
Genotypings of the H of functional label dCAPS-F3 ' 5 ' to Resources of Tea Plant
1. material to be tested
The material that this experiment is studied is listed in Table 2 below, including 28 parts of Resources of Tea Plant.
28 parts of studied Resources of Tea Plant of 2 experiments of table and its dihydroxy catechin content
2. functional label is to different Resources of Tea PlantF3′5′HThe genotype detection of gene SNP 848
Using the H of molecular labeling dCAPS-F3 ' 5 ' to 28 parts of marker gene type analysis of material.DNA extractions, PCR amplification system, journey
Sequence, digestion and Ago-Gel condition are with embodiment 1.Result is as shown in figure 3, be AA, material wherein in the genotype of material 1-10
The genotype of material 11-20 is AG, and the genotype of material 21-28 is GG.
3. 28 parts of tea plant material catechin content identifications
Catechin authentication method high performance liquid chromatography is with described in embodiment 1.In 28 parts of materials, genotype is 10 parts of materials of AA
Dihydroxy catechin content is 32.6 ± 4.6 mg/g, genotype for AG 10 parts of material dihydroxy catechin contents for 42.0 ±
8.9 mg/g, genotype is 84.1 ± 22.2 mg/g for 8 parts of material dihydroxy catechin contents of GG.Statistical analysis shows, GG
The material dihydroxy catechin content pole of genotype is significantly higher than other 2 kinds of materials of genotype, and the H of dCAPS-F3 ' 5 ' can make
To identify and screening the functional label of dihydroxy catechin tea plant material high.
Specific embodiment described herein is only to the spiritual explanation for example of the present invention.Technology neck belonging to of the invention
The technical staff in domain can be made various modifications or supplement to described specific embodiment or be replaced using similar mode
Generation, but without departing from spirit of the invention or surmount scope defined in appended claims.
SEQUENCE LISTING
<110>Tea Inst., Chinese Academy of Agricultural Sciences
<120>Screen 3 ', 5 '-'-hydroxylase gene of flavonoids functional label and its application, the application of dihydroxy catechin tea tree high
Method
<130> 11
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 22
<212> DNA
<213>It is artificial synthesized
<400> 1
gatttcatac catcgattgc gt 22
<210> 2
<211> 23
<212> DNA
<213>It is artificial synthesized
<400> 2
tgagcttctc ttcaccagga att 23
<210> 3
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 3
accaaaacac tcaaccaggt 20
<210> 4
<211> 20
<212> DNA
<213>It is artificial synthesized
<400> 4
tgccttgatg ttggtcgtgt 20
Claims (4)
1. a kind of flavonoids 3 ', 5 '-'-hydroxylase gene functional label for screening dihydroxy catechin tea tree high, it is characterised in that should
As shown in SEQ ID No.1, anti-sense primer is as shown in SEQ ID No.2 for the upstream primer sequence of functional label.
2. a kind of 3 ', 5 '-'-hydroxylase gene of flavonoids functional label for screening dihydroxy catechin tea tree high has height in screening
Application in dihydroxy catechin tea tree, it is characterised in that the upstream primer sequence of the functional label as shown in SEQ ID No.1,
Anti-sense primer is as shown in SEQ ID No.2.
3. a kind of flavonoids 3 ', 5 '-'-hydroxylase gene function of screening dihydroxy catechin tea tree high as claimed in claim 2
Screening is marked at the application process in dihydroxy catechin tea tree high, it is characterised in that comprise the following steps：
Using functional label to the bp to the bp of downstream 871 of the H gene initiation codon ATG of F3 ' 5 ' downstreams 676 in each tea plant material
DNA fragmentation enter performing PCR amplification, digestion and agarose gel analysis, if the fragment for being shown as 196bp after amplified production digestion
The tea tree is defined as low dihydroxy catechin tea tree, and the tea tree determines if the fragment of 176bp is shown as after amplified production digestion
It is dihydroxy catechin tea tree high.
4. application process as claimed in claim 3, it is characterised in that the system and response procedures of described PCR amplifications be：PCR
Reaction system：32 µL ddH2The mM MgCl of O, 2 μ L DNA, 5 μ 10 × PCR of L Buffer, 4 μ L 252, the Mm of 5 μ L 2
DNTP, 1 μ L KOD-Plus-Neo enzymes, 10 μM of each 3 μ L of upstream and downstream primer；PCR amplification programs are：94 DEG C of 2 min, so
After carry out following circulation, 94 DEG C of 15 sec, 58 DEG C of 25 sec, 68 DEG C of 5 sec, totally 35 circulations；Last 68 DEG C of extensions
2min；Described digestion and Ago-Gel condition be：PCR primer 10 μ L, restriction endonuclease EcoRI 10U, the μ of inscribe enzyme buffer liquid 2
L, uses ddH2O polishings are to 20 μ L digestion systems；The endonuclease reaction time is 1 hour, and temperature is 37 DEG C, agar of the amplified production 3%
Carbohydrate gum is separated by electrophoresis.
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| CN115961086A (en) * | 2023-01-17 | 2023-04-14 | 华中农业大学 | Tea tree flavonoid 3-O-methyltransferase gene and application thereof |
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