CN106755308B - Flavonoid 3 ', 5' -hydroxylase gene function marker for screening high-dihydroxy catechin tea tree and application method thereof - Google Patents
Flavonoid 3 ', 5' -hydroxylase gene function marker for screening high-dihydroxy catechin tea tree and application method thereof Download PDFInfo
- Publication number
- CN106755308B CN106755308B CN201611026548.9A CN201611026548A CN106755308B CN 106755308 B CN106755308 B CN 106755308B CN 201611026548 A CN201611026548 A CN 201611026548A CN 106755308 B CN106755308 B CN 106755308B
- Authority
- CN
- China
- Prior art keywords
- catechin
- dihydroxy
- tea
- tea tree
- enzyme digestion
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/156—Polymorphic or mutational markers
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Mycology (AREA)
- Botany (AREA)
- Immunology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a flavonoid 3 ', 5' -hydroxylase gene functional marker for screening high-dihydroxy catechin tea trees, and an application method thereof. The invention discloses a functional marker for identifying tea plant with high-dihydroxy catechin, which is applied to molecular marker-assisted selection and can quickly screen out tea plant materials with high-dihydroxy catechin, thereby accelerating the pace of breeding high-quality tea varieties. The invention has important theoretical significance and economic value for utilizing molecular markers to assist in selecting high-dihydroxy catechin tea tree varieties.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a flavonoid 3 ', 5' -hydroxylase gene functional marker for screening high-dihydroxy catechin tea trees, and an application method thereof.
Background
Catechins (catechins) are the major components of tea tree tea flavonoids (flavanoids) and account for 8-26% of the dry weight of green tea (Chen & Zhou, 2005), and almost all properties of finished tea such as taste, liquor and aroma are directly or indirectly related to catechins (Wang et al, 2000). Catechin has effects of resisting oxidation, resisting mutagenesis, resisting cancer, cardiovascular disease, ultraviolet radiation protection, resisting diabetes, resisting bacteria, diminishing inflammation, reducing weight, and treating Parkinson disease (summer waves and Korean duckweed, 2009), and belongs to flavan-3-ol compound. Catechin is a derivative of 2-phenylbenzopyran, and can be divided into several components according to the number of hydroxyl groups in the B ring of catechin, isomers of 2, 3-position on C ring, and whether gallic acid is connected to 3-position on C ring. Catechins can be mainly classified into B-ring di-and tri-hydroxy catechins according to the number of hydroxyl groups of the B-ring of catechin, in which Epicatechin (EC) and epicatechin gallate (ECG) belong to B-ring dihydroxy catechin, and Epigallocatechin (EGC) and epigallocatechin gallate (EGCG) belong to B-ring tri-hydroxy catechin. Flavonoid 3 ', 5' -hydroxylase (F3 '5' H) is an important enzyme in the synthesis of tea flavan-3-ol. F3 '5' H belongs to CYP75A subfamily, can respectively catalyze flavone, flavanone, flavanonol and flavonol to be converted into 3 ', 4', 5 'trihydroxy products, and is the only enzyme system for catalyzing B-ring 5' hydroxylation reaction in the currently known cytochrome P450 family.
During black tea fermentation, 1 dihydroxy catechin and 1 trihydroxy catechin are required for each theaflavin molecule formation, so fresh leaf raw materials containing equal concentrations of dihydroxy catechin and trihydroxy catechin are most beneficial for forming high quality black tea. However, the patent applicant systematically identifies 403 tea tree resource catechin components in 2010 and 2011, and finds that compared with the countries mainly producing black tea such as kenya, China does not have high catechin resources, but the proportion of EGCG in the total amount of catechin is high, and is averagely 59.3-61.3% (Jin et al, 2014), which is much higher than that of kenya (all varieties are below 32%) (Owuor & Obanda, 2007), and the content of dihydroxycatechin ECG and EC is lower. Therefore, in future improvement of tea plant varieties in China, particularly in breeding of black tea varieties, tea plant resources with high dihydroxy catechin need to be screened and utilized, and then high-quality black tea varieties need to be cultivated.
Until now, biochemical determination methods are mostly adopted for the identification of the catechin content of tea at home and abroad. The method needs a certain amount of tea tree leaves, and the identified plants can be identified after 3-4 years, so that the method consumes too long time and has low efficiency. In addition, the catechin content of the identified plant is greatly influenced by the cultivation environment and the age of the tea tree, and accurate evaluation can be realized only by identifying for many years and multiple points. With the rapid development of molecular biology technology, molecular Marker Assisted Selection (MAS) is widely applied to molecular breeding, and because MAS is not influenced by environment and breeding generation, early selection and prediction can be performed, and the breeding period of tea trees is greatly shortened. The functional markers are developed based on specific gene sequences, and are co-separated from target genes, so that the accuracy of selection is greatly improved.
Disclosure of Invention
Aiming at the problems in the prior art, the invention aims to design and provide a technical scheme for screening a flavonoid 3 ', 5' -hydroxylase gene functional marker of a high-dihydroxy catechin tea tree and an application and application method thereof.
The flavonoid 3 ', 5' -hydroxylase gene function marker for screening the high-dihydroxy catechin tea tree is characterized in that an upstream primer sequence of the function marker is shown as SEQ ID No.1, and a downstream primer is shown as SEQ ID No. 2.
The application of the flavonoid 3 ', 5' -hydroxylase gene functional marker for screening the high-dihydroxy catechin tea tree in screening the tea tree with the high-dihydroxy catechin is characterized in that the sequence of an upstream primer of the functional marker is shown as SEQ ID No.1, and the sequence of a downstream primer is shown as SEQ ID No. 2.
The application method of the flavonoid 3 ', 5' -hydroxylase gene functional marker for screening the tea tree with high-dihydroxy catechin in screening the tea tree with high-dihydroxy catechin is characterized by comprising the following steps:
and carrying out PCR amplification, enzyme digestion and agarose gel analysis on DNA fragments from 676 bp downstream to 871 bp downstream of the ATG (start codon) of the F3 '5' H gene in each tea tree material by using a functional marker, wherein the tea tree is determined to be a low-dihydroxy catechin tea tree if the amplified product shows a 196bp fragment after enzyme digestion, and the tea tree is determined to be a high-dihydroxy catechin tea tree if the amplified product shows a 176bp fragment after enzyme digestion.
The application method is characterized in that the PCR amplification system and the reaction program are as follows: and (3) PCR reaction system: 32 mu L ddH2O，2 µL DNA，5 µL 10×PCR Buffer，4 µL 25 mM MgCl 23 muL of upstream and downstream primers of 5 muL 2 Mm dNTP, 1 muLKOD-Plus-Neo enzyme and 10 muM respectively; the PCR amplification procedure was:94 ℃ for 2min, then 35 cycles of 94 ℃ for 15 sec, 58 ℃ for 25 sec, 68 ℃ for 5 sec; finally extending for 2min at 68 ℃; the conditions of enzyme digestion and agarose gel are as follows: PCR product 10. mu.L, endonuclease EcoRI 10U, endonuclease buffer 2. mu.L, and ddH2Supplementing O to 20 mu L of enzyme digestion system; the enzyme digestion reaction time is 1 hour, the temperature is 37 ℃, and the amplification product is separated by electrophoresis in 3 percent agarose gel.
Compared with the prior art, the invention has the advantages that: the functional marker is applied to molecular marker-assisted selection, and the tea plant material with high-dihydroxy catechin can be quickly screened out, so that the pace of breeding high-quality black tea tree varieties is accelerated. The invention has important theoretical significance and economic value for utilizing molecular markers to assist in selecting high-dihydroxy catechin tea tree varieties.
Drawings
FIG. 1 shows the genes for the flavonoid 3 ', 5' -hydroxylase of Longjing 43 and Fenghuang tea treeF3′ 5′HComparing the spectrogram;
in fig. 1: LJ43 is Longjing 43, FFDCS is Phoenix tea tree.
FIG. 2 is a comparison spectrogram of the difference of enzyme digestion bands of Longjing 43 and Fenghuang tea trees;
in fig. 2: m is Marker; 1 and 2 are DNA of Longjing 43 respectively, and the PCR products amplified by the primer pairs 1 and 2 are treated by endonucleaseEcoRI strips before and after enzyme digestion, 3 and 4 are PCR products amplified by primer pairs 1 and 2 for DNA of phoenix big tea trees respectively through endonucleaseEcoRI bands before and after enzyme cleavage.
FIG. 3 shows the result of genotype detection of 28 tea plant resource SNP848 by functional marker dCAPS-F3 '5' H;
in fig. 3: m is Marker; 1-10 are Fuding big white tea, vine tea, white shiya tea, silver bamboo shoot, Fuan big white tea, Ningzhou thick leaf seed, Jianghua sweet tea, Dayang tea, Shuizu tea and Murili No.4 in sequence, the size of a PCR product after enzyme digestion is 196bp, and the genotype of the SNP848 is AA; 11-20 are Lechang Langgtian bitter tea, Ruyuan Liuzhou No.1, Taishan white Yun tea, Yinghong No.1, Wuling red, Lechang tip leaf white hair, Rodin red bud seed, stannum tea No. 10, big leaf tea group and Honghe Langdao tea in sequence, the size of the PCR product after enzyme digestion is 196bp and 176bp, and the genotype of SNP848 is AG; 21-28 are respectively Fenghuang tea tree, teacher No. 5, Zhen Yuan No.2, Changning No.4, Jing Gu old storehouse group, Ma Li Po No. 8, Yun cha Pupistil and Yun county No.1 in sequence, the size of the PCR product after enzyme digestion is 176bp, and the SNP848 genotype is AG.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
The reagents used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Example 1
1. Test material
Longjing 43 and Fenghuang tea tree were selected as the research materials.
2. Determination of catechin content
Picking two leaves of tea tree spring young sprout, drying with 120 deg.C hot air for 5min to fix sample, drying at 75 deg.C to dry, and storing at low temperature. And grinding by a machine when the sample is to be detected, sieving by a 40-mesh sieve, and storing at low temperature for later use. 0.2g of sample (to the nearest 0.0001 g) was weighed and placed in a 10mL centrifuge tube. Adding 5mL of 70% preheated methanol solution at 70 ℃, uniformly mixing, and carrying out water bath at 70 ℃. Water bath is carried out for 10min, and the middle is shaken for 2 to 3 times. Centrifuging at 3,500r/min for 10min, and transferring the supernatant. And (5) repeating the steps 2-4, combining the supernatants collected twice, and metering to 10 mL. Transferring 2mL of the extractive solution into a volumetric flask, diluting to 10mL with a stable solution (5% 10mg/mL EDTA solution, 5% 10mg/mL ascorbic acid solution, 10% acetonitrile), mixing, and filtering with 0.45 μm filter membrane. Detecting by High Pressure Liquid Chromatography (HPLC), and qualitatively and quantitatively analyzing alkaloid and catechin by external standard method. Liquid chromatography measurement conditions: c12 column 4.6 mm X250 mm (4 μm, Philomo, Guangzhou); the mobile phase A is 0.5% formic acid, the mobile phase B is acetonitrile, the flow rate is 1mL/min, the column temperature is 30 ℃, the detection wavelength is 280nm, the sample injection amount is 10 mu L, and gradient elution is carried out: the mobile phase B is changed from 6.5% linear gradient to 16% in 16min, and from 16% linear gradient to 25% in 16min to 20min, and is maintained for 5min, returned to the initial state, and equilibrated for 5 min. The measurement result shows that the content of trihydroxy catechin of the phoenix big tea tree is equivalent to that of the Longjing 43, but the content of dihydroxy catechin is 2.4 times that of the Longjing 43.
TABLE 12 difference in catechin content (mg/g) of spring tea of tea plant material
| Name of Material | EGC | EC | EGCG | ECG | Trihydroxy catechin (EGC + EGCG) | Dihydroxy catechin (EC + ECG) | Total amount of catechins |
| Dragon well 43 | 8.2 | 5.4 | 69.2 | 32.5 | 77.4 | 37.9 | 115.3 |
| Phoenix tea tree | 16.3 | 22.7 | 65.9 | 68.3 | 82.2 | 91.0 | 173.2 |
3. Extraction of genomic DNA
1g of fresh tender tips are taken, and liquid nitrogen is added to the fresh tender tips to be ground into powder. Placing 0.2g of the powder into a 1.5mL centrifuge tube, adding 700 μ L CTAB extracting solution, mixing well, shaking uniformly once every 20min in a water bath at 65 ℃ for 1 h. Adding equal volume of chloroform/isoamyl alcohol (24: 1), mixing, and standing for 2 min. Centrifuge at 14,000g for 10min at room temperature and collect the supernatant. Adding equal volume of precooled isopropanol, and standing for 1h at-20 ℃. Centrifuge at 14,000g for 10min and discard the supernatant. Add 300. mu.L of high salt solution and incubate at 65 ℃ for 30min until the pellet dissolves. Centrifuge at 10,000g for 10min at room temperature and collect the supernatant. 1/10 volumes of pre-cooled NaAc (pH 5.2), 2/3 volumes of pre-cooled isopropanol were added and mixed well and left at-20 ℃ for 30 min. Centrifuge at 14,000g for 5min and discard the supernatant. The precipitate was washed with 70% ethanol 1 time and with absolute ethanol once. Blow-drying on a clean bench for 30min, dissolving in 200 μ L sterilized water, and storing at-20 deg.C.
4. PCR sequencing and sequencing analysis
Designing specific primers to amplify the contents of tea tree materialF3′5′HThe DNA fragment from 54 bp upstream to 891bp downstream of the initiation codon ATG, primers designed by software Primer5.0, has the following sequence:
an upstream primer (shown as SEQ ID No. 3): 5'-ACCAAAACACTCAACCAGGT-3' the flow of the air in the air conditioner,
the downstream primer (shown as SEQ ID No. 4): 5'-TGCCTTGATGTTGGTCGTGT-3', respectively;
and (3) PCR reaction system: 32 mu L ddH2O，2 µL DNA，5 µL 10×PCR Buffer，4 µL 25 mM MgCl2，5 µL 2 Mm dNTP，1µLKOD-Plus-Neo enzyme, 10 μ M forward and reverse primers each 3 μ L. The PCR amplification procedure was: 94 ℃ for 2min, then 35 cycles of 94 ℃ for 15 sec, 5325 sec, 68 ℃ for 30 sec; finally, extension was carried out at 68 ℃ for 7 min. And (3) carrying out electrophoresis on the PCR amplification product on 1.2% agarose gel, observing under an ultraviolet lamp, cutting and recovering the gel, connecting a carrier, transforming, and carrying out PCR screening positive cloning sequencing on bacterial liquid. Analysis found 3 single nucleotide mutations (SNPs) in the first exon between the two materials, of which only SNP848 was a non-synonymous mutation (fig. 1).
5. Development of functional marker dCAPS-F3 '5' H
Designing specific primers to amplify the contents of tea tree materialF3′5′HDNA fragment from 676 bp downstream to 871 bp downstream of ATG gene initiation codon, and primers were designed by using dCAPS Finder 2.0 (http:// helix. wustl. edu/dCAPS. html). Introducing 1bp mismatch at the 3' end of the downstream primer, and the sequence is as follows:
an upstream primer (shown as SEQ ID No. 1): 5'-GATTTCATACCATCGATTGCGT-3' the flow of the air in the air conditioner,
the downstream primer (shown as SEQ ID No. 2): 5'-TGAGCTTCTCTTCACCAGGAATT-3', respectively;
and respectively carrying out PCR amplification, enzyme digestion and agarose gel analysis by using the primer sequences.
And (3) PCR reaction system: 32 mu L ddH2O，2 µL DNA，5 µL 10×PCR Buffer，4 µL 25 mM MgCl 25 muL 2 Mm dNTP, 1 muL KOD-Plus-Neo enzyme, and 3 muL of each of 10 muM forward and reverse primers.
The PCR amplification procedure was: 94 ℃ for 2min, then 35 cycles of 94 ℃ for 15 sec, 58 ℃ for 25 sec, 68 ℃ for 5 sec; finally, extension was carried out at 68 ℃ for 2 min.
The enzyme digestion system, time, temperature and agarose gel electrophoresis are as follows: 10 μ L of PCR product, endonucleaseEcoRI10U, endonuclease buffer 2. mu.L, in ddH2Supplementing O to 20 μ L; the enzyme digestion reaction time is 1 hour, the temperature is 37 ℃, and the amplification product is separated by electrophoresis in 3 percent agarose gel.
Analysis shows that the size of the band of the PCR product of the Longjing 43 is 196bp after enzyme digestion, which indicates thatThe genotype of SNP848 is AA, and 1 176bp band is generated after the enzyme digestion of the large Fenghuang tea tree, which indicates that the genotype of SNP848 is GG (figure 2). The molecular marker can be used for identifying tea tree resourcesF3′5′HThe gene SNP848, and can screen out tea tree material containing high dihydroxy catechin.
Example 2
Genotype analysis of tea plant resource by functional marker dCAPS-F3 '5' H
1. Test material
The materials studied in this experiment are listed in table 2, including 28 parts of tea plant resource.
TABLE 2 tea plant resource of 28 parts studied in this experiment and its dihydroxycatechin content
2. Functional mark for different tea tree resourcesF3′5′HGenotype detection of Gene SNP848
Analysis of the marker genotype of 28 material samples using the molecular marker dCAPS-F3 '5' H. DNA extraction, PCR amplification system, procedure, digestion and agarose gel conditions were the same as in example 1. The results are shown in FIG. 3, in which the genotypes of materials 1-10 are AA, the genotypes of materials 11-20 are AG, and the genotypes of materials 21-28 are GG.
3.28 tea tree material catechin content identification
The catechin identification method by high performance liquid chromatography was the same as described in example 1. In 28 parts of materials, 10 parts of materials with the genotype of AA have the content of dihydroxycatechin of 32.6 +/-4.6 mg/g, 10 parts of materials with the genotype of AG have the content of dihydroxycatechin of 42.0 +/-8.9 mg/g, and 8 parts of materials with the genotype of GG have the content of dihydroxycatechin of 84.1 +/-22.2 mg/g. Statistical analysis shows that the content of dihydroxy catechin in the GG genotype material is significantly higher than that in other 2 genotypes, and dCAPS-F3 '5' H can be used as a functional marker for identifying and screening high-dihydroxy catechin tea tree materials.
The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> institute of tea leaf of Chinese academy of agricultural sciences
<120> flavonoid 3 ', 5' -hydroxylase gene functional marker for screening high-dihydroxy catechin tea tree, and application method thereof
<130>11
<160>4
<170>PatentIn version 3.3
<210>1
<211>22
<212>DNA
<213> Artificial Synthesis
<400>1
gatttcatac catcgattgc gt 22
<210>2
<211>23
<212>DNA
<213> Artificial Synthesis
<400>2
tgagcttctc ttcaccagga att 23
<210>3
<211>20
<212>DNA
<213> Artificial Synthesis
<400>3
<210>4
<211>20
<212>DNA
<213> Artificial Synthesis
<400>4
Claims (3)
1. An application of a primer pair of flavonoid 3 ', 5' -hydroxylase genes for screening tea trees with high-dihydroxy catechin in screening tea trees with high-dihydroxy catechin is characterized in that an upstream primer sequence of the primer pair is shown as SEQ ID No.1, and a downstream primer is shown as SEQ ID No. 2.
2. Use according to claim 1, characterized in that it comprises the following steps:
and carrying out PCR amplification, enzyme digestion and agarose gel analysis on DNA fragments from 676 bp to 871 bp downstream from the ATG (start codon) of the F3 '5' H gene in each tea tree material by using a primer pair, wherein the tea tree is determined to be a low-dihydroxy catechin tea tree if the amplified product shows a 196bp fragment after enzyme digestion, and the tea tree is determined to be a high-dihydroxy catechin tea tree if the amplified product shows a 176bp fragment after enzyme digestion.
3. The use according to claim 2, wherein the PCR amplification system and reaction sequence is: and (3) PCR reaction system: 32 mu L ddH2O，2 µL DNA，5 µL 10×PCR Buffer，4 µL 25 mM MgCl23 muL of each of upstream and downstream primers of 5 muL 2 Mm dNTP, 1 muL KOD-Plus-Neo enzyme and 10 muM; the PCR amplification procedure was: 94 ℃ for 2min, then 35 cycles of 94 ℃ for 15 sec, 58 ℃ for 25 sec, 68 ℃ for 5 sec; finally extending for 2min at 68 ℃; the conditions of enzyme digestion and agarose gel are as follows: PCR product 10. mu.L, endonuclease EcoRI 10U, endonuclease buffer 2. mu.L, and ddH2Supplementing O to 20 mu L of enzyme digestion system; the enzyme digestion reaction time is 1 hour, the temperature is 37 ℃, and the amplification product is separated by electrophoresis in 3 percent agarose gel.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611026548.9A CN106755308B (en) | 2016-11-22 | 2016-11-22 | Flavonoid 3 ', 5' -hydroxylase gene function marker for screening high-dihydroxy catechin tea tree and application method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611026548.9A CN106755308B (en) | 2016-11-22 | 2016-11-22 | Flavonoid 3 ', 5' -hydroxylase gene function marker for screening high-dihydroxy catechin tea tree and application method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106755308A CN106755308A (en) | 2017-05-31 |
| CN106755308B true CN106755308B (en) | 2020-05-26 |
Family
ID=58971505
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611026548.9A Active CN106755308B (en) | 2016-11-22 | 2016-11-22 | Flavonoid 3 ', 5' -hydroxylase gene function marker for screening high-dihydroxy catechin tea tree and application method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106755308B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110714092B (en) * | 2019-09-04 | 2020-09-08 | 广东省农业科学院茶叶研究所 | Molecular marker locus located in flavonol synthetase gene and linked with (+) -catechin content of tea tree and application thereof |
| CN110835653B (en) * | 2019-09-04 | 2020-11-10 | 广东省农业科学院茶叶研究所 | Molecular marker site located on ABC transporter gene and linked with (+) -catechin content of tea tree and application thereof |
| CN110468231B (en) * | 2019-09-04 | 2020-09-18 | 广东省农业科学院茶叶研究所 | Group of molecular markers linked with (+) -catechin content of tea trees and application thereof |
| CN110438256B (en) * | 2019-09-04 | 2020-09-18 | 广东省农业科学院茶叶研究所 | Molecular marker locus linked with epigallocatechin gallate content of tea tree and application thereof |
| CN110438255B (en) * | 2019-09-04 | 2020-09-18 | 广东省农业科学院茶叶研究所 | Molecular marker locus linked with tea tree epicatechin gallate content and application thereof |
| CN110468230B (en) * | 2019-09-04 | 2020-09-18 | 广东省农业科学院茶叶研究所 | Molecular marker locus linked with tea tree secondary metabolite content and located on receptor-like protein kinase gene and application thereof |
| CN110734996B (en) * | 2019-09-04 | 2020-09-08 | 广东省农业科学院茶叶研究所 | Molecular markers linked with caffeine content of tea tree and application thereof |
| CN110819731B (en) * | 2019-09-04 | 2020-11-10 | 广东省农业科学院茶叶研究所 | SNP molecular markers linked with (+) -catechin content of tea tree and application thereof |
| CN110643729B (en) * | 2019-09-04 | 2020-09-18 | 广东省农业科学院茶叶研究所 | SNP molecular marker locus linked with content of gallocatechin gallate of tea tree and application thereof |
| CN110607383B (en) * | 2019-09-04 | 2020-09-08 | 广东省农业科学院茶叶研究所 | Molecular marker locus linked with tea tree epicatechin content and application thereof |
| US20220267834A1 (en) * | 2019-09-04 | 2022-08-25 | Tea Research Institute, Guangdong Academy Of Agricultural Sciences | Molecular marker combination linked to quantitative traits of tea plant (+)-catechin content |
| CN115961086B (en) * | 2023-01-17 | 2023-07-14 | 华中农业大学 | Tea plant flavonoid 3-O-methyltransferase gene and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1678746A (en) * | 2002-08-30 | 2005-10-05 | 国际花卉开发有限公司 | Flavanoid 3',5'hydroxylase gene sequences and uses therefor |
-
2016
- 2016-11-22 CN CN201611026548.9A patent/CN106755308B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1678746A (en) * | 2002-08-30 | 2005-10-05 | 国际花卉开发有限公司 | Flavanoid 3',5'hydroxylase gene sequences and uses therefor |
Non-Patent Citations (3)
| Title |
|---|
| Camellia sinensis cultivar UPASI-10 flavonoid 3",5"-hydroxylase mRNA, complete cds，GenBank: DQ194358.1，1533bp mRNA linear;Rani,A.等;《NCBI GenBank》;20050928;1 * |
| Camellia sinensis flavonoid 3",5"-hydroxylase mRNA, complete cds，GenBank: AY945842.1，1533bp mRNA linear;Park,J.-S.;《NCBI GenBank》;20070301;1 * |
| 紫芽茶树类黄酮生物合成关键酶基因表达与总儿茶素、花青素含量相关性分析;周天山等;《作物学报》;20160119;第42卷(第4期);525-531 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106755308A (en) | 2017-05-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106755308B (en) | Flavonoid 3 ', 5' -hydroxylase gene function marker for screening high-dihydroxy catechin tea tree and application method thereof | |
| CN107164459B (en) | Functional marker for identifying and screening tea trees with high catechin indexes and application of functional marker | |
| Dutta et al. | Expression of terpenoid indole alkaloid biosynthetic pathway genes corresponds to accumulation of related alkaloids in Catharanthus roseus (L.) G. Don | |
| Yang et al. | Digital gene expression analysis provides insight into the transcript profile of the genes involved in aporphine alkaloid biosynthesis in lotus (Nelumbo nucifera) | |
| CN104894143B (en) | A kind of method for improving tanshinone content in salvia miltiorrhiza hair roots | |
| Zheng et al. | Anthocyanin profile and gene expression in berry skin of two red Vitis vinifera grape cultivars that are sunlight dependent versus sunlight independent | |
| Sun et al. | Metabolome and transcriptome profiling reveals quality variation and underlying regulation of three ecotypes for Cistanche deserticola | |
| Mora-Ortiz et al. | De-novo transcriptome assembly for gene identification, analysis, annotation, and molecular marker discovery in Onobrychis viciifolia | |
| Yang et al. | Metabolic profiles and cDNA-AFLP analysis of Salvia miltiorrhiza and Salvia castanea Diel f. tomentosa Stib | |
| CN110592111B (en) | Ginkgo flavonoid 3 '-hydroxylase gene GbF3' H1 and application thereof | |
| Jiang et al. | Molecular and analysis of a phenylalanine ammonia-lyase gene (LrPAL2) from Lycoris radiata | |
| Deng et al. | Uncovering tea-specific secondary metabolism using transcriptomic and metabolomic analyses in grafts of Camellia sinensis and C. oleifera | |
| Honda et al. | Identification of drought-induced genes in giant leucaena (Leucaena leucocephala subsp. glabrata) | |
| Kim et al. | Accumulation of flavonoids and related gene expressions in different organs of Astragalus membranaceus Bge | |
| Yao et al. | Hormonal and transcriptional analyses provides new insights into the molecular mechanisms underlying root thickening and isoflavonoid biosynthesis in Callerya speciosa (Champ. ex Benth.) Schot | |
| Rostami et al. | The isolation and expression analysis of cinnamate 4-hydroxylase and chalcone synthase genes of Scrophularia striata under different abiotic elicitors | |
| Li et al. | Selection and validation of reference genes desirable for gene expression analysis by qRT-PCR in MeJA-treated ginseng hairy roots | |
| Bai et al. | Transcriptomic analysis and dynamic expression of genes reveal flavonoid synthesis in Scutellaria viscidula | |
| Esfahani et al. | Altered gene expression and root thebaine production in polyploidized and methyl jasmonate-elicited Papaver bracteatum Lindl | |
| Abedini et al. | The effects of promoter variations of the N-Methylcanadine 1-Hydroxylase (CYP82Y1) gene on the noscapine production in opium poppy | |
| Ren et al. | Integrated metabolomics and transcriptome analysis on flavonoid biosynthesis in flowers of safflower (Carthamus tinctorius L.) during colour-transition | |
| Pan et al. | Accumulation of calycosin and its 7-O-β-D-glucoside and related gene expression in seedlings of Astragalus membranaceus Bge. var. mongholicus (Bge.) Hsiao induced by low temperature stress | |
| Wang et al. | Comparative transcriptome analysis revealed the molecular mechanism of the effect of light intensity on the accumulation of rhynchophylline and isorhynchophylline in Uncaria rhynchophylla | |
| Dong et al. | Integration of transcriptome and proteome analyses reveal molecular mechanisms for formation of replant disease in Nelumbo nucifera | |
| Artigot et al. | Expression of flavonoid 6-hydroxylase candidate genes in normal and mutant soybean genotypes for glycitein content |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |