CN108840949A - From the peroxidase POD1 relevant to theaflavin generation of tealeaves and its application - Google Patents
From the peroxidase POD1 relevant to theaflavin generation of tealeaves and its application Download PDFInfo
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Abstract
The invention discloses a kind of from the peroxidase POD1 relevant to theaflavin generation of tealeaves and its application.The present invention provides a kind of protein, are named as POD1 albumen, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.Using POD1 protein level as index, fermentation of black tea parameter can be arranged by objective data, the black tea with expected theaflavin content be obtained, to effectively push the standardization of tea processing, automation and mechanization.The present invention can be used for Tea Breeding.The tea tree breed of low expression POD1 gene is cultivated, convenient for production green tea.The tea tree breed of high expression POD1 gene is cultivated, convenient for making black tea by spreading the leaves on withering racks to dry.The present invention can be used for screening the tea tree breed for being suitable for making black tea by spreading the leaves on withering racks to dry and the tea tree breed suitable for making green tea.If POD1 gene expression dose is high, the tea tree breed is suitable for making black tea by spreading the leaves on withering racks to dry, if the expression of POD1 gene is low, the tea tree breed is suitable for production green tea.
Description
Technical field
The present invention relates to tea processing technical fields, and in particular to a kind of from the relevant to theaflavin generation of tealeaves
Peroxidase POD1 and its application.
Background technique
In order to ensure ester catechin (EGC, EGCG, ECG and CG) and non-ester catechin (EC during Green Tea Processing
And C) not oxidized degradation, the intracorporal oxidizing ferment of tealeaves (predominantly peroxidase and polyphenol oxygen are usually inactivated using water-removing
Change enzyme).Since catalytic activity of the people to these oxidizing ferment in tealeaves lacks enough understandings, and the temperature that finishes and when
Between it is different because of Different Tea Varieties, therefore Green Tea Processing automation and standardisation process be affected and limit.
Theaflavins compound is generated in black tea manufacturing process, content typically constitutes from the 0.3%-1.5% of dry weight of tea leaves, and 4
Planting main theaflavins compound is：TF, TF2A, TF2B and TF3.Theaflavins compound is by catechin compounds
With various combination oxidative condensation under the action of oxidizing ferment in tealeaves.Also due to people are to these oxidations in tealeaves
The catalytic activity of enzyme lacks to be understood enough, and the temperature of black tea processing and fermentation is carried out using the oxidizing ferment of tealeaves itself, wet
The conditions such as degree and time are also different because of Different Tea Varieties, and the producer is mostly processed to control black tea according to the experience of oneself
Journey.This is extremely limited the process automation of black tea and the degree of product standardization.
Theaflavins compound has effects that adjust blood lipid and prevents cardiovascular disease, and has no toxic side effect, certain
Aspect is even better than catechin.Theaflavin plays an important role to the soup look and flavour of black tea.Therefore the height of theaflavin content
The low selling price for determining black tea, and determine one of the key factor of tea drink quality quality.Using theaflavin as raw material
Natural drug and the research and development of functional food have a extensive future.Screen special peroxidase and polyphenol oxidase enzyme
Source synthesizes theaflavins compound using enzymatic oxidation, studies theaflavins compound synthesis mechanism, has important theory
With practical significance.Although peroxidase (POD) and polyphenol oxidase (PPO) are extremely important in black tea manufacturing process, right
Its gene and heredity, which understand, to be lacked.The albumen that fermentation of black tea aoxidizes catechin and synthesizes theaflavin is mainly responsible in parsing tealeaves
Encoding gene can help tea tree molecular breeding, cultivate low expression peroxidase and polyphenol oxidase kind convenient for special
Green tea is made, high expression peroxidase is cultivated and polyphenol oxidase kind can be convenient for making black tea by spreading the leaves on withering racks to dry, convenient for control green tea or red
Stability contorting during tea tea processing, convenient for standardization automation Tea Production.
Oxidizing ferment (predominantly peroxidase and polyphenol oxidase) catalysis oxidation catechin compounds generate o-quinone class
Substance, o-quinone substance further polymerize generation, and even phenol quinones, the latter further aoxidize the list that theaflavins can be generated
Body, monomer are generating theaflavins compound by coupled oxidation.Research has shown that theaflavin and is by catechin and relevant do not have
Gallate-based passes through the dimer compound that oxidation polymerization is formed, and the Forming Mechanism of four kinds of main theaflavin is： (L)-EGC+(L)-
EC→TF：Theaflavin；(L)-EGCG+(L)-EC→TF2A；(L)-ECG+(L)-EGC →TF2B；(L)-EGCG+(L)-ECG→
TF3。
EGC full name is epigallocatechin.EGCG full name is Epigallo-catechin gallate (EGCG).ECG full name is
L-Epicatechin gallate.CG full name is catechin and gallate.EC full name is epicatechin.C full name is catechin.
TF full name is theaflavin.TF2A full name is theaflavin-3-gallate.TF2B full name is the gallate of theaflavin -3 '.
TF3 full name is theaflavih digallate.GC full name is nutgall catechin.GCG full name is nutgall catechin galla turcica
Acid esters.
Summary of the invention
The object of the present invention is to provide a kind of peroxidase POD1 relevant to theaflavin generation from tealeaves and
It is applied.
The present invention provides a kind of protein, are named as POD1 albumen, are any one following of (a1) into (a8)：
(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(a2) contain the fusion protein of (a1)；
(a3) fusion protein that small peptide of the connection containing label obtains in the end of (a1)；
(a4) fusion protein that connection label obtains in the end of (a1)；
(a5) protein that the amino acid sequence shown in sequence 3 in sequence table forms；
(a6) by (a1) or (a2) or (a3) or (a4) or (a5) by the substitution of one or several amino acid residues and/
Or it is deleted and/or added and has protein as derived from it of oxidase function；
(a7) there is 98% or more homology with (a1) obtained from tealeaves and there is as derived from it egg of oxidase function
White matter；
(a8) it is obtained from the early egg as derived from it with (a1) with 98% or more homology with oxidase function of the tea that relaxes
White matter.
The oxidizing ferment is peroxidase and/or polyphenol oxidase.
Label is specifically as shown in table 1.
The sequence of 1 label of table
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
| HA | 9 | YPYDVPDYA |
Protein can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene (POD1 gene) of POD1 albumen also belongs to protection scope of the present invention.
The POD1 gene is following (b1) any described DNA molecular into (b5)：
(b1) code area DNA molecular as shown in sequence 2 in sequence table；
(b2) code area DNA molecular as shown in sequence 4 in sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode the DNA molecule of oxidizing ferment；
(b4) have 90% or more or 95% or more or 98% or more from the DNA sequence dna of tealeaves limited with (b1)
Or 99% or more homology and encode oxidizing ferment DNA molecular；
(b5) from the early DNA sequence dna limited with (b1) of the tea that relaxes have 90% or more or 95% or more or 98% with
Upper or 99% or more homology and the DNA molecular for encoding oxidizing ferment.
The oxidizing ferment is peroxidase and/or polyphenol oxidase.
Above-mentioned stringent condition can be with 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS, in DNA or RNA
Hybridize at 65 DEG C in hybrid experiment and washes film.
The present invention also protects the application of POD1 albumen, at least one of following (c1) to (c16)：
(c1) it is used as oxidizing ferment；
(c2) oxidizing ferment is prepared；
(c3) it is used as peroxidase；
(c4) peroxidase is prepared；
(c5) it is used as polyphenol oxidase；
(c6) polyphenol oxidase is prepared；
(c7) theaflavins compound is prepared；
(c8) theaflavins compound is prepared by substrate of catechin compounds；
(c9) theaflavin is prepared；
(c10) theaflavin is prepared by substrate of catechin compounds；
(c11) theaflavin is prepared using epigallocatechin and epicatechin as substrate；
(c12) promote Catechin in Tea class degradation；
(c13) level of Catechin in Tea class compound is reduced；
(c14) it is horizontal to improve theaflavin in tealeaves；
(c15) it is screened as marker and is suitble to the tea tree breed of production green tea and/or is suitble to the tea tree breed to make black tea by spreading the leaves on withering racks to dry；
(c16) black tea processing technology is controlled as inspection target.
The present invention also protects application of the POD1 gene in Tea Breeding；The Tea Breeding is to cultivate to be suitble to production green tea
Tea tree breed or cultivation be suitble to the tea tree breed to make black tea by spreading the leaves on withering racks to dry.
The present invention is also protected for inhibiting the substance of POD1 gene expression to plant in the transgenosis for preparing the reduction of theaflavin level
Application in object or Transgenic plant tissue.The plant is tea tree.For inhibiting the substance of POD1 gene expression concretely
Aftermentioned specific DNA molecular or recombinant expression carrier with the specific DNA molecular.
The present invention, which also protects, is cultivating what theaflavin level reduced for reducing POD1 protein active and/or the substance of level
Application in plant.The plant is tea tree.
The present invention also protects the application of the substance for detecting POD1 gene expression dose, is following (d1) into (d5)
At least one：
(d1) it evaluates tea tree breed to be measured or individual is suitble to production green tea or black tea；
(d2) ability of tea tree breed to be measured or individual production theaflavin is evaluated；
(d3) disease resistance of tea tree breed to be measured or individual is evaluated；
(d4) screening is suitble to the tea tree breed or individual of production green tea；
(d5) screening is suitble to the tea tree breed or individual to make black tea by spreading the leaves on withering racks to dry.
Substance for detecting POD1 gene expression dose is specifically as follows aftermentioned primer pair.
The present invention also protects the application of the substance for detecting POD1 protein level, for following (d1) into (d5) extremely
Few one kind：
(d1) it evaluates tea tree breed to be measured or individual is suitble to production green tea or black tea；
(d2) ability of tea tree breed to be measured or individual production theaflavin is evaluated；
(d3) disease resistance of tea tree breed to be measured or individual is evaluated；
(d4) screening is suitble to the tea tree breed or individual of production green tea；
(d5) screening is suitble to the tea tree breed or individual to make black tea by spreading the leaves on withering racks to dry.
The present invention also protects a kind of primer pair, the single strand dna as shown in the sequence 5 of sequence table and sequence table
Sequence 6 shown in single strand dna composition.
The present invention also protects a kind of specific DNA molecular, is single strand dna shown in the sequence 7 of sequence table.
The present inventor is analyzed by the transcript profile data of tea tree, in conjunction with prokaryotic expression and functional verification, from easypro tea
The oxidizing ferment that can be catalyzed catechin compounds synthesis theaflavin is had found in morning, has both peroxidase and polyphenol oxidase
The activity of enzyme.
Determining most professional tea making of dependence of black tea processing and fermentation procedure parameter (length of time, temperature, humidity etc.) at present
The judge of teacher is confined to judgement of the feeling (such as vision, tactile) of professional tea making people to tea leaf fermentation.It is this due to people
Different and inaccurate sense significantly limits the standardization and mechanization of tea processing, causes tea quality variation very
Greatly, the repeated consistency between batch is very poor, thus can not be mass produced, cause China's Tea Production quality and stability,
It can not be compared with famous brand in the world in terms of homogeneity.Using POD1 protein level as index, objective data can be passed through
Fermentation of black tea parameter (temperature, humidity, pH, time during tea processing etc.) is set, obtains that there is expected theaflavin content
Black tea, to effectively push the standardization of tea processing, automation and mechanization.
The present invention can be used for Tea Breeding.The tea tree breed of low expression POD1 gene is cultivated, convenient for production green tea.It cultivates
The tea tree breed of height expression POD1 gene, convenient for making black tea by spreading the leaves on withering racks to dry.The conventional breeding period length of xylophyta tea tree (generally requires
The hybrid vigour of 10-20 selects), molecular breeding can greatly shorten breeding time, reduce Large-scale Screening manpower and
Time.
The present invention can be used for screening the tea tree breed for being suitable for making black tea by spreading the leaves on withering racks to dry and the tea tree breed suitable for making green tea.It is specific next
It says, is to be realized by detecting the expression of POD1 gene, if POD1 gene expression dose is high, the tea tree breed is suitable
In making black tea by spreading the leaves on withering racks to dry, if the expression of POD1 gene is low, the tea tree breed is suitable for production green tea.
Based on the prior art, peroxidase levels height implies that the tea tree breed or individual have stronger disease resistance,
Peroxidase activity is higher, and the disease resistance of plant is stronger.So the present invention can be used for cultivating the tea of disease resistance raising
Kind is set, can be used for screening disease resistance high tea tree breed or individual.
Detailed description of the invention
Fig. 1 is the polyphenol oxidase enzyme activity and peroxidase enzyme activity result of the total protein of each sample in embodiment 1.
Fig. 2 is the result of catechin compounds content in embodiment 1.
Fig. 3 is the result of the expression quantity of POD1 gene in embodiment 1.
Fig. 4 is the result of the differential expression of POD1 gene in easypro tea morning different tissues in embodiment 2.
Fig. 5 is the enzyme activity comparison result of Different Tea Varieties in embodiment 3.
Fig. 6 be embodiment 4 in POD1 gene the differential expression of Different Tea Varieties result.
Fig. 7 is the polyacrylamide gel electricity that recombination fungus beetle carries out in destination protein expression and purification process in embodiment 5
Swimming figure.
Fig. 8 is the result of Enzyme activity assay in step 4 in embodiment 5.
Fig. 9 is the photo for reacting finish time in embodiment 5 in step 5.
Figure 10 is the chromatogram of HPLC detection in step 5 in embodiment 5.
Figure 11 is to react finish time in step 5 in embodiment 5, the result of epicatechin concentration in reaction system.
Figure 12 is to react finish time in step 5 in embodiment 5, the result of theaflavin concentration in reaction system.
The result that Figure 13 is LC-MS in step 5 in embodiment 5.
Figure 14 is the result of zymologic property detection in step 6 in embodiment 5.
Figure 15 is the result of the relative expression levels of POD1 gene in embodiment 6.
Figure 16 is in embodiment 6 by inhibiting POD1 gene expression to reduce the result of theaflavin content.
Specific embodiment
Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method is unless otherwise specified conventional method.Test material as used in the following examples is unless otherwise specified
It is commercially available from routine biochemistry reagent shop.Quantitative test in following embodiment is respectively provided with three repeated experiments, as a result
It is averaged.Spectrophotometric used in embodiment is calculated as (5 serial of SoftMax Pro of Spectra MAX 190
number:SMP500-14530-XUSL)。
Local tea variety relaxes, and tea is early, and the tea that referred to as relaxes is early.A new albumen is found from easypro tea morning, is named as POD1 albumen, such as
Shown in the sequence 1 of sequence table.It is POD1 gene, the sequence of open reading frame such as sequence table by the unnamed gene for encoding POD1 albumen
Shown in column 2.POD1 albumen has the function of peroxidase.
The tea that relaxes is early and other each tea tree breeds, and plant plantation is in Anhui Province Luyang District Hefei Agricultural University Of Anhui agricultural
Industrial park, picking blade condition are 25-28 DEG C.
Polyphenol oxidase enzyme activity determination method (colorimetric method)：Reaction system is 400 μ l；Initial system waits for test sample by 50 μ l
The phosphate buffer composition of this (albumen is provided), catechol (as substrate) and pH7.0,0.1mM, the concentration of catechol
For 0.2% (w/v, g/ml)；Initial system is stored at room temperature reaction 3min, as termination system；Using spectrophotometric
Meter detects initial system respectively and cessative aspect ties up to the light absorption value of 460nm, terminates light absorption value-initial system extinction of system
Value=absorbance difference；Polyphenol oxidase enzyme activity=absorbance difference ÷ (total protein content × reaction time), the list of total protein
Position is mg, and the unit in reaction time is min.
Peroxidase enzyme activity determination method (colorimetric method)：Reaction system is 1ml；Initial system is by 50 μ l samples to be tested
The phosphate buffer composition of (albumen is provided), guaiacol (as substrate), 30% hydrogenperoxide steam generator and pH6.0,0.1M,
The concentration of guaiacol is 0.56 μ l/ml, and the concentration of hydrogen peroxide is 0.38 μ l/ml；Initial system is stored at room temperature reaction
3min, as termination system；It detects initial system respectively using spectrophotometer and cessative aspect ties up to the light absorption value of 470nm, eventually
Only light absorption value-initial system light absorption value=absorbance difference of system；Peroxidase enzyme activity=absorbance difference ÷ is (total
Protein content × reaction time), the unit of total protein is mg, and the unit in reaction time is min.
Embodiment 1, black tea/producing green tea Process of Peroxide enzyme and the variation of polyphenol oxidase activity, catechin contain
The variation of variation, the gene expression of amount
One, the acquisition of each sample
The manufacturing process of green tea is predominantly picked, is spread, finishing, rubbing, drying.Specially：Pick tea morning plant of relaxing
One bud, one leaf (being at this time fresh leaf sample)；3h or so (after the completion for spread sample) is spread in room temperature；With 180 DEG C of water-removing 3-
5min (being after the completion the sample that finishes)；Certain shapes (after the completion for rub sample) is rubbed into hand；110 DEG C of dry 15min are left
Right (being after the completion dry sample).
The manufacturing process of black tea is predominantly picked, is withered, rubbing, fermenting, drying.Specially：Pick tea morning plant of relaxing
One bud, one leaf (being at this time fresh leaf sample)；16h or so (being after the completion the sample that withers) is spread in room temperature；Tealeaves is held together into glomeration
It rubs and gently rubs again again after first gently rubbing (after the completion for rub sample)；Encasing 30 DEG C of fermentation 12h of tealeaves with gauze (is after the completion fermentation
Sample), it to scatter every 2h during fermentation and once then proceed to ferment；110 DEG C of dry 15min or so (are after the completion dry sample
This).
Two, enzyme activity
Each sample that step 1 obtains is taken, protein extraction is carried out, obtains the protein extraction solution containing total protein, it will
Protein extraction solution or its dilution (being diluted using the kaliumphosphate buffer of pH6.0,0.1M) are used as sample to be tested, inspection
Polyphenol oxidase enzyme activity and peroxidase enzyme activity are surveyed, the polyphenol oxidase enzyme activity and peroxidase of total protein is calculated
Enzyme activity.
The polyphenol oxidase enzyme activity and peroxidase enzyme activity the result is shown in Figure 1 of the total protein of each sample.In Fig. 1, upper figure
For the photo for terminating system, the following figure is enzyme-activity data.In black tea manufacturing process, fermentation stage POD/PPO activity reaches highest.
During Green Tea Processing, water-removing causes PPO activity sharply to decline.
Three, catechin compounds content
Each sample that step 1 obtains is taken, detects catechin compounds content respectively.
The method for detecting catechin compounds content：
0.1g sample is taken, is added in 1.5ml 80% (volume ratio) methanol aqueous solution, the concussion that is vortexed mixes well, then
Ultrasound 30min in ice bath, then 13000rpm is centrifuged 30min, using 0.22 μm of aperture strainer filtering, collects filtrate；
Filtrate is taken, using HPLC detection (parameter is detected with the HPLC in the step of embodiment 5 five, 15 μ l of sample volume)；Mark
Quasi- product are respectively：GC,EGC,C,EGCG,EC,GCG,ECG.Under identical chromatographic conditions, with standard items go out peak position ±
Peak within 0.5min, is judged as same substance.Under identical chromatographic conditions, the standard of standard items content and peak area is made
Curve obtains the concentration of each catechin compounds in sample (in every mg sample by calculating by reference standard curve
Containing how many μ g catechin compounds).
As a result see Fig. 2.In black tea manufacturing process, each catechin kind compound content declines, and illustrates most of catechu
Chlorins compound generates theaflavins compound in process.During Green Tea Processing, each catechin compounds
Changes of contents is not significant.
Four, the expression quantity of POD1 gene
Each sample that step 1 obtains is taken, the expression quantity for detecting POD1 gene respectively (extracts total serum IgE and reverse transcription is
CDNA uses CsACTIN gene for reference gene, using the relative level of real-time quantitative PCR detection POD1 gene.
Primer pair for detecting POD1 gene is as follows：
Upstream primer (sequence 5 of sequence table)：ATGCTTTGGGGCTACCCTTG；
Downstream primer (sequence 6 of sequence table)：GGGTTCCCTTCACACTTGACT.
As a result see Fig. 3.In black tea manufacturing process, compared with the sample that withers, the expression of POD1 gene in the sample that ferments
Increase 9.4 times.During Green Tea Processing, compared with fresh leaf sample, the expression of POD1 gene is reduced in the sample that finishes
92%.The result shows that POD1 albumen participates in black tea/green tea process.
The differential expression of POD1 gene in embodiment 2, the tea morning different tissues that relax
Root, stem, flower, fruit, bud, a leaf, two leaves, three leaves of tea morning plant of relaxing are picked respectively.
Detect the expression quantity of POD1 gene in each sample respectively (using the method for high-flux sequence).
As a result see Fig. 4.There are differential expressions in easypro tea morning plant different tissues for POD1 gene, as tea raw material
Tissue (young shoot, tender leaf) in expression quantity highest.
Embodiment 3, the enzyme activity of Different Tea Varieties compare
Each tea tree breed wins a leaf and three leaves respectively (leaf and the mixing of three leaves are as sample).
Each sample carries out protein extraction, obtains the protein extraction solution containing total protein, by protein extraction solution or
Its dilution (being diluted using the kaliumphosphate buffer of pH6.0,0.1M) is used as sample to be tested, detects polyphenol oxidase enzyme
Living and peroxidase enzyme activity, is calculated the polyphenol oxidase enzyme activity and peroxidase enzyme activity of total protein.
As a result see Fig. 5.It is suitble to the tea tree breed for making black tea and is suitble to do the POD/PPO activity difference of the tea tree breed of green tea
It is larger.It is more crucial to illustrate that POD/PPO activity is suitble to do which kind of tea for tea tree breed decision.
Embodiment 4, POD1 gene Different Tea Varieties differential expression
Each tea tree breed wins a leaf and three leaves respectively (leaf and the mixing of three leaves are as sample).
Detect the expression quantity of POD1 gene in each sample respectively (using the method for high-flux sequence).
As a result see Fig. 6.POD1 gene is larger in the differential expression of the Typical Tea kind of different suitable black tea green tea.Explanation
POD1 gene is in Different Tea Varieties, to determining that the tea tree breed is suitble to cook green tea or black tea plays key effect.
The purifying and functional verification of embodiment 5, POD1 albumen
One, construction recombination plasmid
1, the leaf of Shu Chazao is taken, total serum IgE is extracted, reverse transcription obtains cDNA.
2, the cDNA obtained using step 1 carries out PCR amplification using the primer pair that F1 and R1 is formed as template.
F1：AGGATCCATGGGGATGGTCTCTTTCATCTC；
R1：CCTCGAG CTATAATTTCAACTTGCTG。
3, the pcr amplification product for taking step 2 to obtain carries out double digestion with restriction enzyme BamH I and Xho I, recycling
Digestion products.
4, pET-28a (+) carrier is taken, double digestion is carried out with restriction enzyme BamH I and Xho I, recycles carrier framework.
5, the digestion products of step 3 are connected with the carrier framework of step 4, obtains recombinant plasmid.According to sequencing result,
Structure is carried out to recombinant plasmid to be described as follows：Sequence is inserted between I restriction enzyme site of BamH I and Xho of pET-28a (+) carrier
Double chain DNA molecule shown in the sequence 2 of list.In recombinant plasmid, fusion shown in the sequence 4 with sequence table, expression
Fusion protein shown in the sequence 3 of sequence table.The fusion protein is also known as the POD1 albumen with 6 × His label.
Two, preparation and reorganization bacterium
The recombinant plasmid of step 1 building is imported into e. coli bl21 (DE3), obtains recombination fungus beetle.
By pET-28a (+) vector introduction e. coli bl21 (DE3), recombinant bacterium second is obtained.
Three, the expression and purifying of destination protein
1, recombinant bacterium is seeded in the LB liquid medium of the kanamycins containing 50mg/L, 37 DEG C, 180rpm shaken cultivation
To OD600nm=0.6.
2, after completing step 1, the concentration 0.5mmol/L that IPTG is added in system and makes it in system, 16 DEG C,
180rpm shaken cultivation 16h.
3, after completing step 2,12000rpm is centrifuged 10min, collects bacterial sediment.
4, the bacterial sediment for taking step 3 to obtain, is suspended with lysis buffer, then in ice bath carry out ultrasonication (300W,
Every ultrasound 3s stops 4s, and 100 times), then 10000rpm is centrifuged 30min, collects supernatant.
Lysis buffer：By 25ml 1M Tris aqueous solution, 30ml 5M NaCl aqueous solution and 945ml ddH2O composition.
5, the supernatant for taking step 4 to obtain is purified using Ni-NTA agaropectin, and Wash is used in purification process
Buffer is washed to remove foreign protein, is used Elution Buffer washing to collect destination protein, is obtained protein solution.
Wash Buffer：By 25ml 1M Tris aqueous solution, 30ml 5M NaCl aqueous solution, 3ml 5M imidazole
Aqueous solution and 942ml ddH2O composition.
Elution Buffer：By 25ml 1M Tris aqueous solution, 20ml 5M imidazole aqueous solution and 955ml
ddH2O composition.
Above-mentioned steps are carried out using recombination fungus beetle, obtained protein solution is named as POD1 protein solution.
Above-mentioned steps are carried out using recombinant bacterium second, obtained protein solution is named as contrast solution.
Following sample during taking recombination fungus beetle to carry out above-mentioned steps respectively carries out polyacrylamide gel electrophoresis：It completes
The supernatant samples (sample 1) collected after step 1, the POD1 that the supernatant samples (sample 2) and step 5 that step 4 is collected obtain
Protein solution (two repetitions of setting, sample 3 and sample 4).Electrophoretogram is shown in Fig. 7.
Four, Enzyme activity assay
The POD1 protein solution or contrast solution for taking step 3 to prepare detect polyphenol oxidase enzyme activity as sample to be tested
With peroxidase enzyme activity, the polyphenol oxidase enzyme activity and peroxidase enzyme activity of total protein is calculated.
The photo of termination system is shown in the left side Fig. 8, and enzyme activity result is shown in the right side Fig. 8.
The polyphenol oxidase enzyme activity of POD1 albumen with 6 × His label be 37.53 absorbance differences/(mg albumen ×
min)。
The peroxidase enzyme activity of POD1 albumen with 6 × His label be 22.50 absorbance differences/(mg albumen ×
min)。
The polyphenol oxidase enzyme activity of contrast solution is 0 absorbance difference/(mg albumen × min).
The peroxidase enzyme activity of contrast solution is 0 absorbance difference/(mg albumen × min).
Five, the ability of theaflavins compound is prepared
Reaction system (100 μ l)：EC and EGC is added in disodium hydrogen phosphate-citrate buffer solution of pH8.0, and makes it
Concentration be 1mM, the POD1 protein solution (total protein content be 30 μ g) and 1 μ l 30% of step 3 preparation is then added
Hydrogenperoxide steam generator.The control systems that POD1 protein solution is replaced with isometric contrast solution are set.
Reaction condition：It is stored at room temperature reaction 10min.
The photo of reaction finish time is shown in Fig. 9.
After reaction, using 0.22 μm of aperture strainer filtering, filtrate is collected.Respectively by filtrate carry out HPLC detection and
LC-MS detection.
HPLC detection parameters：Waters e2695 high performance liquid chromatography, 2489 UV-vis detectors, Empower2 color
Work station is composed, chromatographic column is 5 μm of 250 × 4.6mm of C18 of Phenomenex Gemini；Sample volume is 10 μ l；Mobile phase is A
Phase (0.2% acetic acid aqueous solution), B phase (pure methanol), flow rate of mobile phase 1ml/min.Elution process is shown in Table 2.Standard items are table
Catechin standard items or theaflavin standard items.Under identical chromatographic conditions, go out the peak within the ± 1min of peak position with standard items,
It is judged as same substance.Under identical chromatographic conditions, the standard curve of standard items content and peak area is made, by sighting target
Directrix curve obtains the concentration of respective compound in the system that reaction terminates by calculating.
Table 2
| Time (min) | A% | B% |
| 0 | 5 | 95 |
| 2 | 5 | 95 |
| 14 | 20 | 80 |
| 20 | 25 | 75 |
| 22 | 42 | 58 |
| 28 | 42 | 58 |
| 31 | 100 | 0 |
| 35 | 100 | 0 |
| 38 | 5 | 95 |
The proportion of a certain moment A phase and B phase is shown in table 2, in fact elution is dynamic process, with 0 to 14min
For, it is described as follows：During 0-2min, the volume fraction that A phase accounts for mobile phase is that 5% (corresponding B phase accounts for the body of mobile phase
Fraction is that 95%), during 2-14min, A phase accounts for the volume fraction of mobile phase by (the corresponding B of 5% linear rise to 20%
The volume fraction of mobile phase is mutually accounted for by 95% linear decline to 80%).
LC-MS detection parameters：Agilent Technologies 1290Infinity liquid chromatographic system, Mass Spectrometer Method
Device (Model#G6545A, Serial#SG1637E002, made in Singapore), chromatographic column are Hypersil Gold
column(2.1×100mm,particle size of 1.9μm,Thermo Scientific)；Mobile phase is that A phase (contains
The aqueous solution of 1% acetonitrile and 0.1% acetic acid, % represent volume ratio), (acetonitrile solution containing 0.1% acetic acid, % represent body to B phase
Product ratio), flow rate of mobile phase 0.2ml/min；Elution program is as follows：The volume fraction that 0-1min, B phase account for mobile phase is kept
5%；1-12min, B phase account for the volume fraction of mobile phase from 5% linear rise to 50%；12-13min, B phase account for mobile phase
50% linear rise of volume fraction is to 100%；The volume fraction that 13-18min, B phase account for mobile phase keeps 100%；18-
18.1min, B phase account for the volume fraction of mobile phase from 100% linear decline to 5%；18.1-22min, B phase account for the body of mobile phase
Fraction keeps 5%.
The chromatogram (chromatogram of 369nm) of HPLC detection is shown in Figure 10.It reacts finish time, table catechu in reaction system
The result is shown in Figure 11 of plain concentration, the result is shown in Figure 12 of theaflavin concentration in reaction system.
The result is shown in Figure 13 of LC-MS.The result shows that POD1 albumen can be catalyzed theaflavin generation really.
Six, zymologic property detects
Peroxidase enzyme activity determination method (colorimetric method)：Reaction system is 1ml；Initial system is by 50 μ l samples to be tested
The phosphoric acid of (the POD2 protein solution of step 3 preparation), guaiacol (as substrate), 30% hydrogenperoxide steam generator and 0.1mM
Buffer composition, the concentration of guaiacol are 0.56 μ l/ml, and the concentration of hydrogen peroxide is 0.38 μ l/ml；Initial system is stood
3min is reacted, as termination system；It detects initial system respectively using spectrophotometer and cessative aspect ties up to the extinction of 470nm
Value, terminates light absorption value-initial system light absorption value=absorbance difference of system；Peroxidase enzyme activity=absorbance difference
÷ (total protein content × reaction time), the unit of total protein are mg, and the unit in reaction time is min.
Following pH is respectively adopted in phosphate buffer：pH6.0,6.5,7.0,7.5,8.0.Reaction temperature is room temperature.As a result see
Figure 14 A.
Following reaction temperature is respectively adopted：30,40,50,60,70 or 80 DEG C.Phosphate buffer uses pH6.0.As a result see
Figure 14 B.
The result shows that POD1 albumen peroxidase enzyme activity highest under the conditions of pH8.0,30 DEG C.
Embodiment 6 reduces theaflavin content by inhibition POD1 gene expression
Pick one leaf of a bud for tea morning plant of relaxing, 60 samples of similar size.
60 samples are randomly divided into two groups, every group of 30 samples.
First group：It is immersed in 80mM aqueous sucrose solution, room temperature 96 hours；
Second group：It is immersed in the aqueous solution containing 700 μM of antisense nucleic acids and 80mM sucrose, room temperature 96 hours；
Antisense nucleic acid (sequence 7 of sequence table)：5'-ATCCATTTGGGCCACCAG-3'.
Antisense nucleic acid is single strand dna.
After completion processing, the expression quantity of POD1 gene in sample is detected, method is the same as the step of embodiment 1 four.Relative expression
Level is shown in Figure 15.The result shows that compared with first group of treated sample, the table of the POD1 gene of sample after second group of processing
It is significantly reduced up to amount.
After completion processing, sample is taken out, is successively proceeded as follows：Surface moisture is absorbed, then spreads 16h in room temperature
Then tealeaves is held together after glomeration is first gently rubbed to rub again and gently be rubbed again by left and right, then encase 30 DEG C of fermentation 12h of tealeaves (fermentation with gauze
Period will scatter every 2h once to be then proceeded to ferment), then 110 DEG C of dry 15min or so, obtain dry sample；Then it takes
0.1g dries sample, adds in 1.5ml 80% (volume ratio) methanol aqueous solution, and the concussion that is vortexed mixes well, then in ice bath
Ultrasonic 30min, then 13000rpm is centrifuged 30min, using 0.22 μm of aperture strainer filtering, collects filtrate；Filtrate is taken, is used
HPLC detection (parameter is detected with the HPLC in the step of embodiment 5 five, 15 μ l of sample volume)；Standard items are TF, in identical chromatography
Under the conditions of the peak that goes out within ± the 0.5min of peak position with standard items be judged as same substance, mark is made under identical chromatographic conditions
The standard curve of quasi- product content and peak area obtains the concentration of TF in dry sample by calculating by reference standard curve
(how many μ g TF contained in the dry sample of every g).The result is shown in Figure 16.Compared with the drying sample that first group obtains, second group is obtained
TF concentration in dry sample significantly reduces.
SEQUENCE LISTING
<110>Agricultural University Of Anhui
<120>From the peroxidase POD1 relevant to theaflavin generation of tealeaves and its application
<130> GNCYX181158
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 352
<212> PRT
<213> Camellia sinensis
<400> 1
Met Gly Met Val Ser Phe Ile Ser Thr Val Pro Ser Leu Leu Ala Leu
1 5 10 15
Ala Pro Leu Pro Ser Pro Thr Thr Ser Thr Thr Ser Val Ala Val Ser
20 25 30
Lys Tyr Pro Ile His Ala Ser Thr Val Arg Cys Lys Ile Glu Ala Asp
35 40 45
Asn Gly Ser Glu Asp Gln Phe Gly Arg Arg Asp Ile Leu Lys Cys Phe
50 55 60
Gly Ala Thr Leu Gly Met Glu Ile Ile Ala Ser Ser Gly Ser Phe Val
65 70 75 80
Glu Glu Ala Asn Ala Ala Asp Leu Ile Gln Arg Arg Gln Arg Ser Glu
85 90 95
Phe Gln Ser Ser Val Lys Gly Thr Leu Phe Thr Ala Ile Lys Ala Asn
100 105 110
Pro Asp Leu Val Pro Ser Leu Leu Thr Leu Ala Met Asn Asp Ala Met
115 120 125
Thr Tyr Asp Lys Ala Thr Lys Ser Gly Gly Pro Asn Gly Ser Val Arg
130 135 140
Phe Ser Ser Glu Ile Ser Arg Pro Glu Asn Lys Gly Leu Ser Ala Ala
145 150 155 160
Met Asn Leu Leu Glu Glu Ala Lys Lys Glu Ile Asp Ser Tyr Ser Lys
165 170 175
Gly Gly Pro Ile Ser Phe Ala Asp Leu Ile Gln Phe Ala Ala Gln Ser
180 185 190
Ala Leu Lys Ser Thr Phe Leu Ala Ser Ala Ile Arg Lys Cys Gly Gly
195 200 205
Asn Glu Glu Lys Gly Ser Leu Leu Tyr Thr Ala Tyr Gly Ser Asn Gly
210 215 220
Gln Trp Gly Leu Phe Asp Lys Gln Phe Gly Arg Ser Asp Ala Gln Glu
225 230 235 240
Pro Asp Pro Glu Gly Arg Val Pro Leu Trp Ala Lys Ala Ser Val Gln
245 250 255
Glu Met Lys Asp Lys Phe Ser Ala Ile Gly Phe Gly Pro Arg Gln Leu
260 265 270
Ala Val Met Ser Ala Phe Leu Gly Pro Asp Gln Met Ala Thr Glu Thr
275 280 285
Leu Leu Ala Thr Asp Arg Asp Val Ala Pro Trp Val Glu Lys Tyr Gln
290 295 300
Arg Ser Arg Glu Thr Val Ser Glu Thr Asp Tyr Glu Val Asp Leu Ile
305 310 315 320
Thr Thr Leu Thr Lys Leu Ser Ala Leu Gly Gln Gln Ile Asn Tyr Glu
325 330 335
Ala Tyr Ala Tyr Pro Val Gln Arg Val Asp Phe Ser Lys Leu Lys Leu
340 345 350
<210> 2
<211> 1059
<212> DNA
<213> Camellia sinensis
<400> 2
atggggatgg tctctttcat ctcaactgta ccttccttgc ttgcacttgc tcctctccca 60
tctcccacca ccagcaccac atctgttgct gtctccaaat acccaattca tgctagtaca 120
gttcgctgta aaattgaagc tgacaatggt agtgaggatc aatttggtcg aagagacatt 180
cttaaatgct ttggggctac ccttggcatg gaaataatag caagctcagg ctcatttgtt 240
gaagaggcta atgctgctga tctaatacag cgcagacagc gttctgagtt tcagtcaagt 300
gtgaagggaa ccctttttac agctataaag gcaaacccag atcttgttcc atccttattg 360
actttggcga tgaatgatgc tatgacttat gataaggcca caaaatctgg tggcccaaat 420
ggatctgtac gtttcagctc agagattagc agaccagaga ataaggggct ctctgccgca 480
atgaatttat tagaggaagc aaagaaggaa atagattcat attccaaggg tggtcctatt 540
tcatttgcgg atctcatcca atttgcagca caaagtgcct taaagtctac atttctagct 600
tctgccatcc gtaaatgtgg tggaaatgaa gagaaaggaa gcttactata cactgcatat 660
ggttcaaatg ggcagtgggg cttgtttgac aagcaatttg ggaggtcaga tgctcaagag 720
ccagatccag aagggagggt tcccctgtgg gcgaaagcaa gtgttcagga aatgaaagat 780
aagttctcgg ccataggctt tggtccccgc cagctagctg ttatgtctgc attcttgggt 840
cctgatcaaa tggccactga gaccttattg gccacagatc gagatgttgc tccatgggtt 900
gagaaatacc aaagaagccg agaaacagtc tctgaaactg actacgaggt tgacctaata 960
actaccctca caaaattaag tgctttgggg caacaaatca attatgaggc atacgcatat 1020
cctgttcaaa gagttgattt cagcaagttg aaattatag 1059
<210> 3
<211> 386
<212> PRT
<213> Artificial sequence
<400> 3
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Met Gly Met Val Ser Phe Ile Ser Thr Val Pro Ser Leu Leu
35 40 45
Ala Leu Ala Pro Leu Pro Ser Pro Thr Thr Ser Thr Thr Ser Val Ala
50 55 60
Val Ser Lys Tyr Pro Ile His Ala Ser Thr Val Arg Cys Lys Ile Glu
65 70 75 80
Ala Asp Asn Gly Ser Glu Asp Gln Phe Gly Arg Arg Asp Ile Leu Lys
85 90 95
Cys Phe Gly Ala Thr Leu Gly Met Glu Ile Ile Ala Ser Ser Gly Ser
100 105 110
Phe Val Glu Glu Ala Asn Ala Ala Asp Leu Ile Gln Arg Arg Gln Arg
115 120 125
Ser Glu Phe Gln Ser Ser Val Lys Gly Thr Leu Phe Thr Ala Ile Lys
130 135 140
Ala Asn Pro Asp Leu Val Pro Ser Leu Leu Thr Leu Ala Met Asn Asp
145 150 155 160
Ala Met Thr Tyr Asp Lys Ala Thr Lys Ser Gly Gly Pro Asn Gly Ser
165 170 175
Val Arg Phe Ser Ser Glu Ile Ser Arg Pro Glu Asn Lys Gly Leu Ser
180 185 190
Ala Ala Met Asn Leu Leu Glu Glu Ala Lys Lys Glu Ile Asp Ser Tyr
195 200 205
Ser Lys Gly Gly Pro Ile Ser Phe Ala Asp Leu Ile Gln Phe Ala Ala
210 215 220
Gln Ser Ala Leu Lys Ser Thr Phe Leu Ala Ser Ala Ile Arg Lys Cys
225 230 235 240
Gly Gly Asn Glu Glu Lys Gly Ser Leu Leu Tyr Thr Ala Tyr Gly Ser
245 250 255
Asn Gly Gln Trp Gly Leu Phe Asp Lys Gln Phe Gly Arg Ser Asp Ala
260 265 270
Gln Glu Pro Asp Pro Glu Gly Arg Val Pro Leu Trp Ala Lys Ala Ser
275 280 285
Val Gln Glu Met Lys Asp Lys Phe Ser Ala Ile Gly Phe Gly Pro Arg
290 295 300
Gln Leu Ala Val Met Ser Ala Phe Leu Gly Pro Asp Gln Met Ala Thr
305 310 315 320
Glu Thr Leu Leu Ala Thr Asp Arg Asp Val Ala Pro Trp Val Glu Lys
325 330 335
Tyr Gln Arg Ser Arg Glu Thr Val Ser Glu Thr Asp Tyr Glu Val Asp
340 345 350
Leu Ile Thr Thr Leu Thr Lys Leu Ser Ala Leu Gly Gln Gln Ile Asn
355 360 365
Tyr Glu Ala Tyr Ala Tyr Pro Val Gln Arg Val Asp Phe Ser Lys Leu
370 375 380
Lys Leu
385
<210> 4
<211> 1161
<212> DNA
<213> Artificial sequence
<400> 4
atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60
atggctagca tgactggtgg acagcaaatg ggtcgcggat ccatggggat ggtctctttc 120
atctcaactg taccttcctt gcttgcactt gctcctctcc catctcccac caccagcacc 180
acatctgttg ctgtctccaa atacccaatt catgctagta cagttcgctg taaaattgaa 240
gctgacaatg gtagtgagga tcaatttggt cgaagagaca ttcttaaatg ctttggggct 300
acccttggca tggaaataat agcaagctca ggctcatttg ttgaagaggc taatgctgct 360
gatctaatac agcgcagaca gcgttctgag tttcagtcaa gtgtgaaggg aacccttttt 420
acagctataa aggcaaaccc agatcttgtt ccatccttat tgactttggc gatgaatgat 480
gctatgactt atgataaggc cacaaaatct ggtggcccaa atggatctgt acgtttcagc 540
tcagagatta gcagaccaga gaataagggg ctctctgccg caatgaattt attagaggaa 600
gcaaagaagg aaatagattc atattccaag ggtggtccta tttcatttgc ggatctcatc 660
caatttgcag cacaaagtgc cttaaagtct acatttctag cttctgccat ccgtaaatgt 720
ggtggaaatg aagagaaagg aagcttacta tacactgcat atggttcaaa tgggcagtgg 780
ggcttgtttg acaagcaatt tgggaggtca gatgctcaag agccagatcc agaagggagg 840
gttcccctgt gggcgaaagc aagtgttcag gaaatgaaag ataagttctc ggccataggc 900
tttggtcccc gccagctagc tgttatgtct gcattcttgg gtcctgatca aatggccact 960
gagaccttat tggccacaga tcgagatgtt gctccatggg ttgagaaata ccaaagaagc 1020
cgagaaacag tctctgaaac tgactacgag gttgacctaa taactaccct cacaaaatta 1080
agtgctttgg ggcaacaaat caattatgag gcatacgcat atcctgttca aagagttgat 1140
ttcagcaagt tgaaattata g 1161
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence
<400> 5
atgctttggg gctacccttg 20
<210> 6
<211> 21
<212> DNA
<213> Artificial sequence
<400> 6
gggttccctt cacacttgac t 21
<210> 7
<211> 18
<212> DNA
<213> Artificial sequence
<400> 7
atccatttgg gccaccag 18
Claims (10)
1. protein is any one following of (a1) into (a8)：
(a1) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(a2) contain the fusion protein of (a1)；
(a3) fusion protein that small peptide of the connection containing label obtains in the end of (a1)；
(a4) fusion protein that connection label obtains in the end of (a1)；
(a5) protein that the amino acid sequence shown in sequence 3 in sequence table forms；
(a6) (a1) or (a2) or (a3) or (a4) or (a5) are passed through to the substitution and/or missing of one or several amino acid residues
And/or add and have protein as derived from it of oxidase function；
(a7) there is 98% or more homology with (a1) obtained from tealeaves and there is as derived from it protein of oxidase function；
(a8) it is obtained from the early protein as derived from it with (a1) with 98% or more homology with oxidase function of the tea that relaxes.
2. the encoding gene of protein described in claim 1.
3. gene as claimed in claim 2, it is characterised in that：The gene is following (b1) any described into (b5)
DNA molecular：
(b1) code area DNA molecular as shown in sequence 2 in sequence table；
(b2) code area DNA molecular as shown in sequence 4 in sequence table；
(b3) hybridize under strict conditions with (b1) or (b2) DNA sequence dna limited and encode the DNA molecular of oxidizing ferment；
(b4) have 90% or more or 95% or more or 98% or more or 99% from the DNA sequence dna of tealeaves limited with (b1)
The above homology and the DNA molecular for encoding oxidizing ferment；
(b5) from the early DNA sequence dna limited with (b1) of the tea that relaxes have 90% or more or 95% or more or 98% or more or
99% or more homology and the DNA molecular for encoding oxidizing ferment.
4. the application of protein described in claim 1, at least one of following (c1) to (c16)：
(c1) it is used as oxidizing ferment；
(c2) oxidizing ferment is prepared；
(c3) it is used as peroxidase；
(c4) peroxidase is prepared；
(c5) it is used as polyphenol oxidase；
(c6) polyphenol oxidase is prepared；
(c7) theaflavins compound is prepared；
(c8) theaflavins compound is prepared by substrate of catechin compounds；
(c9) theaflavin is prepared；
(c10) theaflavin is prepared by substrate of catechin compounds；
(c11) theaflavin is prepared using epigallocatechin and epicatechin as substrate；
(c12) promote Catechin in Tea class degradation；
(c13) level of Catechin in Tea class compound is reduced；
(c14) it is horizontal to improve theaflavin in tealeaves；
(c15) it is screened as marker and is suitble to the tea tree breed of production green tea and/or is suitble to the tea tree breed to make black tea by spreading the leaves on withering racks to dry；
(c16) black tea processing technology is controlled as inspection target.
5. application of the gene described in Claims 2 or 3 in Tea Breeding；The Tea Breeding is to cultivate to be suitble to production green tea
Tea tree breed or cultivation are suitble to the tea tree breed to make black tea by spreading the leaves on withering racks to dry.
6. for inhibiting the substance of gene expression described in Claims 2 or 3 in the genetically modified plants for preparing the reduction of theaflavin level
Or the application in Transgenic plant tissue.
7. for reducing protein active described in claim 1 and/or the substance of level in the plant for cultivating the reduction of theaflavin level
In application.
8. the application for the substance for detecting gene expression dose described in Claims 2 or 33, for following (d1) into (d5) extremely
Few one kind：
(d1) it evaluates tea tree breed to be measured or individual is suitble to production green tea or black tea；
(d2) ability of tea tree breed to be measured or individual production theaflavin is evaluated；
(d3) disease resistance of tea tree breed to be measured or individual is evaluated；
(d4) screening is suitble to the tea tree breed or individual of production green tea；
(d5) screening is suitble to the tea tree breed or individual to make black tea by spreading the leaves on withering racks to dry.
9. the application for the substance for detecting protein level described in claim 1, at least one of following (d1) to (d5)：
(d1) it evaluates tea tree breed to be measured or individual is suitble to production green tea or black tea；
(d2) ability of tea tree breed to be measured or individual production theaflavin is evaluated；
(d3) disease resistance of tea tree breed to be measured or individual is evaluated；
(d4) screening is suitble to the tea tree breed or individual of production green tea；
(d5) screening is suitble to the tea tree breed or individual to make black tea by spreading the leaves on withering racks to dry.
10. primer pair or specific DNA molecular；
It is single-stranded shown in primer pair single strand dna as shown in the sequence 5 of sequence table and the sequence 6 of sequence table
DNA molecular composition；
The specific DNA molecular is single strand dna shown in the sequence 7 of sequence table.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111575255A (en) * | 2020-05-14 | 2020-08-25 | 安徽农业大学 | Oxidase, DNA molecule for coding oxidase and application thereof |
| CN111575259A (en) * | 2020-05-12 | 2020-08-25 | 安徽农业大学 | Ester type catechin synthetase, coding gene and application thereof |
| CN114015733A (en) * | 2021-11-12 | 2022-02-08 | 中国农业科学院茶叶研究所 | Enzyme-salt coupling catalytic synthesis method of polyester catechin |
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| CN111575259A (en) * | 2020-05-12 | 2020-08-25 | 安徽农业大学 | Ester type catechin synthetase, coding gene and application thereof |
| CN111575255A (en) * | 2020-05-14 | 2020-08-25 | 安徽农业大学 | Oxidase, DNA molecule for coding oxidase and application thereof |
| CN114015733A (en) * | 2021-11-12 | 2022-02-08 | 中国农业科学院茶叶研究所 | Enzyme-salt coupling catalytic synthesis method of polyester catechin |
| CN114015733B (en) * | 2021-11-12 | 2023-08-08 | 中国农业科学院茶叶研究所 | Enzyme-salt coupling catalytic synthesis method of polyester catechin |
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