CN112760327B - Persimmon procyanidine precursor transmembrane transporter DkMATE5 and application thereof - Google Patents
Persimmon procyanidine precursor transmembrane transporter DkMATE5 and application thereof Download PDFInfo
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- CN112760327B CN112760327B CN202110211287.2A CN202110211287A CN112760327B CN 112760327 B CN112760327 B CN 112760327B CN 202110211287 A CN202110211287 A CN 202110211287A CN 112760327 B CN112760327 B CN 112760327B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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- 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
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6851—Quantitative amplification
Abstract
The invention discloses a persimmon procyanidine precursor transmembrane transporter DkMATE5 and application thereof, belongs to the technical field of plant genetic engineering, and obtains DkMATE4 and DkMATE5 genes through primary screening of a transcriptome database, and finds that DkMATE5 protein has an procyanidine transporting function. The DkMATE5 gene is further proved to promote preferential transmembrane transport of persimmon procyanidine precursor catechin, epicatechin and epicatechin gallate through escherichia coli in vivo complementary experimental analysis. The method provides scientific basis and new gene resources for the analysis of the component and content difference of procyanidine of different deastringent types of persimmons and the genetic improvement of the persimmons.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, and particularly relates to a persimmon procyanidine precursor transmembrane transporter DkMATE5 and application thereof.
Background
Persimmon fruits contain a large amount of Proanthocyanidins (PAs; commonly known as "persimmon tannin") which are important indicators for determining the maturity and commercial quality of fruits. The biosynthesis of procyanidins involves shikimic acid, communal phenylpropane, core flavonoids-anthocyanins and procyanidin specific pathways. Procyanidin precursors are synthesized in the cytoplasm, recognized by transporters localized on the vacuolar membrane and transported to the vacuole for polymerization, thereby forming macromolecular procyanidins. At present, structural genes and regulatory genes for procyanidine biosynthesis are cloned and identified, but transmembrane transport of procyanidine precursors is less researched. Particularly, whether preferential transport of persimmon procyanidin precursor substances causes astringent taste removing type differences of persimmon varieties is not completely clear so far.
Multidrug and toxic compound export (MATE) proteins are present in bacteria, yeast, plants and animals and perform a relatively conserved, fundamental transport function in most prokaryotic and eukaryotic organisms. There are 56 members of the arabidopsis MATE family, of which the MATE transporter encoded by TT12 transports flavonoids into the vacuole. The role of MATE protein in transmembrane transport of persimmon procyanidin precursor substances is not clear.
Disclosure of Invention
One of the purposes of the invention is to provide a persimmon procyanidine precursor transmembrane transporter DkMATE5 and application thereof, wherein the nucleotide sequence of the persimmon procyanidine precursor transmembrane transporter is shown as SEQ ID No. 1.
The invention clones DkMATE5 gene with the function of trans-membrane transport of procyanidine precursor for the first time, and finds that the gene has selectivity in trans-membrane transport of procyanidine precursor of persimmon, and the gene preferentially transports catechin, epicatechin and epicatechin gallate. The preferential transfer is the reason for the difference of the deastringency types of the persimmon varieties, and provides scientific basis and new gene resources for the subsequent genetic improvement of the sweet persimmons.
The second purpose of the invention is to provide the application of the persimmon procyanidin precursor transmembrane transporter DkMATE5 in preferential transporter catechin, epicatechin and epicatechin gallic acid.
The third purpose of the invention is to provide the application of the persimmon procyanidin precursor transmembrane transporter DkMATE5 in procyanidin transport.
The fourth purpose of the invention is to provide an amplification primer for amplifying the DkMATE5 gene, wherein the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 4-5.
The fifth purpose of the invention is to provide a fluorescent quantitative PCR specific primer for detecting DkMATE5 gene, wherein the nucleotide sequence of the fluorescent quantitative PCR specific primer is shown as SEQ ID NO. 6-7.
The sixth object of the present invention is to provide an expression vector containing the DkMATE5 gene.
The seventh purpose of the invention is to provide a genetic engineering bacterium, which comprises the expression vector.
The eighth object of the present invention is to provide the use of the above expression vector or genetically engineered bacterium for promoting transmembrane transport of catechin, epicatechin, and epicatechin gallate.
Compared with the prior art, the invention has the beneficial effects that:
the DkMATE4 and DkMATE5 genes are obtained for the first time through screening of a transcriptome database, and the DkMATE5 protein is found to have a proanthocyanidin precursor transport function. The DkMATE5 gene is further proved to promote preferential transmembrane transport of persimmon procyanidine precursor catechin, epicatechin and epicatechin gallic acid by an escherichia coli in vivo complementation experiment. The method provides scientific basis and new gene resources for the analysis of the component and content difference of procyanidine of different deastringent types of persimmons and the genetic improvement of the persimmons.
Drawings
FIG. 1 shows the results of the gene expression tests of DkMATE4 and DkMATE5 in the development period of different varieties of persimmon fruits in example 1, wherein A is the expression of DkMATE4 and B is the expression of DkMATE 5.
FIG. 2 is the measurement results of the soluble PAs and insoluble PAs content of the instant transformed persimmon leaves of DkMATE4 and DkMATE5 in example 2 of the present invention, wherein, the graph A is the soluble PAs content and the graph B is the insoluble PAs content.
FIG. 3 is the results of the Escherichia coli function complementation test in example 3 of the present invention, wherein Panel A is the expression of DkMATE5 in the Escherichia coli mutant strain supplemented with catechin (C), Epicatechin (EC), epicatechin gallate (ECG), Epigallocatechin (EGC) and epigallocatechin gallate (EGCG), Empty Vector represents Empty Vector control, DkMATE5 represents Escherichia coli transformed with prokaryotic expression plasmid PGEX-6P-1-DkMATE5, wherein 6 plaques in each row are the initial OD600Plaque with values of 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 in order. Panel B is a growth curve of a strain containing no substrate, panel C is a growth curve of a strain to which catechin (C) is added, panel D is a growth curve of a strain to which Epicatechin (EC) is added, and panel E is a growth curve of a strain to which epicatechin gallate (ECG) is added.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments of the present invention, and it should be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1 acquisition and analysis of DkMATE5 and DkMATE4 Gene sequences
1. Acquisition of DkMATE5 and DkMATE4 Gene sequences in ` Esche 1 `fruit
(1) Firstly, downloading MATE gene family HMM information through a Pfam database (http:// Pfam. xfam. org), comparing an oil persimmon genome (http:// www.kakiwi.zju.edu.cn/cgi-bin/persimmon/index. cgi) by using HMMER software, obtaining candidate MATE protein sequences, comparing the candidate protein sequences with the Pfam database, keeping MATE genes with conserved domains as DkMATE family gene members, searching DkMATE differential expression genes by using a transcription group database, and determining the differential expression of the DkMATE4 genes and the DkMATE5 genes;
(2) RACE primers are respectively designed by utilizing Primer Premier 5.0 software, wherein the RACE Primer for amplifying the DkMATE4 gene is shown as SEQ ID NO.13-14, and the RACE Primer for amplifying the DkMATE5 gene is shown as SEQ ID NO. 2-3. And using 'Esche 1' as a template, and amplifying by using a SMARTer RACE cDNAamplification Kit (Clontech, USA) to obtain a target gene sequence.
(3) The extraction of the RNA from the pulp of 'Eben No. 1' persimmon was carried out using the RNAPlant Plus Kit (Tiangen Biotech Ltd., Beijing, China), and PrimeScript was usedTMThe RT reagent Kit with the gDNAeraser (TaKaRa, Japan) reverse transcription Kit synthesized cDNA.
(4) Designing a primer (shown as SEQ ID NO. 15-16) for amplifying the full length of the DkMATE4 gene and a primer (shown as SEQ ID NO. 4-5) for amplifying the full length of the DkMATE5 gene, carrying out PCR amplification by using PrimeSTAR Max Premix (2x) (TaKaRa, Japan) Hi-Fi enzyme and taking 'Eben No. 1' cDNA obtained by the reverse transcription as a template to respectively obtain PCR fragments containing the complete DkMATE4 gene coding region and DkMATE5 gene coding region sequence, and obtaining the DkMATE5 gene sequence shown as SEQ ID NO.1 and the DkMATE4 gene sequence shown as SEQ ID NO.12 after sequencing.
2. Gene expression patterns of DkMATE4 and DkMATE5 in different varieties and fruit development stages:
(1) selecting Ebei persimmon No.1 and Mill persimmon as test material, collecting pulp at 2.5 weeks, 5 weeks, 10 weeks, 15 weeks, 20 weeks and 27.5 weeks after blooming, and storing at-80 deg.C for use.
(2) Collecting persimmon fruits of different varieties at different development stages, extracting RNA by using an RNAPlant Plus Kit (Tiangen Biotech Ltd., Beijing, China), and referring to PrimeScriptTMThe RT reagent Kit with the gDNAeraser reverse transcription Kit (TaKaRa, Japan) synthesized cDNA.
(3) The specific primers of the fluorescent quantitative PCR for quantitatively detecting the DkMATE4 gene and the DkMATE5 gene are respectively designed, wherein the specific primers of the fluorescent quantitative PCR for quantitatively detecting the DkMATE4 gene are shown as SEQ ID NO.17-18, and the specific primers of the fluorescent quantitative PCR for quantitatively detecting the DkMATE5 gene are shown as SEQ ID NO. 6-7.
By using
Quantitative analysis of DkMATE4 and DkMATE5 was performed by Green Real-Time PCR Master Mix fluorescent dye (TaKaRa, Japan) and ABI QuantStaudio 7Flex Real-Time PCR instrument.
Quantitative PCR reaction system: 5 μ L of
Green Real-time PCR Master Mix, 1. mu.L cDNA, 0.4. mu.L forward and reverse primers, plus RNA free H2O to a final volume of 10. mu.L.
Quantitative PCR reaction conditions: the run was run for 40 cycles at 95 ℃ for 30s, then 95 ℃ for 15s, 58 ℃ for 30s, 72 ℃ for 20s, then 95 ℃ for 15s, 40 ℃ for 30 s.
Four replicates were set for each sample and Ct values were read under default conditions with Diospyros kaki thunb. action (GenBank Accession No. ab219402) as an internal reference gene. All results are shown as mean (SE) with corresponding standard error.
The real-time fluorescence quantitative PCR detection result is shown in FIG. 1, wherein, the graph A is the expression condition of DkMATE4, and the graph B is the expression condition of DkMATE 5. The results showed that the expression levels of DkMATE4 and DkMATE5 were gradually decreased in the early fruit development stage, and were expressed in large amounts in the middle and late fruit development stages, and that the expression levels were higher in all the persimmons (Eben 1) than in the non-all-sweet persimmons (Mopan persimmon). And the expression patterns of two genes DkMATE4 and DkMATE5 of the same gene family in fruits of different varieties at different periods show larger difference.
Example 2 analysis of transient transformation of persimmon leaves with DkMATE4 and DkMATE5
(1) According to the DkMATE5 gene sequence shown in SEQ ID NO.1 and the DkMATE4 gene sequence shown in SEQ ID NO.12 obtained in example 1, MATE5-OEF and MATE5-OER shown in SEQ ID NO.8-9 and MATE4-OEF and MATE4-OER shown in SEQ ID NO.19-20 are respectively designed, according to the instructions, the DkMATE5 gene and the DkMATE4 gene are respectively introduced into an entry vector pDONR207 by using a Gateway method, and are introduced into an overexpression vector pKLR 2GW7 through a reaction, respectively construct overexpression recombinant plasmids, and the overexpression recombinant plasmids are transferred into agrobacterium tumefaciens (GV3101) by using a conventional method.
(2) The agrobacterium after transfection is instantaneously transformed into 'Eben No. 1' leaves by adopting a conventional injection and permeation method, the leaves infected by the agrobacterium are collected after 10 days of injection, and the leaves are stored at minus 80 ℃ for use after quick freezing by liquid nitrogen. The soluble, insoluble Procyanidin (PAs) content of transiently transformed leaves was determined according to Folin-Ciocalteau method (Oshida et al, 1996) with empty vector as control. The measurement results are shown in FIG. 2, in which the graph A shows the content of soluble PAs and the graph B shows the content of insoluble PAs.
According to the detection result of fig. 2, after the DkMATE4 overexpression recombinant plasmid is transformed into persimmon leaves, compared with an empty vector Control group (Control), the content of soluble and insoluble PAs in 'Ekuki No. 1' leaves is not significantly changed, which indicates that DkMATE4 is not involved in the transportation of PAs. And the determination and analysis of the content of PAs in the leaves of the transformation overexpression recombinant plasmid pK2GW7-DkMATE5 and the empty vector control group show that after the transformation of the DkMATE5 overexpression recombinant plasmid, the content of soluble and insoluble PAs in the leaves is remarkably increased compared with the content of PAs in the empty vector control group (p is less than 0.01), and the result shows that DkMATE5 in the completely sweet persimmon is involved in the transport of the PAs, while DkMATE4 of the same gene family is not involved in the transport of the PAs.
Example 3 E.coli functional complementation assay determination of DkMATE5 transport preference
1. Construction of PGEX-6P-1-DkMATE5 expression vector
(1) Using PrimeSTAR Max Premix (2x) high fidelity enzyme (TaKaRa, Japan), DkMATE5 gene full-length plasmid was used as a template, primers containing BamH I and Not I restriction sites (shown in SEQ ID NO. 10-11) were used for amplification and cloning to prokaryotic expression vector PGEX-6P-1 to construct prokaryotic expression plasmid PGEX-6P-1-DkMATE 5.
2. Experiment for complementation of functions of Escherichia coli
The constructed prokaryotic expression plasmid PGEX-6P-1-DkMATE5 is transformed into an escherichia coli mutant strain acrB lacking the drug efflux capability according to a conventional method. Successfully transformed single colonies were picked to 5mL LB liquid medium and shaken at 37 ℃ and 200r/min for about 12 h. 500 mu L of the bacterial liquid is taken to be put into 50mL of LB liquid culture medium, and the bacteria is shaken at 37 ℃ and 200 r/min. Measurement of OD of bacterial liquid600The value, when about 0.6 is reached, 1mM IPTG is added until OD is reached600When the value reached 1.0, 1mL of the bacterial solution was taken. Will OD600The bacterial solution with 1.0 is subjected to six continuous descending gradients (OD)600The values are 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 in sequence), and 1.5. mu.L of each bacterial solution was spotted onto six solid LB plates (containing 1mM IPTG) containing different substrates, wherein the substrates were catechin (C), Epicatechin (EC), epicatechin gallate (ECG), Epigallocatechin (EGC) and epigallocatechin gallate (EGCG). The growth of the bacteria was observed after 24h incubation at 37 ℃ and the strain acrB transformed with the empty vector PGEX-6P-1 was used as a control. The measurement results are shown in FIG. 3A. Wherein C (+) is added catechin, EC (+) is added epicatechin, ECG (+) is added epicatechin gallic acid, EGC is added epigallocatechin, and EGCG is added epigallocatechin gallate; empty Vector is the Empty Vector control group; DkMATE5 is transformed with prokaryotic expression plasmid PGEX-6P-1-DkMATE5 Escherichia coli, wherein each row of 6 plaque as initial OD600Plaque with values of 1.0, 0.8, 0.6, 0.4, 0.2, 0.1 in order.
In addition, successfully transformed single colonies were picked, cultured at 37 ℃ and the OD of the bacterial solution was measured600Value as OD of bacterial liquid600When the value reaches about 1.0, diluting to 0.5-0.6. Then, 5. mu.L of the above-mentioned bacterial solution was added to 1ml of LB liquid medium (containing 1mM IPTG) containing different substrates, i.e., catechin, epicatechin, and epicatechin gallate, respectively. Then measuring the light absorption value OD of the bacterial liquid every 1h600Values were recorded and transformed with the empty vector PGEX-6P-1 into the strain acrB as a control. The measurement results are shown in FIG. 3. Wherein, the graph B is a strain growth curve without a substrate, the graph C is a strain growth curve with catechin (C) added, the graph D is a strain growth curve with Epicatechin (EC) added, and the graph E is a strain growth curve with epicatechin gallate (ECG) added.
The results of the E.coli complementation test according to FIG. 3A showed that the bacterial strain containing PGEX-6P-1-DkMATE5 had the same plaque growth status as the control group containing the empty vector PGEX-6P-1 without the addition of substrate. When the substrate catechin (C) or the substrate Epicatechin (EC) or the epicatechin gallate (ECG) is added, the growth condition of the strain containing PGEX-6P-1-DkMATE5, namely the strain expressing DkMATE5 is obviously better than that of a control group, and when the substrate Epigallocatechin (EGC) or the epigallocatechin gallate (EGCG) is added, the growth condition of the strain containing PGEX-6P-1-DkMATE5 is not different from that of the control group.
Further, in the absence of added substrate, there was no difference between the growth curves of the strain containing the prokaryotic expression plasmid PGEX-6P-1-DkMATE5 and the control strain containing the empty vector PGEX-6P-1. When substrate catechin (C) or substrate Epicatechin (EC) or epicatechin gallate (ECG) is added, the growth condition of the strain containing prokaryotic expression plasmid PGEX-6P-1-DkMATE5, namely the strain expressing DkMATE5 is obviously better than that of a control group. This result demonstrates that expression of DkMATE5 complements the function of the escherichia coli mutant strain acrB lacking drug efflux capacity, i.e., DkMATE5 transporter prefers to transport catechin, epicatechin, and epicatechin gallate, but not to transport epigallocatechin and epigallocatechin gallate.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Sequence listing
<110> university of agriculture in Huazhong
<120> persimmon procyanidine precursor transmembrane transporter DkMATE5 and application thereof
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ggagattacc gaccattgag gaccttcaag gaactgacat ctctgttccg gatagagacc 120
gcgaagctct ggagaatctc ggctccgatt atagtgacta ccatctgtaa ctatgcgatt 180
aactccacca ccagcatctt cgtcggccac ctcggcgatg tcgaactctc cgccgtctct 240
atctccgtct ccgtcatcgc cgtcttctct ttcggcttcc tgcttggtat ggggagtgca 300
ctagaaacac tctgtggcca agcttatggt gcagggcaag ttcacatgct tggggtctat 360
atgcagcgct catggataat tctgtttgtc acctgcattc ttctcacgcc aatttacata 420
tttgctacac cggtcctgaa gctcttcggc caagaagacg agatcgcgga tcttgcaggg 480
acattctcca tccaaatcat cccccaattg tttgctcttg ctttcacttt tcctacccaa 540
aagttccttc aggcacagag caaggtcaat gtccttatgt ggattggagc gggggatcta 600
atagtacacg ttgctctgct ttcgctgttt atttacggat tgggttgggg caccacaggc 660
gcggccatag catttgacct ctcgagctgg atgctttcag tggctcaaat tgtttatgca 720
gtggggtggt gcaaggatgc ttggcaagga ttgtcatggg cggcattgac ggatatttgg 780
gcctttgtca ggctctccct tgcctcggct gttatgctat gcctagaggt ctggtatatg 840
acgagtataa tcgtactcgc tggtaatctt gataatgcag tgattgcggt tggatcactt 900
tctatttgta tgaacttcaa tggatatgag ttcatgttgt tcatcggaat aaatgctgcg 960
ataagcgttc gtgtctccaa tgagcttggt ttgggccatc caagagcagc taagtactcc 1020
gtctatgtta cagttcttca atccttggtc attgggattc tctgcatgat tgttgtaatg 1080
gcaactcgag aatatttcgc cgtcatattc acagacagca aggatatgca acaagccgta 1140
tctcacctag cataccttct tggtttcact atgcttctta acagtgtcca gcccgtgata 1200
tccggtgttg ctgtcggggg aggatggcaa gctatggtag cttatatcaa cttgggttgt 1260
tattacattt tcgggcttcc tctaggctat gttcttggtt ttgtagcaaa tctaggagta 1320
cagggacttt ggggtggtat gatagctgga actgcacttc agacactgct ccttttgatc 1380
attctttaca gaaccaactg gaacaaagag gtagagcaga caacggaccg aatgcgaaaa 1440
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ctggatgtgg aagaagccaa gaggcaggtg ctgttttcgc ttcccatgat tcttaccagt 180
gtttcttact atctcatccc ggtcgtcgcc gtcatgttcg ccggccatct cggggagctt 240
cagctcgccg gagccaacct tgccaattct tgggccaccg ttagcggctt tgctttcatg 300
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agaatgatgg gcatatacct gcaagcatct tgcatcatct ccttcttctt cagcatcctc 420
atctccctgt tctggttcta ctccgagccc attctcgtct tcctccgaca agatcctcaa 480
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ttcttgcaga acatcctccg cttcctccag acccaaagcg ttgtttttcc actcctaatc 600
tgctcgctcc tgcctctgct tctccacttc ggaatcacct attttctggt tcactgcacc 660
agcctcggct tcaagggcgc cgcaatggcg gtctcgatct cgctgtggat ctcggtgctc 720
atgctggctg tctacctggt tcttgcgaag cggtttgaga ccacttggac tgggctttct 780
tccgagtcgt tcggccatgt tttctccaac ttgaagttgg ctctgccttc tgcagcaatg 840
gtgtgcttgg agtactgggc ttttgagctg ctggttctat tagctggact aatgcctgat 900
tcagaattaa acacttcgtt acttgcaatg tgtgtgaaca cagaagccat agcctacatg 960
atcacttacg gtctcagtgc tgctgctagc acgagggtgt caaatgagct gggggcaggg 1020
cagccagagc gggccaagca cgccatgggc gtcagcctga agctctccct tctcctcgct 1080
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agctccaaaa ttatacaaca cttcgcttca ttgaccccat ttcttgtggt ttccatcatg 1200
cttgattcca tccaaggggt cttatcaggg gtggctagag gatgtgggtg gcagcactta 1260
gcagtgtaca taaacatggg cacattctac tgcctgggca tgccgattgc catcctcctt 1320
ggatttaagc tcaaactcta cgctcagggc ttgtggttgg gtttactgtg cgggctctcg 1380
tgtcaggcgg gcaccctttt gctgctttca aagcgggcta agtggattgg ggtcaacttg 1440
tgtgacacca ccgacgctgc caacaaacag aaccctcttg ttgcgtga 1488
<210> 13
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
tttccactcc taatctgctc gctcc 25
<210> 14
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
ccaagtggtc tcaaaccgct tcgca 25
<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
aagggagaga tatcgatcca tg 22
<210> 16
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
<210> 17
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
<210> 19
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
ggggacaagt ttgtacaaaa aagcaggcta tgtctcggga ggctactgc 49
<210> 20
<211> 49
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
ggggaccact ttgtacaaga aagctgggtt cacgcaacaa gagggttct 49
Claims (8)
1. A persimmon procyanidine precursor transmembrane transporter DkMATE5 is characterized in that the nucleotide sequence of the DkMATE5 gene is shown in SEQ ID No. 1.
2. The use of the persimmon procyanidin precursor transmembrane transporter DkMATE5 in preferential transport of catechin, epicatechin, and epicatechin gallate, as claimed in claim 1.
3. The use of the persimmon procyanidin precursor transmembrane transporter DkMATE5 in procyanidin transport as claimed in claim 1.
4. An amplification primer for amplifying the DkMATE5 gene as claimed in claim 1, wherein the nucleotide sequence of the amplification primer is shown as SEQ ID NO. 4-5.
5. A fluorescent quantitative PCR specific primer for detecting the DkMATE5 gene as claimed in claim 1, wherein the nucleotide sequence of the fluorescent quantitative PCR specific primer is shown as SEQ ID NO. 6-7.
6. An expression vector comprising the DkMATE5 gene of claim 1.
7. A genetically engineered bacterium comprising the expression vector of claim 6.
8. Use of the expression vector of claim 6 or the genetically engineered bacterium of claim 7 to promote transmembrane transport of catechins, epicatechins, and epicatechin gallate.
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| WO2004096994A2 (en) * | 2003-04-25 | 2004-11-11 | Exelixis Plant Sciences, Inc. | Genes upregulated in a tomato plant having an increased anthocyanin content phenotype |
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| WO2004096994A2 (en) * | 2003-04-25 | 2004-11-11 | Exelixis Plant Sciences, Inc. | Genes upregulated in a tomato plant having an increased anthocyanin content phenotype |
| CA2497087A1 (en) * | 2004-03-09 | 2005-09-09 | Commonwealth Scientific And Industrial Research Organisation | Novel genes encoding proteins involved in proanthocyanidin synthesis |
| AU2009285705A1 (en) * | 2008-08-29 | 2010-03-04 | The Samuel Roberts Noble Foundation, Inc. | Epicatechin glucosyltransferase |
| CN110564704A (en) * | 2019-08-21 | 2019-12-13 | 中国农业科学院蔬菜花卉研究所 | Clone of lily anthocyanin transport LhGST gene and application thereof |
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