Anthocyanin glycosyltransferase and acyltransferase and their encoding genes and applications

Abstract

The present invention discloses anthocyanin glycosyltransferase and acyltransferase and their encoding genes and applications. The amino acid sequences of glycosyltransferases LrGT7, LrGT16 and LrGT22 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7 respectively, and the amino acid sequence of acyltransferase LrAT81 is shown in SEQ ID NO.8. The present invention clones and obtains glycosyltransferase genes LrGT71, LrGT16, LrGT22 and acyltransferase gene LrAT81 from black wolfberry, and the proteins encoded by them can catalyze the glycosylation and acylation of anthocyanin to generate stable anthocyanin with multiple modification groups, and have important application value in the biosynthesis of stable anthocyanin in vivo and in vitro, or have a reference role and important significance for the screening and identification of glycosyltransferase genes and acyltransferase genes from other species.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering and biotechnology, and particularly relates to the application of anthocyanin glycosyltransferase gene and acyltransferase gene of black wolfberry in the biosynthesis of stable anthocyanins with multiple modification groups. Background Art

[0002] Anthocyanin, also known as anthocyanin, is one of the main coloring substances in plant flowers or fruits, and is also an important secondary metabolite in the physiological processes of plant growth and stress resistance. For humans, anthocyanin is a compound that has both pigment and health benefits, and anthocyanin is a water-soluble compound with extremely low toxicity. Therefore, it has broad application prospects in industries such as food color additives or health products.

[0003] Due to the structural characteristics of anthocyanins, poor stability is the biggest problem in their application. Although the industry can use processes such as adding color preservatives and concentration to maintain the stability of anthocyanins, this will also increase costs. Modifying the molecular structure of anthocyanins to improve their stability is the most fundamental way to solve the problem. In terms of molecular structure modification, glycosylation modification is the most important step, and the glycosylation of the 3-OH is the first step in the synthesis of unstable anthocyanidins to stable anthocyanins, and it is also a prerequisite for other subsequent modifications; the acylation of anthocyanins further increases the stability of the molecule on the basis of glycosylation. The reason why the modifying group can make anthocyanins more stable is the formation of molecular stacking, and multiple modified anthocyanins are more stable than single modified ones.

[0004] Therefore, the present invention utilizes the gene resources in the black wolfberry and can synthesize stable anthocyanins with multiple modified groups through biotechnology and other means, which has important economic value. Summary of the invention

[0005] The first object of the present invention is to provide glycosyltransferases LrGT71, LrGT16, LrGT22 and acyltransferase LrAT81 of Lycium ruthenicum.

[0006] In order to achieve the above object, the present invention adopts the following technical measures:

[0007] The RNA of wolfberry is extracted and reverse transcribed into cDNA, which is used as a PCR template, and the primers described in the present invention are used to amplify to obtain SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and the four sequences are connected to a pEASY vector respectively, and BL21 (DE3) is transformed to express and obtain recombinant proteins, and the purified recombinant proteins are used to catalyze anthocyanidins to synthesize anthocyanidin-3-coumaroylrutinoside-5-glucoside.

[0008] The black wolfberry LrGT71, LrGT16, LrGT22 and LrAT81 of the present invention are characterized in that their amino acid sequences are shown as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 and SEQ ID NO.8, respectively.

[0009] The second object of the present invention is to provide genes encoding the above-mentioned LrGT71, LrGT16, LrGT22 and LrAT81.

[0010] The coding genes are preferably LrGT71, LrGT16, LrGT22 and LrAT81, and their nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

[0011] The third object of the present invention is to provide the use of the above-mentioned glycosyltransferase LrGT71, LrGT16, LrGT22 and / or acyltransferase LrAT81 in the biosynthesis of stable anthocyanins with multiple modification groups.

[0012] Preferably, said biosynthesis is in vivo or in vitro.

[0013] Preferably, the glycosyltransferase LrGT71 is used to catalyze the synthesis of anthocyanidin-3-glucoside from anthocyanidin.

[0014] Preferably, the glycosyltransferase LrGT16 is used to catalyze the synthesis of anthocyanidin 3-rutinoside from anthocyanidin 3-glucoside.

[0015] Preferably, the acyltransferase LrAT81 is used to catalyze the synthesis of anthocyanidin-3-coumaroylrutinoside from anthocyanidin-3-rutinoside.

[0016] Preferably, the glycosyltransferase LrGT22 is used to catalyze the synthesis of anthocyanidin-3-coumaroylrutinoside from anthocyanidin-3-coumaroylrutinoside-5-glucoside.

[0017] Preferably, glycosyltransferases LrGT71, LrGT16, acyltransferases LrAT81 and glycosyltransferases LrGT22 are used to jointly catalyze the direct synthesis of anthocyanidin-3-coumaroylrutinoside-5-glucoside from anthocyanidins.

[0018] Compared with the prior art, the present invention has the following advantages and effects:

[0019] The present invention clones glycosyltransferase genes LrGT71, LrGT16, LrGT22 and acyltransferase gene LrAT81 from ruthenium wolfberry, and the proteins encoded by the glycosyltransferase genes can catalyze the glycosylation and acylation of anthocyanins to generate stable anthocyanins with multiple modification groups, and have important application value in the in vivo and in vitro biosynthesis of stable anthocyanins, or have a reference role and important significance for the screening and identification of glycosyltransferase genes and acyltransferase genes from other species.

[0020] The glycosyltransferases LrGT71, LrGT16, acyltransferase LrAT81 and glycosyltransferase LrGT22 of the present invention can directly catalyze anthocyanidin to synthesize anthocyanidin-3-acylrutyl-5-glucoside in the same system.

[0021] The glycosyltransferases LrGT71, LrGT16, acyltransferase LrAT81 and glycosyltransferase LrGT22 of the present invention have a specific catalytic order, wherein LrGT71 is the first step, LrGT16 is the second step, LrAT81 is the third step and LrGT22 is the last step. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 . Target protein purification and SDS-PAGE detection, M: protein marker, 1: LrGT71, 2: LrGT16, 3: LrAT81, 4: LrGT22.

[0023] Figure 2 .HPLC (top) and mass spectrometry (bottom) detection of petunidin reaction catalyzed by recombinant LrGT71.

[0024] Figure 3 .HPLC (upper) and mass spectrometry (lower) detection of petunidin-3-glucoside reaction catalyzed by recombinant LrGT16.

[0025] Figure 4 .HPLC (upper) and mass spectrometry (lower) detection of petunidin-3-rutinoside reaction catalyzed by recombinant LrAT81.

[0026] Figure 5 .HPLC (top) and mass spectrometry (bottom) detection of the reaction of petunidin-3-coumaroylrutinoside catalyzed by recombinant LrGT22.

[0027] Figure 6 .HPLC detection of petunidin reaction catalyzed by recombinant LrGT71, LrGT16, LrAT81 and LrGT22 in the same system. DETAILED DESCRIPTION

[0028] The following examples are provided to illustrate the present invention rather than to limit the present invention.

[0029] The experimental methods not specifically specified in the following examples can be carried out according to conventional methods.

[0030] Example 1: Screening of Lycium ruthenicum anthocyanin glycosyltransferase genes LrGT71, LrGT16, LrGT22 and acyltransferase gene LrAT81

[0031] Through the sequencing and annotation information of the genome and transcriptome of black wolfberry, the glycosyltransferase sequences with the conservative structure of plant secondary metabolites glycosyltransferase PSPG box and the acyltransferase sequences with the conservative sequence of anthocyanin acyltransferase were screened. The content of anthocyanin in the development and ripening process of black wolfberry fruit was positively correlated with the gene expression, and the functions of candidate genes were predicted by combining the phylogenetic tree. The glycosyltransferase genes finally screened were LrGT71, LrGT16, LrGT22 and the acyltransferase gene LrAT81, and their predicted functions were anthocyanin-3-glucosyltransferase, anthocyanin-3-rhamnosyltransferase, anthocyanin-5-glucosyltransferase and anthocyanin-3-acyltransferase, respectively.

[0032] Example 2: Gene cloning, vector construction and sequencing analysis

[0033] RNA was extracted from the fruit of Lycium ruthenicum, and the Prime Script RT Reagent Kit With gDNAEraser (Takara) was used for genomic DNA removal and reverse transcription of RNA. The reaction system for genomic DNA removal was as follows: 5×gDNA Eraser Buffer 2.0μL, gDNA Eraser 1.0μL, total RNA 1μg, RNase Free H2O to 10μL; react at 42℃ for 2 minutes; after the reaction, place the system on ice. The reaction system for reverse transcription was as follows: 10μL of genomic DNA digestion reaction system, including 5×PrimeScript Buffer 2 (for real time) 4.0μL, PrimeScript RT Enzyme Mix I 1.0μL, RT Primer Mix 1.0μL, RNase Free dH2O 4.0μL; react at 37℃ for 15 minutes, and then keep at 85℃ for 5 seconds; the obtained product is the cDNA of Lycium ruthenicum fruit, which is stored in a -20℃ refrigerator.

[0034] Based on the full-length coding sequence in the transcriptome of Lycium barbarum fruit, the primer sequences of LrGT71, LrGT16, LrGT22 and LrAT81 were designed using Primer Primer5.0 software, and the forward primer and reverse primer sequences are shown in Table 1. The cDNA of Lycium barbarum fruit was used as a template, and the forward and reverse primer pairs shown in Table 1 were used to perform PCR amplification using PrimeSTAR max (2×) (Takara). The PCR reaction system was: PrimeSTAR Max (2×) 25μL, cDNA 3μL, 10μM concentration of forward primer and reverse primer 2μL each, ddH2O 18μL. The PCR reaction conditions were: 98℃ 2min; 98℃ 30s, 55℃ 30s, 72℃ 1min; 35 cycles; 72℃ extension 10min. After PCR, the singleness of the bands was detected by gel electrophoresis, and the PCR products were recovered using a product recovery kit (Meiji Company), and the concentration was determined after recovery.

[0035] Table 1. Primer sequences for full-length CDS cloning of candidate genes (underlined vector homology arm sequences)

[0036]

[0037] Vector construction: The plasmid pEASY (retained by this research group, transformed into the pEASY-Blunt E2 vector of Quanshijin Company to DH5α competent state, and the empty plasmid was extracted and stored) was digested with NdeI endonuclease (NEB Company). The reaction system was: 10×Cutsmart Buffer 5μL, pEASY plasmid no more than 1μg, NdeI HF 1μL, and sterilized ddH2O was added to 50μL. The product after the single restriction reaction was purified and recovered using the product recovery kit of Meiji Company. After purification, the concentration was determined, and seamless cloning and connection were performed with a molar ratio of 3:1 between the recovered PCR product and the vector (using the seamless cloning kit of Qingke Company). The connection reaction system was: 2×Connection Mix 2μL, recovered vector: 0.5μL, and recovered PCR product 1.5μL. Reaction at 50℃ for 30min. Prokaryotic expression vector plasmids containing the anthocyanin glycosyltransferase genes LrGT71, LrGT16, LrGT22 and the acyltransferase gene LrAT81 of Lycium ruthenicum were obtained respectively.

[0038] The ligation product was transformed into DH5α cloning competent medium, and the transformed bacterial solution was spread on LB plates containing 100 mg / L ampicillin antibiotics and inverted in a 37°C constant temperature incubator for 12-16 hours. The next day, the forward primer on the vector and the reverse primer of the gene were used for colony PCR analysis (using T5 PCR enzyme from Qingke Biotechnology). The reaction system was: 2×T5 SuperMix 5μL, T7 forward primer (10μM, TAATACGACTCACTATAGGG) 0.5μL, gene reverse primer (10μM, see Table 1) 0.5μL, bacterial solution template 1μL, sterilized ddH2O 3μL. The PCR reaction program was: 98℃2min; 98℃10s, 55℃ (the specific annealing temperature is subject to the synthetic primer) 10s, 72℃30s; 30 cycles; 72℃ extension 3min.

[0039] For each gene, three clones that were positive by colony PCR were selected, and the plasmids were extracted and sent to Sangon Biotech Co., Ltd. for sequencing identification. After sequencing analysis, the nucleotide sequence of the cloned black wolfberry anthocyanin glycosyltransferase gene LrGT71 is shown in SEQ ID NO.1, which contains 1344 bases, and the encoded protein is named black wolfberry anthocyanin glycosyltransferase LrGT71, with a total of 448 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO.5; the nucleotide sequence of the black wolfberry anthocyanin glycosyltransferase gene LrGT16 is shown in SEQ ID NO.2, which contains 1413 bases, and the encoded protein is named black wolfberry anthocyanin glycosyltransferase LrGT16, with a total of 471 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO.6; the nucleotide sequence of the black wolfberry anthocyanin glycosyltransferase gene LrGT22 is shown in SEQ ID NO.3, which contains 1404 bases, and the encoded protein is named as black wolfberry anthocyanin glycosyltransferase LrGT22, with a total of 468 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO.7; the nucleotide sequence of the black wolfberry anthocyanin acyltransferase gene LrAT81 is shown in SEQ ID NO.4, which contains 1362 bases, and the encoded protein is named as black wolfberry anthocyanin acyltransferase LrAT81, with a total of 454 amino acid residues, and the specific amino acid sequence is shown in SEQ ID NO.8.

[0040] Example 3: Target protein induced expression and purification

[0041] Extract the prokaryotic expression vector plasmid containing the anthocyanin glycosyltransferase genes LrGT71, LrGT16, LrGT22 and acyltransferase gene LrAT81 obtained in Example 2, and then transform them into the expression competent state of Escherichia coli BL21 (DE3) respectively. Pick the positive single clone in 5mL LB liquid culture medium containing 100mg / L Amp antibiotic, and culture it at 37℃, 200rpm overnight. The next day, inoculate it in 400mL fresh LB liquid culture medium containing 100mg / L Amp antibiotic at a volume ratio of 1:100, and culture it at 37℃, 200rpm until OD 600 When the concentration reaches 0.6, add the inducer IPTG to a final concentration of 0.1 mM and continue to culture at 16°C for 16-20 hours. Collect the cells by low-temperature centrifugation, wash once with an appropriate amount of sterilized ddH2O, centrifuge again, and discard the supernatant.

[0042] The cells were resuspended in 10 mL of lysis buffer (pH 7.5, containing 20 mM Tris-HCl, 0.5 M NaCl and 10 mM imidazole), and frozen and thawed three times in a 40°C water bath and liquid nitrogen. PMSF was added to a final concentration of 1 mM, and the resuspended cells were incubated in ice water and ultrasonically disrupted. The ultrasonic conditions were: ON 5 s, OFF 5 s, 30 min. After the disruption was completed, the cells were centrifuged at 4°C and 6000 g for 15 min, and the supernatant was taken as the crude enzyme.

[0043] The total protein after induction was purified by Ni-NTA affinity filler (Qiagen) according to the filler instructions. The eluted target protein was desalted and concentrated using an ultrafiltration centrifuge tube. Finally, SDS-PAGE was used to detect the protein purification effect. The purification effects of LrGT71, LrGT16, LrAT81 and LrGT22 were as follows: Figure 1 Thus, purified black wolfberry glycosyltransferases LrGT71, LrGT16 and LrGT22 and black wolfberry acyltransferase LrAT81 were obtained.

[0044] Example 4: Enzyme activity reaction and product identification of LrGT71, LrGT16, LrAT81, and LrGT22

[0045] The reaction was carried out using the purified black wolfberry glycosyltransferase LrGT71, LrGT16 and LrGT22 obtained in Example 3, and the black wolfberry acyltransferase LrAT81. Each 100 μL reaction system included 200 μM substrate (LrGT71 was petunidin, LrGT16 was petunidin-3-glucoside, LrAT81 was the product of the reaction catalyzed by LrGT16, LrGT22 was the product of the reaction catalyzed by LrAT81, and the four enzymes added simultaneously were petunidin), 1 mM glycosyl donor (UDP-glucose or UDP rhamnose) or / and acyl donor (p-coumaryl CoA), 5 μg of purified protein, and finally 100 μL of pH 7.0 phosphate buffer solution was used to make up. An equal amount of heat-inactivated protein was used as a negative control (NC). After gently mixing, the mixture was reacted at 35°C for 1 hour. After the reaction was completed, 100 μL of methanol was added to terminate the reaction. The reaction solution was filtered through a 0.22 μm filter membrane and then analyzed by UHPLC-MS / MS.

[0046] UHPLC-MS / MS conditions were set up, where the UHPLC part: the column was a Hypersil GOLD column (2.1×100 mm, 1.9 μm, Thermo Scientific), phase A was 0.2% formic acid-water, phase B was acetonitrile, the column temperature was 30°C, the injection volume was 1 μL, the flow rate was 0.4 mL / min, and the gradient elution program was: 0-0.5 min, 5% B; 0.5-10 min, 5%-35% B; 10-10.5 min, 90% B; 10.5-12 min, 90% B; 12-12.1 min, 5% B. MS part: positive ion mode, resolution of 12,000, mass-to-charge ratio (m / z) scanning range was set to 100-1500, electrospray ion source (ESI) parameters were as follows: heating temperature and capillary temperature were both set to 350 °C, sheath gas flow rate and auxiliary gas flow rate were set to 40 and 10 respectively, S-lens level was 60%, and collision energy was 20-30 eV.

[0047] UHPLC-MS / MS analysis showed that the molecular ion peak of petunidin produced by LrGT71 was 479.117, and its secondary mass spectrum was 317.065, indicating that the molecular ion peak lost a glucoside, which was consistent with the standard product (petunidin-3-glucoside) ( Figure 2 ), so the product was confirmed to be petunidin-3-glucoside. The molecular ion peak of petunidin-3-glucoside catalyzed by LrGT16 was 625.17, and its secondary mass spectrum was 479.12 and 317.07, which were the molecular weights of the parent ion after one rhamnosyl group (-146) and one glucose group (-146-162) were broken, respectively. Figure 3), so the product was confirmed to be petunidin-3-rhamnosyl glucoside (petunidin-3-rutinoside). The molecular ion peak of petunidin-3-rhamnosyl glucoside catalyzed by LrAT81 was 771.21, and its secondary mass spectrum was 625.18, 479.12 and 317.07, which were the molecular weights after the parent ion cleaved a coumaroyl group (-146), a rhamnosyl group (-146-146) and a glucose group (-146-146-162), respectively. Figure 4 ), so the product was confirmed to be petunidin-3-coumaroyl rhamnosyl glucoside (petunidin-3-coumaroyl rutinoside). The molecular ion peak of petunidin-3-coumaroyl rutinoside catalyzed by LrGT22 was 933.26, and its secondary mass spectrum was 771.21, 479.12 and 317.07, which were the molecular weights after the parent ion broke off a glucose group (-162), broke off a coumaroyl rhamnosyl group (-162-146-146) and further broke off to leave only petunidin (-162-146-146-162), and was consistent with the standard product (petunidin-3-coumaroyl rutinoside-5-glucoside) ( Figure 5 ), thus confirming that the product is petunidin-3-coumaroylrutinoyl-5-glucoside.

[0048] Example 5: Synthesis of multiple modified anthocyanins using LrGT71, LrGT16, LrAT81, and LrGT22 as catalysts and product identification

[0049] The purified black wolfberry glycosyltransferase LrGT71, LrGT16 and LrGT22 obtained in Example 3 and the black wolfberry acyltransferase LrAT81 were used in the same system to catalyze the reaction with petunidin as substrate. The reaction system included 200 μM petunidin, 1 mM UDP-glucose, 1 mM UDP-rhamnose and 1 mM p-coumaroyl CoA, 5 μg of each purified protein, and finally 100 μL of pH 7.0 phosphate buffer solution was used to make up. An equal amount of heat-inactivated protein was used as a negative control (NC), and the mixture was gently mixed and reacted at 35°C for 1 hour. After the reaction was completed, 100 μL of methanol was added to terminate the reaction, and the reaction solution was filtered through a 0.22 μm filter membrane and analyzed by HPLC and UHPLC-MS / MS.

[0050] The results of HPLC analysis and comparison with the standard showed that the glycosyltransferases LrGT71, LrGT16 and LrGT22 of Lycium barbarum and the acyltransferase LrAT81 in the same system can directly catalyze the synthesis of petunidin products such as Figure 6The asterisk in FIG. 4 is used to mark the product, and its retention time is consistent with that of petunidin-3-coumaroylrutinoyl-5-glucoside, and the mass spectrometry result is consistent with that of Example 4, so the product is confirmed to be petunidin-3-coumaroylrutinoyl-5-glucoside.

[0051] >SEQ ID NO.1

[0052] ATGACTACTTCTCAACTTCACATTGCACTCCTTGCTTTCCCTTTTGGCAGCCATGCAGCTCCCTTGCTCACACTTGTCCAAAAACTCTCTC

[0053] CATTTTTACCATCAGACACAATTTTTCCTTCTTCAACACATCACAATCCAACACCTCAATCTTCTCTAAATCTTCAAAACCAGACAACCT

[0054] CAAAATCTACAATGTTTGGGATGGTGTCAAAGAAGGCAATGCCACCCCTTTTGGTCGTGAGGCAATTGAGCTCTTCATACAATCAACTCCT

[0055] AATAATTTTATCAAGTCCATGAAAGAGGCAGAGGAGGAAACAGGGGTAAAATTTTCTTGCATTTTTAGTGATGCATTCTTGTGGTTTTCTT

[0056] GTGAATTGGCTGAGAAAATGAATGTCCCTTGGATTGCTTTTTGGACTGCTGGTTCTTGTTCTTTATCTATTCATTTGTACACTGATTTAAT

[0057] TCGATCGAACGACGAAACATTATCAAAAATCCCAGGATTTTTATCAAGTTTAAAGATGAGTGACATGCCACCAGAAGTTGTGACAGAGAAT

[0058] TTAGAGGGTCCCATGCCATCTATGATATACAACATGGCATTAAATTTGCACAAGCAGATGGTGTTGTACTTAATTCTTTTGAAGAATTGG

[0059] ATCCGATAATCAACAAAGACCTCAAATCCAAGCTCCAAGGTACTCAACATTGGCCCTTTAGCTCTACAATCATCAAAAAAGGTACACTT

[0060] AGATGCTAATTCTGATGAAAGTGGATGCATCAAATGGCTAGAAAAGCAAAATGAAAAATCAGTAGTGTATCTTAGTTTTGGTACCGTCACA

[0061] GCATTACCCCCCTAATGAAATTGTGGCGATATCAGAAGCACTAGAAGCCAAAAGGATACCTTTTATTTGGTCATTAAGAGATAATGGGGTTA

[0062] AAATTTTGCCGAAAGGGTTTCTTGAAAGAACAGAAGAATTTGGGAAAATAGTTTCTTGGGCACCTCAATTGGAAATCTTGGAACATTCATC

[0063] TGTTGGTGTTTTTGTAACACATTGTGGATGGAATTCTATTTTGGAAGGCATATCATATGGTGTGCCTATGATTTGTAGGCCTTTTTTTGGT

[0064] GACCAGAAAATTGAATAGTAGAATGGTGGAGAGTGTTTGGCAAATTGGTTTGCAAATTGAAGGTGGGAGTTTCACTGAAAGTGGAACAATGA

[0065] ATGCATTGGATATATTTTTCAAGGAGGAAAAAGGGAAGGTATTAAGGCAAAATGTTAAAAGGCTAAAAGAAAAAGCATTAGAAGCTGTGAA

[0066] ATCAGATAATGGGAGTTCAACTGAAAATTTCAAGGTTCTAGTGGAGCTGGTTAATTTCACAAGGCCACT

[0067] >SEQ ID NO.2

[0068] ATGGAGAATGAGAACTCAAATGATGTTCTTCATATAGTTATGCTCCCTTTTTTGCTTTTGGTCATATCAGTCCATTGTTCAGCTTGCTA

[0069] ACAAGCTTTCCTCTCATGGTCTCAAAGTTTCTTTTTTCACTGCATCTGGCAATGCTGGCAGAGTCAAATCTATGCTGAATTCTGCTCCCAC

[0070] TACTCATATAGTCCCTCTTACTCTTCCTCAAGTTGAAGGTCTACCTCCTGGGGCAGAAAGTACTGCAGAATTGACACCAGTAACTGCTGAA

[0071] CTTCTCAAAGTTGCTTTAGACCAAATGCAACCACAAATCAAGTCTCTACTTTCCAATCCAAACCCCATTTGTTCTCTTTGATTTTGCTC

[0072] AAGAATGGCTCCCTAAAATGGCTGATGAATTAGGGGATCAGACTGTTTTTTACTCTGTTTTTGTAGCACTTTTCACTGCTTTTCTTACTTG

[0073] CCCTGCTAGAGTTCCTCAACCCAAGAAATATCCAACTCTTGAAGACATGAAGAAACCTCCACCTGGATTTCCTATCACCTCTGTCACCTCA

[0074] GTCAAAACCTTTGAGGCTCAAGATTTTCTATATATTTTCAAGAGCTTCCATGGTGGTCCTACTGTATATGACCGTGTACTCTCAGGACTTA

[0075] AGGGTTGCTCAGCTATACTAGCTAAGACTTGTTCCCAAATGGAGGGGCCTATATAGAATACGTGAAATCGCAGTTCGATAAACCTGTTTT

[0076] TCTAGTAGGACCAGTAGTTCCTGACCCACCTTCAGGAAAATTGGAAGAGAGATGGGCTAGTTGGTTAAACAAGTTTGAAGCTGGAACAGTT

[0077] ATTTACTGTTCTTTTGGAAGTGAAACTTTCTTGAATGATGATCAGATCAAAGAACTAGCTTTAGGTTTGGAACAAACTGGGCTACCTTTCT

[0078] TTCTGGTCTTAAATTTTCCTGCCAATGTCGATGTCCCAGCCGAACTAAACCGAGCTTTACCAGAAGGTTTTCTGGAGAGAGTGAAAGACAA

[0079] GGGAATCATTCATTCAGGTTGGGTGCAGCAACAGAACATACTTGCTCATGCCAGTGTAGGTTGCTATGTATGCCATTCAGGGTTCAGTTCA

[0080] GTGATAGAGGCACTAGTGAATGACTGTCAAGTTGTTATGTTGCCTCAGAAAGGTGATCAGTTCTTGAATGCAAAGCTGGTGAGTGGTGATA

[0081] TGAAAGCTGGGGTGGAGGTAAATAGGAGGGATGAAGATGGCTATTTTGGTAAAGAAGATATTAAAAAAGCTGTGGAGATGGTGATGGTGGA

[0082] GGTTGACAAGCAGCCAGGTAAATTAATTAGAGCAAATCAGAAGAAATGGAAGGAGTTTTTGTTGAACAAGGATATCCAATGCAAGTTTATT

[0083] GAGGATTTAGTTAATGAAATGACAGCAATGGCTAAGGTCTCAAGTAAC

[0084] >SEQ ID NO.3

[0085] ATGGTGCAACCCCATGTCATTTTAACAACATTTCCAGCACAAGGGCATATTAATCCAGCACTTCAATTTGCCAAAATCTTGTCAAAATGG

[0086] GCATAAAAGTGACATTTTCTACAAGCATTTATGCCCAAAGCCTTATGGATGAAAAATCCATTGCTAATTTTCCAAAGGGGTTGATGAATTT

[0087] TGTTCCATTTTCTGATGGATTTGATGATGGCGTTGATCATTCAAAAGACCCTAAATTTTACATGTCACAACTTCGTAAATGTGGGAGTGAG

[0088] ACTGTGAAAAAGATTATTCTCAATTGCTCTGAAAATGGAAGTCCTATAACTTGCCTTCTTTACTCCATTTTTCTTCCTTGGGCAGCAGAGG

[0089] TCGCTCGTGAAGTTAACATCCCTTCTGCTCTCCTTTGGAGTCAACCAGCTACAATATTGGACATATACTATTTTCACTTTAATGGTTATGA

[0090] AGAACAAATGGCTAACGAATCCAATGATCCAAATTGGTCCATTCAACTTCCAGGGCTTCCACTATTGAAAACTAAAGATCTTCCTTCATTT

[0091] TTACTTCCATCAAGTGCAAAAGGAAGCCTTAGAGTTGCACTTCCCCCTTTCAAAGAATTAATAGACACATTGGATGCTGAAATTAATCCTA

[0092] AAATTCTTGTGAATTCATTTGATGAATTAGAGCCTAAGGCACTCAAAGCAATTGAAGGTTACAAGTTTTACGGAATTGGACCACTAATTCC

[0093] TAGTGCTTTTTTAGATGGAAATGACCCTTTGGATTCTTGTTTTGGTGCTGATCTTTTTTGAGAAATCAAATGATTATATGGAATGGTTAAAC

[0094] ACAAAGCCAAATTCAAGTGTTGTTTATATATCATTTGGGAGTCTAATAAATCCATCAATAAGTCAAATGGAGGAGATATCAAAAGGGTTGA

[0095] TAGAAATAGGGGAGGCCATTTTTGTGGATAATGAAAAAAAATGAAAAAGACAAAGAAAATGCGAAAAAGATTGATTGTATTGAAGAGTTGGA

[0096] AAAAATAGGGAAAATTGTCCCATGGTGTTCGCAACTCGAAGTTTTGAGACATCCCTCTCTGGGATGTTTTGTTTCGCACTGTGGATGGAAT

[0097] TCGGCTTTGGAGAGCTTAGCTTGTGGAGTGCCGGTCGTGGCGTTTCCTCAATGGACTGATCAAATGACAAATGCCAAGCAAATTGAAGATG

[0098] TGTGGAAGAGTGGAGTGAGAGTGAATGTGAATGAAGATGGTGTTGTTGAGAGTGAAGAAATGAAAAGGTGCATTGAGTTGGTTATGGATGG

[0099] AGGGGAAAAGGGGAAGAATGAGAAATAATGCTAAGAATTGGAAGATTGGCTAGAGAAGCTGTGAAAGAAGGTGGATCTTCACATAAG

[0100] AATTTAAAGGATTTTATTGATGAAGTCGCTAAAGGTTAC

[0101] >SEQ ID NO.4

[0102] ATGAGCCAAATTACCGAGCAAAATTTGAATGGTTCTTGTATTCAAATTGAAATCTTGAATGAAAAAATTATAAAACCATCATTACCAACTC

[0103] CAAATCACCTCAATTCCTACAAGTTATCCTTCTTTGATCAAATTGTTCCTAATTTTGCTGTGCCCCTTCTTTATTTCTATCCTCCAGTTCC

[0104] ATCTGAAAAATCCAACTTGCAAAGTGATGAAGCAGTTCATACACAACTACAAAACTCATTATCTGAGATTTTAACCAAGTTTTATCCACTT

[0105] GCTGGAAGGTTGTCAGAAGATGGTACTTGCATTGAATGTCAAGATCAAGGGGTTATTTACTTAGAAGCAAAGGTGAATTGCCAATTGACTG

[0106] AATCCTAGACAAAGCTTACAAAGATACTGATCTTGTCAAACTCTTTGTGCCACCTATAAGAATAAGGCTAGCTGAATTGCCTAATAGACC

[0107] AATGATGGCAATTCAGGCCACCATGTTCGAACATGGTGGCCTAGCACTAGCCGTGCAGATGGTTCGTACGTTAGTCGATGGATTCTCTGGC

[0108] TGCGCGTTAACCGATGAATGGGCTAAGGTTAGCCGAATGGAGAAGGGGAATGCTAGAAATTTACAATTCCGTTCCGATTTAGCGGAAATAT

[0109] TTCCACCTAAGGAAAATATTTTTGAGATGATTAAGAAAGGTAGGCCTAAAGGATATGAGATGAAAATTGCTACAAGGATATTTATGTTTTGA

[0110] TGAAGTTGCAATATCTAAGTTGAAGGAAAATGTGAATAAGTCTTTGAGTTATTCTCAAGAGTTGAAGTTGTGACCGCACTTATTTGGAGA

[0111] AGCCTTATGCGTGTGGTGAGGTTGAGGCATGGTCATAATAGGCCATCCATGCTACAATTTGCCATCAATTTAAGGAAGAGGAGATCCAA

[0112] AACTAGTAGGCGAAGACCAGAACTTTTTTGGGAACTTCTCCATTTGACATCCCAATCAAATGTGTACCATCTCAGCAACCAAGATTTGGA

[0113] ACTACATGAAATTGTAACCTTAATTAGGAATGCAAAGAACAAAATCCTATCAAGTATTGCCAATGCTTCAAGTGAAGAGATTTTCTCAATA

[0114] GTGACACAGTCATTGAACCAAAATAAGAAGGATAATGATGATGAAATGGACCTTTATCCTACTTCAAGTTTGTGTAGATTTCCTCTAA

[0115] ATGAGTCAGATTTTGGGTGGGCTGAACCAATTTGGGTTAGCAGAGTAATGTGCCATTTCAATTGTTCTTCTTGATGGATTCAAAAAGTGG

[0116] GATTGAGGCTAGAGTTTGCTTGAATGAAGATGATATGATTAAGCTTGAAAATGATGTTGATATTGTGGAGTTTAGTTCTGTGCTAAAG

[0117] >SEQ ID NO.5

[0118] MTTSQLHIALLAFPGFGSHAAPLLTLVQKLSPFLPSDTIFSFFNTSQSNTSIFSKSSKPDNLKIYNVWDGVKEGNATPFGREAIELFIQSTP

[0119] NNFIKSMKEAEEETGVKFSCIFSDAFLWFSCELAEKMNVPWIAFWTAGSCSLSIHLYTDLIRSNDETLSKIPGFLSSLKMSDMPPEVVVEN

[0120] LEGPMPSMIYNMALNLHKADGVVLNSFEELDPIINKDLKSKLQKVLNIGPLALQSSKKVHLDANSDESGCIKWLEKQNEKSVVYLSFGTVT

[0121] ALPPNEIVAISEALEAKRIPFIWSLRDNGVKILPKGFLERTEEFGKIVSWAPQLEILEHSCVGVFVTHCGWNSISEGISYGVPMICRPFFG

[0122] DQKLNSRMVESVWQIGLQIEGGSFTESGTMNALDIFFKEEKGKVLRQNVKRLKEKALEAVKSDNGSSTENFKVLVELVKCHKAT

[0123] >SEQ ID NO.6

[0124] MENENSNDVLHIVMLPFFAFGHISPFVQLANKLSSHGLKVSFFTASGNAGRVKSMLNSAPTTHIVPLTLPQVEGLPPGAESTAELTPVTAE

[0125] LLKVALDQMQPQIKSLLSNLKPHFVLFDFAQEWLPKMADELGIKTVFYSVFVALSTAFLTCPARVPQPKKYPTLEDMKKPPPGFPITSVTS

[0126] VKTFEAQDFLYIFKSFHGGPTVYDRVLSGLKGCSAILAKTCSQMEGPYIEYVKSQFDKPVFLVGPVVPDPPSGKLEERWASWLNKFEAGTV

[0127] IYCSFGSETFLNDDQIKELALGLEQTGLPFFLVLNFPANVDVPAELNRALPEGFLERVKDKGIIHSGWVQQQNILAHASVGCYVCHSGFSS

[0128] VIEALVNDCQVVMLPQKGDQFLNAKLVSGDMKAGVEVNRRDEDGYFGKEDIKKAVEMVMVEVDKQPGKLIRANQKKWKEFLLNKDIQCKFI

[0129] EDLVNEMTAMAKVSSN

[0130] >SEQ ID NO.7

[0131] MVQPHVILTTFPAQGHINPALQFAKNLVKMGIKVTFSTIYAQSLMDEKSIANFPKGLMNFVPFSDGFDDGVDHSKDPKFYMSQLRKCGSE

[0132] TVKKIILNCSENGSPITCLLYSIFLPWAAEVAREVNIPSALLWSQPATILDIYYFHFNGYEEQMANESNDPNWSIQLPGLPLLKTKDLPSF

[0133] LLPSSAKGSLRVALPPFKELIDTLDAEINPKILVNSFDELEPKALKAIEGYKFYIGPLIPSAFLDGNDPLDSCFGADLFEKSNDYMEWLN

[0134] TKPNSSVVYSFGSLINPSISQMEEISKGLIEIGRPFLWIMKKNEKDKENAKKIDCIEELEKIGKIVPWCSQLEVLRHPSLGCFVSHCGWN

[0135] SALESLACGVPVVAFPQWTDQMTNAKQIEDVWKSGVRVNVNEDGVVESEEMKRCIELVMDGGEKGEEMRNNAKKKWKELAREAVKEGGSSHK

[0136] NLKDFIDEVAKGY

[0137] >SEQ ID NO.8

[0138] MSQITEQNLNGSCIQIEILNEKIIKPSLPTPNHNLSYKLSFFDQIVPNFAVPLLYFYPPPSEKSNLQSDEAVHTQLQNSLSEILTKFYPL

[0139] AGRLSEDGTCIECQDQGVIYLEAKVNCQLTEFLDKAYKDTDLVKLFVPPIRIRLAELPNRPMMAIQATMFEHGGLALAVQMVRTLVDGFSG

[0140] CALTDEWAKVSRMEKGNARNLQFRSDLAEIFPPKENIFEMIKKGRPKGYEMKIATRIFMFDEVAISKLKENVNKSLSYSSRVEVVTALIWR

[0141] SLMRVVRLRHGHNRPSMLQFAINLRGRGDPKLVGEDQNFFGNFSIDIPIKCVPSHSNQDLELHEIVTLIRNAKNKILSSIANASSEEIFSI

[0142] VTQSLNQIREGYNDDEMDLYPTSSLCRFPLNESDFGWAEPIWVSRVNVPFQLFFLMDSKSGIEARVCLNEDDMIKLENDVDIVEFSSVLK

Claims

1. Glycosyltransferase LrGT71, LrGT16, LrGT22 or acyltransferase LrAT81, characterized in that The amino acid sequences of glycosyltransferases LrGT71, LrGT16 and LrGT22 are shown in SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, respectively, and the amino acid sequence of acyltransferase LrAT81 is shown in SEQ ID NO.

8.

2. A gene encoding the glycosyltransferase LrGT71, LrGT16, LrGT22, or acyltransferase LrAT81 according to claim 1.

3. The gene according to claim 2, characterized in that The encoding genes of glycosyltransferase LrGT71, LrGT16, LrGT22 and acyltransferase LrAT81 are genes LrGT71, LrGT16, LrGT22 and LrAT81, respectively, and their nucleotide sequences are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, respectively.

4. Use of the glycosyltransferase LrGT71 according to claim 1 in catalyzing the synthesis of anthocyanidin-3-glucoside from anthocyanidin.

5. Use of the glycosyltransferase LrGT16 according to claim 1 in catalyzing the synthesis of anthocyanidin 3-rutinoside from anthocyanidin 3-glucoside.

6. Use of the acyltransferase LrAT81 according to claim 1 in catalyzing the synthesis of anthocyanidin-3-rutinoside from anthocyanidin-3-coumaroylrutinoside.

7. Use of the glycosyltransferase LrGT22 according to claim 1 in catalyzing the synthesis of anthocyanidin-3-coumaroylrutinoside into anthocyanidin-3-coumaroylrutinoside-5-glucoside.

8. Use of the glycosyltransferases LrGT71, LrGT16, acyltransferase LrAT81 and glycosyltransferase LrGT22 described in claim 1 in co-catalyzing the direct synthesis of anthocyanidin-3-coumaroylrutinoside-5-glucoside from anthocyanidins.

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