NL2029361B1 - LYCIUM RUTHENICUM MURR ANTHOCYANIN SYNTHESIS-RELATED MYB TRANSCRIPTION REPRESSOR LrETC1 AND APPLICATIONS THEREOF - Google Patents

Abstract

Disclosed is a Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription 5 repressor LrETC1 and applications thereof, and belongs to the technical field of genetic engineering. The MYB transcription repressor LrETC1 participating in synthesis of anthocyanin is screened and cloned in the present invention; gene cDNA is 240bp in length totally, encoding 79 amino acids; and the LrETC1 belongs to R3 MYB transcription factors through analysis and display by a protein sequence database. Multiple sequence alignment and evolutionary tree 10 analysis show that the MYB transcription repressor LrETC1 belongs to AtCPC-like transcription repressors. LrETC1 is a transcription factor which is located in a cell nucleus and does not have an activation function. A LrETC1 transgenic Arabidopsis strain is obtained through Agrobacterium—mediated genetic transformation; and compared with a wild type, for the LrETC1 transgenic Arabidopsis strain, a coat of a seed is light brown, no pigment accumulation occurs for 15 an Arabidopsis seedling under high sucrose concentration stress, and the expression level of a gene of a related structure is down regulated.

Description

LYCIUM RUTHENICUM MURR ANTHOCYANIN SYNTHESIS-RELATED MYB TRANSCRIPTION REPRESSOR LrETC1 AND APPLICATIONS THEREOF Technical Field The present invention relates to the technical field of genetic engineering, and particularly relates to a Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC1 and applications thereof. Background Lycium ruthenicum Murr Murry belongs to the deciduous spiny shrubs of Solanaceae lycium barbarum, is rich in nutrition, and can be used as both medicine and food, and people pay wide attention to the Lycium ruthenicum Murr due to the fact that the Lycium ruthenicum Murr is rich in a large amount of anthocyanin. The anthocyanin has strong antioxidant activity, can help plants to resist the stress environment, and is also beneficial to human health.

Transcription factors, known as trans-acting factors, usually refer to proteins encoded by genes, and can be specifically combined with related cis-acting elements of gene promoter regions to activate or repress gene expression, so as to improve the adaptability of the plants to the environment. The MYB transcription factors play an important role in metabolic regulation of the anthocyanin. Although several MYB transcription factors for positively regulating synthesis of the anthocyanin in the Lycium ruthenicum Murr have been reported in existing research results, the MYB transcription factors for repressing synthesis of the anthocyanin have not been reported yet.

Therefore, how to provide a Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor is an urgent problem to be solved by those skilled in the art.

Summary In view of this, the present invention provides a Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC1 and applications thereof.

To achieve the above purpose, the present invention adopts the following technical solutions: The amino acid sequence of the Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC1 is shown as SEQ ID No. 2.

The present invention also provides a gene of the Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC1, and the nucleotide sequence of the gene is shown as SEQ ID No. 1.

The present invention also provides a recombinant vector containing the transcription repressor LrETC1 gene.

The present invention also provides a recombinant strain containing the transcription repressor LrETC1 gene or the recombinant vector.

The present invention also provides applications of the transcription repressor LrETC1 or the gene in Lycium ruthenicum breeding.

According to the technical solution, compared with the prior art, the present invention discloses the Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC17and the applications thereof.

Compared with the prior art, the MYB transcription repressor LrETC1 participating in anthocyanin synthesis is screened and cloned through transcriptome data of Lycium ruthenicum Murr according to annotation results and the expression quantity difference of the MYB transcription factor, the gene cDNA of the LrETC1 is 240bp in total, and 79 amino acids are encoded; protein sequence database analysis shows that the LrETC1 contains one conservative MYB-DNA-binding structural domain and belongs to R3-MYB transcription factors.

Multiple sequence alignment and evolutionary tree analysis show that that the LrETC7 and the anthocyanin synthesis-related MYB transcription repressor in other species are gathered in one class and belong to AtCPC-like transcription repressors. gRT-PCR analysis shows that that the L7ETC1 is expressed in all tissues of the Lycium ruthenicum Murr, and the expression level is gradually increased along with fruit ripening.

Subcellular localization and transcriptional activity detection experiments show that the LrETC1 is a transcription factor which is located in the cell nucleus and does not have activation function.

A plant overexpression vector is constructed, and LrETC1 transgenic Arabidopsis strains are obtained through agrobacterium-mediated genetic transformation.

Compared with a wild type, for the LrETC1 transgenic Arabidopsis strain, a coat of a seed is light brown, no pigment accumulation occurs for an Arabidopsis seedling under high sucrose concentration stress, and the expression level of a gene of a related structure is down regulated.

Description of Drawings To more clearly describe the technical solutions in the embodiments of the present invention or in the prior art, the drawings required to be used in the description of the embodiments or the prior art will be simply presented below.

Apparently, the drawings in the following description are merely the embodiments of the present invention, and for those ordinary skilled in the art, other drawings can also be obtained according to the provided drawings without contributing creative labour.

Fig. 1 is a diagram of the result of target gene LrETC1 gel electrophoresis provided by the present invention.

Fig. 2 is a diagram of hydrophilicity and hydrophobicity analysis of LrETC1 protein provided by the present invention.

Fig. 3 is a diagram of a transmembrane domain of LrETC1 protein provided by the present invention.

Fig. 4 is a prediction diagram of LrETC1 signal peptide provided by the present invention.

Fig. 5 is a diagram of prediction of a tertiary structure of protein provided by the present invention.

Fig. 6 is a diagram of phylogenetic tree analysis of LrETC1 protein provided by the present invention and MYB protein related to the anthocyanin synthesis pathway in other species.

Fig. 7 is a diagram of multi-sequence alignment of an amino acid sequence of LrETC1 provided by the present invention with similar MYB proteins in other species.

Fig. 8 is a diagram of PCR identification of subcellular localization bacterium-carrying solution of LrETC1 provided by the present invention.

Fig. 9 is a diagram of subcellular localization of LrETC1 protein provided by the present invention, in which GFP: GFP fluorescence; DAPI: nucleic acid dye; Bright: bright field; Merged: superposition of bright field, green fluorescence and blue fluorescence.

Fig. 10 is a diagram of transcriptional activity analysis of LrETC1 provided by the present invention, in which, from left to right, they are: (SD/-Trp) culture medium, (SD/-Trp/-His/-Ade) culture medium, and (SD/-Trp/-His/-Ade) culture medium coated with X-a-Gal dye.

Fig. 11 is a diagram of expression level analysis of LrETC1 provided by the present invention in Lycium ruthenicum Murr and white Lycium barbarum fruits.

Fig. 12 is a diagram of selection of transgenic Arabidopsis TO-generation seeds provided by the present invention.

Fig. 13 is a diagram of phenotype of wild-type and transgenic Arabidopsis T1-generation seeds provided by the present invention.

Fig. 14 is a diagram of Arabidopsis seedlings under 6% sucrose stress provided by the present invention.

Fig. 15 is a diagram of expression levels of structural genes in anthocyanin synthesis provided by the present invention.

Detailed Description The technical solutions in the embodiments of the present invention will be described clearly and completely in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those ordinary skilled in the art without creative work shall fall within the protection scope of the present invention.

The embodiment of the present invention discloses a Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC1 and applications thereof.

Part of reagents and raw materials involved in the embodiment are as follows: (1) Plant materials: Lycium ruthenicum and white Lycium barbarum fruits collected from Lycium barbarum resource nursery gardens of Ningxia Academy of Agricultural and Forestry

Sciences in Ningxia Hui Autonomous Region; common tobacco, Nicotiana benthamiana and Arabidopsis Col-0 preserved in the experiment. (2) Vectors: a cloning vector 5SminTM TA/Blunt-Zero Cloning Kit purchased from Vazyme Biotech Co., Ltd.; subcellular localization vector P1300-GFP, yeast PGBKT7 vector and vector PCM 1307. (3) Strains: Escherichia coli DH5a purchased from Tsingke Biotechnology Co., Ltd.; yeast AH109 and Agrobacterium GV3101. The main reagents are shown in the following table 1. Table 1 ~~ "Reagents Manufacturers “RNAprep pure Plant Kit plant total RNA extraction kit ~~ TIANGEN Plant genomic DNA extraction kit TIANGEN PrimeScriptTM RT reagent Kit with gDNA Eraser (Perfect Real Time) Takara 1-5™2xHigh-Fidelity Master Mix MCLAB 2xRapid Tag Master Mix Vazyme Phanta Max Super-Fidelity DNA Polymerase Vazyme Restriction enzyme Takara Universal DNA purification and recovery kit TIANGEN Plasmid miniprep kit TIANGEN Plasmid maxiprep plasmid box Vigorous 10% (wt/vol) BSA Sigma Cellulase R10, Macerozyme R10 Yakult Pharmaceutical Ind Yeast SD culture medium Clontech Nitrogen baes medium Clontech 2xAceQ Universal SYBR qPCR Master Mix Vazyme Solution and culture medium formula: Enzymatic hydrolysate: putting 20mM of MES (pH of 5.7) solution added with 1.5% (wt/vol) of cellulase R10, 0.4% (wt/vol) of macerozyme R10, 0.4M of mannitol and 20mM of KCI into a thermostatic water bath kettle at 55°C, and carrying out water bath for 10min to inactivate the DNA enzyme and proteinase and enhance the solubility of the enzyme.

Taking out the heated enzyme solution, cooling to room temperature (25°C), and adding 10mM of CaCl, and 0.1% of BSA.

The enzyme solution is prepared on site for use.

WI solution: adding 0.5M of mannitol and 20mM of KCI into 4mM of MES (pH of 5.7) solution.

The prepared solution can be stored at room temperature (22-25°C). WS5 solution: adding 154mM of NaCl, 125mM of CaCl, and 5mM of KCI into 2mM of MES (pH of 5.7) solution.

The prepared solution can be stored at room temperature.

MMG solution: adding 0.4M of mannitol and 15mM of MgCl; into 4mM of MES (pH of 5.7) solution. The prepared solution can be stored at room temperature.

PEG-Ca transfection solution: adding 0.2M of mannitol and 100mM of CaCl; into 20-40% (wt/vol) of PEG4000 solution in which ddH»O is completely dissolved.

5 10 x TE: adding 10mL of 0.1M Tris-HCI and 2mL of 10mM EDTA; adding water until reaching 100mL; and sterilizing at 121°C for 20min.

x LIAC: adding 1M of LIAC (weighing 3.3g of anhydrous lithium acetate, and dissolving in 40mL of water, regulating the pH value to 7.5, fixing volume to 50mL, and filtering and sterilizing).

1 x TE/LIAC: adding 0.1mL of 10 x TE and 0.1mL of 10 x LiAC, and adding water to reach 10 1mL.

40% PEG conversion solution: 0.8mL of 50% PEG solution, 0.1mL of 10 x TE, and 0.1mL of 10 x LIAC.

YPAD culture medium: adding 10g/L of yeast extract, 20g/L of peptone, 50mL of 40% glucose solution (independently sterilized for 15min at 115°C), 20g/L of agar (solid culture medium) and 15mL of 0.2% Adenine solution (added when the culture medium is cooled to about 60°C), and sterilizing for 20min at 121°C.

SD culture medium: 26.7g/L of nitrogen base medium and Xg/L of amino acid mix (a kit, added according to the volume of the prepared culture medium by a corresponding amount), regulating PH to be 5.8, then adding 20g/L of agar, and sterilizing for 15min at 121°C. When the culture medium is cooled to about 60°C, adding 15mL of 0.2% adenine solution and 50mL of 40% glucose solution.

LB culture medium: adding 10g/L of sodium chloride, 5g/L of yeast extract, 10g/L of tryptone and 12.5¢/L of agar (solid), and sterilizing at high pressure for 20min at 121°C.

YEB culture medium: adding 5g/L of beef extract, 5g/L of peptone, 1g/L of yeast extract, 5g/L of sucrose, 0.5g/L of MgSO4:7H:2O and 12.5g/L of agar (solid), and sterilizing at high pressure for 20min at 121°C.

Infection liquid: adding 2.215g/L of MS powder, 30g/L of sucrose, 200 pmol/L of acetosyringone (added when the culture medium is cooled to about 60°C), regulating the pH to

5.2, and sterilizing at high pressure for 20min at 121°C.

Co-culture medium: adding 4.43g/L of MS powder, 30g/L of sucrose, 1mg/L of 6-BA, 0.1mg/L of NAA, 7g/L of agar, 200umol/L of acetosyringone, regulating the pH to 5.8, and sterilizing at high pressure for 20min at 121°C.

Induced differentiation culture medium: adding 4.43g/L of MS powder, 30g/L of sucrose, 1mg/L of 6-BA, 0.1mg/L of NAA, 7g/L of agar, regulating the pH to 5.8, and sterilizing at high pressure for 20min at 121°C. Adding 5mg/L of hygromycin and 300mg/L of cef when the culture medium is cooled to not scald hands.

Rooting culture medium: adding 4.43g/L of MS powder and 30g/L of sucrose, regulating the pH to 5.8, sterilizing at high pressure for 20min at 121°C, and adding 5mg/L of hygromycin and 300mg/L of cef when the culture medium is cooled to not scald hands.

Formula of Arabidopsis inflorescence infection liquid: adding 2.215g/L of MS powder and 2% of sucrose, sterilizing for 20min at 121°C, and adding Silwet solution with final concentration of

0.02%-0.03% after the culture medium is cooled to room temperature.

Thus, the above-mentioned steps are no longer repeated.

Embodiment 1 The DNA extraction and RNA extraction refer to the kit instructions and are operated in a conventional manner.

Synthesis of single-stranded cDNA: total RNA of Lycium ruthenicum Murr fruits is taken as a template, and single-stranded cDNA is synthesized through experimental steps given by PrimeScriptTMRT reagent Kit with gDNA Eraser (Perfect Real Time) of Takara.

Cloning of LrETC1 genes: the amplification primer sequence is shown in Table 2, a primer is diluted to be 10 uM, and PCR amplification reaction is carried out by taking the obtained cDNA as a template. The reaction system is shown in Table 3.

Table 2 _ Name Forward/Rever ~~ Primersequence (5—3) Purpose of gene se primer LETC1 Forward ~~ ATGAGAAAACCTTGTTGTGA, like SEQIDNo.3 Target fragment Reverse ATCTCAATTCACTTCCATAG, like SEQ ID No. 4 amplificati on LrETC1 Forward: GAGCTCGGTACCCGGGGATCCATGAGAAAACCTTGTTG Constructi -GFP TGA, like SEQ ID No. 5 on of Reverse: GCTCACCATGTCGACTCTAGATGGAAGTGAATTGAGATC LrETC1 AGGC, like SEQ ID No. 6 subcellular localizatio n vector GAL4B Forward: TATGGCCATGGAGGCCGAATTCATGAGAAAACCTTGTTG Constructi D- TGATC, like SEQ ID No. 7 on of LrETC1 Reverse: CCGCTGCAGGTCGACGGATCCCTATGGAAGTGAATTGA LrETC1 GATCAG, like SEQ ID No. 8 yeast hybrid vector gLrETC Forward: CTAAACATGGTGAAGGTTGCTG, like SEQ ID No. 9 gRT-PCR 1 Reverse: CAGTTCTCCTCGGTAATCTTCC, like SEQ ID No. 10 LrActin Forward: CTCAGCACCTTCCAGCAGAT, like SEQ ID qRT-PCR No. 11 qRT-PCR Reverse: TAACACTGCAACCGCATTTC, like SEQ ID No. 12

Table 3 ~~ Components ~~ Usage Reaction conditions “1-5™2 x High-Fidelity Master Mix ~~ 25a 98°C, 2min; LrETC1-F 2ul 98°C, 10sec; LRETC1-R 2ul 55°C, 15sec; | 34 cycles Template cDNA 2ul 72°C, 10sec; ddH20 19ul 72°C, 5min; Total 50ul 12°C, hold After amplification is completed, the PCR product is taken out and subjected to 1% agarose gel electrophoresis detection; electrophoresis is performed for about 7min, and the PCR product is observed with an ultraviolet gel imager.

If the target strip is single and correct in size, the gel block is cut off and placed in a 2mL centrifugal tube, and gel is recovered through the operation step of a TIANGEN universal DNA purification and recovery kit.

Connection and transformation of Escherichia coli The gene segment of the gel-recovered product L/ETC1 is connected with the T cloning vector, and the reaction system is shown in the Table 4. Table 4 Components Usage Reaction conditions 5xTA/Blunt-Zero Cloning Mix ul React for 5min at room PCR recovery product 4ul temperature (20-37°C), and Total 5ul store at 4°C ee After the reaction is finished, the centrifugal tube is placed on ice, and Escherichia coli DH5a is transformed through a heat shock method, and the steps are as follows: (1) Adding the connection product into a 1.5mL centrifugal tube containing 50 microliters of Escherichia coli competent DH5q, rapidly and gently blowing and beating the centrifugal tube to mix uniformly through a pipette, and placing the centrifugal tube on ice for standing for 30min. (2) After standing is finished, placing the centrifugal tube into a water bath kettle at 42°C; after heat shock is carried out for 1 min, immediately placing the centrifugal tube on ice for 2 min.

Then, adding 1mL of LB liquid culture medium without antibiotics, and shaking the centrifugal tube in a shaking table at the speed of 200rpm and the temperature of 37°C for about 1h. (3) Taking out the centrifugal tube, centrifuging for 2 min at the speed of 5500rpm, removing most of supernate in a clean bench, uniformly mixing remaining bacteria solution, coating on the LB solid culture medium containing the corresponding antibiotics with the bacteria solution, and inverting the mixture into a constant-temperature incubator at the temperature of 37°C for a night.

(4) Taking out the flat plate the next day, picking single bacteria from the clean bench and falling into the culture medium added with 1mL of LB (containing antibiotics) liquid, and shaking the mixture in the shaking table at the temperature of 37°C for 1-2h. Performing colony PCR identification through universal primers M13-F and M13-R of the T cloning vector. The reaction system is shown in Table 5: Table 5 ~~ Components ~~ Usage Reaction conditions “2x Rapid Tag Master Mix ~~ 1oyl 95°C, 3min; M13-F 1pl 95°C, 15sec; M13-R Tul 55°C, 15sec; | 25 cycles Bacteria solution Tul 72°C, 30sec; ddH20 7ul 72°C, 5min; Total 20ul 12°C, hold Extracting of Escherichia coli plasmid DNA Agarose gel electrophoresis is carried out on the colony PCR product; 200l of positive bacteria solution with correct strip size is sucked out and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing; the sequencing result is subjected to alignment with the target gene sequence; 20pl of bacteria solution is sucked into 10mL of LB liquid culture medium (containing antibiotics) after no error; and overnight-culture is carried out at the shaking table at 200rpm in 37°C; On the next day, the bacteria solution is taken out after the bacteria solution has certain concentration; 1mL of bacteria solution is sucked into the 1.5mL centrifuge tube from the clean bench; glycerol is added and uniformly mixed; and the mixture is stored in a refrigerator at -80°C. The plasmid is extracted from the residual bacteria solution according to the steps of the TIANGEN plasmid miniprep kit.

Embodiment 2 The open reading frame and the derived amino acid sequence of the LrETC1 transcription factor can be searched through ORF Finder (https://www.ncbi.nlm.nih.gov/orffinder/y on NCBI. The amino acid composition of the transcription factor, the molecular weight of protein, the theoretical isoelectric point and the stability can be predicted through online software ProtParam (http://web.expasy.org/protparam/). The hydrophobicity and charge distribution condition of the protein can be analysed through online software ProtSca le (https://web.expasy.org/protscale/). The transmembrane domain of the LrETC1 protein can be analysed through TMHMM 2.0 (http://www .cbs.dtu.dk/services/ TMHMM) software. The signal peptide of the protein can be predicted through Cell-PLoc2.0 (http://www.cbs.dtu.dk/services/Sign alP). Subcellular localization of the protein can be predicted through Wolf Psort (https://www.genscript.com/wolf-ps ort.htmL) and online software (https://www.csbio.sjtu.edu.cn/bicinf/Cell-PLoc-2/). Secondary and tertiary structure prediction can be conducted on the protein through SOPMA (https://npsa- prabi.ibcp.fr/cgi-bin/nps a_automat.pl?page=/NPSA/npsa_sopma.html} and SWISS MODEL (https://swis smodel.expasy.org’). The DNMAL domain can be analysed through SMART (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). The homologous sequences of the LrETC1 and other species are subjected to alignment through conserved DNAMAN 8.0, and the phylogenetic tree can be constructed through a Neighbor-Joining method (NJ) by using MEGA7.0 software.

Construction of recombinant vector: Cloning a target gene homologous recombinant fragment.

The cDNA solution obtained in the embodiment is taken as a template, the homologous recombinant fragment of LrETC1-GFP is amplified by using a corresponding primer in a Table 2, and the amplification system is shown in Table 6: Table 6 ~~ Components ~~ Usage Reaction conditions 1-5™2 x High-Fidelity Master Mix 25 ~~ 98°C,2min LrETC1-GFP-F 2ul 98°C, 10sec LRETC1-GFP-R 2ul 55°C, 15sec | 35 cycles Template cDNA 2ul 72°C, 10sec | ddH20 19ul 72°C, 5min Total 50ul 12°C, hold Ee Agarose gel electrophoresis detection and gel recovery are performed on the PCR product, and the PCR product is stored at the temperature of -20°C for later use; Vector enzyme digestion reaction Double enzyme digestion is performed with enzyme digestion sites BamHI and Xbal on the subcellular localization vector which is pCAMBIA 1300-GFP stored in a laboratory.

Double enzyme digestion is performed with enzyme digestion sites ECORI and BamHI on the yeast hybrid vector which is PGBKT7 stored.

The enzyme digestion system is shown in Table 7. Table 7 “Components ~~ Usage Reaction conditions “Restriction enzyme 1 ~~ 254 Restriction enzyme 2 2.5 37°C, 30min Buffer Sul 4°C, hold Vector 40ul Total 50ul ee

Agarose gel electrophoresis is performed on the enzyme digestion product to verify whether the vector is completely open and whether the cut strip is of an expected size so as to ensure the accuracy of enzyme digestion.

The vector gel block is cut off after the accuracy is ensured, the vector is recovered by utilizing the universal DNA purification recovery kit, and the recovered vector is stored at the temperature of -20°C for later use; Homologous recombination connection of fragment and vector Homologous recombination connection is performed on a cloned target fragment with a homologous arm and the corresponding cut vector to construct a subcellular localization vector with LrETC1-GFP and a PGBKT7 yeast recombinant BD vector with GAL4BD-LrETC1. The homologous recombination connection reaction system is shown in Table 8. Table 8 Components ~~ Usage Reaction conditions “Linear vector ~~ wa Insert fragment 2ul 5 x CE II buffer 2ul 37°C, 30min Exnase II Tul 4°C, hold ddH20 4yl Total 10ul Transformation of Escherichia coli DH5a by recombinant vector The constructed subcellular localization vector LrETC 1-GFP with the target fragment and the PGBKT7 vector GAL4BD-LrETC1 with the target fragment are taken out from the PCR instrument and immediately placed on ice to transform Escherichia coli DH5q, and the transformation method is the same as the above embodiment.

On the next day, after the bacterial colony grows out, conventional bacterial colony PCR identification and sequencing are performed to confirm that the vectors are constructed successfully.

Plasmid miniprep is performed on positive bacteria solution of GAL4BD-LrETC1 (see the kit instructions, and treat in a conventional manner). 100 pl of positive bacteria solution of LFETC1-GFP is added into 200mL of LB (containing kanamycin) liquid culture medium and shaken for 24h; after the bacteria solution shows a certain concentration, and plasmid maxiprep is performed through the TIANGEN endotoxin-free plasmid maxiprep kit to obtain high-concentration and high-purity plasmids for subsequent protoplast conversion experiments.

Extraction of protoplast (1) Cutting off tender green, thick and well-grown tobacco leaves which grow for 3-4 weeks (generally 5-7 true leaves exist), putting the tobacco leaves on clean filter paper, immediately dipping a small amount of carborundum to gently and quickly rub the lower epidermis of the leaves until the leaves are smooth and slight tissue fluid seeps out, cutting the leaves into small pieces of 5 x 5mm by using a sharp blade, and immediately, quickly and mildly spreading the small pieces in a prepared fresh enzymatic hydrolysate (the side with the lower epidermis being ground off is downward), so that the leaves can be fully contacted with the enzymatic hydrolysate and fully digested.

(2) Wrapping a culture dish filled with the enzymatic hydrolysate by using tinfoil, putting the culture dish into a constant-temperature shaking table at 37°C to perform shake culture in dark for 2-3 hours, observing the digestion condition of the culture dish every 1 hour during the period, and lightly rotating the enzyme solution until the enzyme solution gradually becomes green, which indicates that the protoplast starts to be released.

(3) After the enzymatic hydrolysate becomes thick green and the leaves become relatively transparent, lightly taking out the undigested leaves by using tweezers, sucking 20ul of the undigested leaves on a glass slide, and checking the release condition of the protoplast under a microscope.

(4) When the mass and concentration of the protoplast are determined to be used for subsequent experiments, lightly transferring the cracked solution into a 50mL flat-bottom centrifuge tube, and diluting the protoplast solution by using W5 solution of the same volume. Centrifuging for 2min with 100g, removing the supernatant as much as possible, adding about 3- 5mL of the W5 solution, mildly rotating the centrifuge tube, and resuspending the protoplast precipitated at the bottom of the tube.

(5) Sucking 20pl of the resuspended protoplast solution in a cell counter, observing the concentration of the protoplast, observing about 2 x 10° protoplast solutions under a 100X microscope. Standing the protoplast solution on ice for 30 min.

(6) Centrifuging the centrifuge tube for 1min with 100g, removing the supernatant as much as possible (not contacting the supernatant), adding 2mL of MMG solution, mildly rotating the centrifuge tube, resuspending the protoplast solution, and uniformly mixing the protoplast solution and the ice for subsequent transformation experiments.

Protoplast transformation steps (DNA-PEG-Ca transformation method): (1) Adding 10ul (10-20 ug) of recombinant subcellular localization plasmid DNA into a 2mL centrifuge tube, adding 100 microliters of protoplast which is resuspended and uniformly mixed inthe MMG solution, and gently rotating the centrifuge tube to uniformly mix.

(2) Adding 110 pl of fresh PEG-CaCl: solution into the centrifuge tube, and lightly knocking the wall of the centrifuge tube to uniformly mix. Incubating at room temperature for 6 min. After the incubation is finished, adding 440ul of WS solution, gently reversing the centrifuge tube, and terminating the transfection process. Centrifuging for 2 min with 100g, and removing the supernate. Adding 1M of W5 solution to clean the protoplast, centrifuging for 2min with 100g, and removing the supernate.

(3) Coating 0.1% BSA (sterilized) solution on the surface of a 6-pore cell culture dish to prevent the protoplast from being attached to the surface of the culture dish. Gently transferring the protoplast resuspended with the WI solution into the culture dish, wrapping with tinfoil, and culturing overnight under dark conditions at 25°C. Observation of GFP fluorescence signals under microscope: (1) Transferring the protoplast cultured overnight into the 2mL centrifuge tube from the cell culture dish, centrifuging for 2 min with 100g, removing most of the supernate, and gently rotating the centrifuge tube to resuspend the protoplast. (2) Absorbing the converted protoplast solution onto the glass slide, and observing the GFP fluorescence signals under a fluorescence microscope. (3) In order to determine the position of the cell nucleus, carrying out DAPI dyeing on the protoplast. That is, uniformly mixing the protoplast solution and ready-to-use DAPI ice solution which have the same volume, dyeing on the ice in a dark place for 5-10 min, diluting the protoplast with 1 x phosphate buffer, and terminating the dyeing. Absorbing the protoplast dyed by DAPI and placing on the glass slide, and observing the blue fluorescence signals under the fluorescence microscope.

Yeast transformation (1) Scratching the yeast AH109 strain in an YPAD culture medium, and inversely culturing in a 28°C constant-temperature incubator for 3 days. After the colony grows out, picking a single colony from the clean bench, adding into a conical flask containing 5mL of YPAD liquid culture medium, and culturing in a constant-temperature shaking table at 28°C overnight.

(2) In the morning of the next day, adding 10 times by volume of fresh YPAD liquid culture medium into the conical flask, and continuing culturing until the OD is 0.3-0.6. After culturing to a proper concentration, taking out, pouring into a 50mL centrifuge tube in the clean bench, and centrifuging at the room temperature at the rate of 5000 rpm for 5min.

(3) Pouring out the liquid in the centrifuge tube, resuspending with 20mL of sterile water, and centrifuging at the room temperature at the rate of 5000rpm for 5min.

(4) Preparing 1 x TE/LIAC solution (which is used immediately after preparation) as required. Pouring out the liquid in the centrifuge tube, cleaning the 2mL centrifuge tube with 1mL of 1 x TE/LIAC solution, and centrifuging at the room temperature for 3min. Resuspending with 300- 500pl of 1 x TE/LIAC solution until the yeast competence is finished.

(5) Sequentially adding 5pl of plasmid DNA, 10pl of salmon sperm DNA (which is boiled with boiling water for 10min before use and then put on ice) and 100ul of yeast competence into the 2mL centrifuge tube, and uniformly mixing. Carrying out shake culture on the shaking table at 28°C for 30 min. Then, putting the centrifuge tube in a 42°C water bath kettle, carrying out water bath for 15min, and uniformly mixing once during the water bath.

(6) Taking out the centrifuge tube, instantaneously centrifuging at room temperature, collecting the yeast precipitate, resuspending with 1mL of sterile water, and centrifuging at the rate of 5000rpm for 1min. Removing the supernatant, adding 100pul of sterile water (determined according to concentration) resuspension, taking 50pl of resuspension, coating on the (SD/-Trp) culture medium, and inversely culturing in the constant-temperature incubator at 28°C for 3 days. (7) Dotting the yeast colony: picking the grown single colony to 30ul of sterile water, resuspending, respectively sucking 2ul and respectively dotting on the yeast (SD/-Trp) and (SD/- Trp/-His/-Ade) culture mediums. Inversely culturing in the constant-temperature incubator at 28°C for 3 days. Real-time fluorescent quantitative PCR analysis The cDNA solution of each tissue obtained by the method in the embodiment 1 is diluted by four times by utilizing the primer sequence in Table 2 according to the LrETC1 gene sequence obtained by cloning; the operation is performed by adopting the steps of the Vazyme high- specificity SYBR dye method quantitative PCR detection kit; PCR reaction is performed in a Bio- Rad CFX96 ™ real-time PCR detection system; and the reaction system is shown in Table 9. Table 9 “Components ~~ Usage Reaction conditions 2xAceQ Universal SYBRqPCR ~~ 541 95°C, 5min; Master Mix cDNA 0.5ul 95°C, 10sec qQLrETC1-F 0.5ul 55°C, 30sec | 40 cycles GLRETC1-R 0.5ul 72°C, 20sec ddH:=0 3.5ul 65°C, 5sec Total 10ul 95°C, 15sec ee The qPCR result is that the relative expression level of the gene is calculated by adopting a 2-AACt method, and three biological repeats are set in each experiment. Results indicate that: PCR amplification is carried out by taking cDNA as the template so as to obtain 240bp band; the T cloning vector is connected; sequencing is carried out by using the universal primer; the sequencing result is basically consistent with the gene sequence obtained by transcriptome; and the band is determined as the target gene segment. The gel electrophoresis results are shown in Fig. 1: Bioinformatics analysis of the LrETC1 gene: the gene Open Reading Frame (ORF) of the LrETC1 is 240bp (ATGGCTGATTTGGACCGTTCAAGCACATCAGATAAATCCTTTATGGACTC

GTCAGCCGCATTTGAGGCCAACAACGTAGAAACCTCGAAGCTTGAATTTTCAGAAGACGAG GAAATCCTCGTTACTAAAATGTTCAACTTGGTTGGTGAGAGGTGGTCATTAATTGCTGGAA

GAATTCCAGGTAGAACTGCAGAGGAAATTGAGAAGTACTGGAACTCAAGACACTCTACCAG CCAGTAA, as shown in SEQ ID No. 1), and 79 amino acids (MADLDRSSTSDK

SFMDSSAAFEANNVETSKLEFSEDEEILVTKMFNLVGERWSLIAGRIPGRTAEEIEKYWNSRHS TSQ*, as shown in SEQ ID No. 2) are encoded.

Hydrophily and hydrophobicity analysis, transmembrane analysis and tertiary structure prediction of LrETC1 coded protein: The analysis through online software ProtParam and ProtScale shows that the theoretical molecular weight is 8978.86D, the theoretical isoelectric point is 4.64, and the protein instability coefficient is 61.61, which indicates that the protein is an unstable protein. The average hydrophilicity (GRAVY) is -0.671 (Table 10), which indicates that the protein is hydrophilic protein (Fig. 2). Table 10 _ Protein Theoretical molecular Theoretical PI Instability ~~ Total average weight (D) value Coefficient hydrophilicity (GRAVY) ~ LrETCT ~~ gurssee 484 BIB 0e7t TMHMM and SignalP4.1 are adopted to predict that the LrETC1 protein {Fig. 3 and Fig. 4) does not have transmembrane region or signal peptide, which indicates that the protein belongs to non-transmembrane proteins and non-secretory proteins.

SOPMA is utilized to predict that the secondary structure of the L/ETC1 protein comprises

50.63% of a-helix, 8.86% of B-turn, 7.58% of B lamella and 32.91% of random curl (Table 11).

Table 11 “Protein ahelix = Btun Blamella = Randomourl LETC1 5063% 8.86% 7.59% 3291% The similarity of the tertiary structure model, constructed by SWISS-MODEL, of the LrETC1 protein and the WER transcription factor is 40.74% (see Fig. 5).

(3) SMART is utilized to perform conservative structure domain analysis on the protein, and the result shows that the LrETC7 only has one R3 conservative structure domain from 29-77 sites, so that the LrETC1 protein belongs to the R3 subfamily.

A Cell-PLoc 2.0 online tool is utilized to perform subcellular localization prediction on the LrETC1, and the result shows that the LrETC1 is localized in the cell nucleus.

Software MEGA-X is utilized to perform Clustalw alignment on the LrETC1 protein and the MYB protein sequence related to anthocyanin synthesis in Arabidopsis, apple, grape and other species. The alignment results are analysed through a Neighbor-Joining method, Bootstrap is set to be 1000, and the phylogenetic tree is constructed. The analysis indicates that the LrETC1 protein is gathered with the AtCPC-like transcription repressor of the R3-MYB subgroup, such as SIMYB-ATV of tomato, PhMYBx of Petunia hybrida, and AtETC1 of Arabidopsis (Fig. 8). Therefore, it can be presumed that the LrETC1 belongs to the R3-MYB transcription repressor and has similar functions with the AtCPC-like transcription repressor.

Multi-sequence alignment of LrETC 1 protein DNAMAN 8.0 software is utilized to carry out multi-sequence alignment analysis on the amino acid sequence of LrETC1 and the homologous MYB protein in other species, and the result shows that the LrETC1 has only one R3 repetitive sequence without any inhibitory amino acid motif, but has a motif linking with a bHLH transcription factor and belongs to MYB transcription factors of R3-MYB family (Fig. 7). Subcellular localization analysis of LrETC1 protein The recombinant vector LrETC1-GFP (Fig. 8) and an empty vector P1300-GFP as a control are transferred into the protoplast of tobacco under the driving of a CaMV35S constitutive promoter. The observation under the fluorescence microscope shows that the control vector distributes green fluorescence in the whole cell, while LrETC1-GFP only emits a strong fluorescence signal in the cell nucleus (Fig. 9). Therefore, the transcription factor is located in the cell nucleus and plays a role in regulating the cell nucleus as a transcription regulator.

Transcription activity analysis of LrETCT: In order to verify whether the LrETC1 has the transcription activation activity, a PGBKT7 recombinant plasmid containing the LrETC1 is constructed, empty PGBKT7 is taken as a negative control, and the PGBKT7 connected with LrAN11 (having the transcription activation activity) is taken as a positive control. The result shows that the control and the experimental group can normally grow on the (SD/-Trp) culture medium, which indicates that the plasmid is successfully transferred into yeast. The rest, except the positive control, does not grow on the (SD/-Trp/-His/- Ade) culture medium, which indicates that the LrETC7 does not have the transcription activation activity, and does not turn blue in the (SD/-Trp/-His/-Ade) culture medium coated with X-A/pha- Gal dye liquor, and the positive control has light blue (Fig. 10). Therefore, the conclusion is preliminarily drawn that the LrETC1 does not have the transcription activation activity in yeast cells, and it is guessed that the transcription factor is combined with other transcription factors to form a compound so as to regulate and control the expression of the gene.

Expression analysis of LrETC1 in Lycium ruthenicum Murr and white Lycium barbarum fruits In order to verify the expression quantity condition of LrETC1 obtained by transcriptome data, the expression quantity of LrETC1 is analysed by utilizing qRT-PCR in different development stages (51-85) of the Lycium ruthenicum Murr and white Lycium barbarum fruits. The result shows that the expression quantity of LrETC1 in Lycium ruthenicum Murr is obviously higher than that of white-fruit Lycium barbarum, and the expression quantity of LrETC1 is gradually increased along with increase of the anthocyanin content in the fruit ripening process, but the expression quantity starts to be reduced in the S5 stage of complete ripening of the fruits. However, in the white Lycium barbarum fruits, from the S1 stage of starting development of the fruits to the S5 stage of complete ripening of the fruits, LrETC1 is hardly expressed (Fig. 11).

Tissue specific expression analysis of LrETC1 in Lycium ruthenicum Murr

The qRT-PCR analysis is performed by taking cDNA of different tissues of Lycium ruthenicum Murr as a template. The result shows that LrETC1 has expression in stems, leaves, flowers and fruits, does not have expression specificity and has the highest expression quantity in leaves and fruits.

The qRT-PCR analysis shows that the expression quantity of LrETC1 is in positive correlation with accumulation of anthocyanin and has high-level expression when anthocyanin is greatly accumulated at the beginning of the fruits, which indicates that LrETC1 is activated at the beginning of accumulation of anthocyanin and indicates that the R3-MYB transcription repressor can be activated at the beginning of synthesis of anthocyanin so as to provide feedback regulation and play an important role in the process of balancing accumulation of anthocyanin.

Embodiment 3 Construction of plant overexpression recombinant vector Cloning of target gene homologous recombinant fragment: The cDNA solution obtained in the step of the above embodiment is taken as a template, homologous recombinant fragment amplification is carried out on the plant overexpression vector by using corresponding primers in Table 12, and the amplification system and PCR product detection and recovery are the same as those of the above embodiment.

Table 12 “Name of Forward/Rever ~~ Primersequence (5—3) Purpose gene se primer “LrETC1 Forward ~~ ACCGTCGACGAGCTCTCTAGAATGAGAAAACCTTGT Construction of TGTGA, like SEQ ID No. 13 LrETC1- Reverse TTTGCGGAGTACCCGGGTACCCTATGGAAGTGAAT PCM1307 TGAGAT, like SEQ ID No. 14 overexpression vector LrETC1 Forward: CACCATCACCATCATCCCGGGATGAGAAAACCTTGT Construction of TGTGA, like SEQ ID No. 15 LrETC1-pEAQ- Reverse: TGAAACCAGAGTTAAAGGCCTCTATGGAAGTG HT AATTGAGAT, like SEQ ID No. 16 overexpression vector Vector enzyme digestion reaction Double enzyme digestion is carried out on a vector PCM1307 by using Xba | and Kpnl restriction enzymes; and double enzyme digestion is carried out on a vector pZYB9-pEAQ-HT by using Smal and Stul restriction enzymes. The enzyme digestion system and product recovery are the same as those of the above embodiment.

The homologous recombinant connecting fragment and vector, Escherichia coli DH5a conversion, bacteria solution PCR identification and plasmid extraction are the same as those in the above embodiment.

Transformation of Agrobacterium tumefaciens Operation steps comprise: (1) Taking out the prepared competent Agrobacterium tumefaciens from the temperature of -80°C, and placing on ice.

(2) Adding 10 pl of recombinant plasmids and 100ul of competent Agrobacterium tumefaciens into a 1.5mL precooled centrifugal tube, and performing standing reaction on ice for 30min.

(3) Placing the centrifugal tube into liquid nitrogen to be quickly frozen for 5min, then taking out and placing into a water bath kettle at the temperature of 37°C to heat in a water bath way for 5 min, and placing the centrifugal tube on ice for 2 min.

(4) Adding 1mL of antibiotic-free LB liquid culture medium into the centrifugal tube in the ultra-clean workbench, and culturing in the shaking table at the temperature of 28°C for about 4h. Taking out the centrifugal tube and coating an LB flat plate (antibiotics kanamycin and rifampicin are added), and inversely culturing in an incubator at the temperature of 28°C for 2 days.

(5) After bacterial colonies grow out, picking single bacterial colonies and placing into the LB resistant liquid culture medium to culture in the shaking table at the temperature of 28°C overnight.

Taking out the single bacterial colonies next day for bacterial colony PCR verification. Storing the positive bacterial liquid in a refrigerator at the temperature of -80°C.

Genetic transformation of Arabidopsis Agrobacterium-mediated Arabidopsis inflorescence infection method (1) Picking fresh agrobacterium single colony into 1mL of LB resistant liquid culture medium, and culturing in the shaking table at the temperature of 28°C overnight. On the next day, taking out the product at a ratio of 1: 50, culturing until ODsoo is approximately equal to 0.8-1.0, centrifuging at 5500rpm for 5 min, removing the supernatant, and resuspending with infection liquid until ODsgo is approximately equal to 0.8.

(2) Thoroughly watering wild Arabidopsis plants one day before infection, selecting the Arabidopsis plants with good growth conditions in the flowering phase for transformation, and completely cutting off pods of seedlings before transformation. Putting all inflorescences of the Arabidopsis into a beaker filled with bacterial liquid for infection for 30s.

(3) Moisturizing the infected plants with a preservative film, and performing light-shielding culture in a greenhouse at 23°C for 24 hours.

(4) After performing light-shielding culture for 24 hours, removing the preservative film, and culturing under normal illumination conditions. In order to improve the transformation efficiency, repeating infection once 10 days later, and performing normal culture after the infection is ended until seeds are collected.

(5) Collecting TO-generation mature seeds, and performing positive plant screening by utilizing a hygromycin-containing resistant culture medium. Culturing the screened positive plants until T1-generation seeds are collected, and properly drying the seeds and storing for subsequent experiments.

Sugar stress of transgenic Arabidopsis The dried Arabidopsis seeds are disinfected and sterilized in the clean bench; the seeds are sown in 1/2MS with the sucrose concentration of 3% and 1/2MS with the sucrose concentration of 6%; the seeds are vernalized in a refrigerator at 4°C for 3 days and then taken out after 3 days; and the taken-out seeds are normally cultured in a greenhouse at 25°C. After 2-3 weeks, observing the accumulation of anthocyanin is observed.

Anthocyanin content determination (1) Grinding about 0.1g of material to be determined in liquid nitrogen into powder, immediately adding 600ul of 1% HCI methanol solution, and shaking at 4°C for 6 hours.

(2) Taking out after 6 hours, adding 400 of water and 400! of chloroform, and centrifuging in a refrigerated centrifuge at 4°C at 14000rpm for 5 min.

(3) Transferring the supernatant solution in which the anthocyanin is dissolved into a clean

1.5mL centrifuge tube, sucking 200pul of solution, and determining the light absorption values of A530nm and AB857nm. Calculating the anthocyanin content (mg/g) =A530nm-0.33 x A657nm by a formula. Each sample is subjected to biological repetition three times.

Results indicate that: After seeds of the obtained LrETC1 transgenic TO-generation plants are collected, the seeds are sown in the 1/2MS culture medium containing 35mg/L of hygromycin, and the positive plants have tender green leaves, normal growth and development and good growth vigor due to the resistance of hygromycin. However, the wild type Arabidopsis does not contain resistance genes, so that the growth is slow, and the wild type Arabidopsis slowly becomes yellow and stops growing (Fig. 12). After the seeds grow in the culture medium for about 10 days, the plants with good growth state and large body shape are selected and transferred into nutrient soil for culture. PCR identification and sequencing are carried out after 2 weeks; the culture is continued after the positive plants are determined; and the mature T1-generation seeds are collected. The result is shown in Fig. 13. Compared with the wild type Arabidopsis, the seed coat of the seed of the transgenic T1-generation Arabidopsis of the LrETC1 gene is light in colour and is light brown, and the seed coat of the wild type Arabidopsis is dark brown. The result shows that the content of procyanidin of the LrETC1 transgenic line is low, and the transcription factor may participate in repressing accumulation of procyanidin in the seed coat.

Existing researches show that sucrose can promote accumulation of anthocyanin, so that high-concentration sucrose is utilized to perform stress treatment on the Arabidopsis so as to explore the effect of genes in anthocyanin synthesis regulation. According to previous researches, the Arabidopsis accumulates the anthocyanin under the sucrose concentration of 6%. Therefore,

in order to verify whether the LrETC1 can repress the accumulation of the anthocyanin of the Arabidopsis under the sugar stress condition, three (OE #1, OE #2 and OE #3) transgenic positive plants are selected respectively to be subjected to high-sugar stress treatment of the Arabidopsis. The results show that wild type Arabidopsis seedlings growing for 2-3 weeks on the 1/2MS culture medium with 8% of sucrose accumulate partial pigments on leaves, petioles and stem top parts, and transgenic LrETC1 plants hardly have pigment accumulation (Fig. 14). Wild type and transgenic seedling strains are subjected to anthocyanin content determination, and the pigment content is consistent with the phenotypic change. The expression levels of LrETC1 genes in the transgenic plants and structural genes (AtCHS, AtCHI, AtDFR, AtANS and the like) in an Arabidopsis anthocyanin synthesis path are analysed by utilizing qRT-PCR. The expression levels of the structural genes related to anthocyanin synthesis in the transgenic strains are obviously reduced, which indicates that the LrETC1 (Fig. 15) transcription factor can repress the accumulation of the anthocyanin of the Arabidopsis under high-sugar stress.

Each embodiment in the description is described in a progressive way. The difference of each embodiment from each other is the focus of explanation. The same and similar parts among all of the embodiments can be referred to each other.

The above description of the disclosed embodiments enables those skilled in the art to realize or use the present invention. Many modifications to these embodiments will be apparent to those skilled in the art. The general principle defined herein can be realized in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principle and novel features disclosed herein.

SEQUENCE LISTING

<110> Institute of Wolfberry Sciences, Ningxia Academy of

Agricultural and Forestry Sciences <120> LYCIUM RUTHENICUM MURR ANTHOCYANIN SYNTHESIS-RELATED MYB

TRANSCRIPTION REPRESSOR LrETC1 AND APPLICATIONS THEREOF <130> Lycium LrETC1 NL <150> CN202110679791.5 <151> 2021-06-18 <160> 16 <170> PatentIn version 3.5 <210> 1 <211> 240 <212> DNA <213> Lycium ruthenium Murr <400> 1 atggctgatt tggaccgttc aagcacatca gataaatcct ttatggactc gtcagccgca 60 tttgaggcca acaacgtaga aacctcgaag cttgaatttt cagaagacga ggaaatcctc 120 gttactaaaa tgttcaactt ggttggtgag aggtggtcat taattgctgg aagaattcca 180 ggtagaactg cagaggaaat tgagaagtac tggaactcaa gacactctac cagccagtaa 240 <210> 2 <211> 79 <212> PRT <213> Lycium ruthenium Murr <400> 2 Met Ala Asp Leu Asp Arg Ser Ser Thr Ser Asp Lys Ser Phe Met Asp 1 5 10 15 Ser Ser Ala Ala Phe Glu Ala Asn Asn Val Glu Thr Ser Lys Leu Glu

Phe Ser Glu Asp Glu Glu Ile Leu Val Thr Lys Met Phe Asn Leu Val 40 45 Gly Glu Arg Trp Ser Leu Ile Ala Gly Arg Ile Pro Gly Arg Thr Ala 50 55 60

Glu Glu Ile Glu Lys Tyr Trp Asn Ser Arg His Ser Thr Ser Gln 65 70 75 <210> 3 <211> 20 <212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 3 atgagaaaac cttgttgtga 20 <210> 4

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 4 atctcaattc acttccatag 20 <210> 5

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 5 gagctcggta cccggggatc catgagaaaa ccttgttgtg a 41 <210> 6

<211> 43

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 6 gctcaccatg tcgactctag atggaagtga attgagatca ggc 43 <210> 7

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 7 tatggccatg gaggccgaat tcatgagaaa accttgttgt gatc 44 <210> 8

<211> 44

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 8 ccgctgcagg tcgacggatc cctatggaag tgaattgaga tcag 44 <210> 9

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 9 ctaaacatgg tgaaggttgc tg 22 <210> 10

<211> 22

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 10 cagttctcct cggtaatctt cc 22 <210> 11

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 11 ctcagcacct tccagcagat 20 <210> 12

<211> 20

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 12 taacactgca accgcatttc 20 <210> 13

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 13 accgtcgacg agctctctag aatgagaaaa ccttgttgtg a 41 <210> 14

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 14 tttgcggagt acccgggtac cctatggaag tgaattgaga t 41 <210> 15

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 15 caccatcacc atcatcccgg gatgagaaaa ccttgttgtg a 41 <210> 16

<211> 41

<212> DNA

<213> Artificial Sequence

<220>

<223> Primer

<400> 16 tgaaaccaga gttaaaggcc tctatggaag tgaattgaga t 41

Claims (5)

CONCLUSIONS 1. A Lycium ruthenicum Murr anthocyan synthesis-related MYB transcriptional repressor LrETC1, wherein the amino acid sequence of the MYB transcriptional repressor LrETC1 is shown as SEQ ID NO: 2. A gene of the Lycium ruthenicum Murr anthocyanin synthesis-related MYB transcription repressor LrETC1 according to claim 1, wherein the nucleotide sequence of the gene is represented as SEQ ID NO: 1. A recombinant vector containing the transcriptional repressor LrETC1 gene according to claim 2. A recombinant strain containing the transcriptional repressor gene LrETC17 according to claim 2 or the recombinant vector according to claim 3. Use of the transcriptional repressor LrETC1 according to claim 1 or the gene according to claim 2 for the breeding of Lycium barbarum.