Lycoris radiata LlMYB6 gene as well as expression protein and application thereof

Abstract

The invention discloses a lycoris radiata LlMYB6 gene as well as an expression protein and application thereof, and belongs to the technical field of plant molecular biology. The invention provides a lycoris radiata LlMYB6 gene, the nucleotide sequence of the lycoris radiata LlMYB6 gene is shown as SEQ ID NO.1 in a sequence table, and the amino acid sequence of the lycoris radiata LlMYB6 gene is shown as SEQ ID NO.2 in the sequence table. Expression modes of LlMYB6 in three different development stages of pink lycoris longtube flower slices are quantitatively determined in real time through fluorescence, and functions of the gene are identified through subcellular localization, stable tobacco transformation, dual-luciferase detection and GUS activity detection. It is determined that the transcription factor LlMYB6 can negatively regulate synthesis and accumulation of lycoris radiata anthocyanin by inhibiting expression of the enzyme gene LlPAL, and an effective molecular tool is provided for improving plant flower color through genetic engineering.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology, and particularly relates to the function and identification of a Lycoris radiata LlMYB6 gene. Background Art

[0002] Lycoris longituba, a species of Lycoris genus in the Amaryllidaceae family, is primarily found in Jiangsu and Anhui provinces and is endemic to my country. Its rich intraspecific flower color variation makes it highly ornamental and holds broad promise for landscape applications. The pink color of longituba species gradually fades during the three stages of petal development (early bud, mid-bud, and peak bloom), significantly impacting their ornamental value. Molecular research is underway to uncover transcription factors involved in anthocyanin metabolism in longituba, potentially further elucidating the molecular mechanisms underlying the formation of these vibrant flower colors and petal fading.

[0003] Anthocyanins are important pigments that determine flower color. Their synthesis and accumulation in plants are influenced not only by environmental factors but also by key enzyme genes and transcription factors in metabolic pathways. The MYB transcription factor family, one of the most influential in plant life, has been systematically validated in numerous plants. MYB transcription factors have been identified that positively regulate anthocyanin synthesis and accumulation, as well as those that negatively regulate anthocyanin synthesis and accumulation, resulting in lighter flower color. Currently, research on anthocyanin synthesis and accumulation in Lycoris radiata has primarily focused on structural genes, with relatively little research on transcriptional regulation. Therefore, investigating the role of MYB transcription factors in flower color regulation in Lycoris radiata will not only further refine the molecular mechanisms of flower color formation in Lycoris radiata but also provide insights for flower color improvement and molecular breeding. Summary of the Invention

[0004] One of the technical problems to be solved by the present invention is to provide a long-tube Lycoris radiata LlMYB6 gene. Another technical problem to be solved by the present invention is to provide a specific application of the long-tube Lycoris radiata LlMYB6 gene, providing an effective molecular tool for improving the ornamental traits of plants using genetic engineering.

[0005] In view of the above problems existing in the prior art, the technical solutions adopted by the present invention are as follows:

[0006] A Lycoris radiata LlMYB6 gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0007] The amino acid sequence of the expressed protein of the Lycoris radiata LlMYB6 gene is shown in SEQ ID NO.2.

[0008] A biological material, which is an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell containing the Lycoris radiata LlMYB6 gene.

[0009] The application of the long-tube Lycoris radiata LlMYB6 gene or the expression protein of the long-tube Lycoris radiata LlMYB6 gene or the biological material in long-tube Lycoris radiata breeding.

[0010] The application of the long-tube Lycoris radiata LlMYB6 gene or the expression protein of the long-tube Lycoris radiata LlMYB6 gene or the biological material in inhibiting plant anthocyanin synthesis.

[0011] A method for inhibiting anthocyanin synthesis in plants, specifically comprising overexpressing the Lycoris radiata LlMYB6 gene in the plants.

[0012] In some embodiments, the method comprises the following steps:

[0013] (1) constructing an expression vector of the long-tube Lycoris radiata L1MYB6 gene;

[0014] (2) transforming the constructed expression vector of Lycoris radiata LlMYB6 gene into plants or plant tissues;

[0015] (3) Cultivate and screen transgenic plants or plant tissues with reduced anthocyanin content or lighter flower color.

[0016] In some embodiments, the method described herein is directed to a plant selected from the group consisting of tobacco and Lycoris radiata.

[0017] In some embodiments, the overexpression vector of the Lycoris radiata LlMYB6 gene described in the method is Super1300-LlMYB6.

[0018] In some embodiments, the transformation described in the methods is mediated by Agrobacterium.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present application experimentally discovered a long-tube Lycoris radiata LlMYB6 gene. After analyzing the expression pattern of the gene, the gene was functionally identified through subcellular localization, stable transformation of tobacco, dual luciferase detection, and GUS activity detection experiments, and it was determined that it is a transcription factor gene that negatively regulates the synthesis and accumulation of anthocyanins in the pink long-tube Lycoris radiata. The results of transgenic experiments showed that overexpressing LlMYB6 in tobacco can reduce the anthocyanin content of tobacco corolla and make its flower color lighter. The results of dual luciferase detection and GUS activity detection experiments showed that LlMYB6 can bind to the promoter of LlPAL and inhibit its transcription, ultimately negatively regulating the synthesis and accumulation of anthocyanins in the petals of long-tube Lycoris radiata. The present application provides an effective molecular tool for improving the ornamental traits of plants using genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The phenotype of pink long-tube Lycoris at three different flowering stages; S1: early bud stage; S2: mid-bud stage; S3: full flowering stage;

[0022] Figure 2 This is the gel image of the full-length amplification of the LlMYB6 gene;

[0023] Figure 3 This is the expression level of LlMYB6 in three different flowering stages of pink Lycoris radiata;

[0024] Figure 4 The subcellular localization of LlMYB6 in Nicotiana benthamiana leaves; 35s::GFP: Expression in the lower epidermal cells of Nicotiana benthamiana leaves after injection of Agrobacterium GV3101 carrying the empty vector Super1300; 35s::GFP-LlMYB6: Expression in the lower epidermal cells of Nicotiana benthamiana leaves after injection of Agrobacterium GV3101 carrying the recombinant plasmid Super1300-LlMYB6;

[0025] Figure 5 The difference in phenotype and total anthocyanin content between the control (WT and EV) and transgenic tobacco petals; WT: wild-type tobacco; EV: empty-transformed tobacco; OE-1: line 1 with stable overexpression of LlMYB6; OE-2: line 2 with stable overexpression of LlMYB6;

[0026] Figure 6 The expression levels of anthocyanin metabolic pathway-related enzyme genes in control (WT and EV) and transgenic tobacco petals;

[0027] Figure 7 This is the result of dual luciferase assay for the interaction between LlMYB6 and LlPAL promoter;

[0028] Figure 8This is the GUS activity detection result of the interaction between LlMYB6 and LlPAL promoter. DETAILED DESCRIPTION

[0029] The present invention is further described below with reference to specific examples. In the following examples, any operations not described in detail are routine biological experimental procedures and can be performed with reference to molecular biology laboratory manuals and existing published journals, or according to the kits and product instructions. Unless otherwise specified, the materials and reagents used in the following examples are all commercially available.

[0030] Example 1:

[0031] 1 Plant material

[0032] The materials used in this experiment were collected from the Lycoris Germplasm Resource Bank of the Garden Plant Discipline of Nanjing Forestry University. In August 2020, the perianth pieces of flowers of pink long-tube Lycoris at three different developmental stages (S1 early bud stage, S2 mid-bud stage, S3 full flowering stage) were selected. Figure 1 ), placed in a sterile centrifuge tube, and immediately placed in liquid nitrogen for quick freezing, and then stored in a -80℃ refrigerator.

[0033] 2 Experimental methods

[0034] 2.1 Total RNA extraction from plant tissues and cDNA synthesis

[0035] RNA was extracted from the petals of three pink Lycoris radiata plants at different flowering stages using the Aidlab EASYspin Plus Plant RNA Kit. Three independent biological replicates were performed for each flowering stage. For detailed procedures, see the manufacturer's instructions. RNA concentration was measured using a nucleic acid analyzer. RNA was then reverse-transcribed into cDNA using the TransScript® One-Step RT-PCR SuperMix Reverse Transcription Kit. The resulting cDNA was diluted 10-fold with water and stored at -20°C.

[0036] 2.2 Obtaining the full-length sequence of the target gene

[0037] 2.2.1 Target gene amplification

[0038] Based on the unpublished transcriptome database of Lycoris radiata petals, the research team screened out a MYB gene family member that was differentially expressed in the three different developmental stages of the pink Lycoris radiata tepals and named it LlMYB6.

[0039] The reference sequence of the LlMYB6 gene was analyzed for restriction enzyme sites using BioXM software. Based on the map of the plant overexpression vector Super1300, restriction enzyme sites SmaI and KpnI were selected. Specific primers (LlMYB6-S1300-F: 5′-aagcttctgcaggggcccgggATGGGTAGATCTCCATGTTGTGAGA-3′; LlMYB6-S1300-R: 5′-gcccttgctcaccatggtaccTCTAATTACATGTGGATTGATAGACCTTA-3′) were designed using CE design software. cDNA from S3 perianth segments was used as a template for PCR amplification using Takara's PrimeSTAR® Max DNA Polymerase. The reaction system was as follows:

[0040]

[0041] Reaction conditions: 94°C, 4 min; (98°C, 10 s; 59°C, 30 s; 72°C, 35 s, 35 cycles); 72°C, 10 min; termination at 16°C.

[0042] The obtained product was detected by 1.2% agarose gel electrophoresis and then recovered using the EasyPure Quick Gel Extraction Kit.

[0043] 2.2.2 Construction of recombinant plasmid and transformation of E. coli

[0044] After gel excision and recovery of the PCR product, it was constructed into the Super1300 vector using homologous recombination. The Super1300-LlMYB6 recombinant plasmid was added to DH5α competent E. coli cells and incubated on ice for 20 minutes. The cells were then incubated at 42°C for 30 seconds, removed, and placed on ice for 2 minutes. 250 µL of LB liquid medium (without resistance) was then added and incubated at 200 rpm at 37°C for 1 hour. The culture was then centrifuged, and 200 µL of the supernatant was discarded. The remaining culture was pipetted to mix thoroughly, then spread onto a solid LB plate (with kana resistance) and incubated upside down at 37°C for 14-18 hours until a single colony appeared.

[0045] 2.2.3 Bacterial testing and sequencing

[0046] White monoclonal colonies were selected from the plates for bacterial detection. The bacterial detection primers were the forward primer S1300-F (5'-AACGCTTTACAGCAAGAACGGAATG-3') of the Super1300 vector and the reverse primer LlMYB6-S1300-R of the LlMYB6 gene. The reaction system was as follows:

[0047]

[0048] Reaction conditions: 94°C, 5 min; (94°C, 30 s; 59°C, 30 s; 72°C, 2 min, 35 cycles); 72°C, 10 min; terminate the reaction at 16°C.

[0049] PCR products were examined on a 1.2% agarose gel, and three positive clones of the correct length were selected for sequencing. The nucleotide sequence of the LlMYB6 gene was determined as SEQ ID NO. 1, and the amino acid sequence was determined as SEQ ID NO. 2. Positive single colonies with the lowest base mismatch rate and repeatable sequencing were selected for plasmid extraction and subsequent experiments.

[0050] 2.3 Real-time fluorescence quantification

[0051] Based on the full-length cDNA sequence of LlMYB6, fluorescent quantitative primers were designed in the non-conserved region (LlMYB6-qPCR-F: 5′-GAGACAATGAGATGCCCGGAT-3′; LlMYB6-qPCR-R: 5′-CAACTGCCACAACCTTCACTGC-3′). The LleIF gene of Lycoris radiata was used as an internal reference gene (LleIF-qPCR-F: 5′-CGAAAGAACGGGTACTTAGTCA-3′; LleIF-qPCR-R: 5′-ATGCGGAACATCACAGTTATG-3′). cDNA from three different developmental stages of pink Lycoris radiata perianth discs was diluted 10-fold and used as template. A total of 10 µL of the reaction system was prepared according to the following ratios:

[0052]

[0053] The reaction program was 95°C, 30 s; (95°C, 5 s; 60°C, 30 s, 40 cycles); 95°C, 15 s; 60°C, 1 min.

[0054] Each sample was repeated 3 times biologically and 3 times technically to ensure good repeatability of experimental data. The obtained reliable data were used 2 -ΔΔCT The expression differences of target genes were calculated by SPSS 20.0 software, and the obtained data were analyzed for significance.

[0055] 2.4 Cloning and expression analysis of the pink Lycoris radiata LlMYB6 gene

[0056] The petals of the pink long-tube Lycoris radiata appear dark pink in the small bud stage, and the anthocyanin content is the highest; however, the petal color becomes significantly lighter in the mid-bud stage, and the anthocyanin content decreases significantly; the petal color is the lightest in the full bloom stage, and the anthocyanin content is the lowest ( Figure 1 By analyzing the transcriptome of petals of Lycoris radiata at three different flowering stages, a candidate MYB transcription factor gene, LlMYB6, was identified that may negatively regulate the synthesis and accumulation of anthocyanins in Lycoris radiata. Using the cDNA of petals of the pink Lycoris radiata at S3 as a template, the full-length sequence of LlMYB6 was cloned using specific primers ( Figure 2 ). Sequencing results showed that the full-length sequence of LlMYB6 was 669 bp, encoding 222 amino acids. qRT-PCR was used to measure the expression level of LlMYB6 during the three flowering stages. The results showed that the expression level of LlMYB6 was lowest in the small bud stage of pink Lycoris radiata; however, the expression level increased significantly in the middle bud stage, about 10.12 times that of the small bud stage; the expression level in the full flowering stage did not increase significantly compared with the middle bud stage ( Figure 3 ). Thus, the changing trend of LIMYB6 expression is opposite to the decreasing trend of anthocyanin content in the petals of pink Lycoris radiata, indicating that LIMYB6 may be a key transcription factor that negatively regulates the synthesis and accumulation of anthocyanins in Lycoris radiata.

[0057] 2.4 Subcellular localization

[0058] 2.4.1 Freeze-thaw transformation of Agrobacterium tumefaciens GV3101

[0059] After double enzyme digestion, 1 µL of the recombinant plasmid Super1300-LlMYB6 was added to 33 µL of Agrobacterium GV3101 competent cells. The cells were pipetted and mixed thoroughly, then placed in an ice bath for 20 min, quick-frozen in liquid nitrogen for 5 min, and then in a 28°C water bath for 5 min and an ice bath for 5 min. 250 µL of LB liquid medium (no resistance) was then added and cultured at 200 rpm and 28°C for 1 h. After the culture was complete, the bacterial solution was centrifuged at 6000 rpm for 1 min, part of the supernatant was discarded, and 100 µL was evenly spread on LB solid medium (containing 50 mg / L Kana). The culture was inverted and cultured in a 28°C incubator for 40-48 h. Positive single colonies were picked for bacterial examination, and the single colonies with correct bacterial examination results were kept for later use.

[0060] 2.4.2 Transient Expression Injection into Nicotiana benthamiana

[0061] Agrobacterium containing Super1300 empty vector, Agrobacterium containing Super1300-LlMYB6 recombinant plasmid, and Agrobacterium containing auxiliary expression vector P19 were cultured in LB liquid medium (containing 50 mg / L Kana) in the dark with shaking (28°C, 200 rpm) until the bacterial solution OD 600 The bacterial solution was centrifuged at 4°C, 5000 rpm for 10 min, and the cells were collected and washed with a buffer solution (containing 10 mmol·L -1 MgCl2, 10 mmol·L -1 MES, 150 μmol·L -1 The resuspended bacteria were resuspended in acetosyringone. Finally, the resuspended bacteria solution was mixed according to the proportion (V P19辅助载体 :V 空载体 =5:7, V P19辅助载体 :V 重组质粒 =5:7), after thorough shaking and mixing, activate in the dark at 28°C for 3 h.

[0062] The mixed bacterial solution was injected into the underside of Nicotiana benthamiana leaves, which had been watered for three days beforehand, using a 1 mL medical syringe. After injection, the leaves were watered thoroughly and incubated in an incubator for two days. The injected tobacco leaves were then stained with DYPI and slides were prepared. Fluorescence localization of the lower epidermal cells of tobacco leaves transiently expressing LlMYB6 was observed under an LSM710 laser confocal microscope. The subcellular localization of the LlMYB6 protein was determined using the lower epidermal cells of tobacco leaves injected with an empty Super1300 vector as a control.

[0063] 2.4.3 Observation of LlMYB6 subcellular localization

[0064] Agrobacterium GV3101 carrying the recombinant plasmid Super1300-LlMYB6 was injected into Nicotiana benthamiana leaves and cultured in an incubator for 2 days. Laser confocal microscopy was used to observe the results, showing that GFP signals could only be detected in the nuclei of the lower epidermal cells of Nicotiana benthamiana ( Figure 4 ), preliminarily proved that LlMYB6 protein is mainly located in the cell nucleus, and it is speculated that it can play a role in transcriptional regulation in the cell nucleus.

[0065] 2.5 Verification of LlMYB6 Stable Transformation Function

[0066] 2.5.1 Stable transformation of tobacco using the leaf disc method

[0067] Agrobacterium containing Super1300 empty vector and Super1300-LlMYB6 recombinant plasmid were inoculated into 50 mL of LB liquid medium (containing 100 mg / L Kana) and cultured at 28°C, 200 rpm in the dark until the bacterial solution OD reached 0. 600 Reaching 0.4. Young, tender leaves of Nicotiana tabacum K326 were used as explants. After washing, they were disinfected in a laminar flow hood with 75% alcohol for 30 seconds, then with 0.1% disinfectant for 8 minutes, and finally rinsed 3-5 times with sterile ddH2O. The disinfected leaves were cut into 1 cm × 1 cm leaf discs and immersed in the bacterial solution for infection with manual shaking for 10 minutes. The leaf discs were then transferred to symbiotic medium and incubated in the dark for 3 days. After dark incubation, the leaf discs were transferred to screening medium and incubated at 25°C under normal light conditions (16 h light / 8 h dark) until resistant buds differentiated from the callus surrounding the leaf discs. The resistant buds were then transferred to bud-strengthening medium for further culture. When the resistant buds reached approximately 2 cm in length, they were excised and transferred to rooting medium. Once the roots reached a certain extent, the seedlings were hardened and transplanted to a greenhouse for growth.

[0068] 2.5.2 Positive detection of genetically modified tobacco

[0069] After the transgenic tobacco plants reached stable growth, leaves were collected from the wild-type, untransfected, and transgenic lines. Leaf DNA was extracted using the Tiangen Plant Genomic DNA Extraction Kit (see the instructions for detailed procedures). Transformed plants were tested for positive results using the DNA template, using the Super1300 vector forward primer S1300-F and the LlMYB6 gene reverse primer LlMYB6-S1300-R.

[0070] 2.5.3 Extraction and preservation of anthocyanins

[0071] The transgenic tobacco corolla material was thoroughly ground in liquid nitrogen, and 100 mg of dry powder was weighed. 1.5 ml of 1% hydrochloric acid methanol extract was added and shaken thoroughly for 1 min. The extract was extracted at 4°C in the dark, and shaken once every 8 h. After 24 h of extraction, the extract was centrifuged at 4°C, 10,000 rpm, and 10 min. The supernatant was filtered with a 0.22 μm organic phase filter membrane and stored in a 1.5 ml brown chromatographic bottle and stored at -20°C.

[0072] 2.5.4 Determination of anthocyanin content

[0073] The anthocyanin content in transgenic tobacco was determined by high performance liquid chromatography (HPLC). The chromatographic column used for liquid chromatography was C18, the diode array detector was used, the mobile phase A was acetonitrile, the mobile phase B was 0.4% phosphoric acid solution, the mobile phase was isocratic, the volume ratio of A to B was 20:80, the flow rate was 1.0 ml / min, the column temperature was 30℃, the injection volume was 10 μL, and the detection wavelength was 526 nm.

[0074] Using cyanidin-3-O-glucoside as a standard, it was dissolved in 1% hydrochloric acid methanol solution to prepare standard solutions of 0.01, 0.025, 0.05, 0.075, and 0.1 mg / ml, respectively. The peak area was measured by HPLC. A standard curve was plotted with the mass concentration of the standard (X) as the abscissa and the peak area (Y) as the ordinate. The linear regression equation and correlation coefficient were obtained, which were used to quantitatively analyze the total anthocyanin content in transgenic tobacco corolla.

[0075] 2.5.5 Real-time fluorescence quantitative analysis of transgenic Nicotiana tabacum

[0076] Corollas of wild-type, untransfected, and LlMYB6-positive transgenic lines were collected for RNA extraction and reverse transcription into cDNA, following the same procedures as in 2.1. Using NtEFlα as an internal reference gene, the primers listed in Table 1 were used to analyze the expression of enzyme genes involved in the anthocyanin metabolic pathway in each line, following the same procedures as in 2.3.

[0077] Table 1 Primers for qRT-PCR

[0078]

[0079] 2.5.6 Functional Verification Results of Stable Transformation of LlMYB6 Gene into Tobacco

[0080] The gene was introduced into tobacco through Agrobacterium transformation, and finally 5 T0 generation positive plants with phenotypes were obtained. Transgenic lines (OE-1 and OE-2) with strong growth and high LlMYB6 expression were selected for comparison with the wild type (WT) and empty vector line (EV). The phenotypic observation results showed that the color of the corolla of the two transgenic lines was significantly lighter than that of the control group (WT and EV), and the color of the corolla of the OE-1 line was the lightest, approaching white. The results of the total anthocyanin content determination showed that the total anthocyanin content of the corolla of the two transgenic lines was significantly lower than that of the control group (WT and EV), and the total anthocyanin content of the OE-1 line was the lowest, which was consistent with the phenotypic observation results ( Figure 5 ).

[0081] The expression levels of enzyme genes related to anthocyanin metabolism in transgenic tobacco petals were determined by qRT-PCR. It was found that compared with the control, the expression levels of three key enzyme genes (NtPAL, NtC4H, and Nt4CL) in the early stage of the anthocyanin synthesis pathway were significantly inhibited in the two transgenic lines. At the same time, the expression level of the key enzyme gene (NtDFR) in the late stage of the anthocyanin synthesis pathway was also significantly decreased. In addition, the expression levels of NtFLS, which is directly related to flavonol synthesis, and NtANR, which is directly related to proanthocyanidin synthesis, were significantly upregulated in the two transgenic lines ( Figure 6 The results showed that LlMYB6 could affect the accumulation of anthocyanins by regulating the expression of enzyme genes related to anthocyanin metabolism in tobacco.

[0082] 2.6 Dual-luciferase assay to verify the interaction between LlMYB6 and the LlPAL promoter

[0083] 2.6.1 Dual-luciferase vector construction and Agrobacterium transformation

[0084] Based on the promoter sequence of the LlPAL gene, the restriction enzyme sites were analyzed using BioXM software. Combined with the pGreenII 0800-LUC vector map, the restriction enzyme sites SmaI and SalI were selected. Specific primers (proLIPAL-LUC-F: 5′-ctatagggcgaattgggtaccCAAACAAAATTTTCTTTGTCAAGAAAA-3′; proLIPAL-LUC-R: 5′-agaactagtggatcccccgggTGTATGAAAATTTAGTTGATGAATGATTAA-3′) were designed using CE design software. DNA from the perianth segments of the pink Lycoris radiata at stage S3 was used as a template for amplification. The amplification reaction system was the same as in 2.2.1. After gel excision and recovery, the amplified product was constructed into the pGreenII 0800-LUC vector by homologous recombination as a reporter. The recombinant plasmid proLIPAL-LUC was transformed into competent E. coli and the bacterial test steps were the same as 2.2.2 and 2.2.3. The bacterial test primers were the forward primer LUC-F (5'-GTTCCTTATATGTAGCTTTCGACA-3') of the pGreenII 0800-LUC vector and proLIPAL-LUC-R. After the bacterial test, positive single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.

[0085] The sequence of the LlMYB6 gene was analyzed for restriction enzyme sites using BioXM software. The restriction enzyme sites SmaI and XhoI were selected based on the map of the vector pGreenII62-SK. Specific primers (LlMYB6-SK-F: 5′-agaactagtggatcccccgggATGGGTAGATCTCCATGTTGTGAGA-3′; LlMYB6-SK-R: 5′-gcccttgctcaccatggtaccTCTAATTACATGTGGATTGATAGACCTTA-3′) were designed using CE design software for amplification. The amplification reaction system and procedure were the same as in 2.2.1. After gel excision and recovery, the PCR product was constructed into the pGreenII62-SK vector by homologous recombination as an effector. The recombinant plasmid LlMYB6-SK was transformed into competent Escherichia coli and the bacterial testing steps were the same as 2.2.2 and 2.2.3. The bacterial testing primers were the forward primer SK-F (5'-TAATACGACTCACTATAGGGCGAGCGCCGCCATG-3') of the pGreenII 62-SK vector and LlMYB6-SK-R. After the bacterial testing, positive single colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing.

[0086] All correctly sequenced recombinant plasmids were transformed into Agrobacterium GV3101 containing the pSoup vector. The specific transformation and bacterial testing steps were the same as 2.4.1.

[0087] 2.6.2 Injection of Nicotiana benthamiana and dual-luciferase assay

[0088] Agrobacterium containing pGreenII 62-SK empty vector (SK), LlMYB6-SK, and proLIPAL-LUC recombinant plasmids was inoculated into LB liquid medium (containing 100 mg / L Kana) and cultured at 28°C with shaking until OD 600 = 0.7, then resuspend in an equal volume of infection aid. Mix the resuspended bacterial suspension containing the transcription factor and promoter at a ratio of 4:3. Activate the mixed bacterial suspension at 28°C in the dark for 3 hours before infection. Infect Nicotiana benthamiana leaves with the same steps as in 2.4.2.

[0089] After culturing for 2 days, the leaf blade near the injection hole was removed using a 0.8 cm diameter cork punch, placed in a centrifuge tube containing steel balls, quickly frozen in liquid nitrogen, and then ground using a sample grinder. The ratio of LUC activity to REN activity was then determined using the Dual Luciferase Reporter Gene Assay Kit.

[0090] 2.6.3 Dual luciferase assay verification results

[0091] The mixed bacterial solution carrying the recombinant plasmids LlMYB6-SK and proLIPAL-LUC was injected into Nicotiana benthamiana leaves. After the culture was completed, samples near the injection hole were collected to measure the ratio of LUC activity to REN activity. The results showed that the ratio of LUC activity to REN activity after co-injection of LlMYB6-SK and proLlPAL-LUC was significantly reduced compared with the control group (co-injection of SK empty vector and proLlPAL-LUC) ( Figure 7 ). This indicates that LlMYB6 can bind to the LlPAL promoter and inhibit its promoter activity on downstream genes.

[0092] 2.7 GUS activity analysis

[0093] 2.7.1 Construction of GUS activity detection vector and transformation of Agrobacterium

[0094] Based on the promoter sequence of the LlPAL gene, restriction enzyme sites were analyzed using BioXM software. Combined with the pBI121-GUS vector map, the restriction enzyme sites SbfI and SmaI were selected. Specific primers (proLIPAL-GUS-F: 5′- cgccaagcttgcatgcctgcaggCAAACAAAATTTTCTTTGTCAAGAAAA-3′; proLIPAL-GUS-R: 5′-ataagggactgaccacccgggTGTATGAAAATTTAGTTGATGAATGATTAA-3′) were designed using CE design software. DNA from S3 pink Lycoris radiata perianth segments was used as a template for amplification. The amplification reaction system was the same as in 2.2.1.

[0095] After gel excision and recovery of the PCR product, construct it into the pBI121-GUS vector using homologous recombination. Subsequent transformation of the recombinant plasmid pBI121-proLlPAL into competent E. coli and bacterial testing were performed as in 2.2.2 and 2.2.3. The bacterial testing primers were proLIPAL-GUS-F and the reverse primer GUS-R (5'-CGAGGTACGGTAGGAGTTGG-3'), respectively. After bacterial testing, single positive colonies were selected and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing. The sequenced recombinant plasmid pBI121-proLlPAL was transformed into Agrobacterium tumefaciens GV3101, following the same transformation and bacterial testing procedures as in 2.4.1.

[0096] 2.7.2 Injection of Nicotiana benthamiana and GUS activity detection

[0097] Agrobacterium containing Super1300 empty vector (EV), Super1300-LlMYB6 (35S::LlMYB6), and pBI121-proLlPAL (proLlPAL-GUS) recombinant plasmids were inoculated into LB liquid medium (containing 100 mg / L Kana) and cultured at 28°C with shaking until the OD 600 = 0.7, then resuspend in an equal volume of infection aid solution. Mix the resuspended bacterial suspension containing the transcription factor and promoter at a 1:1 ratio. Activate the mixed solution in the dark at 28°C for 3 hours. Injection infection into Nicotiana benthamiana leaves and subsequent culture procedures are the same as in 2.4.2.

[0098] Three days after infection, a circular leaf disc was punched near the injection site using an 8 mm diameter borer. The disc was then placed in a test tube and GUS stain was added to cover the sample. After staining at 37°C in the dark for 1-2 days, the disc was decolorized with 75% ethanol, which was replaced 3-5 times. The disc was then decolorized with 95% anhydrous ethanol until the leaf was completely decolorized. The disc was then photographed and observed using a stereomicroscope. GUS activity was detected using the Coolable GUS gene quantitative detection kit.

[0099] 2.7.3 GUS activity detection results

[0100] The interaction between LlMYB6 and the LlPAL promoter was further examined by GUS activity detection experiments. After the Agrobacterium containing proLlPAL-GUS and the Agrobacterium containing 35S::LlMYB6 were mixed and injected into Nicotiana benthamiana leaves, the GUS staining results showed a lighter blue color compared to the control (co-injection of Super1300 empty vector and proLlPAL-GUS), and their GUS activity was also significantly lower than that of the control ( Figure 8 These results indicate that LlMYB6 can effectively inhibit the expression of the LlPAL gene by binding to the LlPAL promoter, thereby negatively regulating the anthocyanin synthesis in Lycoris radiata.

Claims

1. A Lycoris radiata LlMYB6 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The expressed protein of the Lycoris radiata LlMYB6 gene according to claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that The biological material is an expression cassette, a recombinant vector, a recombinant bacterium or a recombinant cell containing the Lycoris radiata LlMYB6 gene according to claim 1.

4. Use of the long-barrel Lycoris radiata L1MYB6 gene according to claim 1 or the expression protein of the long-barrel Lycoris radiata L1MYB6 gene according to claim 2 or the biological material according to claim 3 in long-barrel Lycoris radiata breeding.

5. Use of the long-tube Lycoris radiata L1MYB6 gene according to claim 1 or the expression protein of the long-tube Lycoris radiata L1MYB6 gene according to claim 2 or the biomaterial according to claim 3 in inhibiting plant anthocyanin synthesis.

6. A method for inhibiting anthocyanin synthesis in plants, characterized in that: Overexpressing the Lycoris radiata LlMYB6 gene according to claim 1 in a plant.

7. The method according to claim 6, characterized in that The following steps are involved: (1) constructing an expression vector for the Lycoris radiata LlMYB6 gene according to claim 1; (2) transforming the constructed expression vector of Lycoris radiata LlMYB6 gene into plants or plant tissues; (3) Cultivate and screen transgenic plants or plant tissues with reduced anthocyanin content or lighter flower color.

8. The method according to claim 7, characterized in that The plant is tobacco or Lycoris radiata.

9. The method according to claim 7, characterized in that The overexpression vector of the Lycoris radiata LlMYB6 gene is Super1300-LlMYB6.

10. The method according to claim 7, characterized in that The transformation is mediated by Agrobacterium.

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