A gene for promoting anthocyanin synthesis in plants and uses thereof

Abstract

The application discloses a gene for promoting anthocyanin synthesis of plants. The application finds a new gene LrbHLH42 for promoting anthocyanin biosynthesis, which can be synergistically regulated with LrAN2 to promote anthocyanin synthesis. The LrbHLH42 promoter has obvious allelic variation in Lycium ruthenicum Murr and Lycium chinense Miller, and is one of main reasons for causing difference in expression of the LrbHLH42 gene in the Lycium ruthenicum Murr and the Lycium chinense Miller. Therefore, the LrbHLH42 promoter in the Lycium ruthenicum Murr is used as a basic element of the gene LrbHLH42, which can significantly improve anthocyanin synthesis and accumulation in plants, improve the anthocyanin content in plants, and has practical production popularization and application value.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a gene that promotes anthocyanin synthesis in plants and its uses. Background Technology

[0002] Black goji berries are a unique wild plant resource native to the saline-alkali lands of Northwest my country. Recorded in classic Tibetan medicine texts such as *Jingzhu Materia Medica* and *Four Medical Classics*, black goji berries have been used as a traditional Chinese medicine for over a thousand years, primarily for treating heart disease, menstrual irregularities, bleeding gums, and for improving eyesight and overall health. Currently, most black goji berries in my country are distributed in Qinghai and Tibet, with the concentration in the Qinghai Lake and Keluke Lake areas of Qinghai Province. This region is a major area of ​​saline-alkali soil in Qinghai Province, which has endowed black goji berries with special physiological characteristics of drought and salt tolerance. The unique environment of the Qaidam Basin, including large temperature differences, salt absorption, high altitude, and long hours of sunshine, contributes to the high anthocyanin accumulation and black phenotype of black goji berries, giving them their renowned medicinal value and commercial appeal. Black goji berries not only help restore the ecosystem but also boost the income of surrounding residents, making them an important component of the "Northwest Organic Agricultural and Livestock Product Export Base."

[0003] With the improvement of people's living standards, the demand for highly nutritious, green, and organic foods is increasing. Driven by e-commerce, black goji berries, as a berry used for both food and medicine, have gradually gained widespread popularity in the eastern coastal areas and southern China. Black goji berries contain over 4% anthocyanins, far exceeding the content in blueberries. Anthocyanins possess various pharmacological effects, including antioxidant, anti-aging, anti-inflammatory, anti-tumor, anti-fatigue, and blood sugar and lipid-lowering properties. Research on the preparation and pharmacological activity of anthocyanin monomers in black goji berries is also increasing, but the biosynthetic molecular mechanism of high anthocyanin content in black goji berries has only been reported in the last two years. The high expression of structural genes LrF3'5'H, LrDFR, and LrANS in the anthocyanin biosynthesis pathway activates anthocyanin synthesis in black goji berries. The transcription factor LrAN2 has also been identified as a key candidate gene regulating anthocyanin biosynthesis in black goji berries. Population scanning has also revealed that LrAN2 is a necessary but not sufficient condition for anthocyanin synthesis in black goji berries. Whether other genes synergistically regulate anthocyanin accumulation remains unclear. Furthermore, the content levels of various compounds and key nutrients in black goji berries are unknown. It is necessary to identify key genes that promote anthocyanin synthesis in black goji berries to increase the anthocyanin content in the plant and enhance the efficacy of black goji berries. Summary of the Invention

[0004] To address the above problems, the present invention provides a gene that promotes anthocyanin synthesis in plants, the coding sequence of which is the nucleotide sequence shown in SEQ ID NO:1.

[0005] Furthermore, the promoter sequence of the gene is the nucleotide sequence shown in SEQ ID NO:2.

[0006] Furthermore, the nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0007] The present invention also provides a recombinant vector, which is a vector connected with a gene that promotes anthocyanin synthesis in plants;

[0008] The coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO:1; the promoter sequence of the gene is the nucleotide sequence shown in SEQ ID NO:2, and the preferred nucleotide sequence of the gene is shown in SEQ ID NO:4.

[0009] Furthermore, the vector is a transgenic vector with a constitutive CaMV 35S promoter; the transgenic vector is preferably the pC2300s vector.

[0010] The present invention also provides a recombinant bacterium comprising the aforementioned recombinant vector; the recombinant bacterium includes recombinant Escherichia coli and recombinant Agrobacterium.

[0011] The present invention also provides the use of the aforementioned gene, the aforementioned recombinant vector, and the aforementioned recombinant bacteria in increasing the anthocyanin content of plants.

[0012] Furthermore, the plant includes wolfberry, preferably black wolfberry.

[0013] Finally, this invention provides a method for increasing the anthocyanin content of plants, comprising the following steps:

[0014] The aforementioned gene, recombinant vector, or recombinant bacteria can be transferred into a plant.

[0015] Furthermore, the plant includes wolfberry, preferably black wolfberry.

[0016] This invention discovers a novel gene, LrbHLH42, that promotes anthocyanin biosynthesis in wolfberry, which can synergistically regulate anthocyanin synthesis with LrAN2. Significant allelic variations exist in the LrbHLH42 promoter between black and white wolfberry, which is one of the main reasons for the differential expression of the LrbHLH42 gene in these two varieties. Therefore, using the LrbHLH42 promoter from black wolfberry as a basic element in the LrbHLH42 gene can significantly improve the synthesis and accumulation of anthocyanins in plants, thereby increasing the anthocyanin content. This has practical value for production and widespread application.

[0017] Obviously, based on the above description of the present invention, and according to common technical knowledge and conventional methods in the field, various other modifications, substitutions or alterations can be made without departing from the basic technical concept of the present invention.

[0018] The following detailed embodiments further illustrate the above-described content of the present invention. However, this should not be construed as limiting the scope of the present invention to the following examples. All technologies implemented based on the above-described content of the present invention fall within the scope of the present invention. Attached Figure Description

[0019] Figure 1 Phenotypes of black goji berries and white goji berries ( Figure 1 A), PCA diagram of metabolomics ( Figure 1 B), differential metabolite heatmap ( Figure 1 C) and KEGG enrichment map of differential metabolites ( Figure 1 D);

[0020] Figure 2 To predict the distribution of protein homologous species ( Figure 2 A), Volcano diagram of differential genes between ginkgo and black goji berries (A) Figure 2 B), differential gene KEGG enrichment map ( Figure 2 C), Diagram of differential expression of structural genes in the anthocyanin synthesis pathway ( Figure 2 D) and qRT-PCR determination of the relative expression levels of structural genes in the anthocyanin synthesis pathway ( Figure 2 E);

[0021] Figure 3 The structure diagram of the LrbHLH42 gene ( Figure 3 A), phylogenetic tree analysis ( Figure 3 B), Subcellular localization ( Figure 3 C), Dual-luciferase reporter assay ( Figure 3 D), yeast two-hybrid experiment ( Figure 3 E) and transient expression in tobacco ( Figure 3 F);

[0022] Figure 4 Analysis of cis-acting elements for the LrbHLH42 promoter ( Figure 4 A), Construction of plant expression vectors ( Figure 4 B), Dual-luciferase reporter assay ( Figure 4 C) and GUS staining of various tissue parts of tomato ( Figure 4 D). Detailed Implementation

[0023] Example 1: Study on genes promoting anthocyanin biosynthesis in black wolfberry

[0024] (I) Research Methods

[0025] 1. Metabolomics analysis

[0026] (1) Materials

[0027] During field sampling, a type of white goji berry was discovered that coexists with black goji berry in nature. Its plant type and fruit shape are consistent with those of black goji berry. Therefore, white goji berry was used as a negative control in the study to explore the nutritional factors and coloring mechanism of black goji berry.

[0028] The plant materials used in the study were ginkgo and black goji berries, collected in September 2021 at Nuomuhong Farm, Golmud City, Qinghai Province (96°27.2856'N, 36°26.9625'E). The freshly picked fruits were frozen in liquid nitrogen and then stored at -80°C.

[0029] (2) Sample extraction

[0030] The sample was freeze-dried in a Scientz-100F freeze dryer under vacuum and then ground into powder using a grinder (MM 400, Retsch) (30 Hz, 1.5 min). 100 mg of the powder was weighed and dissolved in 1.2 mL of 70% methanol extract. The sample was vortexed every 30 min for 30 s each time, for a total of 6 vortexes. The sample was then placed in a 4°C freezer overnight. After centrifugation (12000 rpm, 10 min), the supernatant was collected, filtered through a 0.22 μm microporous membrane, and stored in a sample vial for UPLC-MS / MS analysis.

[0031] (3) Metabolomics analysis of wolfberry

[0032] The data acquisition system mainly included ultra-high performance liquid chromatography (UPLC) (SHIMADZU Nexera X2) and tandem mass spectrometry (MS / MS) (Applied Biosystems 4500QTRAP). Chromatographic separation was performed using an Agilent SB-C18 column (2.1 mm × 100 mm); flow rate: 0.35 mL / min; column temperature: 40℃; mobile phase: phase A was ultrapure water (0.1% formic acid), phase B was acetonitrile (0.1% formic acid); elution gradient: at 0.00 min, phase B proportion was 5%; within 9.00 min, the proportion of phase B linearly increased to 95% and maintained at 95% for 1 min; from 10.00 to 11.10 min, the proportion of phase B decreased to 5% and equilibrated to 5% for 14 min; injection volume was 4 μL.

[0033] Mass spectrometry (MS) conditions were as follows: ESI source operating parameters were: ion source, turbine spray; source temperature 550℃; ion spray voltage (IS) 5500V (positive ion mode) / -4500V (negative ion mode); ion source gas I (GSI), gas II (GSII), and curtain gas (CUR) were set to 50, 60, and 25.0 psi, respectively; collision-induced ionization parameter was set to high. Instrument tuning and mass calibration were performed in QQQ and LIT modes using 10 and 100 μmol / L polypropylene glycol solutions, respectively. QQQ scans used MRM mode with the collision gas (nitrogen) set to medium. DP and CE optimizations for each MRM ion pair were completed through further optimization. A specific set of MRM ion pairs was monitored for each epoch based on the eluted metabolites. Data processing was performed using Analyst 1.6.3 software.

[0034] 2. Transcriptomics analysis

[0035] Total RNA was extracted from black and white wolfberries using the Tiangen RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (centrifugal column type). Total RNA concentration was measured using a Nanodro analyzer, and the quality of total RNA bands was detected by 1.0% agarose gel electrophoresis. After constructing cDNA libraries from black and white wolfberries, the cDNA libraries were sequenced using an Illumina HiSeq 2000 instrument, with a read length of 100 bp and three biological replicates.

[0036] This study employed a parameter-free transcriptome analysis strategy. Raw sequencing data was filtered, removing low-quality and low-complexity sequences at both ends to obtain high-quality clean reads. These clean reads were then assembled, and reliable transcripts were processed and spliced ​​to obtain unigenes. Functional annotation and differential expression analysis were performed on the assembled unigenes.

[0037] Structural genes and transcription factors related to anthocyanin biosynthesis were identified from the annotated genes. Using ginkgo biloba as a reference, the fold change in gene expression levels was calculated. Furthermore, qRT-PCR was used to determine the transcriptional levels of key structural genes related to anthocyanin biosynthesis in both black and ginkgo biloba.

[0038] 3. Cloning and bioinformatics analysis of the LrbHLH42 gene

[0039] Genomic DNA was extracted from black and white wolfberry fruits using the Tiangen DNA Extraction Kit (DP360), and total RNA was extracted from the fruits using the Tiangen Total RNA Extraction Kit (DP441). The total RNA was then reverse transcribed into cDNA using a reverse transcription kit (TaKaRa, Code No. 6201A). Using DNA and cDNA as templates, specific primers for the LrbHLH42 gene were designed (LrbHLH42-F: ATGATGATGGCGATCATACAGA; LrbHLH42-R: TTAAACTCTAGGGATTATC). The gDNA and cDNA sequences of the LrbHLH42 gene in black and white wolfberry fruits were amplified using a high-fidelity polymerase (TaKaRa, Code No. R045). PCR products were detected by 1.0% agarose gel electrophoresis (200V, 250mA, 10min). Purification of the target fragment was performed using an agarose gel recovery kit, and the concentration of the recovered product was determined using a Nanodro analyzer. Attach the recycled products to the carrier - Blunt vector (TransGen) was used to transform E. coli DH5α, followed by culture, sequencing, and plasmid extraction.

[0040] Sequencing results were assembled and aligned using AlignX software. Gene structure maps were drawn using online software (http: / / gsds.gao-lab.org / ), and open reading frames and amino acid sequences of candidate genes were analyzed using ExPaSy-Protparam software (http: / / expasy.org / tools / protparam.html). A phylogenetic tree of the LrbHLH42 protein was constructed using the Neighbour-joining method in MEGA 5.10.

[0041] A plant expression vector, LrbHLH42-CAMEGFP, for subcellular localization was constructed using homologous recombination. This vector was transformed into *E. coli* DH5α and plated on streptomycin-resistant streptavidin-containing solid medium. Positive clones were identified by colony PCR and sequenced. Plasmid LrbHLH42-CAMEGFP was extracted from correctly sequenced single clones and transformed into *Agrobacterium tumefaciens* GV3101. *Agrobacterium tumefaciens* was injected into healthy tobacco leaves and cultured in the dark for 2-3 days. The distribution of GFP signal within the cells was observed using a laser confocal microscope.

[0042] 4. Expression profile analysis of LrbHLH42

[0043] Total RNA was extracted from black and white wolfberry fruits at different growth stages. The total RNA was reverse transcribed into cDNA using a reverse transcription kit (Takara, Code No. RR037A), diluted 100-fold, and set aside for later use. The spatiotemporal specificity of LrbHLH42 was determined by qRT-PCR.

[0044] 5. Construction of LrbHLH42 plant expression vector

[0045] Using the TA clone recombinant plasmid of LrbHLH42 as a template, restriction enzyme primers were designed for PCR amplification, followed by gel recovery and concentration determination. Simultaneously, restriction endonucleases were used to digest the plant expression vector pC2300s (i.e., the transgenic vector containing the constitutive CaMV35S promoter) and the recovered product of LrbHLH42 at 37℃ for 70 min. The digested products of LrbHLH42 and the vector CaMV35S were ligated using T4 DNA ligase at 4℃ for 12–18 h. The ligation products were transformed into E. coli DH5α, and positive single clones were screened by colony PCR. Twelve positive single clones were sent to Sangon Biotech (Shanghai) for sequencing. Plasmids (CaMV35S:LrbHLH42) were extracted from the correctly sequenced single clones after culture and were used for further processing. The recombinant plasmid CaMV35S:LrbHLH42 was transformed into Agrobacterium competent cells LBA4404 using the freeze-thaw method. Agrobacterium positive monoclonal cells containing the plant expression vector CaMV35S:LrbHLH42 were inoculated into LB liquid medium containing Kana (100.00 mg / mL) and Rif (100.00 mg / mL) and cultured at 28℃ and 200 r / min for 48 h until the OD600 of the culture medium was 0.80.

[0046] 6. Verification of the interaction between LrbHLH42 and LrAN2

[0047] To utilize transient expression in tobacco, Agrobacterium tumefaciens strains of CaMV35S:LrbHLH42 and CaMV35S:LrAN2 were injected individually or together. Injected leaves were cultured in a 24°C dark room for one day, followed by growth under 16h light / 8h dark conditions, and phenotypic observation was performed. Six to seven days after Agrobacterium injection, leaves of the same size were harvested for total anthocyanin content determination.

[0048] The interaction strength between LrbHLH42 and LrAN2 proteins was verified using a yeast dual-hybrid, dual-luciferase assay.

[0049] 7. Amplification of the LrbHLH42 gene promoter sequence

[0050] Using black goji berry gDNA as a template, Tail-PCR amplification was performed using the Genome Walking Kit (Takara, Code No. 6108). Three R-terminal specific primers (bHLH42-R1: AGCTTGCCCGGTAATCCGA; bHLH42-R2: GAATCCAATGTCTTCTTGA; bHLH42-R3: ACGTCGTTCTGCAAGTACATG) were designed based on the known LrbHLH42 gene sequence. Three rounds of nested PCR amplification were performed with the upstream primers provided in the kit. The reaction system and reaction conditions were referenced in the literature (Tomohiro Nakayama, Masayoshi Soma, Dolkun Rahmutula, Yukio Ozawa, Katsuo Kanmatsuse (2001) Med Sci Monit, 7(3):345-349). Based on the results of Tail-PCR, specific primers (bHLH42pro-F: GGCCCTTGGCAGTGAGCGCA; bHLH42pro-R: TCTACTGCACAATATTGCAA) were designed. Using gDNA from black and white wolfberries as templates, PCR amplification was performed using the high-fidelity enzyme PrimeSTARMax Premix (2×). The PCR reaction system consisted of 25 μL PrimeSTAR Max Premix (2×), 0.5 μL each of forward and reverse primers, 23 μL ddH2O, and 1 μL cDNA. The PCR reaction program was 30 cycles: 98℃ for 10 s, 55℃ for 15 s, and 72℃ for 1 min. Tail-PCR and PCR products were recovered using a gel extraction kit (Tiangen), and the target gene was ligated into a T vector. On the Blunt Cloning Kit, E. coli DH5α was transformed. Positive single clones identified by colony PCR were sent to Sangon Biotech (Shanghai) for sequencing. After shaking the culture, plasmids were extracted from the correctly sequenced positive single clones and stored for later use.

[0051] Sequence alignment and assembly were performed using AlignX software; promoter active region prediction was performed using the BDGP neural network promoter prediction online software (https: / / www.fruitfly.org / seq_tools / promoter.html); promoter cis-acting element analysis was performed using Plant CARE (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / htmL / ).

[0052] 8. Functional verification of the LrbHLH42 gene promoter

[0053] To investigate the promoter activity of the LrbHLH42 gene in black and white wolfberries, plant expression vectors pKGWFS7:LrbHLH42pro-black and pKGWFS7:LrbHLH42pro-white were constructed using homologous recombination technology. The promoter and vectors were amplified by PCR using specific primers (gus-F: GACTCTTGACCATGGTAGATCTG; gus-R: caaatgtttgaacgatccccTCACCTGTAATTCACACG; amp-LrbHLH42-F: cttcaacgttgcggttccccTTTCAGACGAGGTAGGGGTAGGCCGA; amp-LrbHLH42: ATCTACCATGGTCAAGAGTCCTGTATGATCGCCATCATTCTAC). The expression vectors were then amplified using the restriction endonuclease Sma. Linearization of the vector was achieved by reacting at 37℃ for 5 min and then at 65℃ for 10 min, followed by homologous recombination. 5 μL of the reaction solution was used to transform *E. coli* DH5α, and positive single clones were identified by colony PCR. After sequencing, the plasmid was extracted for later use. The recombinant plasmid was transformed into *Agrobacterium* competent cells GV3101, and tomatoes were infected using the *Agrobacterium*-mediated tomato genetic transformation system. Positive transgenic tomatoes were stained with GUS.

[0054] II. Research Results

[0055] This embodiment used black and white wolfberry fruits as experimental subjects, and analyzed key metabolites and candidate genes for color differentiation in black and white wolfberry fruits using combined metabolomics and transcriptomics analysis. Furthermore, the genome, coding region, and promoter region sequences of LrbHLH42, a key candidate gene that may inhibit anthocyanin synthesis in white wolfberry, were isolated and cloned, and bioinformatics analysis and functional verification were performed. See [link to results]. Figures 1-4 .

[0056] Metabolomics results showed that flavonoids were the main compounds in both black and white wolfberries. Preliminary identification revealed that spermidine B / A / I was the most abundant in black wolfberry, followed by amino acids and alkaloids. The flavonoids phenanthrene-7-O-glucoside and naringenin-4'-O-glucoside were among the top 20 most abundant compounds. Bis(dihydrocaffeoyl)spermine and bis(dihydrocaffeoyl)spermine were detected only in black and white wolfberries, and not in yellow or hybrid yellow wolfberries. All compounds were mainly divided into two categories: Class 1 (375 compounds) showed higher accumulation in black wolfberry, while Class 2 (399 compounds) showed higher accumulation in white wolfberry. The pH value of black goji berries is lower than that of white goji berries, and the anthocyanin content is much higher. A total of 11 different structural forms of anthocyanins were detected, among which petunia pigment-3-O-[6”-O-(Z) p-coumaryl rutin]-7-O-glucoside had the highest content.

[0057] Transcriptome results revealed differential expression of 25,279 genes between black and white wolfberries, with 12,381 genes upregulated and 12,898 genes downregulated in black wolfberry. KEGG enrichment showed that 571 differentially expressed genes were involved in the synthesis of plant secondary metabolites, with the most significant differences observed in flavonoid and anthocyanin biosynthetic pathways. Eight structural genes involved in anthocyanin synthesis were expressed at higher levels in black wolfberry than in white wolfberry, suggesting that anthocyanin biosynthesis may be related to the black fruit trait of black wolfberry. The expression level of the bHLH transcription factor CL8159.Contig5_All (LrbHLH42), which regulates anthocyanin biosynthesis, was 2139.57 times higher in black wolfberry than in white wolfberry.

[0058] The open reading frame (ORF) of LrbHLH42 is 2019 bp, encoding 672 amino acids (amino acid sequence shown in SEQ ID No. 5), and the CDS base sequence is shown in SEQ ID No. 1. Phylogenetic analysis showed that LrbHLH42 has high homology with bHLH42 of pepper and potato. Subcellular localization results showed that LrbHLH42 could be detected with clear GFP fluorescence signals in the cell nucleus and cell membrane, indicating that LrbHLH42 is localized in the cell nucleus and cell membrane.

[0059] The nucleotide sequence of SEQ ID No. 1 is as follows:

[0060] ATGATGGCGATCATACAGACTAGCAGCCTGCAGCTGCAAACTATGTTGCAAAATTCAGTGCAATC

[0061] GGTTAAATGGACATATAGTCTTTTCTGGAAATTCTGTCCCGAACAAGGGGTGTTAGTGTGGAGAG

[0062] ATGGATATTACAATGGGCCTATAAAGACCAGGAAGACTCTGCAGCCAATGGAAGTTAGTGCTGAA

[0063] GAAGCTTCTCTTCATAGAAGCCAACAACTTAAAGAACTTTACGAATCACTTTCTGCCGGTGAGTC

[0064] AAATCAGCCGGCAAGAAGGCCGTCAGCAGCCTTGTCGCCGGAGGACTTGACGGAATCTGAGTGG

[0065] TTTTATCTCATGTGTGTTTCTTTCTCTTTTCCTCCCGGCATCGGATTACCGGGCAAGGCTTATACAA

[0066] AGAAACATCACGTATGGATAATGGGCGCAAACGATGTCGATAGCAAAATCTTCTGTAGAACCATT

[0067] CTTGCAAAGAGTGCCCACATACAGACTGTCGTCTGTATCCCTCTATTGGACGGTGTACTGGAACT

[0068] GGGAACCACAGAAAGGGTTCAAGAAGACATTGGATTCATACACCATATAAAGAGCATCTTCACTG

[0069] AGCAACAACCACCTCAGCCAACAAAGCCAGCTTTATCTGAGCACTCCACCTCCAATCCCCCCACC

[0070] TTTTCGGAGCCAAACTTTTACTCAGGCAATACTCTGCCGTCCGCCAGTATCTACCTGGCGGATCAA

[0071] GACGGTAGAATTGCCAGAGAAGAAGAAGAGGACGAGGATCAAGAAGATGATGATGACGACGAC

[0072] GACGACTCAGATAGTATAGCAATTCAAAATGGGCTTGGCGCGGCTGAGGCTAGTGAGCTCATGCA

[0073] GCTTGATGTGTCTGAAGCTATACGGCTCGGCTCACCGGAAGATGGCTCGAATAATATGGACTCGG

[0074] ATTTTCATTTGGTTGGCGTTAGCCAAGCAGGAAATACAGCTGAGTCTTTCAAAGCTGAAACTGCC

[0075] ATTAGTTGGGCTCACTTCCAAGACCTTCAACATTTACCAGGTGTCCCTAGTAATGATGAATTATCA

[0076] CAAGAAGACACACACTATTCTCAAACAGTGTCAACCGTTCTGGAGCACCTCTCAAACACAAGCT

[0077] CCAAAGTTTGCTCTAATTCCATAATGGGCTTTATTTCTCACGACTCAGCCCAATCCGCCTTCACATT

[0078] ATGTCCCAACAACGCCACCGTTTGCAGCCCAAACCCGGACCACTGCCACCTCCCCGACCCGTTT

[0079] GCCTCCTCTCAGCGGCTGCTCAAAAACATACTCTTCACTGTCCCATTTCTACACACTAAATTATAC

[0080] CAAGGTGAACCTAATTCTCCTCCAAAGTCACGTGGTGACGTGGCGACTGTTGATTCGTCCTCGAC

[0081] TGCCTCTCGGTTCGGTAAAGGATGTACGATAACCAACCAAGTAGAGCCAAGTGGAAACCATGTAC

[0082] TTGCAGAACGACGTCGTAGAGAAAAGCTCAATGAACGGTTTATCATATTAAGGTCACTTGTACCA

[0083] TTCGTGACAAAAATGGATAAAGCATCAATTCTTGGTGACACTATTGAGTATGTCAAGCAGTTACGT

[0084] AAGAAAGTGAAGGATCTTGAAGCACGTGATCCTCACACGGAGGGGACCAAAGATGCAGATGAA

[0085] AAGAGCGGTACAGCAAAAGTAGTGAAGGCGTTGCAAGGGAGGGGTAAGAGGACAATGAAGACT

[0086] GTGGAAGGAAGTGTTGGCGGAGCCCCGGCAAAGATCACGGTGGAGACACCGTGTACAGGGGAG

[0087] GATGAGGAGGTTGTGCAAGTACAAGTTTCAATTATCGAAAACGATGCATTGGTGGAGCTCCGGTG

[0088] TCCGTACAAAGAAGGGTTGCTGCTAGATGTAATGCAGATGCTAAGGGAACTTAAGGTTGAAGTTG

[0089] TGGCCATTCAATCATCACTTAATAGTGGTATCCTTTTGGCTGAGTTAAGAGCTAAGGTAAAAGAAA

[0090] ATATATATGGAAGGAAAGCAAGCATTCTGGAAGTCAAAAAGTCAATACATCGGATAATCCCTAGA

[0091] GTTTAA

[0092] The amino acid sequence of SEQ ID No. 5 is:

[0093] MMAIIQTSSLQLQTMLQNSVQSVKWTYSLFWKFCPEQGVLVWRDGYYNGPIKTRKTLQPMEVSAE

[0094] EASLHRSQQLKELYESLSAGESNQPARRPSAALSPEDLTESEWFYLMCVSFSFPPGIGLPGKAYTKKH

[0095] HVWIMGANDVDSKIFCRTILAKSAHIQTVVCIPLLDGVLELGTTERVQEDIGFIHHIKSIFTEQQPPQPT

[0096] KPALSEHSTSNPPTFSEPNFYSGNTLPSASIYLADQDGRIAREEEEDEDQEDDDDDDDDSSIAIQNGL

[0097] GAAEASELMQLDVSEAIRLGSPEDGSNNMDSDFHLVGVSQAGNTAESFKAETAISWAHFQDLQHLP

[0098] GVPSNDELSQEDTHYSQTVSTVLEHLSNTSSKVCSNSIMGFISHDSAQSAFTLCPNNATVCSPNPDHC

[0099] HLPDPFASSQRLLKNILFTVPFLHTKLYQGEPNSPPKSRGDVATVDSSSTASRFGKGCTITNQVEPSGN

[0100] HVLAERRRREKLNERFIILRSLVPFVTKMDKASILGDTIEYVKQLRKKVKDLEARDPHTEGTKDADE

[0101] KSGTAKVVKALQGRGKRTMKTVEGSVGGAPAKITVETPCTGEDEEVVQVQVSIIENDALVELRCPY

[0102] KEGLLLDVMQMLRELKVEVVAIQSSLNSGILLAELRAKVKENIYGRKASILEVKKSIHRIIPRV

[0103] qRT-PCR results showed that the expression level of the LrbHLH42 gene increased with the development and maturation of wolfberry fruits, with significantly higher expression levels in mature fruits compared to other stages. The transcriptional level of LrbHLH42 in black wolfberry was higher than that in white wolfberry at different growth stages, indicating that the expression of the LrbHLH42 gene exhibits spatiotemporal specificity. This suggests that high expression of the LrbHLH42 gene can promote anthocyanin accumulation in black wolfberry.

[0104] Yeast dual-hybrid and dual-luciferase reporter assays showed that LrbHLH42 interacts with LrAN2 protein. Transient expression results in tobacco indicated that LrbHLH42 alone did not accumulate anthocyanins in leaves; however, co-injection of LrbHLH42 and LrAN2 resulted in tobacco leaves exhibiting a distinct purple hue, more pronounced than with either treatment, and higher anthocyanin content. In conclusion, LrbHLH42 interacts with LrAN2, thereby promoting anthocyanin synthesis.

[0105] The promoter sequence upstream of the LrbHLH42 gene was isolated using Tail-PCR. The promoter sequence of the LrbHLH42 gene from black goji berries is shown in SEQ ID No. 2:

[0106] GGCCCTTGGCAGTGAGCGCAACGCAATTTTCAGACGAAGTAGGGATAGGCCGAAGAAGTATTGG

[0107] GGAGAGGTAATTAGACACTACATGGCGCAATTACAGCTTACCGAGGACATAACCTTAGATAAGAG

[0108] GGTTTGGAGGACTCAAATTAGGGTAGAAGGCTAGTAGATAGTCTCGTTATCCGTTCTTATTAGTAG

[0109] TCGTATTATTGCAATATAATTTCTTGTGCTCCGATTTCTGCTACTATCTGTTATTATCTGTTATTTCCT

[0110] GTGTTTTGGTTATCCTGTGTTATCTGCTCCGATTTATGCTATTATCTGTTATTTCATGTGCTCTGATTA

[0111] TTCTGTGTTATCTGTGTCGCTTGCATTATTTCATTTCCATATCGCCTTAAATATCTTATCCGAATCTCT

[0112] TAACCTTATTCGACTTCTTTTTATGCTTTATTGAGCCGAGGGTCTTTTGGAAACAGCCATCCTACC

[0113] TTGGTAGGAGTCAGGTCTGCGTACACTCTACCCACCCCAGACCTCACGATGTGGGATTTCACTGG

[0114] GTTGTTGTTGTTTGTTTGTTGTATTAGCATAATTTATAGAATAATTCTTATGGATATTATAATTATTATG

[0115] CTTTTAATATTTCAAAAGAAGAAGTAAGAAATCAATTTCCAGATATTGAAAATAAAAACAATTT

[0116] TACCAATTTACTCTGAACCCTTTCTCTTTTCTTAGTTTATACAGGTATTCAAAATTTACTAACGAG

[0117] GTTAACCTCAATTCGCTTTATGTAAGAGTTACGTTGTAGAAATAGCATCTCTAGTTGCTTTTCGGCC

[0118] AATGTTTTAAAAAATAGCCATTATTATAGAGTTACAAATTCTACATGTAAAACTCCACGAAGATTAG

[0119] TTGTAACTTTTACCCATAAACGTTATGTACCAAAGAAGATTTTCACTCTCAATCTTAGGATGAAATT

[0120] TTTTTTATCTAGGGCGAAGAAATCCCAAACCATCCACCAAACTCCTCGGTGGCGAACCCTTTAATT

[0121] CACTTTAAAATTGAGGTTCTTAATTAATGATTTAGGAAAGTTTACTCACCACATTTAGTTAGTTTTC

[0122] CAACAAAGACGAAGCCTATATAATTAAACACAGATGTTGAAATATATAGCCAGAGATAGTAAATAA

[0123] CTTAGGGTCAAAGCATTTGTGGTTAAATAGCATTCTAATAGTAGGATATTTCCAATGGACCACATAT

[0124] TAAAATCCAACTCCCAGTCATCATTTTTTTTTTTCACGGATTGCCCTTCGTTTGGGAAGGTCTTTAA

[0125] ATTTTGCCCCTCAAATTTGTGGTCTTTAAATTTTGCCCTTCATATTTGTGATCTTTAAATTTTGCCCT

[0126] TTGCTTGGAAAGATGAGCGAATACATGAAGTTCTGGGTTTGAACCCCCACTCAGGCATAAAATAA

[0127] AAAAATGATTTCGCAAGGCAGCACAAGGGGAGTGTATGCCGGATCCAGCATGCGATCTTTAAGA

[0128] AAAAGTTAAAGTTATGCCCGGATCCGGCATAACTAAATGTCTGCCCCTCCGGCAGACTTTTAGTTAT

[0129] GTTTAACTAAAAGTCTGCCCCTCCGGCAGACAAAATTTAAACTTTACCTTATAAGGCAATCTGGTA

[0130] GTTTACTCATAATTGTGGTTTGGACCAAAACAAATTTCTGTGGTTTATAAAGATGCTTTAAGGATT

[0131] AATTGGCTCTGAAATATGGACTTGAAGTGATCTGCTTTGTTTAGATTCTAAAGGTGTACATATTTG

[0132] AAGGCATCAGGAAGATATTATGTGGGAATTGAAAAAAGAAATTAAGTACATCACTGATTCAATATT

[0133] ACATAGGTTTTTTTGGATGTTTGTAACTCTGTTCTAACACAGTTGAAGTGGTTAAAAAAAAAATACA

[0134] TATATATGCTTCAAGACAAAGTTTGCCCATAAGGCATAAGTATGCCCCCATCGGCATAAGTTTGGTA

[0135] ATTCCTAAACAAGTTTATGTCGATGGGGGCATACTTTTAATGGACAAACTTTTATGCCGGATCCGG

[0136] CATAAACTTGTGAAGGAATTATCAAAGTTATGTCGGAACCGACATACTTATGCCAAGTCTGCCCCA

[0137] TAAGGCATACGTATGCCGGGTCCGGCATAACTTTGCTAATTTCATCACAAGTGTATGCCGATGGGA

[0138] GCATAGCGAAATTTAAACTCTGCCTTGTGAATTTTTTTTTAAATTTTGACTGTGTGGGGTTCGAAC

[0139] CTGAAACCCATGAAATTTAGCGAAGAGTAAAATTTAAAGATTTTAAATATGAGGGGCAAAATTTA

[0140] AAGACCACCCCAGAAGAAGGGCAATTCTGAGAATTTTCCCCCCAGTCATTCGTCTCTGTCTCTTA

[0141] ACAAAAACGTGACTTGTTAGAAAACATAGCTTATAATGGTTACAAAATCTAATAAATGGCAAAAC

[0142] TCATGACCAGAACCAAGACCTTAACCGTCTGGTAGTCACCAACCAGTCAATATCAACAACCATCT

[0143] ACTCTCTTCAATCTCTCTCTCTCTCTCTCAAAACAAAAAACAAAAAAAAAATCACTTCTTTACAA

[0144] ATTCCAAAAACTTATTTGCTTCTCTCTCCTAATGTGTTGATCAACTTAACTTTTTATGTTCTAAGGT

[0145] GTAGAATATAATAATATAGTGCAATTTCAAAGTTTTCACATATCTCCTCCTTTGCAATATTGTGCAGT

[0146] BUT

[0147]

[0148] Sequencing results, after assembly and alignment, showed that the LrbHLH42pro-black sequence was 2660 bp, the LrbHLH42pro-white sequence was 2455 bp, and the LrbHLH42pro-white sequence was missing a 230 bp base sequence. Plant CARE analysis indicated that three MYB-responsive elements were present in the 230 bp sequence missing from the LrbHLH42pro-white sequence.

[0149] A plant expression vector for the LrbHLH42 promoter was constructed, and transgenic tomato lines were obtained using an Agrobacterium-mediated tomato genetic transformation system. GUS staining results showed that LrbHLH42pro-black could drive GUS protein expression in tomato, with its fruit, flowers, stigma, pollen, and parts of the root all showing a blue tinge, exhibiting stronger promoter activity than LrbHLH42pro-white. This indicates that there is significant allelic variation of the LrbHLH42 promoter in black and white wolfberry, which is one of the main reasons for the differential expression of the LrbHLH42 gene in these two varieties.

[0150] The full sequence of the LrbHLH42 gene in black goji berries was determined by sequencing, and the complete nucleotide sequence of the LrbHLH42 gene is shown in SEQ ID No. 4:

[0151] GGCCCTTGGCAGTGAGCGCAACGCAATTTTCAGACGAAGTAGGGATAGGCCGAAGAAGTATTGGGGAGAGGTAATTAGACACTACATGGCGCAATTACAGCTTACCGAGGACATAACCTTAGATAAGAGGGTTTGGAGGACTCAAATTAGGGTAGAAGGCTAGTAGATAGTCTCGTTATCCGTTCTTATTAGTAGTCGTATTATTGCAATATAATTTCTTGTGCTCCGATTTCTGCTACTATCTGTTATTATCT

[0152] GTTATTTCCTGTGTTTTGGTTATCCTGTGTTATCTGCTCCGATTTATGCTATTATCTGTTATTTCATGTGCTCTGATTATTCTGTGTTAT

[0153] CTTGTCGCTTGCATTATTTCATTTCCATATCGCCTTAAATATCTTATCCGAATCTCTTAACCTTATCTGACTTCTTTTTATGCTTTTATT

[0154] GAGCCGAGGGTCTTTTGGAAACAGCCATCCTACCTTGGTAGGAGTCAGGTCTGCGTACACTCTACCCACCCCAGACCTCACGATG

[0155] TGGGATTTCACTGGGTTGTTGTTGTTTGTTTGTTGTATTAGCATAATTTATAGAATAATTCTTATGGATATTATAATTATTATGCTTTT

[0156] AATATTTCAAAAAAGAAAAAGTAAGAAATCAATTTCCAGATATTGAAAATAAAAACAATTTTACCAATTTACTCTGAACCCTTTCTC

[0157] TTTTCTTTAGTTTATACAGGTATTCAAAATTTACTAACGAGGTTAACCTCAATTCGCTTTATGTAAGAGTTACGTTGTAGAAATAGCA

[0158] TTCCTAGTGCTTTTCGGCCAATGTTTTAAAAAATAGCCATTATTATAGAGTTACAAATTCTACATGTAAAACTCCACGAAGATTAGT

[0159] TGTAACTTTTACCCATAAACGTTATGTACCAAAGAAGATTTTCACTCTCAATCTTAGGATGAAATTTTTTTTATCTAGGGCGAAGAA

[0160] ATCCAAACCATCCACCAAACTCCTCGGTGGCGAACCCTTTAATTCACTTTAAAATTGAGGTTCTTAATTAATGATTTAGGAAAGTT

[0161] TACTCACCACATTTAGTTGTTTTCCAACAAAGACGAAGCCTATATAATTAAACACAGATGTTGAAATATATAGCCAGAGATAGTAA

[0162] ATAACTTAGGGTCAAAGCATTTGTGGTTAAATAGCATTCTAATAGTAGGATATTTCCAATGGACCACATATTAAAATCCAACTCCCA

[0163] GTCATCATTTTTTTTTTTCACGGATTGCCCTTCGTTTGGGAAGGTCTTTAAATTTTGCCCCTCAAATTTGTGGTCTTTAAATTTTGC

[0164] CCTTCATATTTGTGATCTTTAAATTTTGCCCTTTGCTTGGAAAGATGAGCGAATACATGAAGTTCTGGGTTTGAACCCCCACTCAG

[0165] GCATAAAATAAAAAAATGATTTCGCAAGGCAGCACAAGGGGAGTGTATGCCGGATCCAGCATGCGATCTTTAAGAAAAGTTAA

[0166] AGTTATGCCGGATCCGGCATAACTAAATGTTCTGCCCCTCCGGCAGACTTTTAGTTATGTTTAACTAAGTCTGCCCCTCCGGCAG

[0167] ACAAAATTTAAACTTTACCTTATAAGGCAATCTGGTAGTTTACTCATAATTGTGGTTTGGACCAAAAAAACAAATTTCTGTGGTTTATA

[0168] AAGATGCTTTAAGGATTAATTGGCTCTGAAATATGGACTTGAAGTGATCTGCTTTGTTTAGATTCTAAAGAGTGTACATATTTGAA

[0169] GGCATCAGGAAGATATTATGTGGGAATTGAAAAAAGAAATTAAGTACATCACTGATTCAATATTTACATAGGTTTTTTTTGGATGTTT

[0170] GTAACTCTGTTCTAACACAGTTGAAGTGGTTAAAAAAAAATACATATATATGCTTCAAGACAAAGTTTGCCCATAAGGCATAAGTAT

[0171] GCCCCCATCGGCATAAGTTTGGTAATTCCTAAACAAGTTTATGTCGATGGGGGCATACTTTTAATGGACAAACTTTTATGCCGGAT

[0172] CCGGCATAAACTTGTGAAGGAATTATCAAAGTTATGTCGGGAACCGACATACTTATGCCAAGTCTGCCCCATAAGGCATACGTATGC

[0173] CGGGTCCGGCATAACTTTGCTAATTTCATCACAAGTGTATGCCGAATGGGAGCATAGCGAAATTTAAACTCTGCCTTGTGAATTTTT

[0174] TTTTAAATTTTGACTGTGTGGGGTTCGAACCTGAAACCCATGAAATTTAGCGAAGAGTAAAATTTAAAGATTTTAAAATATGAGGG

[0175] GCAAAATTTAAAGACCACCCCAGAAGAAGGGCAATTCTGAGAATTTTCCCCCCAGTCATTCGTCTCTGTCTCTTAACAACAAACGT

[0176] GACTTGTTAGAAAACATAGCTTATAATGGTTACAAAATCTAATAAATGGCAAAACTCATGACCAGAACCAAGACCTTAACCGTCTG

[0177] GTAGTCACCAACCAGTCAATATCAACAACCATCTACTCTCTTCAATCTCTCTCTCTCTCTCTCTCAAACAAAACAACAAAAAAAAAATC

[0178] ACTTCTTTACAAATTCCAAAAACTTATTTGCTTCTCTCTCCTAATGTGTTGATCAACTTAACTTTTTATGTTCTAAGGTGTAGAATATA

[0179] ATAATATAGTGCAATTTCAAAGTTTTCACATATCTCCTCCTTTGCAATATTGTGCAGTAGAATGATGGCGATCATACAGACTAGCAG

[0180] CCTGCAGCTGCAAACTATGTTGCAAAATTCAGTGCAATCGGTTAAATGGACATATAGTCTTTTCTGGAAATTCTGTCCCGAACAA

[0181] GGGTAAGCATTAACGGATTCAGAATTTAAACATTCTGAATGTAATTTTTCAAATATATATATCTAAGGCTATATACTTATTACTTGCA

[0182] CTGAATTTCGCTAAACGTACAAAACTCAAGATTGGATCTACCACCTCCATTGAATAAATATCTTCCCCTTGTCTAAATTTTTATCGCA

[0183] TTCTTTATTTCAAAATATGGCTGGAAATATGATTGACTCCTTAACAAGGTGTGATCTTTCTTTCATATCAAGAATTTTCCTGGAGGA

[0184] TTCAAATTGAGACGTCTTGATGAGTTGTAAGTTGGGAGAGTTCAATGGATACTTTTCTTGCAATGAATTTTATGAACTCGCAAAC

[0185] TCGTAATTGAATCTTGTCACTATCATTGAGTAAAATCTCTTTCCCGGGTAACTCTTTTACGTCTTCTTTCATATCAAGAATCATCTTAT

[0186] TAAATAGGGGATGATTCAGAATTTAAATGTCCTGAGTTCGAATTAAGTTATAGAGTTAATTGACTCTGAAATATATATCTAGCTCTAT

[0187] CTATAATTATTTTTTATTTATATCAAATTTTGCGAAATTTGCCAACTCATGATTGAATCTGCCAATGTCGACGTCCAATATAGATTCTTC

[0188] CACTTTGCTAAATGATTGCACTTTTCATTTCAAAACATGTGTGAAGTAATATGATAATAAAGTCGTTACTGTATTGCTCTTACACTG

[0189] GATATGTTGTTGTTGCTGTTATATAACTCTTTGGTACTATTTCATAGGGTGTTAGTGTGGAGAGATGGATATTACAATGGGCCTATA

[0190] AAGACCAGGAAGACTCTGCAGCCAATGGAAGTTAGTGCTGAAGAAGCTTCTCTTCATAGAAGCCAACAACTTAAAGAACTTTA

[0191] CGAATCACTTTCTGCCGGTGAGTCAAATCAGCCGGGCGAGAAGGCCGTCAGCAGCCTTGTCGCCGGAGGACTTGACGGAATCT

[0192] GAGTGGTTTTATCTCATGTGTGTTTCTTTCTCTTTTCCTCCCGGCATCGGGTAAGTTCCTCAGCTTGCATCATTCACTTCATTTTGT

[0193] TGATCGCTGTTTGACTTGACAGAAAGTTTAATAAAGAAAAAAAGAGAATTGAAACTTGTAGTTTTGCATATATGATAACATTTGTA

[0194] TGATTATAAAACTCTTGAAACTTGTGATGTTAAACATGTCATAACATTTATGTTGCTATAAAAGTTTATTAAATAAATATAGGAACGT

[0195] GCCATCCTTTTCTTAGTGGACTTGTAAAGAAATACGTATATCCAATCAAAATGGTGCGGACAAGTAGTAATAAGTTTTAACTGCAT

[0196] AATTATATTGCAAAATCAAAATTACTACAAATAACTACTTAATTATTGTAAGTAATCTTAACATGGTGATATAGAAATTATACAATATC

[0197] GTAGCATGACTTGCCTTATTTCTCATGTTACTAAACCATTTATTTTGGACGGTTAATTAGTTGTAATTATTTTTAAGAGGATTTAATG

[0198] TGTGTGAATTTTTTTAAACTATATCAATGCATACAAATTTTTTTAAAATAAATAAATAAACCACCTCAATGGTGAGGGGTTAGGCAG

[0199] ACACCTACGGCCAGTTCATTTCATCAGCAAAAGTTACAAGAGGTGGAAGAAGTTCAAAAAATATATAGTTAAATAGTGCTACTTT

[0200] TCTTGTTTTTCATTTTCACATTGTATTACTATATACACTATTTTCATCCCGACAACGAATTAGGGTAACAACCATATTCAATTCAGTGT

[0201] ACCCCTTTCAATCCATGCCACTATGAATTAACTAATTAGGTTGAGAGCCAAATGAATTAAAATCTCAGTTACCTTCCTAGGTAGTG

[0202] CTAGTAGAATTTTCAATAGAGTGAGCACACGATAGCAATATTCACCATACAAAGTAATTTGGAGAGTCAGTGAGGTTGTTACAT

[0203] CGAAATAAAAGCAAGAATATACAAAAGAAGCAACTTCAACTGTGACTTTCCTAAGTCATTGATTAATGAGATACCATATATTCATA

[0204] GAAACTCTTGCTAGTCTGTTTTGCTAAACATATAATATGCTGATAAAATAAGTTAATGTGGTAACTTAAATGCCTACTCTTCTTCCTT

[0205] GTTGAGGAGCTAATTAATTTTGTTGTATTTATGGGTTGCTTATTACTTTGGGAATAGTGGTAACTAATAAAAAAAATTGTTAGTTAT

[0206] TGAAGTCTAAGAGAAATGCTTGTTTTAACAGTGGGGATCTGCTTTAATTAACATCCATATAAAAGATGGTGTTACTTCCGATAAGG

[0207] ATAAATTTGGAACATTTCGTATAGTTCAAGGACAAAATTGACCTTTTATTCTTTGTAAAAAATAAAATTAAATTTGTGGTATTAAAA

[0208] ATATTATTAATATTTGTGCAATTTCATTTTGTAAAATGAGAAGTTTGTCAAGTCATTTTCAAACAAATAAGTGTATACTAGTAATTAT

[0209] TTTCAAAATAGATTAACAAAGAAATAATGTCACGTAGACCGAAAAAGAGGGATCATTTGGTACTGAAGTGCTTTCGATCTAGTAT

[0210] GCTATAGCTAGTGCAGTCAACCAAAAATCAGGGCTATAATTAAACAGTGGCGTATTGATTAATGCCAATTTTACCTTGGGAAGGA

[0211] TCAGTTACGTTTCTTTGGTGCAGTAAATAAATTTATAGTACTAGGGTCTAGGATTCCTTTTCTCCAATTGCATAAGCTCAGTTTGAA

[0212] TTATTTCTTGTGTACTTAACTTGAAACAAATCTTTATTTTGATGAAACAAGATTACCGGGCAAGGCTTATACAAAGAAACATCACG

[0213] TATGGATAATGGGCGCAAACGATGTCGATAGCAAAATCTTCTGTAGAACCATTCTTGCAAAGG-TATAATA-CTGGTACTTTTTTGT

[0214] TTTGTACTCGAGCAAAGAAAACAT-AGCATGGGGAATTTATTTTGTTAAATCATTT-CACTTG-GTTCCT-GTTTATTGTTAGTCTAG

[0215] GATCGTTGCATAAGCGTTTTTCTTTAG-CTACAAGACAGTGTATACC-CGTGCGTCCAACACTTTGAATTATAATGTATTTATGTGTA

[0216] TATAATAGTGTTTGGATCATGATAATACATATATTTTATATTACTAATAAATATACATAAGTTTATACTATTTTTATGCATAAGTTAGTGA

[0217] ATCATGCTGCTTCTATATTACTACTCTATATAATCAAGGATTCACAAAATTTATCGTCCTCATCGCCCAAAGAGCCCCACAATAGTAA

[0218] GTTCTTTTTTTGGGGGGGTGGGGTAATACACACAGTGGTGGTTGCATACCTTGGAGTATTACTTTTGGCTTGCTAATTTCTTTTTTTT

[0219] TT-CTTTTTTTTTAACAAGACAAACTCTCTTTCTTAAGCATTTCAACATGGGCCTTTATTCGTATATATTAATATCATAAGTACATATTG

[0220] TGACCTCTTATTTTGCTACTTATTAAATTAATTTTTAATCCTTCATCTCAAAATATTTATCACGTTTTACTTTTCGAGGGTCAAATTAA

[0221] ATAAATATTAATCAATAATTTTTTTAAAAAAAAATCAATTATTAATAGGAGAATTGCAATTTATAGTATATTTCAAATAGGCTTTT

[0222] AGTATCCAAATTTTAATTTTGAAACATTGAATTAATCTAATCTATTTTATTCTCCGAAAATTAATTAAAATTGTCTTTTGAAAAACGAA

[0223] ATGTGAAAAATATTTTAGGACAAACGGAATATATTACTTGTCCTTATTTACAATTGGAGCATTTATTAAGTCATACTATATCTTTTGTT

[0224] AAACCTTAATATTTTACTTCTTTAGTGAGCTTATATGTGCTTATAAAGATTTGTCCTAAAATTATTTTTAGGTATAGTTTTATATCTTTA

[0225] TTTTATTAATGTATTTTAATTATTATTTATTCATATTTTTGTAAGATAGTTAATCAGGTTAATATGTGTAAAAACTCAAAATTAAGGTTA

[0226] TAAATTGTAGTATAAATCTAGAGTTATTGAAATTTTTTGTTGTGTTAGATGATTCTAGAAGATAGAAATATGAGTGTCTAATGTGGA

[0227] TGAAAATAAAATACATCATTAAATCTTAGATGATTTCAAGATAAGCACAAAATGAGTTTGAAGCAAGGCGAAATTAAGTGTCAGC

[0228] TAGCCACATGTTTGGACGGGTTCAGTAGCGTTTTGGTTCAAACTCTTTATTTGTCTTAAGAGTTCATAGATTATGTATAGATTATTA

[0229] ATTTATAACCAAGTAACTTAAAATAATTAGATTCAGAATCCATAAACTTCAAATTTTGGCTCAGCATCTAACTTCAAGTAAATAATTT

[0230] CATTAAAAAATAAAGTTAAAGATAATGAGTGAAATGAAAGTTTTGCTTATATATTGAAAATATCCTTATTTGAAATTTTAATTATTAT

[0231] GTAGTAAAAAGGTGTAAAATGAACTATTTTTTTAATTTTAATATCTATTTTATATTTTGATTTTGGGGTCGGGCTTAGCACAGGCCT

[0232] CGTGAAACTAGTAGTAGTAGATGGCGTATGCCCGTGCTGTGTACAGCCCAATGCTTTGGATTATAGTGTATCTATGTATATGTAGT

[0233] TGTGTTTGGGTCGTGATAATATATATATTTTATTGCTAATAAACATACATAAGTTTGCACTATTTTTATGCGCGCGGGCGCGCGCGC

[0234] GCACACACACACACACACACACACGCGCACACACACACACACACACACACACACACATATATATATATATAGATAGATAGA

[0235] GAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGACATCTTATGTTTAAAACACGATTAATAATAACATTGTAGTTTGTGCTCCG

[0236] TACCCAAAACTTTATTATATTATTGTTTTCTACGAATACAAAGTTTGCAAA-TTTATTAATAATTTTCAAAAGAGAAGACTT-TTTAA

[0237] AAGGAAACTATTTTCCTCTCTTTGAGATAAAACAATAACAATATTTAAGCATCAGTTGATACTTTGAATTTTAACTTGATTAATTTAA

[0238] AGGTGTAAAATATTCTATTGTTAATGTATGTAGATTGGACCTAAACATAATTATTATTTTTTTTTATCAAATTTTTATTTGACTAATTC

[0239] TAATTCAAATTATTAAATTTAATTTTACATGTTTAAAACGAACAAAGTAGAAATTTGATTTTCTATTTTAAACGAAGAACTATTATTTT

[0240] TTAATTTTTGGTAAATATTCTTTGGTTTAGCTCATTTTACTTGTCATGTTATATTTTGCACGATTTTTTTATCGAAACATCAATTAGAAT

[0241] TATAATTTGACTAATTTACCTTATTCATATTATTGATCTCCATTTAATATTATTTTTTCTTTTACGACATTAATCTTTTTTTCACATATATTAG

[0242] AGTAAGGATAAAATGAAAAGGTAATTAAATTTTATCTTATTTTAAAATATAAATATTTTAAGTATATTATTGTAAACATAAAAAATA

[0243] AATGACATCGTGGCATAGCAAATACAACAGTTAAATATCTAAATTAGACTTCAGGCTAATATAAGAAAGAAAAATTATATGG

[0244] TTTGATTACTTAACCTTTGAAAAAAAAGCAATAATATAGGTCCATGTTTTTTGCTGTATAATAATTTCATTTGCTCCCATTAATGG

[0245] ATTGATACGCATATGGCAATGAATCCACCATTATTGACTTAATAAGGATATGATTTTTTAATATGGGGGTGCAAACCAGGACACATA

[0246] ACTATAAGAGAGTAGCTGCCAAAATACTGCCAAAATAAGCTCCCTAAGGTCGCACCTAAGATCCGATTTTTTTTTTTTTTTTTGAC

[0247] ATTGCTTTTTTTTTTTTTTGCTATTACTTGATTTTTTTAATATGTGGTTCATCTTTTTTTTAATGAGTTTGGAGAGGATGGGGTTACT

[0248] TTTGTTTTTATTTTTTTTAATATTACTCAGTGCTTTTAGTATGGGGCCCACCTTATATTTTTATTTTTTTTAAGAGACTGACGACAAAG

[0249] CATGATTAAAACCCATGCTTCTATATAGTAATTTTTTTTTAAAATATTGCTTGGTATTTTTAATATAAGACTCACCTTATAAAATTTTTT

[0250] TTCTTTTTTTAAATGACTGATGATGAAGCATGGGAATAGAAAAATATAGTTAACATGCATTTCTTCAACCGGTAGTTATACCTAAAA

[0251] TTACTATAGGAATTGTATCCTAGGGGGTCGTTTGGTTTAAGGTATAAGTTGGGTTATCCCAATATTAATTTTTATACCATGTTTGATA

[0252] TAAGGTATAAATTAGTGCTGGGATAAATTTATACCTTGTATCAAATATTGTATAAAAATTAGTGCTGGGATAACCCAACTTATACCTT

[0253] TTTAACCCACTTATACTGAGATTATTTTATACCATCTTTTAAATGGTATAAAATAATTCCAATAAATAAAATAAATTAATCCTGAGATTA

[0254] TAATTCAGGAACTAATGAGTCCTTAAACCAAACCACCCCTAAGAATTAAGATAAATGTTATTTTCTCTCTCTTGTTTTTCGTCGTTG

[0255] AAAGCAATTTAATTAACTGACTCTTTTCTTTTTGTTTATCCACGGAACTGACTTGCCTATATAGAGTGCCCACATACAGGTAATAGC

[0256] AGACCGTTTGACATACTCTTAATTTACTCCCTCCGCACTATAATGAGTGTCATTTTAATCAAAAATATGCATATTAAAAAATTAATGA

[0257] TAAAATATAAACTTTAGCAATTTACCCTTATGTAAAAAAAAAAAATCTTGATGATTCATGCACAAGTAGGCTATCATTTGACATTGA

[0258] GAATCCTATCGTCATCATTTAAAGTACGGAAAAGGATCAAAACTACCCCTGAACTTTAAAAAATAATTCATTCATACCCCTTCTTTAT

[0259] ACTTTAGGGGCCAATTATACCCTTACAGTTATACTATGAGGTCAATTATACCCTTCTGTCTAACTGTTGCCGCGTGGCATCATCCCAG

[0260] CGCTTCAAAATTATTTTTACCTCAAATAATTTTTTACTCACTAAAATAACTCAATCCGACCCGATTTTTTTTACAGCAAAAATAATAC

[0261] GGATAATTTTTCCAGAGAAAAATATAAAAATTATCTGTATTATTTTTGCTGAAAAAAAAATTCGGTCAGATTGAGTTATTTTAGTG

[0262] AGTAAAAAATTATTTGAGAGTAAAATAATTTTGAAGGGCTCGGATGATGCCACGTGGGAACGGTTTGACATAAGGGTATAATTG

[0263] ACCCTATAGTATAACTGTAAAGGTATAATTGTCCATAAAGTATAACTAAGGGTGTAGATAAACTATTTTTCAAAGTTCAGGGGTAAT

[0264] TTTGGCCCTTTTCCGTTTAAAGTATTACTACTTCTTTTGTCCAAGGGCAAAGTTGAAAAAAAAACTAGTCAATATATGCATTGATG

[0265] TTTCAAAATGACACTTATTATGAGACAAAAAAATTAATTAAGATGACACTTATTACGGGTCGGATGGAGTGTATAGCTTTCCAATT

[0266] ATTATTTAAATATATTTCATTTTCCCAAAAAGAAAAACTTAATTATTTTTCCTGTCATTACTTTATCATTTTGTTGCAGACTGTCGTCT

[0267] GTATCCCTCTCTTGGACGGTGTACTGGAACTGGGAACCACAGAAAGGGTAAAACGCAAAAAAAAAAATCTCAATATATCTATATT

[0268] AATACACTTTGCAGTAATTCAATTTTATAGATTTAAATGATAGATATTGTCAATGTAAATTTTGACACTATCAGTATAATTTAATCGAT

[0269] TGAAGAATATCGTACATATCCATGTCATGACATATATAGGTTCAAGAAGACATTGGATTCATACACCATATAAAGAGCATCTTCACT

[0270] GAGCAACAACAACCTCAGCCAACAAAGCCAGCTTTATCTGAGCACTCCACCTCCAATCCCCCCACCTTTTCGGAGCCAAACTTT

[0271] TACTCAGGCAATACTCTGCCGTCCGCCAGTATCTACCTGGCGGATCAAGACGGTAGAATTGCCAGAGAAGAAGAAGAGGACGA

[0272] GGATCAAGAAGATGATGATGATGACGACGACGACGACTCAGATAGTATAGCAATTCAAAATGGGCTTGGCGCGGCTGAGGCTA

[0273] GTGAGCTCATGCAGCTTGATGTGTTCGAAGCTATACGGCTCGGCTCACCGGAAGATGGCTCGAATAATATGGACTCGGATTTTCA

[0274] TTTGGTTGGCGTTAGCCAAGCAGGAAATACAGCTGAGTCTTTCAAAGCTGAAACTGCCATTAGTTGGGCTCACTTCCAAGACCT

[0275] TCAACATTTACCAGGTACTCTATTCAATTATTTATTACCTCTTCCGTTTCAATTTAAGTGTGTCACTTTTTTTTCAATTAGTTATAAAA

[0276] ATAGTGTTTTTTTATATTTAGTAACTCTTAAATTATAACATTTTATGAAATATTTAAGACATGATTGAAAGGTCATTCGGTACATTACA

[0277] TACTTTTTTTTTAACACACAGTTGTTAGCTATGGAGTTGTTAGCGAACATATTAAAGACAAAATTCGTAGCTAATTCATATTTTTTCTTT

[0278] ATAGCGCACATTTTTTGTTTAAGACTCGCAAGATTTAGAAGTCCCTCTTATTTACTTAAAATATTCCTCTAGTCAAATTAAAATACTT

[0279] ATTCAAATAGTGTACGTAGAATAAATTATATATTATTAATTAAACATCCCTATAATATGACTGAATTGCTGATATATGTATTTCATGTATTT

[0280] TCAAATCAAACAGTTACACAGTGACACCCTACGTTATGGAATTTTACTTATATGAACTTAATTTATAGTAATTCCGTGAAGAAGA

[0281] AAAGACTTTTTGTTAAAGTTGATGATCCAATTTTGGCAGTTTGGTCGAGGGGTTAGATTATATTTTCTCTTGTTATAGAAGTCGTA

[0282] ATAAATTGAGGTAAAATTGAGGTCCCTGTACTACGACAGAAAAACATTCTGTTTCTCGTGGTTATGTTTTCTTATGCATGTTTTATT

[0283] ACCCTTTACCACAAATCCCTGTTGTCTGCCTATTTTATGCATGCTACTTCTACTTCCCACACAACATATCACCTTCACACGATTGATC

[0284] TGGTCCTTTAGCTACTTTGGAAAAAGCATCAAAGTTTATCCATATAATTTTAAACTAGAACTAAACGATCACCATTCGAAGAAA

[0285] AAAAAAAAGAGATATTTGAACTTGATACGATTGAAGTCAAATATTTGTCTGACGAATTCTAAGTGAGTTTATTTTTGTTCGCTAAG

[0286] GATTGTGACATAGTAAAATTGAATTTAACTTCTATACACTTATAATATAAATGATTTGTTATATCAGACTACTAATATAAAACAATTAC

[0287] ACGTAAATGTTCAGAAAAAGTGAGACTAATAATCTAGAAAATAAGGCAAATTACTTTCTCACACAGGCTAAAATAAACTTATAGTGC

[0288] AAAAATTATACATCAATGTATAAGGTATATTCATTAAAATTAAGAGTTTACGTTTTGTGATTGTTAGTATAAAAAATGTTTACACAAT

[0289] CAAGTTATTACTTGATTTATAAGGTAATATAAGTACAAATTACAAGGATAATGTACAAAAAAATCTTTTAAGTCATTGATAATGTATA

[0290] CACATTATGTACACAAAATTTGGCAGTTATTCTATCGATAAAAAGAATCAGAGTTGTTCTAATTTGTAACTTCAATGAAATTGGAATA

[0291] ATTACTATATCACCACAATCTGTATGCATAAATTAAATCCTTAAATTAAAAGCCAAAATTTTGCAAATGCAGGTGTCCCTAGTAATGA

[0292] TGAATTATCACAAGAAGACACACACTATTCTCAAACAGTGTCAACCGTTCTTGAGCACCTCTCAAACACAAGCTCCAAAGTTTGC

[0293] TCTAATTCCATAATGGGCTTTATTTCTCACGACTCAGCCCAATCCGCCTTCACATTATGTCCCAACAACGCCACCGTTTGCAGCCC

[0294] AAACCCGGACCACTGCCACCTCCCCGACCCGCTTGCCTCCTCTCAGCGGCTGCTCAAAAACATACTCTTCACTGTCCCATTTCTA

[0295] CACACTAAATTATACCAAGGTGAACCTAATTCTCCTCCAAAGTCACGTGGTGACGTGGCGACTGTTGATTCGTCCTCGACTGCCT

[0296] CTCGGTTCGGTAAAGGATGTACGATAACCAACCAAGTAGAGCCAAGTGGAAACCATGTACTTGCAGAACGACGTCGTAGAGAA

[0297] AAGCTCAATGAACGGTTTATCATATTAAGGTCACTTGTACCATTCGTGACAAAAATGGATAAAGCATCAATTCTTGGTGACACTAT

[0298] TGAGTATGTCAAGCAGTTACGTAAGAAAGTTCAGGATCTTGAAGCACGTGATCCTCACACGGAGGGGACCAAAATATGCAGGTAT

[0299] AATTTTTCACGCATAGACAAGTTAAATTTCACATAATCACGTAACTGCATGTTTATGATAGTTTGGCATTACCCGTTTTGTTATTACG

[0300] CATAAAATAATAGTGTCATCGTGTTTAAAATATGACCGTTAATTTTTATCATTCATCCTCCCAAATTTTCAGGAAGATGTCTCAAAAA

[0301] AATTATTTAAGCCTACTTTTTTCTTTGACATGTGAATTGCGGGTCCTAAATGTTCGACATTAAGTAACTAAAAGAAAGGTCAACCA

[0302] GGAGAACAGAGGAAAGAGAGGGAGAAAAAAAGGAGAGAAGGTTGCTTTCTTTAGAGTTGTAACGAAAGGTCCGTTTTGGCCA

[0303] TGAATATTGTGAGTATTTGAAAGAAATAGTTTTTTGTTTTCAAAATGAAAAATGATATTTCAAAATTGGAGTAGTGTGGCCATGAA

[0304] TATAAATTGGAGTAGTTTTTGAATTTTTGTGAGTGATTTGGAGTGAAAAAGTGTTTTTTTAATACTTGCCAAATTCCAGACCAAGA

[0305] TTTTCTAGAATTTTATGGTCAAACAGGATTTTCAGAATTTAACTCCCGAAAAAAGTAAAAAAAAAAAATAACCATGGCCAAACGG

[0306] CCACTAAATTAAGGAGAGTCAAAAACATTCTTTAAACTATGAGTTTAATTCCAATGCACTAATCATAGTTATTTATGTAATTAAATTA

[0307] CTTCAAGAATAATTGTAGATAAGTTTATTTAATACTAGCATTGATTGAAAATTTCGAATAGAATAAATAACCTATTATACCATGTTACATTAAACTTATGAAAAATAATT-ACTGTAGTATTAGACTATGAATGTATATAATTTAGATAACATAATTCTAGAAAACTAAAAATAGGAC CAAATTTAACCTACTGCATATCATATATTACTCACTCGACTGGCTCTAATTTTGG-TCGTGACAAAAGATGAAAAGAGCGGTACAG CAAAAGTAGTGAAGGCGTTGCAAGGGAGGGGTAAGAGGACAATGAAGACTGTGGAAGGAAGTGTTGGCGGAGCCCCGGCAAAGATCACGGTGGAGACACCGTGTACAAGGGAGGATGAGGAGGTTGTGCAAGTACAAGTTTCAATTATCGAAAACGATGCATTGGTGGAGCTCCGGTGTCCGTACAAAGAAGGGTTGCTGCTAGATGTAATGCAGATGCTAAGGGAACTTAAGGTTGAAGTTGTGGCCATTCAATCATCACTTAATAGTGGTATCCTTTTGGCTGAGTTAAGAGCTAAGGTATGCCTAAAATACTTTCCTGCATGACTTTATATTTTTTAACAATGATGACATACTCGGTACAAGAGAGGATTTGAATTTTATGAGATTTGGATTCTAGAACAACAATCTCCGGTGCTAGTAACTAGTTTGTACCTATTTTGTGAATTTCCTTACACATACTTAATATGAAAAGCTATTGGGTACTGTCGAACTTGTATATTCAAGCCTCCATCCACCCTGCAGACAAATATGAGGTTTTT-CGGCCGATTCCAATTATATACAAGCACAAAAACACTTTTAGTATTAAAAAAAT AGATATTGTCAATATAATTTAA-CCTGTTATATCAAGTTATTTACTATACCATTTATTCTAAATTATTAAATAGAGTTAATTACCTGTAAATCTTTT-AGTAAACTGATTATTGTAAACACAATTATGTCATAACTGTATATAACTTAAATTATTTTTTAATTTGTTTTGTTATTTGTTTTTC CAGGTAAAAGAAAATATATATGGAAGGAAAGCAAGCATTCTGGAAGTCAAAAAGTCAATACATCGGATAATCCCTAGAGTTTAA

[0308] In summary, the LrbHLH42 gene in black goji berries can synergistically regulate and promote anthocyanin synthesis with LrAN2. Significant allelic variations exist in the LrbHLH42 promoter between black and white goji berries, which is one of the main reasons for the differential expression of the LrbHLH42 gene in these two varieties. Therefore, using the LrbHLH42 promoter from black goji berries as a basic element in the LrbHLH42 gene composition can significantly improve the synthesis and accumulation of anthocyanins in plants, thereby increasing the anthocyanin content and demonstrating practical value for production and application.

Claims

1. A gene that promotes anthocyanin synthesis in plants, characterized in that: The coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO:

1.

2. The gene according to claim 1, characterized in that: The promoter sequence of the gene is the nucleotide sequence shown in SEQ ID NO:

2.

3. The gene according to claim 1 or 2, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

4. A recombinant vector, characterized in that: It is a vector that connects genes that promote anthocyanin synthesis in plants; The coding sequence of the gene is the nucleotide sequence shown in SEQ ID NO:1; the promoter sequence of the gene is the nucleotide sequence shown in SEQ ID NO:

2.

5. The recombinant vector according to claim 4, characterized in that: The nucleotide sequence of the gene is shown in SEQ ID NO:

4.

6. The recombinant vector according to claim 4, characterized in that: The vector is a transgenic vector carrying a constitutive CaMV 35S promoter.

7. The recombinant vector according to claim 6, characterized in that: The transgenic vector is the pC2300s vector.

8. A recombinant bacterium, characterized in that: It comprises the recombinant vector as described in any one of claims 4 to 7; the recombinant bacteria include recombinant Escherichia coli and recombinant Agrobacterium.

9. The use of the gene according to claims 1-3, the recombinant vector according to claims 4-7, and the recombinant bacteria according to claim 8 in increasing the anthocyanin content of plants, characterized in that: The plant in question is black goji berry.

10. A method for increasing the anthocyanin content of plants, characterized in that: Includes the following steps: Take any one of the biological materials described in claims 1 to 3, the recombinant vector described in claims 4 to 7, or the recombinant bacteria described in claim 8, and transfer it into a plant; The plant in question is black goji berry.

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