Application of begonia sempervirens BsMYB15 gene in anthocyanin regulation

Abstract

The invention belongs to the technical field of begonia new variety cultivation, and particularly relates to application of begonia sempervirens BsMYB15 in anthocyanin regulation and control. The nucleotide sequence of the begonia sempervirens BsMYB15 gene is as shown in SEQ ID No. 1. According to the application, on the basis of preliminary research on begonia sempervirens transcriptome in the earlier stage, further screening is carried out to obtain the BsMYB15 gene related to anthocyanin regulation, and the preliminary research result of the gene shows that the gene is combined with a BsDFR promoter to mediate the change of the anthocyanin content, and meanwhile, the gene also participates in anthocyanin synthesis regulation through interaction with BsDREB1B. A direct gene expression verification result shows that after overexpression, accumulation of anthocyanin can be obviously promoted, and after silence, the anthocyanin content can be correspondingly reduced. Based on the results, a certain technical foundation can be laid for the cultivation of new varieties of begonia in four seasons and the cultivation of new varieties of other plants.

Description

Technical Field

[0001] This application belongs to the field of begonia breeding technology, specifically involving the application of Begonia semperflorens BsMYB15 in anthocyanin regulation. Background Technology

[0002] Begonia semperflorens is a species of begonia belonging to the family Begoniaceae and the genus Begonia. Begonia Begonia semperflorens is a perennial evergreen herbaceous plant widely used in landscaping due to its rich flower colors (which can be divided into white, pink, peach, and red series) and its ability to bloom in all four seasons. Furthermore, in horticultural plant research, the green leaves of Begonia semperflorens are easily affected by the environment, leading to changes in leaf color; therefore, it is also a commonly used plant material for research on anthocyanin metabolites.

[0003] Anthocyanidin, a derivative of benzopyran, is an important reason why plant leaves, flowers, and fruits exhibit different colors. Different levels of anthocyanidin accumulation endow different plants and different tissue parts with different colors, thus making it an important determining factor in the ornamental value of garden plants.

[0004] Further research on anthocyanin regulatory genes has shown that anthocyanin synthesis is directly regulated by functional genes such as DFR and CHS. However, transcription factors such as MYB and bHLH have a significant impact on the expression and regulation of these functional genes. Since there are many types and functions of transcription factors, in-depth research on the specific types and actual functions of transcription factors related to anthocyanin regulation is of great technical and theoretical significance and practical breeding guidance for the cultivation of new plant varieties. Summary of the Invention

[0005] The purpose of this application is to provide a BsMYB15 gene for Begonia semperflorens and to conduct a preliminary study on its application in anthocyanin regulation, thereby laying a certain technical foundation for further breeding of new varieties of Begonia semperflorens or other new plant varieties.

[0006] The specific technical solution adopted in this application is as follows.

[0007] The BsMYB15 gene of Begonia semperflorens has the nucleotide sequence shown in SEQ ID No. 1 and the corresponding amino acid sequence shown in SEQ ID No. 2.

[0008] The BsMYB15 gene of Begonia semperflorens is used in the regulation of anthocyanins in plants. When applied, the BsMYB15 gene regulates the changes in anthocyanin content in plants by mediating BsDFR. Overexpression of the BsMYB15 gene can promote an increase in anthocyanin content in plants, thereby promoting the reddening of the surface color of plant leaves or fruit peels. Reducing the expression level of the BsMYB15 gene can reduce the anthocyanin content in plants. When applied, the BsMYB15 gene can interact with Begonia semperflorens. BsDREB1B Gene co-overexpression is used to promote the increase of anthocyanin content in plant tissues and to promote the reddening of leaf color or fruit peel surface color; The plant in question is, for example, a begonia, an apple, or a tobacco plant.

[0009] Begonia BsDREB1B The gene, whose nucleotide sequence is shown in SEQ ID No. 3, and whose corresponding amino acid sequence is shown in SEQ ID No. 4.

[0010] Begonia BsDREB1B The gene is used in the regulation of anthocyanins in plants. After overexpression of the gene, it is used to increase the anthocyanin content in plants and promote the reddening of plant leaves or fruit peels; the plants mentioned are, for example, begonias or apples. The Begonia BsDREB1B The gene, whose nucleotide sequence is shown in SEQ ID No. 3, and whose corresponding amino acid sequence is shown in SEQ ID No. 4.

[0011] MYB transcription factors constitute a large class of gene regulators with diverse functions. They participate in the metabolic synthesis of phenylpropanoids in plants and are also involved in various physiological activities such as epidermal cell development and stress responses. Therefore, in-depth research on MYB transcription factors has always been one of the key focuses and challenges in plant research.

[0012] In this application, based on preliminary studies of the transcriptome of Begonia semperflorens, the BsMYB15 gene, associated with anthocyanin regulation, was further screened and identified. Preliminary results indicate that this gene mediates changes in anthocyanin content by binding to the BsDFR promoter, and also participates in the regulation of anthocyanin synthesis through its interaction with BsDREB1B. Direct gene expression validation results show that overexpression significantly promotes anthocyanin accumulation, while silencing it correspondingly reduces anthocyanin content. These results provide a technical foundation for the breeding of new Begonia semperflorens varieties and other plant varieties. Attached Figure Description

[0013] Figure 1Figures show the results of cloning the BsMYB15 gene and PCR detection of bacterial culture after ligation with plasmid; A represents the electrophoresis result after PCR amplification; B represents the PCR identification result of bacterial culture after ligation with plasmid; M in figures A and B represents DL2000 plus DNA Maker; 1-4 represent the target gene bands. Figure 2 The figures show the predicted secondary structure of the protein encoded by the BsMYB15 gene. The top figure is a schematic diagram of the distribution of protein secondary structure elements, and the bottom figure is a curve showing the change in protein secondary structure content with sequence position. In the figures, blue represents α-helices, and yellow represents random coils. Figure 3 The results show the predicted tertiary structure of the protein encoded by the BsMYB15 gene; Figure 4 The results of PCR detection of Agrobacterium colonies containing recombinant plasmid pCanG-BsMYB15 are shown in the figure; band M in the figure is DL2000 DNA Maker, and bands 1-5 are the detection results of different samples. Figure 5 The results of subcellular localization experiments after tobacco transformation with BsMYB15; Figure 6 A comparative graph showing typical phenotypic changes after BsMYB15 is instantaneously transformed into an apple; Figure 7 This image shows a comparison of changes in anthocyanin content in apple peel. The left image is a comparison of the instantaneous rotation and control groups before detection, while the right image shows the specific detection results. Lowercase letters in the images indicate significant differences (P<0.05). Figure 8 The changes in the expression levels of functional genes related to anthocyanin synthesis in apple peel after transient transformation (P<0.05). Figure 9 The results show the expression levels of the BsMYB15 gene in some transgenic lines (P<0.05). Figure 10 Phenotypic comparison of different transgenic lines of BsMYB15 Begonia semperflorens; Figure 11 The figures show the changes in anthocyanin content (P<0.05) in different transgenic lines of Begonia semperflorens; the left figure is a comparison of anthocyanin content after extraction in different lines; the right figure shows the specific measurement results. Figure 12 The results of detecting the expression levels of functional genes related to anthocyanin synthesis regulation in different Begonia semperflorens strains (P<0.05). Figure 13The results of yeast single-hybrid experiments using BsMYB15 and BsDFR promoters are shown; the left figure shows the self-activation verification results of the BsDFR promoter before screening and verification; the right figure shows the experimental results after adding 3-AT. Figure 14 The results of dual-luciferase assays for BsMYB15 and BsDFR promoters are shown (P<0.05); the left figure shows the fluorescence imaging results, and the right figure shows the enzyme activity detection results. Figure 15 The results of the self-activation verification experiment for BsMYB15 are as follows; Figure 16 The results of the double hybridization test of pGADT7-BsDREB1B and pGBKT7-BsMYB15 yeasts; Figure 17 Phenotypes of BsMYB15 and BsDREB1B after instantaneous conversion to an apple; Figure 18 The changes in anthocyanin content after BsMYB15 and BsDREB1B were transiently transferred to apple peel, either individually or together (P<0.05); the left figure shows a comparison of extracts after apple peel sampling; the right figure shows the anthocyanin content detection results. Figure 19 The expression levels of functional genes related to anthocyanin synthesis in apples after transient transfection of BsMYB15 and BsDREB1B into the apple epidermis, either individually or jointly (P<0.05). Detailed Implementation

[0014] The present application will be further explained below with reference to the embodiments. Before introducing the specific embodiments, the experimental background of some embodiments is briefly described below.

[0015] Biomaterials: The 'Super Olympia' begonia is a commonly used plant material in existing begonia research and can be obtained from the open market. Because the inventor has been engaged in begonia-related research for a long time, he has cultivated and preserved related germplasm materials. The 'Super Olympia' material used in the following examples has the following conventional culture environment conditions: 25°C for 14 hours during the day and 25°C for 10 hours at night, light intensity of 10000 lx, and relative humidity of 65%. During genetic transformation, the experimental material was selected after the seeds were sown and had grown to about 10 unfolded leaves. The apples used for instantaneous verification (green-skinned apples) were randomly purchased from the market. Nicotiana benthamiana is a commonly used plant material in botanical research. In experiments such as subcellular localization and dual-luciferase assays, robust leaves that have grown for about four weeks after seed sowing are selected as experimental materials. Escherichia coli DH5α strain, Agrobacterium tumefaciens strain GV3101, GV3101 (pSOUP), yeast strains Y187, Y2H, etc., products of Zhuangmeng (Beijing Zhuangmeng International Biogene Technology Co., Ltd.). RNAi- BsMYB15 Interference expression vector, according to the inventor BsMYB15 The gene sequence was constructed and provided by Sangon Biotech; the synthesis and sequencing of related primers were also provided and completed by Sangon Biotech. Main experimental reagents: Restriction endonucleases such as BamHI, EcoRI, SaCI, and ApaiI, from TaKaRa (Baori Biotechnology Co., Ltd.); High-fidelity enzymes 2 × Phanta Flash Master Mix, validating enzyme Green Taq Mix, gene cloning enzyme 5min TA / Blunt-Zero Cloning Kit and homologous recombinase enzyme 2 × CE Mix, DNA extraction kit (FastPurePlant DNA Isolation Mini Kit, DC104), RNA extraction kit (FastPure UniversalPlant Total RNA Isolation Kit, RC411-01), and dual-luciferase reporter gene assay kit (Duo-Lite™ Luciferase Assay System, DD1205), from Nanjing Novizan Biotechnology Co., Ltd.; Acetylsyringone (AS), Rifampin (Rif), Kanamycin (Kan), Carbenicillin (Carb), 6-Benzylaminopurine (6-BA), Naphthaleneacetic acid (NAA), MES, 3-AT, and Auricularyl acetate AbA, from Beijing Solarbio Science & Technology Co., Ltd. Plasmid extraction kit (HighPure Plasmid Mini Kit, PL03), small-volume agar gel DNA recovery kit, PCR purification kit (Cycle Pure Kit, D6492-01), rapid-type high-efficiency competent cell preparation kit for Saccharomyces cerevisiae (EX-Yeast Transformation Kit), and rapid identification kit for yeast positive clones (Cat.NO.ZC221A) are products of Zhuangmeng Biotechnology Co., Ltd.; Reverse transcription kit (TOROBlue® qRT Premix with gDNAEraser2.0, RTQ-202P) and quantitative real-time PCR kit (TOROGreen® qPCR Master Mix, QST-100P) are products of Hangzhou Shuomeng Biotechnology Co., Ltd. 1% hydrochloric acid methanol solution, LB medium, YPDA medium, SD / -Trp / -Leu medium, SD / -His / -Leu / -Trp medium, SD / -Trp / -Leu / -His / -Ade medium, etc., are prepared according to existing techniques.

[0016] Example 1 Based on the previous transcriptome analysis of Begonia semperflorens, and based on the correlation between expression level differences and corresponding phenotypes, the inventors hypothesized that the BsMYB15 gene may be related to the regulation of anthocyanin synthesis in Begonia semperflorens. Therefore, the inventors further cloned and studied the gene. This embodiment briefly introduces the cloning process of the gene as follows.

[0017] (a) Extracting total RNA and reverse transcribing it to prepare cDNA Wild-type Begonia semperflorens leaf tissue was used as a sample, flash-frozen in liquid nitrogen and then thoroughly ground. Subsequently, total RNA was extracted from the leaves using an RNA extraction kit and further reverse-transcribed into cDNA for later use (the relevant procedures were performed according to the instructions of the relevant kits or existing technical procedures, and will not be described in detail here). (ii) Design primers and perform PCR amplification The following primer pairs were designed for PCR amplification: BsMYB15-F: 5'-ATGGTGAGGGCTCCTTGCTG-3', BsMYB15-R: 5'-TCAAAATTCTCGCAACTCAGGC-3'; Using the cDNA prepared in step (I) above as a template, PCR amplification was performed using a high-fidelity enzyme and the primer pair designed above. The 50µL amplification system was designed as follows: 2× Phanta Flash Master Mix (Dye Plus), 25 µL; BsMYB15-F primer, 2 µL (10 µM); BsMYB15-R primer, 2 µL (10 µM); cDNA template, 2 µL; ddH2O, 19 µL; The PCR reaction program was set as follows: 98℃ pre-denaturation for 30s; 98℃ denaturation for 10s, annealing for 5s, 72℃ extension for 4s / kb, 35 cycles; final extension at 72℃ for 1min.

[0018] (III) Ligation and Sequencing The amplified product obtained in step (II) was detected by 1% agarose gel electrophoresis, and the target band was recovered using a small-volume DNA product purification kit (the relevant operations can be performed according to the kit instructions, and will not be repeated here).

[0019] Following the instructions for the 5 × TA / Blunt-Zero Cloning Mix, the recovered product was ligated and then transformed into E. coli competent cells DH5α (refer to the relevant product instructions or existing routine procedures; details will not be repeated here). After transformation, positive monoclonal bacteria were picked and inoculated into 1 mL of LB liquid medium containing 50 mg / L Kan antibiotic. After shaking at 37℃ and 220 r / min for 6 h, bacterial culture was tested by PCR and sequencing to verify the transformation and ensure that it was correct.

[0020] The results of electrophoresis detection of PCR amplification products and the results of PCR verification of bacterial culture are as follows: Figure 1 As shown, the results are in line with expectations. For the correctly transformed strains, the Plasmid Miniprep Kit (ZP101) was used to extract the correct recombinant plasmid containing the BsMYB15 gene, and sequencing was performed to obtain the BsMYB15 gene sequence.

[0021] Sequencing results show that the BsMYB15 gene sequence (753 bp, as shown in SEQ ID No. 1) is as follows: ATGGTGAGGGCTCCTTGCTGTGACAAGATGGGTCTGAAAAGAGGACCCTGGACGCCTGAAGAAGACGAGCTTCTGATAGCTCATATCAACAAGTGGGGACACAGCAACTGGCGCGCTCTTCCGAAGCAAGCTGGGCTTACGAGATGTGGGAAGAGTTGCAGACTACGTTGGACGAACTATCTGAGGCCAGATATCAAGAGAGGAAACTTCAGCAGAGAAGAGGAGGATACGATCATCAGGCTGCATTCCACGCTTGGAAATAGGTGGTCGGCCATTGCTGCAAGGCTGCCTGGGCGAACCGACAATGAGATAAAGAACGTGTGGCACACCCATTTGAAGAAGAGGCTCAAACAAACCAATTCCAAGATTAGTTCTTCCAATGTGATCAGGGAAGTAAAGTCTGAGAAGATAGATTTCCGTGGTGGCTCAGATTTCTGCTTCGAATCAACTTCCCCTCAGCCATCTTCTTCCGCCACAGACGCCTCTGTTATCTCCTGGGAAACATATGCAAGTTTTGATGCCGGGCCCAGGGGAGCTGCCGAGCCGATTCCCTCCATCGTTCGAAGTTTTTCGATCGAGTCGTCGTCATGGGTAGAAAATTCTTATCAGACAATCCGATTGGCAGATGAATCATCATCATCCATGGTAATTGAATCGGACTGTTTAGAGAGTTCAAATCTGGATGATTGCATGGATTTTTGGCTCAATCTTTTGCTGAGAGCAGATGAAGTGCCTGAGTTGCGAGAATTTTGA; The corresponding amino acid sequence (250 aa, as shown in SEQ ID No. 2) is as follows: MVRAPCCDKMGLKRGPWTPEEDELLIAHINKWGHSNWRALPKQAGLTRCGKSCRLRWTNYLRPDIKRGNFSREEEDTIIRLHSTLGNRWSAIAARLPGRTDNEIKNVWHTHLKKRLKQTNSKISSS NVIREVKSEKIDFRGGSDFCFESTSPQPSSSATDASVISWETYASFDAGPRGAAEPIPSIVRSFSIESSSWVENSYQTIRLADESSSSMVIESDCLESSNLDDCMDFWLNLLLRADEVPELREF*.

[0022] (iv) BsMYB15 gene structure Based on the BsMYB15 gene sequence and related sequence alignment results, analysis shows that BsMYB15 has two conserved MYB-type DNA-binding domains (at the N-terminus 40-183 bp and 199-336 bp) and belongs to the R2R3 type MYB.

[0023] Secondary and tertiary structure analyses of the protein encoded by the BsMYB15 gene indicate (e.g.) Figure 2 , Figure 3 As shown in the figure, its secondary structure is relatively simple, containing 69 alpha helixes (27.60%) and 181 random coils (72.40%). The tertiary structure is similar to the secondary structure, both primarily consisting of alpha helixes and random coils. Further analysis... BsMYB15 The results of promoter element analysis show that, BsMYB15 It may be involved in the regulation of responses to abiotic stresses such as high light, drought, and low temperature, which also suggests that the function of the BsMYB15 gene may be diverse.

[0024] Example 2 Based on Example 1, the inventors further constructed an overexpression vector for the BsMYB15 gene and performed related genetic transformations to further study and verify the function of this gene. The relevant experimental procedures and results are briefly described below.

[0025] (I) Construction of overexpression vectors The recombinant BsMYB15 gene was ligated using the pCanG plasmid as a vector. The specific process is as follows.

[0026] First, using BamH The pCanG vector plasmid was digested with restriction endonuclease I. The 50 μL digestion system was designed as follows: pCanG plasmid, 1 µg; 10× QuickCut Green Buffer, 5 µL; QuickCut BamH I. 1 µL; add ddH2O to 50 µL; Enzyme digestion was performed at 30℃ for 2 hours; after digestion, the digestion products were detected by 1% agarose gel electrophoresis and then recovered and purified. Simultaneously, using the cloning vector containing the BsMYB15 gene from Example 1 as a template, PCR amplification was performed to obtain the BsMYB15 gene. For PCR amplification, the primer sequences were designed as follows: OE- BsMYB15 -F:5'-GGGTTCGAAATCGATGGATCCATGGTGAGGGCTCCTTGCTG-3', OE- BsMYB15 -R: 5'-GTCCTAGGCTACGTAGGATCCTCAAAATTCTCGCAACTCAGGC-3'; Subsequently, using homologous recombinase 2 × CE Mix, the recovered linearized vector after digestion was ligated with the BsMYB15 gene containing homologous arms obtained after PCR amplification. The 10 μL ligation system was designed as follows: 2 × CE Mix, 5 µL; vector recovered after enzyme digestion, 2 µL; target gene obtained by PCR amplification, 3 µL; Connect at 50℃ for 10 minutes; Next, the ligation product was transformed into *E. coli* competent cells DH5α, and the correctly transformed strains were further screened and identified. Plasmids were extracted from the correctly identified recombinant strains to obtain the correctly constructed recombinant plasmid: pCanG- BsMYB15 ; Finally, following standard operating procedures of existing technologies, the recombinant plasmid: pCanG- BsMYB15 Agrobacterium competent cells GV3101 were transformed, and correctly transformed recombinant strains were screened and identified (partial experimental results are shown in the figure). Figure 4 (As shown) Save for later use.

[0027] Referring to the above description, simultaneously, using pGreen II 62-sk plasmid as a vector, the recombinant plasmid pGreen II 62-sk- was constructed to obtain: BsMYB15 (by Sac I, Apa I represents the restriction enzyme site); and the recombinant strain was obtained by transforming Agrobacterium GV3101.

[0028] (ii) Subcellular localization The aforementioned preparation containing the recombinant plasmid pCanG- BsMYB15 After amplification of Agrobacterium tumefaciens culture (using LB medium containing 50 mg / L each of Kan+ and Rif), the cells were centrifuged, collected, and the bacterial concentration was adjusted to OD using distilled water. 600 After reaching a concentration of 0.8, add 10 mM MES, 10 mM MgCl2, and 200 µM AS to each 100 mL, let stand in the dark for 3 h, and use as the inoculum. Simultaneously, referring to the above instructions, an infection solution was prepared using Agrobacterium strains transformed with the empty vector plasmid pCanG as a control.

[0029] When performing subcellular localization experiments on transformed tobacco, healthy leaves of Tobacco Benedict were selected, and 1 mL of sterile syringe was slowly injected into the lower epidermis of the tobacco leaf to mark the infected area. After 3 days of dark incubation, leaves with a diameter of about 2 mm were selected to prepare slides. After DAPI staining, the fluorescence distribution was observed using a laser confocal microscope.

[0030] Based on bioinformatics analysis, it is predicted that the BsMYB15 protein, after expression, may be localized in the cell nucleus. Further validation results after transformation in tobacco (e.g.) Figure 5 As shown in the figure, pCanG-BsMYB15 only showed fluorescence in the nuclei of tobacco leaf cells, indicating that BsMYB15 is located in the nucleus, which is consistent with the previous prediction and also shows that BsMYB15 is a typical transcription factor.

[0031] (III) Validation of heterogeneous transformation Referring to the foregoing description, a plasmid containing recombinant pGreenⅡ62-sk- was prepared. BsMYB15 The infection solution and reference standard.

[0032] When verifying the infection of apple peel, a 1 mL sterile syringe needle was inserted obliquely into the apple peel about 1 mm, and the needle hole position was marked. Then, an equal volume of the infection solution was slowly injected into the apple peel using a needleless syringe. After being placed in the dark for 1 day, the apple peel was irradiated under a red and blue light lamp with a light intensity of about 100 µmol / m2 / s for 2 days. The color change of the apple peel was observed and recorded, and the anthocyanin content was further determined. For the anthocyanin content determination, the following procedure was referred to: Take 0.1 g of sample, freeze it in liquid nitrogen and grind it thoroughly, add 1 mL of 1% hydrochloric acid methanol solution, and let it stand at 4℃ in the dark for 24 h; then, centrifuge at 4℃ and 10000 rpm for 20 min; collect the supernatant, and measure the absorbance at 530 nm and 600 nm using a UV spectrophotometer. The difference between the two values ​​is the relative content of anthocyanins, and each increase of 0.01 in the difference is defined as one unit U. The anthocyanin content is calculated as follows: (OD530-OD600)×100 g / FW.

[0033] Relevant typical phenotypic results are as follows Figure 6 As shown, the statistical results of further anthocyanin content determination are as follows: Figure 7 As shown in the figure. Analysis shows that after BsMYB15 transiently transforms apples, the apple peel around the injection point turns significantly red. Further anthocyanin content detection results also show that the anthocyanin content in the experimental group is significantly increased, about 5 times that of the control group. This result indicates that BsMYB15 can significantly promote the accumulation of anthocyanins in apple peel.

[0034] Based on the above results, apple peel samples were taken, and following standard procedures, q-PCR technology was used to detect the expression levels of functional genes related to anthocyanin synthesis regulation (MdCHS, MdLDOX, MdF3H, MdDFR, MdUFGT, etc.) in the apple peel. The results are as follows: Figure 8 As shown, the expression levels of functional genes related to apple anthocyanin synthesis, such as MdCHS, MdLDOX, MdF3H, MdDFR, and MdUFGT, all increased significantly, indicating that BsMYB15 may promote the accumulation of anthocyanins in apple epidermis by regulating the expression of downstream functional genes.

[0035] (iv) Verification of the origin of genetic transformation Using healthy, sterile seedlings of Begonia semperflorens as explants, leaves were cut into approximately 1 cm × 1 cm pieces using sterile scissors. After pre-culturing, the explants were inoculated for 8 min with infection solutions prepared from Agrobacterium tumefaciens bacterial suspensions containing recombinant plasmids pCanG-BsMYB15 and RNAi-BsMYB15, respectively (refer to the aforementioned instructions or existing technologies for the construction of relevant strains and preparation of infection solutions). After infection, the samples were cultured in the dark for 4 days, followed by normal culture and induction of differentiation and rooting (the relevant process can be referred to the inventor's previously published literature and other existing technologies, which will not be repeated here). The positively transformed plants were further identified and tested for various indicators.

[0036] In the real-time quantitative PCR detection, Bs18S was used as an internal control, and 2... -ΔΔCT Method Calculation BsMYB15 The relative expression level of the gene; the specific primer sequences for detection are designed as follows: qPCR-BsMYB15-F: 5'-AACCGACAATGAGATAAAGAACG-3', qPCR-BsMYB15-R: 5'-TAAACTTTGAACGATGGAGGGA-3'; Bs18S-F: 5'-GCTACCACATCCAAGGAAGG-3', Bs18S-R: 5'-CAATGGATCCTCGTTAAGGG-3'.

[0037] Among the successfully transformed plants BsMYB15 Gene expression level detection results as follows Figure 9 As shown in the figure. It can be seen that, compared with the wild type, the overexpression lines (OE-1, OE-2) have... BsMYB15 Gene expression levels increased by approximately 10-fold and 6-fold, respectively. Simultaneously, in the interference lines (RNAi-1, RNAi-2)... BsMYB15 Gene expression levels were also significantly reduced. That is, in the obtained transgenic lines... BsMYB15 The gene expression levels showed a significant difference, indicating that the relevant transgenic lines were successfully obtained.

[0038] Further phenotypic observations after culture (e.g.) Figure 10 As shown in the figure, the leaves of the overexpressing strain were significantly redder than those of the wild type, while the leaves of the interference strain remained green, showing no obvious color change. This also preliminarily indicates that... BsMYB15 Gene overexpression can significantly promote the reddening of Begonia semperflorens leaves.

[0039] Samples were taken from different transgenic lines of Begonia semperflorens (leaf samples), and the anthocyanin content was measured according to the aforementioned instructions. The results are as follows. Figure 11 As shown in the figure, the analysis reveals that compared to the wild type, the anthocyanin content in the overexpression lines increased by 4 times and 3 times, respectively, indicating significant anthocyanin accumulation. Conversely, while the leaf color phenotype of the interference lines did not change significantly, the anthocyanin content decreased by approximately 4 times and 2 times, respectively. This again demonstrates that overexpression of BsMYB15 significantly promotes anthocyanin content, leading to the red phenotype change in Begonia semperflorens leaves; while inhibiting BsMYB15, although reducing anthocyanin content, does not lead to significant phenotypic changes in leaf color due to the complexity of the leaf color presentation mechanism.

[0040] Based on the above results, and referring to existing technologies and routine procedures, the inventors further investigated the expression levels of functional genes related to anthocyanin synthesis in Begonia semperflorens using real-time quantitative PCR technology. The results are as follows: Figure 12 As shown. It can be seen that: Compared to the wild type, the expression levels of functional genes in the overexpression lines were significantly higher, while the expression levels of functional genes in the interference lines were significantly lower. Furthermore, the OE-1 overexpression line showed the highest BsUFGT expression level, with a relative expression level exceeding 30. Meanwhile, previous studies have also indicated that BsDFR in Begonia semperflorens is mainly involved in anthocyanin synthesis, and sample analysis results showed that BsDFR increased approximately 4-fold and 3-fold in the overexpression lines OE-1 and OE-2, respectively. These results suggest that BsMYB15 may regulate anthocyanin content by modulating functional genes such as BsDFR and BsUFGT.

[0041] Example 3 Based on the aforementioned embodiments, in order to further explore the regulatory mechanism of BsMYB15 in the synthesis of anthocyanins in Begonia semperflorens, the inventors conducted further in-depth research using yeast single-hybrid and double-hybrid techniques. The relevant experimental process and results are briefly described below.

[0042] (a) Yeast mono-hybrid Referring to the published research literature and results, prior art, and the foregoing description, the pGADT7 vector (selected) EcoR I. BamH I. Recombinant plasmid pGADT7- was constructed by cutting enzyme sites. BsMYB15 ; Subsequently, referring to the instructions of the rapid-type high-efficiency competent cell preparation kit for Saccharomyces cerevisiae, yeast competent cells were prepared, and the above-mentioned recombinant plasmid pGADT7-BsMYB15 and the recombinant plasmid pHis2- constructed by the inventors in the previous stage were used to prepare the cells. BsDFR Y187 competent cells prepared by co-transformation with promter; After transformation, the bacterial culture was spread onto pre-prepared SD / -Trp / -Leu deficient medium and incubated upside down at 30°C for about 3 days.

[0043] After culturing, pick positive monoclonal colonies and place them in 500 μL of sterile water, adjusting the bacterial concentration to OD200. 600 After being diluted to 0.2, the final concentrations were 1 and 10, respectively. -1 10 -2 10 -3 Then, 6 μL of the diluted bacterial culture was inoculated onto SD / -His / -Leu / -Trp deficient medium, and the interaction was screened using different concentrations of 3-AT.

[0044] The results are as follows Figure 13As shown, the transformed colonies can be seen to grow normally on SD / -His / -Leu / -Trp (0 mM 3-AT) deficient medium, indicating that the above plasmids were successfully transformed into yeast cells. However, the 3-AT-based screening results show that "pGADT7 + pHis2-BsDFR promoter" cannot grow normally on SD / -His / -Leu / -Trp (70 mM 3-AT) medium, indicating that BsDFR promoter activity has been inhibited. In contrast, "pGADT7-BsMYB15 + pHis2-BsDFR promoter" can grow on SD / -His / -Leu / -Trp (70 mM 3-AT) medium. This result indicates that BsMYB15 exerts its transcription factor regulatory function by binding to the BsDFR promoter; that is, BsMYB15 regulates anthocyanin content through BsDFR.

[0045] Furthermore, referring to the foregoing description and conventional operation of existing technology, pGreen Ⅱ 62-sk, pGreen Ⅱ 62-sk-BsMYB15, and pGreen Ⅱ 0800 LUC-BsDFRpromter previously constructed by the inventor were used to jointly infect tobacco leaves in different experimental combinations. After infecting, the leaves were placed in a dark environment for 1 day, and then placed under light for 2 days. The relevant experimental results were observed and recorded.

[0046] The results of the above dual-luciferase experiment are as follows: Figure 14 As shown in the figure, pGreen II 62-sk + pGreen II 0800LUC-BsDFR promoter served as the control group, while pGreen II 62-sk-BsMYB15 + pGreen II 0800LUC-BsDFR promoter served as the experimental group (BsMYB15). Imaging fluorescence observations showed that the fluorescence of the experimental group on the right side of the tobacco leaf was stronger than that of the control group on the left. Further sampling of leaves from the infected area and measurement of enzyme activity using a microplate reader revealed that the enzyme activity in the BsMYB15 experimental group was significantly increased compared to the control. This result further demonstrates that BsMYB15 can activate the BsDFR promoter and, consequently, regulate anthocyanin content changes by mediating this functional gene.

[0047] (ii) Yeast hybrid Referring to the foregoing description and existing technical procedures, pGBKT7 plasmid was selected as the vector (selected). EcoR I. BamH I. Recombinant plasmid obtained by constructing the restriction enzyme site: pGBKT7- BsMYB15 ; Subsequently, pGADT7 and pGBKT7- BsMYB15 The plasmids were co-transformed into competent yeast cells Y2H. After transformation, the transformed bacterial culture was spread onto SD / -Trp / -Leu deficient medium and incubated at 30°C for 3 days. Positive monoclonal yeast colonies were then selected to adjust the OD of the culture. 600 After reaching a concentration of 0.2, 6 μL was inoculated onto SD / -Trp / -Leu / -His / -Ade-deficient medium and cultured at 30°C for 3 days. During the experiment, different concentrations of 3-AT were used to inhibit pGBKT7- BsMYB15 Its self-activating activity.

[0048] In the self-activation verification experiment, pGADT7-T + pGBKT7-53 was used as a positive control, pGADT7-T + pGBKT7-Lam as a negative control, and pGAD7 + pGBKT7-BsMYB15 as an empty vector control. The relevant yeast growth results are as follows: Figure 15 As shown in the figure. Analysis shows that the colony growth is good, indicating that the plasmid has been successfully transferred into the competent yeast cells. Screening with SD / -Trp / -Leu / -His / -Ade (0 mM 3-AT+X-α-Gal) deficient medium results show that all cells except the negative control can grow. The growth of the empty vector control colony is weaker than that of the positive control, indicating that the BsMYB15 gene has self-activation. Using different concentrations of 3-AT for screening, it was found that the empty vector control was inhibited when the 3-AT concentration was 20 mM.

[0049] Based on the above results, the cDNA library of Begonia semperflorens leaves previously constructed by the inventors was subjected to yeast two-hybrid screening. Yeast two-hybrid interaction genes were screened using SD / -Trp / -Leu / -His / -Ade / 3-AT / X-α-Gal deficient medium. Combined with relevant sequencing and comparison with the Arabidopsis thaliana genome, preliminary screening showed that: BsDREB1B It can interact with BsMYB15.

[0050] Based on this, and in conjunction with the foregoing description, the inventors further cloned and obtained... BsDREB1B Gene sequence. The aforementioned BsDREB1B The specific nucleotide sequence of the gene is shown in SEQ ID No. 3, corresponding to... BsDREB1B The sequence of the gene-encoded protein is shown in SEQ ID No. 4; specifically as follows: BsDREB1B The specific base sequence of the gene (636 bp): ATGGAGATTTTCTCGACTTTGTTTGATTCTAGCTACACTTCTTCGGCAAGAAGTGATATCTGCCGGAGGGGAAATTTTTCCGACGAGGACCTTATTCTGGCGGCGAGCTCTCCCAAGAAGAGAGCCGGGAGGAGGAAGTTTAAGGAGACGAGGCACCCC GTTTTCCGAGGCGTGAGGAGGAGGAACTCCGGCAAGTGGGTTTGCGAAGTAAGGGAGCCGAGTAAGCAGGCTAGGATATGGCTCGGGACCTTTCCTACGGCGGAAATGGCGGCCAGGGCCCATGACGTGGCAGCACTTGCTCTCCGCGGCCGGGCTGCT TGCTTGAACTTTGCAGACTCTGCATGGCGGCTTCCTGCCCCGCCTTCGACGGAGCCTAAGGATATTCAGAAGGCAGCGGCGGAAGCTGCGGAGGCGTTCCGACCACGCCCAGAAGACGAGGAAATGAAAGCCGTTAAATTCTCGGCAGAAACGCCGGAA AACGTGTTCTACATGGACGAGGAGGCTGTTTTCGGGATGCCGGGGCTGCTGGCAAACATGGCGGAGGGAATGCTATTGCCCGCCACTGGGAGACGACGGCAGAACGGATGACGCCGATTTCGACGCTGACGTGTCATTATGGAGTTATTCTATATAA The corresponding amino acid sequence of the encoded protein (211aa): MEIFSTLFDSSYTSSARSDICRRGNFSDEDLILAASSPKKRAGRRKFKETRHPVFRGVRRRNSGKWVCEVREEPSKQARIWLGTFTPTAEMAARAHDVAALALRGRAACLNFADSAWRLPAPPSTEPKDIQKAAAEAAEAFRPRPEDEEMKAVKFSAETPENVFYMDEEAVFGMPGLLANMAEGMLLPPPLGDDGRTDDADFDADVSLWSYSI*.

[0051] Based on the above results, using pGADT7 plasmid as a vector, the inventors further constructed and obtained the recombinant plasmid pGADT7- BsDREB1BUsing this recombinant plasmid, pGADT7-T + pGBKT7-53 was used as a positive control, and pGADT7-T + pGBKT7-Lam was used as a negative control. BsMYB15 For no-load control, pGADT7- BsDREB1B +pGBKT7- BsMYB15 As the experimental group, Y2H yeast competent cells were co-transfected with Northwest yeast cells, and yeast double hybridization was verified according to the aforementioned procedures.

[0052] The results are as follows Figure 16 As shown in the diagram. Analysis reveals that the negative control and empty control failed to grow normally, and the yeast growth in the experimental group was weaker than that in the positive control. This result indicates that... BsMYB15 Genes can be with BsDREB1B Interacting.

[0053] To further verify BsMYB15 Genes and BsDREB1B Regarding the gene interaction effects, referring to the foregoing description, the inventors conducted transient transformation verification in apples. During the verification process, pGreenⅡ62-sk + pGreenⅡ62-sk- BsMYB15 and pGreen Ⅱ 62-sk + pGreen Ⅱ 62-sk- BsDREB1B As a control, pGreen II 62-sk- BsMYB15 +pGreen Ⅱ 62-sk- BsDREB1B This is the experimental group. The results are as follows: Figure 17 As shown in the figure, when BsMYB15 and BsDREB1B are expressed individually, the apple peel turns red. However, when BsMYB15 and BsDREB1B are expressed together, the apple peel around the injection site is redder than when they are expressed individually. This indicates that BsMYB15 and BsDREB1B work together to promote the reddening of the apple peel.

[0054] Further sampling of apple peel from different parts was conducted to detect anthocyanin content, and the results were as follows: Figure 18 As shown in the figure, the anthocyanin content in apple peel is basically consistent with the phenotypic changes. When BsMYB15 and BsDREB1B are expressed alone, anthocyanins accumulate to a certain extent. However, when BsMYB15 and BsDREB1B are co-expressed, the anthocyanin content in apple peel around the injection site increases significantly, indicating that BsMYB15 and BsDREB1B can jointly promote the accumulation of anthocyanins in apple peel.

[0055] Furthermore, regarding the instantaneous rotation of the apple... MdDFR , MdCHS , MdF 3 H , MdLDOXThe results of functional gene expression level detection (such as...) Figure 19 (as shown) BsMYB15 and BsDREB1B When these genes are co-expressed in the apple peel, they may promote the accumulation of anthocyanins in the apple peel by co-regulating the expression of downstream functional genes.

Claims

1. The BsMYB15 gene of Begonia semperflorens is characterized by, The BsMYB15 gene of Begonia semperflorens has the nucleotide sequence shown in SEQ ID No. 1 and the corresponding amino acid sequence shown in SEQ ID No.

2.

2. The application of the BsMYB15 gene of Begonia semperflorens as described in claim 1 in the regulation of anthocyanins in plants.

3. The application of the BsMYB15 gene of Begonia semperflorens in the regulation of anthocyanins as described in claim 1, characterized in that, In application, the BsMYB15 gene regulates the changes in anthocyanin content in plants by mediating BsDFR; after overexpression of the BsMYB15 gene, it is used to promote the increase of anthocyanin content in plants, thereby promoting the reddening of the surface color of plant leaves or fruit peels; after reducing the expression level of the BsMYB15 gene, it is used to reduce the anthocyanin content in plants.

4. The application of the BsMYB15 gene of Begonia semperflorens in the regulation of anthocyanins as described in claim 3, characterized in that, When applied, the BsMYB15 gene and Begonia semperflorens... BsDREB1B Gene co-overexpression is used to promote the increase of anthocyanin content in plant tissues and to promote the reddening of leaf color or fruit peel surface color.

5. The application of the BsMYB15 gene of Begonia semperflorens in the regulation of anthocyanins as described in claim 3, characterized in that, The plant in question is specifically begonia, apple, or tobacco.

6. Begonia BsDREB1B Genes, characterized by, The Begonia BsDREB1B The gene, with its nucleotide sequence as shown in SEQ ID No. 3 and its corresponding amino acid sequence as shown in SEQ ID No.

4.

7. The Begonia semperflorens as described in claim 6 BsDREB1B The application of genes in the regulation of anthocyanins in plants is characterized by, After overexpression of this gene, it was used to increase the anthocyanin content in plants, promoting the reddening of plant leaves or fruit peels.

8. The Begonia as described in claim 7 BsDREB1B The application of genes in the regulation of anthocyanins in plants is characterized by, The plant in question is either a begonia or an apple.

Images