Application and method of radish rsbbx9 gene in regulating anthocyanin synthesis

Abstract

The application discloses application and a method of a radish RsBBX9 gene in regulation of anthocyanin synthesis, and belongs to the technical field of plant genetic engineering. The application verifies the regulation of RsBBX9 on anthocyanin content by cloning the RsBBX9 gene, constructing an overexpression vector pFGC1008-RsBBX9 and a VIGS silencing vector pTY-S-RsBBX9, combining transient expression and gene silencing technology. The application further verifies the interaction of RsBBX9 and RsMYB1 by a yeast double-hybrid experiment, a BiFC experiment and a pull-down experiment, and the interaction cooperatively activates an anthocyanin synthesis path. The application provides an efficient technical scheme for radish variety improvement and anthocyanin production.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant genetic engineering, and particularly relates to application and method of radish RsBBX9 gene in regulation of anthocyanin synthesis. BACKGROUND

[0002] Transcription factors (TFs) are key regulators of gene expression, coordinating different biological processes through interactions with DNA, RNA, and proteins. Among them, zinc finger TFs are one of the most widely distributed families in plants, playing important roles in growth, development, and stress responses. The B-box (BBX) protein family is a subfamily of zinc finger TFs, characterized by one or two conserved B-box domains at the N-terminus, which can achieve protein interaction and transcriptional regulation. BBX proteins play a role by directly binding to gene promoters or by heterodimerization to regulate the activity of other TFs, affecting key physiological processes. They are related to plant development and response to environmental stimuli, including photomorphogenesis, flowering, leaf senescence, and stress adaptation.

[0003] Although BBX proteins play a recognized role in plants, the specific role of BBX proteins in anthocyanin biosynthesis has largely remained unexplored. Anthocyanins are a class of water-soluble flavonoids that are widely present in plants, providing vibrant colors to flowers, fruits, and roots, while also providing health benefits as potent antioxidants. The exact role of BBX proteins in radish is not yet clear. SUMMARY

[0004] The purpose of the present application is to provide application and method of radish RsBBX9 gene in regulation of anthocyanin synthesis, in order to solve the problems existing in the prior art. The present application confirms the regulatory effect of RsBBX9 gene on anthocyanin synthesis in radish leaves and fleshy roots.

[0005] To achieve the above purpose, the present application provides the following solutions:

[0006] The present application provides application of RsBBX9 gene or protein coded by the same in regulation of anthocyanin synthesis in radish, and the nucleotide sequence of the RsBBX9 gene is shown in SEQ ID NO. 1.

[0007] Further, the amino acid sequence of the protein coded by the RsBBX9 gene is shown in SEQ ID NO. 2.

[0008] The present application also provides a recombinant vector comprising the RsBBX9 gene.

[0009] The present application provides a host bacterium comprising the recombinant vector.

[0010] The application further provides the application of the recombinant vector in regulating anthocyanin synthesis of radish.

[0011] The application provides a method for regulating anthocyanin content, comprising the step of regulating expression of the RsBBX9 gene.

[0012] Further, the regulation comprises overexpression of the RsBBX9 gene to increase the content of the anthocyanin.

[0013] Further, the regulation comprises inhibition of expression of the RsBBX9 gene to decrease the content of the anthocyanin.

[0014] The application further provides a method for breeding a high-anthocyanin radish variety, comprising the step of overexpressing the RsBBX9 gene.

[0015] The application further provides the application of the RsBBX9 gene or the protein encoded by the RsBBX9 gene in breeding a high-anthocyanin radish variety, and the amino acid sequence of the protein encoded by the RsBBX9 gene is shown in SEQ ID NO. 2.

[0016] The application discloses the following technical effects:

[0017] The application discloses the following technical effects: BRIEF DESCRIPTION OF DRAWINGS

[0018] In order to more clearly illustrate the technical solutions of the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0019] Figure 1Figure 6. Phenotypes of OE-RsBBX9 and Empty-vector plants. a, Phenotypes of OE-RsBBX9 and Empty-vector plants; b, Anthocyanin content of OE-RsBBX9 and Empty-vector plants; c, Anthocyanin content of OE-RsBBX9 and Empty-vector plants; d, Phenotypes of OE-RsBBX9 and Empty-vector plants.

[0020] Figure 2 Figure 9. Interaction between RsMYB1 and RsBBX9. a, Growth of all yeast strains on SD / -Trp / -Leu and SD / -Trpp / -Leu / -His / -Ade / -AbA- / X-α-Gal medium; b, Results of pull-down analysis; c, BiFC assay, scale bar, 20 μm. DETAILED DESCRIPTION

[0021] The following detailed description is provided to understand certain aspects, features and embodiments of the present application. It is not intended that the application be limited to the described embodiments, but rather that the application should be given broad consideration.

[0022] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the present application. In addition, for a range of values of, for example, a parameter, an intermediate value of the parameter is encompassed if the intermediate value is not expressly excluded from the range. Any smaller range of values of the parameter is also encompassed.

[0023] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice of the present application. All documents mentioned herein are incorporated by reference to disclose and describe in full the methods and / or materials which are described therein. In the case of conflict between the present description and the document incorporated by reference, the present description controls.

[0024] Many modifications and variations of the present disclosure described in the specification are possible without departing from the scope or spirit of the present disclosure. Other implementations of the present disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the present disclosure. The specification and examples are illustrative only.

[0025] As used herein, "comprise", "comprising", "include", "including", "have" and "having" and the like are open-ended terms that are intended to mean including but not limited to.

[0026] Example 1

[0027] The nucleotide sequence of RsBBX9 gene was obtained by searching the public genome data of radish through NCBI database.

[0028] The nucleotide sequence of RsBBX9 gene is as follows:

[0029] ATGGAGAAGTTGAAGTGTGAGCTATGCGACGGTGTGGCGTGGATGTTCTGTGAATCAGACCAGGCGAGTTTATGCTGGGACTGCGATGGTTACGTTCACGGAGCTAACTTTCTGGTGGCTAAACACGCGCGCTGCCTTCTTTGTAGCGTTTGCCAGTCTCCTACGCCTTGGAAAGCTTCGGGTCTTCGACTATGCCCAACAGTTTCCAACTGCGAGCCTTGCCTTGCTCGTAAGAATAGTAACGGCAGAAATGGTTACGGAGAAGACGACGGCGCAGAATCTTATGATGAGGATGAGGAGGAAGAAGAAGAAGAAAGTGATGATGAGGAAGACGAAGAAGAAGAAGAAGGGGAGAATCAGGTGGTGCCGTGGGATGCGGCTGCGGCGGTGCAAGAACCTCCAGTGATGAGTTCTTGGTGTTCCGTTAGCAGCGGAGGAGAGGAACGTTTCAGTTTGGTAGAGAAAAGGACGCGACAAGATTCAGATCTTAACTCCTCCCATGATGAATCAAACGAGTCACGACCGTTGAAACGGCTAGCGAGAGATGAAACCTGGCCAAGATCAACGGCTATGATGAAATCAACCTTAAAAATCAAACGACTGTGA (SEQ ID NO. 1);

[0030] The amino acid sequence of the protein encoded by the RsBBX9 gene is as follows:

[0031] MEKLKCELCDGVAWMFCESDQASLCWDCDGYVHGANFLVAKHARCLLCSVCQSPTPWKASGLRLCPTVSNCEPCLARKNSNGRNGYGEDDGAESYDEDEEEEEEESDDEEDEEEEEGENQVVPWDAAAAVQEPPVMSSWCSVSSGGEERFSLVEKRTRQDSDLNSSHDESNESRPLKRLARDETWPRSTAMMKSTLKIKRL (SEQ ID NO. 2);

[0032] 1. Cloning of the radish RsBBX9 gene and construction of an expression vector

[0033] According to the RsBBX9 sequence (SEQ ID NO. 1) and the characteristics of the pFGC1008 vector, specific primers (SEQ ID NO. 3 and SEQ ID NO. 4) with homologous arms were designed. The red-skinned red-fleshed radish cDNA was used as a template for PCR amplification. The reaction system and program were set according to the 2xHieffCanace Advance Fast PCR Master MIX kit instructions (YESEN, China). The PCR amplification program was 98°C pre-denaturation for 30s; 98°C denaturation for 10s, 65°C annealing for 5s, 72°C extension for 10s, 35 cycles; 72°C extension for 2min; the total volume of PCR amplification was 20μL: 1μL of forward primer, 1μL of reverse primer, 2μL of template cDNA, 10μL of high-fidelity enzyme Mix, 6μL of ddH2O; the PCR amplification product was stored at 4°C.

[0034] The primers are as follows:

[0035] Forward primer: TTACAATTACCATGGGGCGCGCCATGGAGAAGTTGAAGTGTGAGCTAT (SEQ ID NO. 3);

[0036] Reverse primer: AACATCGTATGGGTAGGTACCCAGTCGTTTGATTTTTAAGGTTGAT (SEQ ID NO. 4).

[0037] The amplified product was purified by agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing. After successful sequencing, the obtained RsBBX9 gene was ligated into the plant overexpression vector pFGC1008 via homologous recombination. The prepared reaction system (10 μL linearized vector, 4 μL amplified fragment, 4 μL 5x UFO Buffer, 24 μL UvsXase) was incubated at 37℃ for 30 min for recombination, and then transformed into E. coli DH5α competent cells. After screening for the target gene, single clones were selected and cultured. Positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, forming the plant recombinant plasmid pFGC1008-RsBBX9. The pFGC1008-RsMYB308 vector plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101. After screening with CRM and Rif antibiotics, positive Agrobacterium clones were obtained.

[0038] 2. Transient overexpression of radish RsBBX9 in radish leaves

[0039] Preparation of resuspension: The transformed Agrobacterium was shaken until turbid, collected in a sterile 50 mL centrifuge tube, centrifuged for 10 min (12000 rpm), and the supernatant was discarded. 10 mL of the resuspension was added, vortexed to mix (protected from light), and its OD was measured using a micro spectrophotometer. 600 The pH was adjusted to approximately 0.7-0.8, and the plants were incubated at 28℃ and 90 rpm in the dark for 4 hours. The material to be transformed was thoroughly watered the day before transformation. Radish leaves at the two-leaf, two-heart stage with good growth were selected. Before injection, the leaf surface was ensured to be clean and dry. 500 μL of the infection solution was drawn and injected into the radish leaves using a 1 mL disposable sterile syringe. The injected radish seedlings were placed in a light incubator at 28℃ in the dark for 2 days. The injected radish plants were covered with plastic wrap and then transferred to a normal artificial climate chamber for cultivation. After 10 days, the color change at the injection site was closely observed.

[0040] 3. Construction of a virus-induced gene silencing (VIGS) vector for the radish RsBBX9 gene

[0041] A TYMV-based virus-induced gene silencing (VIGS) system was used to characterize the function of RsBBX9 (Yu, J., Yang, X., Wang, Q., Gao, L., Yang, Y., Xiao, D., Liu, T., Li, Y., Hou, X., Zhang, C., 2018. Efficient virus-induced gene silencing in Brassica rapa using a turnip yellow mosaicvirus vector. Biol Plantarum 62. https: / / doi.org / 10.1007 / s10535-018-0803-6). An 80bp palindromic DNA fragment of RsBBX9 was selected, and the pTY-S (Nanjing Genscript Biotech Co., Ltd.) VIGS vector plasmid was digested with SnaB I restriction endonuclease. The synthesized oligonucleotides were ligated to the linearized vector using the Clon Express Ultra One Step Cloning Kit V2 homologous recombination kit (Loviza, Nanjing). Ten milliliters of purified pTY-S vector carrying the RsBBX9 gene sequence plasmid DNA were then infiltrated into the fleshy roots of 'Zidan' radish. 'Zidan' radish fleshy roots injected with the empty pTY-S vector were selected as controls. The injected plants were kept in a growth chamber at 25℃ / 22℃ with a light / dark cycle of 16 hours / 8 hours, and phenotypes were assessed at week four.

[0042] 4. Determination of anthocyanin content

[0043] Place 0.1 g of sample in a test tube and add 10 mL of 0.1 mol / L hydrochloric acid to the ethanol solution. In a 60°C water bath, add 10 mL of extraction buffer for 30 minutes. Pour the solution into a 25 mL volumetric flask, add 5 mL of extraction buffer for 15 minutes, then pour the solution into another 25 mL volumetric flask. Add 5 mL of extraction buffer for 15 minutes, then pour the solution into a 25 mL volumetric flask and extract for 1 hour. Finally, dissolve the extract to a final volume of 25 mL. The reference solution is 0.1 mol / L ethanol-hydrochloric acid. Measure the optical density of the extract at 530 nm, 620 nm, and 650 nm using spectrophotometry.

[0044] 5. Yeast two-hybrid system (Y2H)

[0045] Given that RsMYB1 is a key transcription factor regulating anthocyanin synthesis in radish, this invention uses a yeast two-hybrid assay to study the interaction between RsBBX9 and RsMYB1. First, the complete coding sequences of RsBBX9 and RsMYB1 were cloned. Second, their CDS sequences were constructed into the pGADT7 and pGBKT7 vectors, respectively. The plasmid pairs pGADT7-RsMYB1 and pGBKT7-RsBBX9 were co-transformed into the yeast strain Y2HGold (strain sourced from Shanghai Vidi Biotechnology Co., Ltd.). The yeast strain co-transformed with pGBKT7-lam and pGADT7-T served as negative control 1. The yeast strain Y2HGold transformed with pGBKT7-RsBBX9 and an empty pGADT7 vector served as negative control 2. Yeast strain Y2HGold, co-transformed with empty pGBKT7 and empty pGADT7-RsBBX9, was used as a negative control (3), while yeast strain Y2HGold, co-transformed with pGADT7-T and pGBKT7-53, was used as a positive control. All yeast transformants were grown on SD / Leu / -Trp (from Beijing Cooler Master Technology Co., Ltd.) and SD / -Leu / -Trp / -His / -Ade / AbA / X-α-Gal media (from Beijing Cooler Master Technology Co., Ltd.).

[0046] 7. Drop-down analysis

[0047] To verify the interaction between RsBBX9 and RsMYB1, the sequences encoding RsBBX9 and RsMYB1 were cloned and inserted into vectors carrying His and GST tags respectively (from Wuhan Boyuan Biotechnology). Proteins with His or GST tags were expressed in BL21(DE3) strain (from Wuhan Boyuan Biotechnology). Proteins with His or GST tags were purified using Ni-NTA beads. Both types of proteins were incubated at 4°C for 16 hours. The beads were then harvested and washed four times. Proteins bound to the beads were eluted and separated using SDSPAGE. The presence of pull-down proteins was detected using Western blotting with anti-GST (YEASEN) or anti-His (BlotCycler) antibodies.

[0048] 8. Bimolecular Fluorescence Complementary (BiFC)

[0049] For bimolecular complementary fluorescence (BiFC) detection, sequences encoding RsBBX9 and RsMYB1 were cloned and inserted into YFP-N and YFP-C vectors, respectively. Empty YFP-N and YFP-C vectors were used as negative controls. The different vector combinations were then transformed into Agrobacterium tumefaciens strain GV3101 (from Wuhan Boyuan Biotechnology) and transiently transformed in tobacco leaves. Finally, images of the transcriptional fluorescence signals were obtained using laser scanning confocal microscopy.

[0050] Observation of the leaf phenotype of "Zidan" radish Figure 1 a; the determination of anthocyanin content in the leaves of empty vectors and transgenic plants is shown in [reference 1]. Figure 1 b; the determination of anthocyanin content in the fleshy roots of empty vectors and transgenic plants is shown in section b; Figure 1 c; Observations on root phenotypes of wild-type, empty vector, and transgenic plants are shown in [reference needed]. Figure 1 The results of the yeast two-hybrid experiment are shown in Figure d. Figure 2 As shown in 'a', all yeast strains grew on SD / -Trp / -Leu and SD / -Trpp / -Leu / -His / -Ade- / AbA- / X-α-Gal media; this invention also confirmed the physical interaction between RsMYB1 and RsBBX9 by pull-down analysis. Figure 2 (b) ; This invention also confirmed the physical interaction between RsMYB1 and RsBBX9 in tobacco leaf epidermal cells using BiFC assays. Figure 2 (c)

[0051] In summary, transient overexpression of the RsBBX9 gene in radish leaves can increase the accumulation of anthocyanins and the expression level of anthocyanin biosynthesis-related genes in radish leaves. Silencing the expression of RsBBX9 in radish root can reduce the accumulation of anthocyanins and the expression level of anthocyanin biosynthesis genes in the root.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. The application of the RsBBX9 gene or its encoded protein in enhancing anthocyanin synthesis in radish, characterized in that, The nucleotide sequence of the RsBBX9 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The amino acid sequence of the protein encoded by the RsBBX9 gene is shown in SEQ ID NO.

2.

3. The application of a recombinant vector in enhancing anthocyanin synthesis in radish, characterized in that, The recombinant vector includes the RsBBX9 gene as described in claim 1.

4. A method for increasing the anthocyanin content of radishes, characterized in that, Includes the step of increasing the expression of the RsBBX9 gene as described in claim 1.

5. The method according to claim 4, characterized in that, The enhancement includes overexpressing the RsBBX9 gene to increase the anthocyanin content of the radish.

6. A method for cultivating high-anthocyanin radish varieties, characterized in that, Includes the step of overexpressing the RsBBX9 gene as described in claim 1.

7. The application of the RsBBX9 gene or its encoded protein as described in claim 1 in the breeding of high anthocyanin radish varieties, characterized in that, The amino acid sequence of the protein encoded by the RsBBX9 gene is shown in SEQ ID NO.2.

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