Application and method of radish RsBBX9 gene in regulation and control of anthocyanin synthesis

Abstract

The invention discloses application of a radish RsBBX9 gene in regulation and control of anthocyanin synthesis and a method, and belongs to the technical field of plant genetic engineering. According to the invention, an RsBBX9 gene is cloned, an overexpression vector pFGC1008-RsBBX9 and a VIGS silence vector pTY-S-RsBBX9 are constructed, and transient expression and gene silencing technologies are combined, so that the regulation and control effect of the RsBBX9 on the anthocyanin content is verified. Yeast two-hybridization, BiFC and pull-down experiments further prove that RsBBX9 and RsMYB1 interact to synergistically activate an anthocyanin synthesis pathway. The invention provides an efficient technical scheme for radish variety improvement and anthocyanin production.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an application and method of a radish RsBBX9 gene in regulating anthocyanin synthesis. Background Art

[0002] Transcription factors (TFs) are key regulators of gene expression, coordinating diverse biological processes by interacting with DNA, RNA, and proteins. Among them, zinc-finger TFs are one of the most widespread 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 located at the N-terminus, which enable protein-protein interactions and transcriptional regulation. BBX proteins exert their effects by directly binding to gene promoters or regulating the activity of other TFs through heterodimerization, influencing key physiological processes. They are involved in plant development and responses to environmental stimuli, including photomorphogenesis, flowering, leaf senescence, and stress adaptation.

[0003] Despite their well-established roles in plants, the specific contributions of BBX proteins to anthocyanin biosynthesis remain largely unexplored. Anthocyanins are a class of water-soluble flavonoids widely found in plants that provide vibrant color to flowers, fruits, and roots while also providing health benefits as potent antioxidants. The precise role of BBX proteins in radish remains unclear. Summary of the Invention

[0004] The present invention aims to provide an application and method of the radish RsBBX9 gene in regulating anthocyanin synthesis to solve the problems of the prior art. The present invention confirms the regulatory effect of the RsBBX9 gene on anthocyanin synthesis in radish leaves and fleshy roots.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides an application of the RsBBX9 gene or a protein encoded thereby in regulating the synthesis of radish anthocyanins. The nucleotide sequence of the RsBBX9 gene is shown in SEQ ID NO.1.

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

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

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

[0010] The present invention also provides application of the recombinant vector in regulating the synthesis of radish anthocyanins.

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

[0012] Furthermore, the regulation includes overexpressing the RsBBX9 gene to increase the content of anthocyanins in the radish.

[0013] Furthermore, the regulation includes inhibiting the expression of the RsBBX9 gene to reduce the content of anthocyanins in the radish.

[0014] The present invention also provides a method for cultivating a high-anthocyanin radish variety, comprising the step of overexpressing the RsBBX9 gene.

[0015] The present invention also provides the use of the RsBBX9 gene or the protein encoded thereby in cultivating high anthocyanin radish varieties. The amino acid sequence of the protein encoded by the RsBBX9 gene is shown in SEQ ID NO.2.

[0016] The present invention discloses the following technical effects:

[0017] The present invention reveals for the first time that overexpression of RsBBX9 in radish leaves can significantly increase anthocyanin content and synthesis gene expression levels, while silencing the gene in fleshy roots leads to reduced anthocyanin accumulation. The present invention also confirmed the physical interaction between RsBBX9 and RsMYB1 through yeast two-hybrid, pull-down analysis and bimolecular fluorescence complementation experiments. Specifically, the present invention locked the radish RsBBX9 gene through bioinformatics analysis, combined with transient expression, VIGS silencing and molecular interaction experiments, to reveal the mechanism by which RsBBX9 and RsMYB1 synergistically regulate anthocyanin synthesis, filling the gap in the study of BBX function in radish and providing a new strategy for efficient and low-cost genetic engineering breeding. Compared with the existing technology, the present invention reveals for the first time the regulatory mechanism of BBX protein in radish, and overcomes the problem of low efficiency of single genes through the synergistic action of multiple factors, providing technical support for the selection and breeding of high-anthocyanin radish varieties and the development of functional foods. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1Figure 2 shows the expression of the transiently expressed RsBBX9 gene in radish leaves and the silenced RsBBX9 gene in radish fleshy roots; a shows the observation of the leaf phenotype of "Zidan" radish, including one leaf of an empty vector plant (Empty-vector) and two leaves of transgenic plants (OE-RsBBX9); b shows the determination of anthocyanin content in leaves of empty vector (Empty-vector) and transgenic plants (OE-RsBBX9); c shows the determination of anthocyanin content in fleshy roots of empty vector (Empty-vector) and transgenic plants (pTV1-RsBBX9); d shows the observation of the root phenotype of empty vector (Empty-vector) and transgenic plants (pTV1-RsBBX9);

[0020] Figure 2 The interaction between RsMYB1 and RsBBX9; a is the growth of all yeast strains in SD / -Trp / -Leu and SD / -Trpp / -Leu / -His / -Ade- / AbA- / X-α-Gal culture medium in the yeast two-hybrid assay; b is the pull-down analysis result; c is the BiFC determination result, and the scale bar is 20 μm. DETAILED DESCRIPTION

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0023] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0024] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0025] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0026] Example 1

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

[0028] Nucleotide sequence of the RsBBX9 gene:

[0029] (SEQ ID NO. 1);

[0030] Amino acid sequence of the protein encoded by the RsBBX9 gene:

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

[0032] 1. Radish RsBBX9 gene cloning and expression vector construction

[0033] Based on the RsBBX9 sequence (SEQ ID NO. 1) and the characteristics of the pFGC1008 vector, RsBBX9-specific primers with homology arms (SEQ ID NO. 3 and SEQ ID NO. 4) were designed. PCR amplification was performed using red-skinned, red-fleshed radish cDNA as a template. The reaction system and protocol were set up according to the instructions of the 2× HieffCanace Advance Fast PCR Master MIX kit (YESEN, China). The PCR amplification program was as follows: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 65°C annealing for 5 s, and 72°C extension for 10 s, for 35 cycles; and 72°C extension for 2 min. The total PCR amplification volume was 20 μL: 1 μL forward primer, 1 μL reverse primer, 2 μL template cDNA, 10 μL high-fidelity enzyme mix, and 6 μL ddH2O. The PCR products were 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 resulting amplified product was purified by agarose gel electrophoresis and sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing. Once sequencing was confirmed, the RsBBX9 gene was ligated into the plant overexpression vector pFGC1008 via homologous recombination. The prepared reaction system (specifically, 10 μL of linearized vector, 4 μL of amplified fragment, 4 μL of 5x UFO Buffer, and 24 μL of UvsXase) was incubated at 37°C for 30 minutes for recombination, followed by transformation into competent Escherichia coli DH5α cells. After screening for the target gene, single colonies were isolated and positive clones were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing, resulting in the plant recombinant plasmid pFGC1008-RsBBX9. The pFGC1008-RsMYB308 vector plasmid was extracted and transformed into Agrobacterium tumefaciens GV3101. Positive clones were isolated using CRM and Rif antibiotics.

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

[0039] Preparation of resuspension: Shake the transformed Agrobacterium until turbid, collect it in a sterilized 50mL centrifuge tube, centrifuge for 10min (12000rpm), and discard the supernatant. First add 10mL of resuspension, vortex mix (protect from light), and measure the OD with an ultramicro spectrophotometer. 600 Adjust the value to around 0.7-0.8 and incubate in the dark at 28°C, 90 rpm in a shaker for 4 hours. Water the material to be transformed well the day before transformation. Select radish leaves that are in the two-leaf, two-heart stage and in good condition. Ensure the leaf surface is clean and free of moisture before injection. Draw up 500 μL of infection solution and inject it into the radish leaf using a 1 mL disposable sterile syringe. After injection, place the radish seedlings in a light incubator at 28°C in the dark for 2 days. Cover the injected radish plants with plastic wrap and transfer them to a normal artificial climate chamber for incubation. After 10 days, closely observe the color changes at the injection site.

[0040] 3. Construction of virus-induced gene silencing (VIGS) vector for 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 mosaic virus vector. Biol Plantarum 62. https: / / doi.org / 10.1007 / s10535-018-0803-6). A palindromic 80-bp DNA fragment of RsBBX9 was selected, and the pTY-S (Nanjing GenScript Biotechnology Co., Ltd.) VIGS vector plasmid was digested with SnaB I restriction endonuclease. The synthesized oligonucleotides were ligated into the linearized vector using the Clon Express Ultra One Step Cloning Kit V2 (Luoweizan, Nanjing). Ten milliliters of purified pTY-S plasmid DNA carrying the RsBBX9 gene sequence was then infiltrated into the fleshy roots of "Zidan" radish plants. Roots of "Zidan" radish plants injected with an empty pTY-S vector were selected as controls. The injected plants were maintained in a growth chamber at 25°C / 22°C with a 16-hour / 8-hour light / dark cycle, and phenotypes were assessed in the fourth week.

[0042] 4. Determination of anthocyanin content

[0043] Place 0.1g of sample in a test tube and add 10mL of 0.1mol / L hydrochloric acid to the ethanol solution. Add 10mL of the extract in a 60°C water bath for 30 minutes. Pour the solution into a 25mL volumetric flask and add 5mL of the extract for 15 minutes. Pour the solution into a 25mm volumetric flask and add 5mL of the extract for 15 minutes. Then pour the solution into a 5mL volumetric flask. Add 5mL of the extract for 15 minutes, pour it into a 25mL volumetric flask, extract for 1 hour, and finally dissolve it to 25mL. A reference solution is 0.1mol / L ethanolic hydrochloric acid. Measure the optical density of the extract at 530nm, 620nm, and 650nm using spectrophotometry.

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

[0045] Given that RsMYB1 is a key transcription factor regulating the synthesis of radish anthocyanins. The present invention used a yeast two-hybrid assay to study the interaction between RsBBX9 and RsMYB1. First, the entire coding sequences of RsBBX9 and RsMYB1 were cloned. Secondly, their CDS sequences were constructed into pGADT7 and pGBKT7 vectors, respectively. The plasmid pair pGADT7-RsMYB1 and pGBKT7-RsBBX9 were co-transformed into the yeast strain Y2HGold (the strain was obtained from Shanghai Weidi Biotechnology Co., Ltd.). The yeast strain co-transformed with pGBKT7-lam and pGADT7-T was used as negative control 1. The yeast strain Y2HGold transformed with pGBKT7-RsBBX9 and empty pGADT7 was used as negative control 2. Yeast strain Y2HGold co-transformed with empty pGBKT7 and empty pGADT7-RsBBX9 was used as negative control 3, and yeast strain Y2HGold co-transformed with pGADT7-T and pGBKT7-53 was selected as a positive control. All yeast transformants were grown in SD / Leu / -Trp (provided by Beijing Coolbo Technology Co., Ltd.) and SD / -Leu / -Trp / -His / -Ade / AbA / X-α-Gal medium (provided by Beijing Coolbo Technology Co., Ltd.).

[0046] 7. Pull-down analysis

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

[0048] 8. Bimolecular Fluorescence Complementation (BiFC)

[0049] For bimolecular fluorescence complementation (BiFC) detection, the sequences encoding RsBBX9 and RsMYB1 were cloned and inserted into YFP-N and YFP-C vectors, respectively. YFP-N and YFP-C empty vectors were used as negative controls. Then, the different combination vectors were transformed into Agrobacterium tumefaciens strain GV3101 (from Wuhan Boyuan Biological) and transiently transformed in tobacco leaves. Finally, the fluorescence signal images were transcribed using a laser scanning confocal microscope.

[0050] Observation of the leaf phenotype of "Zidan" radish Figure 1 a in the figure; the anthocyanin content in the leaves of empty vector and transgenic plants was determined as shown in Figure 1 b in the figure; Determination of anthocyanin content in the fleshy roots of empty vector and transgenic plants is shown in Figure 1 c in Figure 2; Observation of root phenotypes of wild-type, empty vector, and transgenic plants is shown in Figure 2. Figure 1 The yeast two-hybrid assay results are shown in 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; the present invention also confirmed the physical interaction between RsMYB1 and RsBBX9 by pull-down analysis ( Figure 2 b); The present invention also confirmed the physical interaction between RsMYB1 and RsBBX9 in tobacco leaf epidermal cells using BiFC assay ( Figure 2 c) in the above example.

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

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

Claims

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

1.

2. The use 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. A recombinant vector, characterized in that The invention comprises the RsBBX9 gene described in claim 1.

4. A host bacterium, characterized in that Comprising the recombinant vector according to claim 3.

5. Use of the recombinant vector according to claim 3 in regulating the synthesis of radish anthocyanins.

6. A method for regulating the anthocyanin content of radish, characterized in that: The method comprises the step of regulating the expression of the RsBBX9 gene according to claim 1.

7. The method according to claim 6, characterized in that The regulation includes overexpressing the RsBBX9 gene to increase the content of anthocyanins in the radish.

8. The method according to claim 6, characterized in that The regulation includes inhibiting the expression of the RsBBX9 gene to reduce the content of anthocyanins in the radish.

9. A method for cultivating high anthocyanin radish varieties, characterized in that: The method comprises the step of overexpressing the RsBBX9 gene according to claim 1.

10. Use of the RsBBX9 gene or the protein encoded by it in claim 1 in breeding 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.

Images