Red-stemmed kale bovit1 protein and application thereof

Abstract

The application discloses a BoVIT1 protein of red-stemmed Chinese kale and application of the BoVIT1 protein. By knocking out the BoVIT1 gene in red-stemmed Chinese kale, the transgenic red-stemmed Chinese kale shows the increase of plant height and the inhibition of stem color formation of Chinese kale, which indicates that the BoVIT1 protein of red-stemmed Chinese kale has an important role in improving the quality of Chinese kale. The application provides a new gene resource for the quality improvement of Chinese kale.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the BoVIT1 protein of red mustard greens and its applications. Background Technology

[0002] Chinese kale (Brassica oleracea var. alboglabra Bailey) is a herbaceous plant belonging to the genus Brassica in the family Brassicaceae. It is a variety of cabbage and has become one of the vegetables introduced and widely cultivated in northern regions. It has also spread to Europe, America, Oceania, Japan, and Southeast Asian countries. The edible parts of Chinese kale are the tender flower stalks and young leaves. The flower stalks have high content of vitamins, soluble proteins, soluble sugars, minerals, and mustard oil, giving them good flavor and nutritional value. However, there are significant differences in flavor and nutritional content among different varieties. In addition, Chinese kale also has health benefits, containing various antioxidants, mainly beta-carotene, flavonoids, and sulforaphane.

[0003] Ideal plant type generally refers to the plant type that is conducive to photosynthesis, growth and development, and grain yield. This breeding concept was first proposed by Donald in 1968. In crop production, ideal plant type is a key factor in increasing yield. Creating ideal plant type materials that combine morphology and function for breeding to break the stagnation of crop yield is an urgent research goal. Therefore, identifying and utilizing functional genes that control the plant type of Chinese kale is of great significance. Summary of the Invention

[0004] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes the application of BoVIT1 protein in red-stemmed mustard greens.

[0005] The present invention also proposes the application of nucleic acid molecules encoding the aforementioned BoVIT1 protein from red mustard greens.

[0006] This invention also proposes biomaterials related to the aforementioned nucleic acid molecules.

[0007] This invention also proposes a primer set for amplifying nucleic acid molecules encoding the BoVIT1 protein of red mustard greens.

[0008] This invention also proposes a method for regulating the height of Chinese kale plants.

[0009] This invention also proposes a method for cultivating tall mustard plants.

[0010] This invention also proposes a method for regulating the color formation of Chinese kale stems.

[0011] The present invention also proposes a product.

[0012] The present invention also proposes applications of the above-mentioned products.

[0013] In a first aspect of the invention, the use of BoVIT1 protein from red mustard greens is proposed in any of the following:

[0014] (a1) Regulating the height of Chinese kale plants;

[0015] (a2) Regulates the color formation of Chinese kale stems;

[0016] The BoVIT1 protein in red mustard greens is a protein that is either a), b), c), or d) of the following:

[0017] a) A protein with the amino acid sequence shown in SEQ ID NO:1;

[0018] b) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of a protein with an amino acid sequence as shown in SEQ ID NO:1;

[0019] c) Proteins with the same function obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO:1.

[0020] d) An amino acid sequence having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, or 70% identity with the amino acid sequence shown in SEQ ID NO:1 and having the same function as the protein shown in SEQ ID NO:1.

[0021] In a second aspect of the invention, the use of the nucleic acid molecule encoding the aforementioned BoVIT1 protein from red mustard greens is proposed in any of the following:

[0022] (a1) Application in regulating the height of Chinese kale plants;

[0023] (a2) Application in regulating the color formation of Chinese kale stems.

[0024] In some embodiments of the present invention, the nucleic acid molecule encoding the BoVIT1 protein has the following nucleotide sequence:

[0025] (b1) A nucleotide sequence or a degenerate sequence thereof as shown in SEQ ID NO:2;

[0026] (b2) A nucleotide sequence derived from the nucleotide sequence defined in (b1) by substitution, deletion or addition of at least one nucleotide, and which encodes a polypeptide with the same or similar function as the sequence of SEQ ID NO:2.

[0027] In some embodiments of the present invention, (b2) above refers to a nucleotide sequence derived from the nucleotide sequence defined in (b1) by substitution, deletion or addition of one or more (up to 10) nucleotides, which encodes a polypeptide with the same or similar sequence function as SEQ ID NO:1.

[0028] In a third aspect of the invention, a biomaterial related to the above-described nucleic acid molecule is provided, which is any one of 1) to 7) below:

[0029] 1) Expression cassettes containing the above-mentioned nucleic acid molecules;

[0030] 2) Recombinant vectors containing the above-mentioned nucleic acid molecules;

[0031] 3) A recombinant vector containing the expression cassette described in 1);

[0032] 4) Recombinant microorganisms containing the above-mentioned nucleic acid molecules;

[0033] 5) Recombinant microorganisms containing the expression cassette described in 1);

[0034] 6) Recombinant microorganisms containing the recombinant vector described in 2);

[0035] 7) Recombinant microorganisms containing the recombinant vector described in 3).

[0036] In a fourth aspect of the invention, a primer set for amplifying the nucleic acid molecule encoding the aforementioned BoVIT1 protein of red mustard greens is proposed.

[0037] In some embodiments of the present invention, the primer set includes a forward primer sequence as shown in SEQ ID NO:3 and a reverse primer sequence as shown in SEQ ID NO:4.

[0038] In a fifth aspect of the invention, a method for regulating the height of Chinese kale plants is proposed, the method comprising the steps of: promoting or inhibiting the expression of the BoVIT1 gene in Chinese kale to regulate the height of Chinese kale plants.

[0039] In some embodiments of the present invention, the height of kale plants can be increased by inhibiting the expression of the BoVIT1 gene in kale.

[0040] In a sixth aspect of the invention, a method for cultivating tall Chinese kale plants is provided, the method comprising the step of: inhibiting the expression of the BoVIT1 gene in Chinese kale.

[0041] In a seventh aspect of the present invention, a method for regulating the color formation of kale stems is proposed, the method comprising the following steps: promoting or inhibiting the expression of the BoVIT1 gene in kale to regulate the color formation of kale stems.

[0042] In some embodiments of the present invention, the inhibition of BoVIT1 gene expression in red-stemmed mustard greens is achieved using the VIGS viral vector system.

[0043] In some embodiments of the present invention, the VIGS viral vector system contains a target sequence for silencing the BoVIT1 gene.

[0044] In some embodiments of the present invention, the target sequence of the silenced BoVIT1 gene is shown in SEQ ID NO:11.

[0045] In an eighth aspect of the invention, a product is provided, comprising a target sequence for silencing the BoVIT1 gene, or a biological material comprising a target sequence for silencing the BoVIT1 gene.

[0046] In some embodiments of the present invention, the target sequence includes a sequence as shown in SEQ ID NO:11.

[0047] In some embodiments of the present invention, the biomaterial includes a VIGS viral vector system.

[0048] In a ninth aspect of the invention, the application of the above-described product in any of the following is proposed:

[0049] (c1) Plant breeding;

[0050] (c2) Prepare products for plant-assisted plant breeding;

[0051] (c3) Regulating plant height;

[0052] (c4) Regulates the formation of plant stem color.

[0053] In some embodiments of the present invention, the plant includes Chinese kale.

[0054] In some embodiments of the present invention, the kale includes red-stemmed kale.

[0055] According to embodiments of the present invention, at least the following beneficial effects are achieved: The present invention discloses the application of the BoVIT1 protein in red-stemmed kale in regulating plant height and stem color formation. Red-stemmed kale with the BoVIT1 gene knocked out exhibits increased plant height and inhibited stem color formation, indicating that the BoVIT1 protein plays an important role in improving kale quality. The present invention provides a new gene resource for improving kale quality. Attached Figure Description

[0056] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0057] Figure 1 This is a schematic diagram of the growth of Chinese mustard greens in Embodiment 1 of the present invention;

[0058] Figure 2 This is a pNC-TRV2 vector spectrum from Example 2 of the present invention;

[0059] Figure 3 This is a phenotypic analysis diagram of the stem of the transgenic plant in the test example of this invention;

[0060] Figure 4 This is a diagram showing the results of pigment accumulation analysis in the stem cross section of *Gnaphalium affine* after BoVIT1 gene silencing in the test examples of this invention.

[0061] Figure 5 This is a graph showing the results of the analysis of the expression level of the target gene in the stem after BoVIT1 gene silencing in the test examples of this invention. Detailed Implementation

[0062] The following will describe the concept and technical effects of the present invention clearly and completely with reference to the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0063] Example 1: BoVIT1 protein in red-stemmed mustard greens

[0064] This embodiment provides a BoVIT1 protein from red-stemmed mustard greens, with the following amino acid sequence:

[0065] BoVIT1 protein sequence of red-legged mustard:

[0066] MATDEDMITRISIEPEKQSLLDHHTEKHFTAGEVVRDIIIGVSDGLTVPFALAAGLSGANASSSIVLTAGIAEVAAGAISMGLGGYLAAKSEADHYAREMKREQEEIVAVPETEAAEVAEILAQYGVEPHEYSPVVNALRKNPQAWLDFMMRFELGLEKPDPKRALQSAFTIAIAYVLGGLVPLFPYMFIPQALNAVVASAAITLIALFIFGYAKGHFTGSRPFRSAFETTFIGAIASAAAFCLAKVVQH*(SEQ ID NO:1).

[0067] Nucleotide sequence encoding the BoVIT1 protein:

[0068] ATGGCTACGGACGAAGATATGATCACGAGGATCTCTATAGAGCCTGAGAAGCAATCACTTCTCGATCATCACACCGAGAAACACTTCACCGCCGGAGAAGTCGTCCGTGACATCATCATCGG CGTCTCCGATGGTTTAACCGTTCCTTTTGCACTCGCCGCCGGACTCTCCGGTGCTAATGCCTCTTCTTCCATCGTACTCACCGCTGGTATCGCTGAAGTCGCAGCCGGTGCTATCTCCATGGG ACTCGGCGGGTACTTAGCGGCCAAGAGTGAAGCGGATCATTACGCGAGAGATGAAACGAGAACAAGAAGAGATCGTGGCCGTTCCTGAGACTGAGGCAGAGCTGAGGTGGCAGAGATTCTGGCACAGTATGGAGTAGAGCCACATGAATATTCACCTGTTGTTAATGCTCTTCGTAAGAATCCTCAAGCATGGCTTGATTTTATGATGAGGTTTGAGCTGGGATTAGAGAAGCCAGATCCAAAGA GAGCGTTACAAAGCGCGTTCACGATTGCAATCGCTTATGTTCTTGGCGGTTTAGTACCGCTATTTCCCTACATGTTCATACCACAAGCCTTAAATGCGGTAGTGGCGTCTGCGGCTATAACTCTGATCGCTCT CTTCATATTTGGCTATGCAAAAGGGCATTTCACCGGCAGCAGACCCTTTAGGAGTGCGTTTGAAACCACTTTTATAGGAGCGATAGCTTCAGCAGCTGCTTTCTGTTTGGCTAAAGTGGTGCAACATTAA(SEQ ID NO:2).

[0069] Example 2: Construction of BoVIT1 gene silencing vector and transgenic plants in red-stemmed mustard greens

[0070] 1. Construction of a vector for silencing the BoVIT1 gene in red-stemmed mustard greens

[0071] The specific steps for constructing the BoVIT1 gene silencing expression vector in red-stemmed mustard greens are as follows:

[0072] (1) Collection of Red-stemmed Mustard Greens

[0073] Red-stemmed Chinese kale material (illustration of Chinese kale at different stages of growth, as shown in the figure) Figure 1 (As shown) The plants were collected from the Key Laboratory of Landscape Plants and Ornamental Horticulture, Zhongkai University of Agriculture and Engineering. The plant culture conditions were: 22℃, 14h light / 10h dark, and 85% relative humidity.

[0074] (2) Extraction of total RNA

[0075] RNA was extracted from the stems of normally grown 1-month-old red-stemmed mustard greens. The RNA extraction and purification process followed the instructions of the Plant RNA Rapid Extraction Kit (Beijing Huayueyang Biotechnology Co., Ltd., Beijing). 1 μg of total RNA was taken and reverse transcribed according to the instructions of the Hifair III 1st Stand cDNA Synthesis Super Mix for qPCR (gDNAdigester Plus) kit (YEASEN). The resulting cDNA product was diluted 3-fold and used for real-time quantitative PCR.

[0076] (3) Cloning experiment of BoVIT1 in red-stemmed mustard greens

[0077] Red-legged mustard BoVIT1 cloning primers:

[0078] Upstream primer: 5'-ATGGCTACGGACGAAGATATGATCAC-3' (SEQ ID NO: 3);

[0079] Downstream primer: 5'-TTAATGTTGCACCACTTTAGCCAAACA-3' (SEQ ID NO: 4).

[0080] PCR amplification was performed using a MyCycler™ PCR instrument (BIO-RAD, USA).

[0081] PCR amplification system: containing 0.5 μL Phusion Taq (Thermo Scientific), 0.5 μM upstream / downstream primers, 2 μL cDNA template, 5 μL HF Buffer, 5 μL GC Buffer, 1.5 μL MgCl2, 2 μL dNTPs, and purified water to a final volume of 50 μL.

[0082] The PCR cloning program was as follows: pre-denaturation at 98℃ for 3 min; followed by cycling: denaturation at 98℃ for 45 s, annealing at 56℃ for 30 s, extension at 72℃ for 90 s (33 cycles in total); extension at 72℃ for 6 min.

[0083] Post-amplification gel electrophoresis confirmed that the amplification strips were clear and uniform, referring to Omega Bio-Tek's [technology / method / instructions]. The Cycle-Pure Kit (D6492) PCR product purification and extraction kit instructions were followed to purify the PCR product, detect its concentration, and store the solution at -20°C for later use. Simultaneously, the purified PCR product was sent to Shanghai Sangon Biotech Co., Ltd. for sequencing. The sequence was correct, and the BoVIT1 gene of red-stemmed mustard greens was amplified in this invention.

[0084] (4) Viral silencing (VIGS) experiment of BoVIT1 in red-stemmed mustard greens

[0085] Gene silencing assays were performed using the Nimble Cloning kit from Hainan Yitian Biotechnology Co., Ltd. PCR products were cloned into circular expression variants of the NC system using Nimble Mix via Nimble Cloning (NC cloning). The pNC-TRV2 vector was donated by Dr. Yan Pu (Yan et al., 2020), and its image is shown below. Figure 2 As shown.

[0086] 1) Constructing the vector and designing primers

[0087] Based on the cloned BoVIT1 sequence of Chinese mustard greens, online tools (https: / / vigs.solgenomics.net / ) were used to select target fragments and design primers according to functional domains (following the requirements of NC cloning experiments, 20bp universal adapter sequences were added before and after the primer sequences):

[0088] Upstream primer:

[0089] 5'-agtggtctctgtccagtcctATGGCTACGGACGAAGATATGATCAC-3' (SEQ ID NO: 5;

[0090] Downstream primer:

[0091] 5'-ggtctcagcagaccacaagtTTAATGTTGCACCACTTTAGCCAAACA-3' (SEQ ID NO: 6).

[0092] Target sequence:

[0093] GAGGATCTCTATAGAGCCTGAGAAGCAATCACTTCTCGATCATCACACCGAGAAACACTTCACCGCCGGAGAAGTCGTCCGTGACATCATCGGCGTCTCCGATGGTTTAACCGTTCCTTTTGCACTCGCCGCCGGACTCTCCGGTGCTAATGCCTCTTCT TCCATCGTACTCACCGCTGGTATCGCTGAAGTCGCAGCCGGTGCTATCTCCATGGGACTCGGCGGGTACTTAGCGGCCAAGAGTGAAGCGGATCATTACGCGAGAGATGAAACGAGAACAAGAAGAGATCGTGGCCGTTCCTGAGACTGAGGCAGCTGAGGT GGCAGAGATTCTGGCACAGTATGGAGTAGAGCCACATGAATATTCACCTGTTGTTAATGCTCTTCGTAAGAATCCTCAAGCATGGCTTGATTTTATGATGAGGTTTGAGCTGGGATTAGAGAAGCCAGATCCAAAGAGAGCGTTACAAAGCGCGTTCACGATTGCAATCGCTTATGTTCTTGGCGGTT TAGTACCGCTATTTCCCTACATGTTCATACCACAAGCCTTAAATGCGGTAGTGGCGTCTGCGGCTATAACTCTGATCGCTCTCTTCATATTTGGCTATGCAAAAGGGCATTTCACCGGCAGCAGACCCTTTAGGAGTGCGTTTGAAACCACTTTTATAGGAGCGATAGCTTCAGCAGCTGCTTTC(SEQ ID NO: 11).

[0094] Using cDNA from *Gnaphalium affine* as a template, PCR amplification was performed on the designed adapter-containing primers using a high-fidelity enzyme. The PCR procedure employed a gradient amplification method, following the instructions from Omega Bio-Tek. The primers were purified by PCR using the Cycle-Pure Kit (D6492) PCR product purification and extraction kit. The product was then washed and centrifuged according to the instructions. Finally, 30 μL of Elution Buffer was added and centrifuged to collect the product.

[0095] 2) Construction of the VIGS-VIT1 recombinant vector

[0096] The purified PCR product was mixed with the NC system expression vector TRV2 to prepare a 10 μL system, specifically containing 10-80 ng of PCR product, 20-120 ng of NC system expression vector, 5 μL of Nimble Mix, and ddH2O to a final volume of 10 μL. The mixture was pipetted 10-20 times to ensure thorough mixing, and then incubated at 50°C for 45 min using standard PCR to construct the VIGS-VIT1 recombinant vector.

[0097] The VIGS-VIT1 recombinant vector was transfected into competent cells using DH5α Chemically Cell competent cells (product specification: CAT#: DL1001) from Shanghai Weidi Biotechnology Co., Ltd. The specific steps are as follows:

[0098] Step 1: Follow the instructions for transfection. Then, centrifuge at 5000 rpm for 1 min to collect the recombinant cells, reserving 100 μL of supernatant. In a clean bench, resuspend the bacterial culture and mix thoroughly. Then, spread it evenly on LB solid medium (containing Kan antibiotics). Invert the plate and incubate overnight in a 37°C mold incubator.

[0099] Step 2: After overnight incubation, in a sterilized laminar flow hood, scrape the positive clone single colony from the culture dish and put it into a shaker tube containing 4 mL LB liquid medium (containing Kan antibiotics). Stir well, seal and label, and then place in a constant temperature shaker at 37°C and 280 rpm for overnight incubation.

[0100] Step 3: After the bacterial culture is completed, proceed with... After the PCR bands of the Robust PCR Master Mix (with Dye) are clear, the product can be sequenced for detection (all sequencing in this experiment was performed by Sangon Biotech (Shanghai) Co., Ltd.).

[0101] Plasmids were extracted from bacterial cultures that successfully transformed into the target sequence, using samples from Omega Bio-Tek. The Plasmid Mini KitⅠ(200) was used for the procedure. Approximately 3 mL of bacterial culture was added according to the instructions, and finally 55 μL of Elution Buffer was added and centrifuged at 12000 rpm for 2 min to thoroughly elute the DNA.

[0102] 2. Obtaining transgenic plants

[0103] (1) Recombinant plasmid transformation of Agrobacterium tumefaciens

[0104] In this experiment, GV3101 Chemically Competent Cell competent cells (product specification: CAT#: AC1001) from Shanghai Weidi Biotechnology Co., Ltd. were used for transfection of competent cells. 1 μL of recombinant plasmid was transfected into 50 μL of competent cells. The specific operation method is as follows:

[0105] 1) Follow the instructions for transfection procedures. Then, centrifuge at 6000 rpm for 1 min to collect the bacterial cells, reserve 100 μL of supernatant, resuspend the bacterial culture and mix well, and spread it evenly on YEB solid medium (containing antibiotics Kan and Rif, kanamycin and rifamycin). Invert the plate and incubate it in a 28℃ biochemical incubator for 2-3 days.

[0106] 2) After overnight incubation, scrape a single positive colony from the culture dish into a shaker containing 4 mL of YEB liquid medium (containing Kan and Rif antibiotics) in a sterilized laminar flow hood. Mix well, seal, label, and incubate at 28°C and 220 rpm for 2-3 days. After the bacterial culture is complete, proceed with... Robust PCR Master Mix (with Dye) bacterial culture PCR identification.

[0107] 3) In a clean bench, take 800 μL of bacterial culture and add it to a centrifuge tube containing 800 μL of 50% sterile glycerol. After completing the Agrobacterium preservation operation, store it in a -80℃ freezer.

[0108] (2) The VIGS-VIT1 recombinant vector was transformed into red-stemmed kale via Agrobacterium tumefaciens.

[0109] Following and optimizing the method of Peng Lihua (2019), Agrobacterium-mediated VIGS-VIT1 recombinant vector was used to infect red-stemmed kale plants. Two groups of gene silencing vectors were transfected with Agrobacterium: TRV2-VIT1 and TRV2 empty vector control group; and one uninfected plant was used as a normal control.

[0110] 1) Preparation stage

[0111] Sterilization of containers and treatment of materials: Sterilize 500mL and 1L blue-mouth bottles, 500mL, 1L and 2L conical flasks, ddH2O, 500mL beakers and other containers; withhold water from infected plants two days in advance.

[0112] Solution preparation:

[0113] a. 1M MES stock solution: 19.52g MES powder was used to prepare a solution in 100mL ddH2O (MW: 195.6, pH adjusted to 5.6 with 1M KOH solution, and sterilized by filtration with a water system filter).

[0114] b. 0.2M Acetosyringone stock solution: 39.24 mg Acetosyringone powder was dissolved in 1 mL DMSO (dimethyl sulfoxide) to prepare a solution (MW: 196.2, sterilized by filtration through a 0.22 μm organic pinhole filter).

[0115] c. Preparation of Agrobacterium initial shaking solution: 50 mL of YEB liquid culture medium (containing 50 μg / mL Kan and 25 μg / mL Rif resistance) in a small Erlenmeyer flask, with the flask mouth covered using Erlenmeyer flask sealing film. Preparation of Agrobacterium expansion shaking solution: 500 mL of YEB liquid culture medium (containing 50 μg / mL Kan and 25 μg / mL Rif resistance) in an Erlenmeyer flask, with the flask mouth covered using Erlenmeyer flask sealing film. Both must be sterilized in an autoclave.

[0116] d. Induction Buffer: Prepare a solution by mixing 1 mL of 1M MES stock solution, 200 μL of 0.2M Acetosyringone stock solution and 98.8 mL of ddH2O, adjust the pH to 5.5 with 1M NaOH, and sterilize by filtration through a 0.22 μm filter membrane in a clean bench (prepare immediately before use).

[0117] e. Infection Buffer: Prepare a solution by mixing 0.5 mL of 1 M MES stock solution and 99.5 mL of ddH2O, adjust the pH to 5.5 with 1 M NaOH, and sterilize by filtration through a 0.22 μm aqueous filter membrane in a clean bench (prepare immediately before use).

[0118] 2) Infection process

[0119] This experiment used the leaf injection method, and the culture solution was infused with the following key gene vectors: VIGS-VIT1 recombinant vector, TRV2 (TRV2 vector without gene transfer), and TRV1 (TRV1 vector without gene transfer).

[0120] a. Initial shaking of single Agrobacterium colonies: After culturing single Agrobacterium colonies in a petri dish into a bacterial suspension, take 1 mL of the bacterial suspension and add it to 50 mL of YEB Agrobacterium initial shaking solution. Place the solution in a shaker and shake at 28℃ and 220 rpm for 16-18 h.

[0121] b. Agrobacterium amplification / secondary culture: Take 6 mL of the bacterial culture from the previous step and add it to 500 mL of Agrobacterium amplification solution. Place the mixture on a shaker at 28℃ and 220 rpm for 6-8 hours. During this period, use a UV-Vis spectrophotometer (UV-2600, Shimadzu Instruments (Suzhou) Co., Ltd.) to measure the transmittance of the bacterial culture and obtain the absorbance A of the bacterial culture. 600 When the concentration reaches 0.5-0.6, stop the culture and store in a 4℃ refrigerator for later use (using the shaken solution without added bacterial solution as the standard substrate).

[0122] c. Induction culture: After pre-cooling the secondary bacterial culture to 4°C using a refrigerated centrifuge, A... 600 When the bacterial concentration is 0.5-0.6, collect the Agrobacterium cultured in the previous step by centrifuging at 4°C and 5000 rpm for 10 min using a refrigerated centrifuge into centrifuge tubes. After collection, resuspend the bacterial culture in an induction buffer of equal volume to the secondary bacterial culture, mix well, and then place it on a shaker for incubation at 28°C and 500 rpm for at least 3 h.

[0123] d. Preparation of infection buffer: After pre-cooling the refrigerated centrifuge to 4°C, collect the Agrobacterium bacteria that have completed induction culture by centrifuging at 4°C and 5000 rpm for 10 min in centrifuge tubes. After collection, resuspend the bacterial solution in infection buffer and adjust the concentration of the infection buffer to A. 600 The values ​​are consistent. (Use the contaminated buffer as a blank control).

[0124] e. TRV1 incorporation and bacterial culture: After all three bacterial cultures have been re-vortexed in the infection buffer to the required level, the TRV1 culture is added to the other bacterial cultures at a 1:1 ratio and mixed thoroughly (ensuring A...). 600 (Do not exceed 0.8%), mix well and use directly.

[0125] f. The plant material was Chinese mustard greens. The infected material was deprived of water two days in advance, and then the plant material was grouped and labeled.

[0126] g. Injection method for plant infection: Using a 1mL syringe, the infection solution was drawn up and injected into the cotyledons and two true leaves from the underside of the leaves to obtain TRV2-VIT1 transgenic Chinese mustard green plants.

[0127] h. Post-infection management: Do not water within 3 days after infection. After infection, incubate in the dark at 18℃ for 2 days, then incubate at 1±2℃ for 16 hours of light / 8 hours of darkness, with a relative humidity of 85%.

[0128] Test case

[0129] 1. Application of BoVIT1 protein in regulating the growth rate of red-stemmed kale plants

[0130] The experimental group used TRV2-VIT1 transgenic Chinese mustard greens plants obtained using the method in Example 2, while the control group used wild-type Chinese mustard greens. Each group had 20 plants. The phenotypes of the Chinese mustard greens in both groups were analyzed. The plants were planted in soil and photographed one month later.

[0131] Table 1

[0132] Group Plant height (cm) WT 16.3±0.1 TRV2-VIT1#2 17.6±0.15***

[0133] The results are shown in Table 1. As can be seen from Table 1, inhibiting BoVIT1 protein expression can effectively increase the plant height of Chinese kale, and BoVIT1 protein can be used to regulate the growth of Chinese kale.

[0134] 2. Application of BoVIT1 protein in regulating the color formation of red-stemmed kale stems

[0135] The detection method for the application of BoVIT1 protein in regulating the color formation of red-stemmed kale stems is as follows:

[0136] (1) After infection, observe the changes in stem color phenotype of TRV2-VIT1 infected plants obtained by the method in Example 2, and compare the stem color with that of the TRV2 group (empty control group). After 4 weeks of culture, the stems of the above-mentioned infected groups and the TRV2 group were sampled with liquid nitrogen and stored in a -80℃ freezer.

[0137] (2) Observe the pigment accumulation in the cross section of the stem under a microscope.

[0138] On the day of sample collection, temporary cross-sectional sections of the stems of red-stemmed mustard greens from each group of the VIGS experiment (TRV2-VIT1 group, TRV2 empty control group, and blank control group) were prepared, and the pigment accumulation was observed and photographed using an optical microscope.

[0139] (3) Detection of BoVIT1 gene expression level by real-time PCR

[0140] Total RNA was extracted from the stems of *Gynostemma pentaphyllum*, and cDNA was obtained through reverse transcription. Based on the cloned BoVIT1 gene sequence, specific primers were designed using Primer 3 web version 4.1.0 (https: / / primer3.ut.ee / ) and following the principles of real-time quantitative PCR primer design.

[0141] Upstream primer F: TGATGAGGTTTGAGCTGGGA (SEQ ID NO: 7);

[0142] Downstream primer R: GACGCCACTACCGCATTTAA (SEQ ID NO: 8);

[0143] The internal reference gene is the BoUBI gene (Genbank accession number: XM_013779408):

[0144] upstream primer F for internal reference gene: GGCTGACTACAACATCCAGAAAGAG (SEQ ID NO: 9);

[0145] Downstream primer R for internal reference gene: CTTGGGTGCAGACGAGCATAACAC (SEQ ID NO: 10).

[0146] Using BoUBI as an internal reference gene, quantitative real-time PCR was performed to determine the expression level of the target gene in gene-silenced plants. Melting curves and standard curves were analyzed using relative quantification software. The relative expression level was determined using a 2-1 ratio. -ΔΔCt Legal comparison analysis.

[0147] Primer validation: The amplification efficiency and correlation coefficient of each primer pair were determined using the standard curve method. The experiment used a BIO-RAD real-time PCR system (BIO-RAD, CFX Connect, USA), with a reaction volume of 50 μL containing 25 μL of... Gold Mix (YEASEN), 5 μM upstream / downstream primers, 1.5 μL cDNA template, and sufficient pure water were used. The reaction program was 98℃ pre-denaturation for 3 min, followed by cycling: 98℃ denaturation for 10 s + 68℃ annealing for 15 s (35 cycles in total), and finally extension at 72℃ for 5 min. Post-amplification gel electrophoresis confirmed that the amplified strips were clear and monotonous.

[0148] Real-time quantitative PCR experiment: The reaction procedure mainly consists of two steps: First, pre-denaturation at 95℃ for 5 min; second, denaturation at 95℃ for 10 s, followed by annealing at 60℃ for 30 s. This step is repeated for 40 cycles. A melting curve is generated using the default settings of the quantitative PCR instrument. The cDNA concentration is adjusted according to the Δct cycle value, with fold changes of 2. -ΔΔct For reference, cDNA was diluted with ddH2O, followed by q-PCR to check if Δct was consistently between 20 and 21. Specific primers, the internal reference gene BoUBI, and the diluted cDNA were used for real-time quantitative PCR (3 technical replicates for each gene, 3 biological replicates for each cDNA template, with the internal reference gene used as a control in each reaction). Gene expression levels were calculated using a 2-1... -ΔΔCT The method is used for calculation.

[0149] The detection results of BoVIT1 protein in red-stemmed kale regulating the color formation of kale stems are as follows: Figure 3-4 As shown, from Figure 3-4 As can be seen, after injecting the virus carrying the target gene into the leaves of red-stemmed kale and culturing for 4 weeks, the stem treated with the virus silencing the BoVIT1 gene showed obvious discoloration, and the pigment accumulation in the outermost cells of the stem was significantly reduced, which was in stark contrast to the empty control and the uninfected group. The results indicate that inhibiting the BoVIT1 protein in red-stemmed kale affects the color formation of the kale stem.

[0150] Real-time quantitative RT-PCR detection results are as follows Figure 5 As shown, from Figure 5As can be seen from the real-time quantitative RT-PCR experiment, the expression level of the target gene in the gene-silenced plant was significantly lower than that in the empty vector control.

[0151] The embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the above embodiments, and various changes can be made within the scope of knowledge possessed by those skilled in the art without departing from the spirit of the present invention. Furthermore, the embodiments of the present invention and the features thereof can be combined with each other unless otherwise specified.

Claims

1. Application of BoVIT1 protein in red-stemmed mustard greens in any of the following: (a1) Increase the height of Chinese mustard green plants; (a2) Inhibits the formation of color in the stems of Chinese mustard greens; The amino acid sequence of the BoVIT1 protein from the red-stemmed mustard green is shown in SEQ ID NO:1; The increase in plant height or inhibition of stem color formation in *Gnaphalium affine* is achieved by inhibiting the expression of the gene encoding the BoVIT1 protein in *Gnaphalium affine*; the inhibition of the expression of the gene encoding the BoVIT1 protein in *Gnaphalium affine* is achieved using the VIGS viral vector system. The VIGS viral vector system contains a silencing agent. BoVIT1 Gene target sequences; The silence BoVIT1 The target sequence of the gene is shown in SEQ ID NO:

11.

2. The use of the nucleic acid molecule encoding the BoVIT1 protein of red mustard greens as described in claim 1 in any of the following: (a1) Increase the height of Chinese mustard green plants; (a2) Inhibits the formation of color in the stems of Chinese mustard greens; The nucleotide sequence of the nucleic acid molecule encoding the BoVIT1 protein is shown in SEQ ID NO:2; The increase in plant height or inhibition of stem color formation in *Gnaphalium affine* is achieved by inhibiting the expression of the gene encoding the BoVIT1 protein in *Gnaphalium affine*; the inhibition of the expression of the gene encoding the BoVIT1 protein in *Gnaphalium affine* is achieved using the VIGS viral vector system. The VIGS viral vector system contains a silencing agent. BoVIT1 Gene target sequences; The silence BoVIT1 The target sequence of the gene is shown in SEQ ID NO:

11.

3. A method for increasing the height of Chinese mustard green plants, characterized in that, The method includes the following steps: inhibiting the growth of *Gynostemma pentaphyllum* in *Gynostemma pentaphyllum*. BoVIT1 Gene expression is used to regulate and increase the height of Chinese mustard green plants; The BoVIT1 The nucleotide sequence of the gene is shown in SEQ ID NO:2; The inhibition of Chinese mustard greens BoVIT1 Gene expression is achieved via the VIGS viral vector system; The VIGS viral vector system contains a silencing agent. BoVIT1 Gene target sequences; The silence BoVIT1 The target sequence of the gene is shown in SEQ ID NO:

11.

4. A method for cultivating tall-leafed Chinese mustard green plants, characterized in that, The method includes the following steps: inhibiting the growth of *Gynostemma pentaphyllum* in *Gynostemma pentaphyllum*. BoVIT1 Gene expression; The BoVIT1 The nucleotide sequence of the gene is shown in SEQ ID NO:2; The inhibition of Chinese mustard greens BoVIT1 Gene expression is achieved via the VIGS viral vector system; The VIGS viral vector system contains a silencing agent. BoVIT1 Gene target sequences; The silence BoVIT1 The target sequence of the gene is shown in SEQ ID NO:

11.

5. A method for inhibiting color formation in the stems of Chinese mustard greens, characterized in that, The method includes the following steps: inhibiting the growth of *Gynostemma pentaphyllum* in *Gynostemma pentaphyllum*. BoVIT1 Gene expression is used to suppress the formation of color in the stem of Chinese mustard greens; The BoVIT1 The nucleotide sequence of the gene is shown in SEQ ID NO:2; The inhibition of Chinese mustard greens BoVIT1 Gene expression is achieved via the VIGS viral vector system; The VIGS viral vector system contains a silencing agent. BoVIT1 Gene target sequences; The silence BoVIT1 The target sequence of the gene is shown in SEQ ID NO:11.

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