Brassica oleracea BoVIT1 protein and application thereof

Abstract

The invention discloses a brassica oleracea BoVIT1 protein and an application thereof. The BoVIT1 gene in the brassica alboglabra is knocked out, the transgenic brassica alboglabra shows that the plant height is increased, and the formation of the color of the stem of the brassica alboglabra is inhibited, so that the BoVIT1 protein of the brassica alboglabra plays an important role in improving the quality of the brassica alboglabra. The invention provides a new gene resource for improving the quality of cabbage mustard.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to red-legged kale BoVIT1 protein and application thereof. Background Art

[0002] Brassica oleracea var.alboglabra Bailey is a herbaceous plant of the genus Brassica in the family Cruciferae. It is a variant of cabbage and has become one of the vegetable species introduced and expanded in northern China. It has also been introduced to Europe, America, Oceania, Japan, and Southeast Asian countries. The edible organs of kale are the plump and tender flower stalks and tender leaves. The stalks have a high content of vitamins, soluble proteins, soluble sugars, minerals, mustard oil, etc., and have good edible flavor and nutritional value. However, there are great differences in the flavor and nutrition of different varieties of kale. In addition, kale also has health care functions. It contains a variety of antioxidants, mainly beta-carotene, flavonoids, glucoraphanin, etc.

[0003] Ideal plant type (ideotype) usually refers to the plant type that is conducive to plant 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 take both morphology and function into consideration and applying them to breeding to break the stagnant crop yield is an urgent research goal at present. Therefore, it is of great significance to explore and utilize the functional genes that control the plant type of kale plants. Summary of the invention

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

[0005] The present invention also provides the application of the nucleic acid molecule encoding the red-legged kale BoVIT1 protein.

[0006] The present invention also provides biological materials related to the above nucleic acid molecules.

[0007] The present invention also provides a primer set for amplifying a nucleic acid molecule encoding the red-legged kale BoVIT1 protein.

[0008] The invention also provides a method for regulating the height of kale plants.

[0009] The invention also provides a method for cultivating tall kale plants.

[0010] The invention also provides a method for regulating the color formation of kale stems.

[0011] The invention also provides a product.

[0012] The invention also proposes the application of the product.

[0013] In the first aspect of the present invention, the use of red-footed kale BoVIT1 protein in any of the following items is proposed:

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

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

[0016] The red-foot kale BoVIT1 protein is the following protein a) or b) or c) or d):

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

[0018] b) a fusion protein obtained by connecting 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) a protein having the same function obtained by replacing 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 that is 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%, 70% identical to the amino acid sequence as shown in SEQ ID NO:1 and has the same function as the protein as shown in SEQ ID NO:1.

[0021] In the second aspect of the present invention, the use of a nucleic acid molecule encoding the red-legged kale BoVIT1 protein in any of the following items is proposed:

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

[0023] (a2) Application in regulating the color formation of 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) the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate sequence thereof;

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

[0027] In some embodiments of the present invention, the above (b2) is: a nucleotide sequence derived from the nucleotide sequence defined in (b1) by replacing, deleting or adding one or several (within 10) nucleotides, and encoding a polypeptide having the same or similar function as the sequence of SEQ ID NO:1.

[0028] In the third aspect of the present invention, a biological material related to the above-mentioned nucleic acid molecule is provided, which is any one of the following 1) to 7):

[0029] 1) An expression cassette containing the above nucleic acid molecule;

[0030] 2) a recombinant vector containing the above nucleic acid molecule;

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

[0032] 4) A recombinant microorganism containing the above nucleic acid molecule;

[0033] 5) a recombinant microorganism containing the expression cassette described in 1);

[0034] 6) a recombinant microorganism containing the recombinant vector described in 2);

[0035] 7) A recombinant microorganism containing the recombinant vector described in 3).

[0036] In a fourth aspect of the present invention, a primer set for amplifying a nucleic acid molecule encoding the above-mentioned red-legged kale BoVIT1 protein is provided.

[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 present invention, a method for regulating the height of kale plants is proposed, the method comprising the following steps: promoting or inhibiting the expression of BoVIT1 gene in kale to regulate the height of kale plants.

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

[0040] In a sixth aspect of the present invention, a method for cultivating tall Chinese kale plants is provided, the method comprising the following steps: 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 BoVIT1 gene in kale to regulate the color formation of kale stems.

[0042] In some embodiments of the present invention, the inhibition of the expression of the BoVIT1 gene in red-legged kale is achieved through a 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 sequence of the target sequence for silencing the BoVIT1 gene is shown as SEQ ID NO:11.

[0045] In an eighth aspect of the present 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 invention, the biological material comprises a VIGS viral vector system.

[0048] In the ninth aspect of the present invention, the application of the above product in any of the following items is proposed:

[0049] (c1) plant breeding;

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

[0051] (c3) regulating plant height;

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

[0053] In some embodiments of the invention, the plant comprises Brassica juncea.

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

[0055] According to the embodiments of the present invention, at least the following beneficial effects are achieved: the present invention discloses the application of red-foot kale BoVIT1 protein in regulating kale plant height and stem color formation, and the red-foot kale with the red-foot kale BoVIT1 gene knocked out shows an increase in plant height and inhibits kale stem color formation, indicating that the red-foot kale BoVIT1 protein plays an important role in improving kale quality. The present invention provides a new gene resource for kale quality improvement. BRIEF DESCRIPTION OF THE DRAWINGS

[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 kale in Example 1 of the present invention;

[0058] Figure 2 This is the pNC-TRV2 vector map in Example 2 of the present invention;

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

[0060] Figure 4 This is a graph showing the results of analyzing the pigment accumulation in the cross section of the stem of Brassica juncea after the BoVIT1 gene was silenced in the test example of the present invention;

[0061] Figure 5 This is a graph showing the analysis results of the expression level of the target gene in the stem after silencing of the BoVIT1 gene in the test example of the present invention. DETAILED DESCRIPTION

[0062] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0063] Example 1 Red-footed kale BoVIT1 protein

[0064] This example provides a red-foot kale BoVIT1 protein, the amino acid sequence of which is as follows:

[0065] Red kale BoVIT1 protein sequence:

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

[0067] Nucleotide sequence encoding the BoVIT1 protein:

[0068] (SEQ ID NO:2).

[0069] Example 2 Construction of Silencing Expression Vector of BoVIT1 Gene of Red-footed Kale and Transgenic Plants

[0070] 1. Construction of silencing expression vector of BoVIT1 gene of red-foot kale

[0071] The specific steps for constructing the silencing expression vector of the red-foot kale BoVIT1 gene are as follows:

[0072] (1) Collection of red-foot kale materials

[0073] Red-footed kale material (growth diagram of small-leaf kale at different stages) Figure 1 The plant culture room conditions were as follows: the temperature was 22°C, the photoperiod was 14h light / 10h dark, and the relative humidity was 85%.

[0074] (2) Extraction of total RNA

[0075] RNA was extracted from the stems of red kale seedlings of normal growth for 1 month. The RNA extraction and purification process was carried out according to the plant RNA rapid extraction kit (Beijing Huayueyang Biotechnology Co., Ltd., Beijing), and the specific steps were carried out according to the instructions. 1 μg of total RNA was taken and reverse transcribed according to the instructions of the HifairⅢ1st Stand cDNA Synthesis Super Mix for qPCR (gDNAdigester Plus) kit (YEASEN Company). The cDNA product obtained by reverse transcription was diluted 3 times and used for real-time fluorescence quantitative PCR.

[0076] (3) Cloning experiment of red kale BoVIT1

[0077] Red-footed kale 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 detected using MyCyclerTM PCR instrument (BIO-RAD, USA).

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

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

[0083] After amplification, gel electrophoresis confirmed that the amplified bands were clear and single, referring to the Cycle-Pure Kit (D6492) PCR product purification and extraction kit instructions purify the PCR product, detect the concentration and store the solution in a -20°C refrigerator for use; at the same time, the purified PCR product is sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequence is correct, and the red-legged kale BoVIT1 gene is amplified by the present invention.

[0084] (4) Virus silencing (VIGS) experiment of red kale BoVIT1

[0085] The gene silencing experiment was performed using the Nimble Cloning kit of Hainan Yitian Biotechnology Co., Ltd. The PCR product was cloned into the circular expression body of the NC system through Nimble Cloning (NC cloning) using Nimble Mix. The pNC-TRV2 vector was donated by Dr. Yan Pu (Yan et al., 2020). The vector map is shown in Figure 2 shown.

[0086] 1) Construction of vector primer design

[0087] Based on the cloned K. oleracea BoVIT1 sequence, an online tool (https: / / vigs.solgenomics.net / ) was used to select target fragments and design primers according to the functional domain (according to the requirements of the NC cloning experiment, a 20 bp universal adapter sequence was added before and after the primer sequence):

[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] (SEQ ID NO: 11).

[0094] Using the cDNA of red-footed kale as a template, the designed primers with adapters were PCR amplified using a high-fidelity enzyme, and the PCR step was amplified using the gradient method. The primer PCR was purified using the Cycle-Pure Kit (D6492) PCR product purification and extraction kit, and the product was then washed and centrifuged according to the instructions. Finally, 30 μL Elution Buffer was added and collected by centrifugation.

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

[0096] The purified PCR product was mixed with the NC system expression vector TRV2 to form a 10 μL system. The specific system is as follows: 10-80 ng of PCR product, 20-120 ng of NC system expression vector, 5 μL of Nimble Mix, and ddH2O was added to 10 μL. The above system was pipetted 10-20 times to mix it thoroughly, and ordinary PCR was used to react at 50°C for 45 minutes to construct the VIGS-VIT1 recombinant vector.

[0097] The DH5ɑ Chemically Cell competent cells (product specification: CAT#: DL1001) from Shanghai Weidi Biotechnology Co., Ltd. were used to transfect the VIGS-VIT1 recombinant vector competent cells. The specific steps are as follows:

[0098] Step 1: Transfection The specific operation method is carried out according to the instructions, and then the recombinant bacteria are collected by centrifugation at 5000rpm for 1min, and 100μL of supernatant is reserved. Operate in a clean bench, resuspend the bacterial solution and mix it thoroughly, then evenly apply it on the LB solid medium (containing Kan antibiotics), and place the plate upside down in a 37℃ mold incubator for overnight culture.

[0099] Step 2: After overnight culture, scrape out the positive clone colony from the culture dish in a sterilized clean bench and put it into a shaking tube containing 4 mL LB liquid culture medium (containing Kan antibiotics), shake it evenly, seal it, mark it, and put it into a constant temperature shaker at 37°C, 280 rpm for overnight culture.

[0100] Step 3: After the bacterial liquid is cultivated PCR Master Mix (with Dye) PCR identification bands are clear and the products can be sequenced (all sequencing in this experiment was performed by Sangon Biotech (Shanghai) Co., Ltd.).

[0101] The plasmid was extracted from the bacterial solution that successfully transferred the target sequence in the sequencing test results. The experiment used Omega Bio-Tek's Plasmid Mini KitⅠ(200) was used for the operation. About 3 mL of bacterial solution was operated according to the instructions, and finally 55 μL of Elution Buffer was added and centrifuged at 12000 rpm for 2 min to fully elute the DNA.

[0102] 2. Obtaining transgenic plants

[0103] (1) Transformation of Agrobacterium tumefaciens with recombinant plasmid

[0104] This experiment uses GV3101 Chemically Competent Cell (product specification: CAT#: AC1001) from Shanghai Weidi Biotechnology Co., Ltd. as the competent cell for transfection. Take 1 μL of recombinant plasmid and transfer it into 50 μL competent cell. The specific operation method is as follows:

[0105] 1) The specific operation method of transfection was carried out according to the instructions. Then, the cells were collected by centrifugation at 6000 rpm for 1 min, 100 μL of supernatant was reserved, the bacterial solution was resuspended and mixed, and evenly applied to the YEB solid culture medium (containing antibiotics Kan and Rif, kanamycin and rifamycin), and the plate was inverted and placed in a 28°C biochemical incubator for 2-3 days.

[0106] 2) After overnight culture, scrape out the positive clone colony from the culture dish in a sterilized clean bench and put it into a shaking tube containing 4 mL YEB liquid culture medium (containing Kan and Rif antibiotics), shake it evenly, seal it, mark it, and put it into a constant temperature shaker at 28°C and 220rpm for 2-3 days. PCR Master Mix (with Dye) bacterial solution PCR identification.

[0107] 3) Take 800 μL of bacterial solution in a clean bench and add it to a centrifuge tube containing 800 μL of 50% sterilized glycerol. After completing the Agrobacterium seed preservation operation, store it in a -80°C refrigerator.

[0108] (2) Transformation of VIGS-VIT1 recombinant vector into red kale via Agrobacterium

[0109] Referring to the method of Peng Lihua (2019) and optimizing it, the Agrobacterium-mediated VIGS-VIT1 recombinant vector was used to infect red-legged kale plants. A total of two groups of gene silencing vector transfection experiments with Agrobacterium were conducted, namely: TRV2-VIT1, TRV2 empty control group; and one uninfected plant as a normal control.

[0110] 1) Preparation

[0111] Sterilization of utensils and material treatment: Sterilize 500mL, 1L blue-mouth bottles, 500mL, 1L, 2L conical flasks, ddH2O, 500mL beakers and other utensils; cut off water for 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 with 100mL ddH2O (MW: 195.6, pH 5.6 was adjusted with 1M KOH solution, and sterilized by filtration with a water filter);

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

[0115] c. Preparation of Agrobacterium initial shaking solution: 50mL (containing 50μg / mL Kan and 25μg / mL Rif resistance) YEB liquid culture medium in a small conical bottle, cover the bottle mouth with a conical bottle sealing film. Preparation of Agrobacterium expansion shaking solution: 500mL (containing 50μg / mL Kan and 25μg / mL Rif resistance) YEB liquid culture medium, cover the bottle mouth with a conical bottle sealing film. Both need to be sterilized in a high-temperature sterilizer.

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

[0117] e. Infection Buffer: 0.5 mL 1 M MES stock solution and 99.5 mL ddH2O are prepared into a solution, and the pH is adjusted to 5.5 with 1 M NaOH. Sterilize the solution by filtering with a 0.22 μm water filter membrane in a clean bench (prepare and use immediately).

[0118] 2) Infection process

[0119] The experiment was conducted using the leaf injection method, and key gene vectors were added to the culture medium: VIGS-VIT1 recombinant vector, TRV2 (TRV2 vector without gene transfer), and TRV1 (TRV1 vector without gene transfer).

[0120] a. Initial shaking of a single Agrobacterium colony: After the single Agrobacterium colony cultured in the culture dish is cultured into a bacterial liquid, 1 mL of the bacterial liquid is added to 50 mL of YEB Agrobacterium initial shaking solution, and the mixture is placed in a shaker and shaken at 28°C and 220 rpm for 16-18 hours.

[0121] b. Agrobacterium propagation / secondary bacterial solution: 6 mL of the bacterial solution cultured in the previous step was added to 500 mL of Agrobacterium propagation solution, and the solution was placed on a shaker at 28°C and 220 rpm for 6-8 h. During this period, the transmittance of the bacterial solution was detected using a UV-visible spectrophotometer (UV-2600, Shimadzu Instruments (Suzhou) Co., Ltd.) to obtain an absorbance of 1:1. 600 =0.5-0.6, the culture was stopped and placed in a 4°C refrigerator for later use (the propagation solution without bacterial solution was used as the standard substrate).

[0122] c. Induction culture: Precool the refrigerated centrifuge to 4°C in advance, and then 600 =0.5-0.6, use a refrigerated centrifuge to collect the Agrobacterium cultured in the previous step in a centrifuge tube at 4°C, 5000rpm for 10min. After the bacterial liquid is collected, resuspend the bacterial liquid with an equal volume of induction buffer as the secondary bacterial liquid, mix well and place on a shaker for culture at 28°C, 500rpm, and induce for at least 3h.

[0123] d. Preparation of infection solution: Pre-cool the refrigerated centrifuge to 4°C in advance, and use the refrigerated centrifuge to centrifuge the induced Agrobacterium at 4°C, 5000 rpm for 10 minutes and collect it in a centrifuge tube. After the bacterial solution is collected, resuspend it in the infection buffer and adjust the concentration of the infection solution to A. 600 The values ​​were consistent. (Infection Buffer was used as blank control).

[0124] e. Mixing of TRV1 and culture of bacterial solution: After all three bacterial solutions are re-spinned in the infection buffer to meet the requirements, add TRV1 bacterial solution to the other bacterial solutions at a ratio of 1:1 and mix well (to ensure that A 600 Not more than 0.8), mix well and use directly.

[0125] f. The plant materials are: small leaf kale. The infected materials were deprived of water two days in advance, and then the plant materials were grouped and marked.

[0126] g. Injection method to infect plants: Use a 1 mL syringe to draw up the infection solution, and inject the cotyledon and two true leaves from the back of the leaf to obtain TRV2-VIT1 transgenic kale plants.

[0127] h. Post-infection management: watering is strictly prohibited within 3 days after infection. Incubate in dark at 18℃ for 2 days after infection, then incubate at 1±2℃ with 16h light / 8h dark, relative humidity 85%.

[0128] Test Case

[0129] 1. Application of BoVIT1 protein in regulating the growth of kale strains

[0130] The experimental group used the TRV2-VIT1 transgenic kale plants obtained by the method of Example 2, and the control group used wild-type kale plants, with 20 plants in each group, and the phenotypes of the kale plants in the experimental group and the control group were analyzed. The plants were planted in soil and photographed after 1 month.

[0131] Table 1

[0132] Group Plant heightcm WT 16.3±0.1 TRV2-VIT1#2 17.6±0.15***

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

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

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

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

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

[0138] On the day of sampling, temporary sections of the stems of red-footed kale in each group of the VIGS experiment (TRV2-VIT1 group, TRV2 empty control group and blank control group) were made, and the pigment accumulation was observed under an optical microscope and photographed.

[0139] (3) Detection of BoVIT1 gene expression by fluorescence quantitative PCR

[0140] Total RNA was extracted from the stems of red-footed kale, and cDNA was obtained by reverse transcription. Based on the cloned BoVIT1 gene sequence, specific primers were designed using the webpage Primer 3web version 4.1.0 (https: / / primer3.ut.ee / ) according to the real-time fluorescence quantitative primer design principle:

[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] Internal reference gene upstream primer F (SEQ ID NO: 8): GGCTGACTACAACATCCAGAAAGAG (SEQ ID NO: 9);

[0145] Internal reference gene downstream primer R (SEQ ID NO: 9): CTTGGGTGCAGACGAGCATAACAC (SEQ ID NO: 10).

[0146] Using BoUBI as the internal reference gene, a fluorescence quantitative PCR experiment was performed to obtain the target gene expression level of the gene silenced plants, and the melting curve and standard curve of the experiment were analyzed using relative quantitative software. The relative expression level was calculated using 2 -ΔΔCt Comparative analysis.

[0147] Primer verification: The standard curve method was used to detect the amplification efficiency and correlation coefficient of each primer pair. The experiment used the BIO-RAD fluorescent quantitative PCR system (BIO-RAD, CFX Connect, USA), and the reaction system was 50 μL, including 25 μL 2×Hieff Mix (YEASEN), 5μM upstream / downstream primers, 1.5μL cDNA template and appropriate amount of pure water. The reaction procedure was 98℃ pre-denaturation for 3min, followed by a cycle of: 98℃ denaturation for 10s + 68℃ annealing for 15s (35 cycles in total), and finally 72℃ extension for 5min. After amplification, gel electrophoresis confirmed that the amplified strip was clear and single.

[0148] Real-time fluorescence quantitative experiment: The reaction procedure is mainly divided into two steps: the first step is 95℃ high temperature pre-denaturation for 5min; the second step is 95℃ denaturation for 10s, 60℃ annealing for 30s, and this step is repeated for 40 cycles; the melting curve is made by the default setting of the fluorescence quantitative PCR instrument. The cDNA concentration is adjusted according to the Δct cycle value, with a multiple change of: 2 -ΔΔct For reference, cDNA was diluted with ddH2O, and then q-PCR was performed to detect whether Δct was between 20 and 21. Specific primers, internal reference gene BoUBI and diluted cDNA were used for real-time fluorescence quantitative detection reaction (each gene was repeated 3 times, each cDNA template was repeated 3 times, and each reaction used the internal reference gene as a control). Gene expression was calculated using 2 -ΔΔCT Method to calculate.

[0149] The results of the detection of red-foot kale BoVIT1 protein in regulating the color formation of kale stems are as follows Figure 3-4 As shown, from Figure 3-4 It can be seen that after the virus carrying the target gene was injected into the leaves of red-legged kale, after 4 weeks of culture, the stems treated with the virus silencing the BoVIT1 gene showed obvious fading, and the pigment accumulation in the outermost layers of cells in the stems was significantly reduced, which was in sharp contrast to the empty control treatment and the uninfected group. The results showed that inhibiting the BoVIT1 protein of red-legged kale would affect the color formation of the kale stems.

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

[0151] The embodiments of the present invention are described in detail above in conjunction with the accompanying drawings, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. Use of red-footed kale BoVIT1 protein in any of the following: (a1) regulating the height of kale plants; (a2) regulating the color formation of kale stems; The red-foot kale BoVIT1 protein is a protein described in any one of the following a)-d): a) a protein with an amino acid sequence as shown in SEQ ID NO: 1; b) a fusion protein obtained by connecting 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; c) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 1; d) an amino acid sequence that is 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%, 70% identical to the amino acid sequence as shown in SEQ ID NO:1 and has the same function as the protein as shown in SEQ ID NO:

1.

2. Use of the nucleic acid molecule encoding the red-legged kale BoVIT1 protein according to claim 1 in any of the following: (a1) regulating the height of kale plants; (a2) regulating the color formation of kale stems; Preferably, the nucleic acid molecule encoding the BoVIT1 protein has the following nucleotide sequence: (b1) the nucleotide sequence shown in SEQ ID NO: 2 or a degenerate sequence thereof; (b2) A nucleotide sequence derived from the nucleotide sequence defined in (b1) by substitution, deletion or addition of at least one nucleotide, and encoding a polypeptide having the same or similar function as the sequence of SEQ ID NO:

2.

3. The biological material related to the nucleic acid molecule according to claim 2, which is any one of the following 1) to 7): 1) An expression cassette containing the nucleic acid molecule according to claim 2; 2) a recombinant vector containing the above nucleic acid molecule; 3) a recombinant vector containing the expression cassette described in 1); 4) A recombinant microorganism containing the above nucleic acid molecule; 5) a recombinant microorganism containing the expression cassette described in 1); 6) a recombinant microorganism containing the recombinant vector described in 2); 7) A recombinant microorganism containing the recombinant vector described in 3).

4. A primer set for amplifying the nucleic acid molecule according to claim 2; Preferably, 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.

5. A method for regulating the height of kale plants, characterized in that: The method comprises the following steps: promoting or inhibiting the expression of BoVIT1 gene in kale to regulate the height of kale plants.

6. A method for cultivating tall Chinese kale plants, characterized in that: The method comprises the following steps: inhibiting the expression of BoVIT1 gene in kale.

7. A method for regulating the color formation of kale stems, characterized in that: The method comprises the following steps: promoting or inhibiting the expression of BoVIT1 gene in kale to regulate the color formation of kale stem.

8. The method according to any one of claims 5 to 7, characterized in that: The inhibition of the expression of the BoVIT1 gene in red-legged kale is achieved through a VIGS virus vector system. Preferably, the VIGS viral vector system contains a target sequence for silencing the BoVIT1 gene; More preferably, the sequence of the target sequence for silencing the BoVIT1 gene is as shown in SEQ ID NO:

11.

9. A product, characterized in that A biological material containing a target sequence for silencing the BoVIT1 gene or containing a target sequence for silencing the BoVIT1 gene; Preferably, the sequence of the target sequence for silencing the BoVIT1 gene is as shown in SEQ ID NO:

11.

10. Use of the product according to claim 9 in any of the following: (c1) plant breeding; (c2) preparing products for plant-assisted plant breeding; (c3) regulating plant height; (c4) Regulates the color formation of plant stems.

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