Method for preparing self-compatible Chinese cabbage by inhibiting BrTHE1 gene expression

Abstract

The invention discloses a method for preparing self-compatible Chinese cabbage by inhibiting BrTHE1 gene expression, and belongs to the technical field of gene engineering. The method disclosed by the invention comprises the following steps: down-regulating, inhibiting or reducing coding gene expression of BrTHE1 protein in a receptor Chinese cabbage or regulating and controlling the activity and / or content of the protein, so that the receptor Chinese cabbage is self-compatible; the amino acid sequence of the BrTHE1 protein is SEQ ID NO: 1. The material obtained by the method is a self-compatible plant, and the self-compatible Chinese cabbage material has important value for Chinese cabbage crossbreeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to a method for preparing self-compatible Chinese cabbage by inhibiting the expression of BrTHE1 gene. Background Art

[0002] Chinese cabbage is one of the important vegetable crops in China. In production, Chinese cabbage is bred through heterosis breeding, and self-incompatible line breeding is one of the main breeding methods. However, self-incompatible line breeding often requires a strong self-incompatible line as a parent, which greatly increases the difficulty of breeding the parent itself. The commonly used method for breeding self-incompatible lines is to spray brine, but such a rough breeding method easily leads to impure seeds. Pollination by removing flower buds at the bud stage can overcome the disadvantage of impure seeds, but this will consume huge human and material costs.

[0003] Therefore, providing a method for creating self-compatible Chinese cabbage materials is an urgent problem to be solved by those skilled in the art. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a method for creating self-compatible Chinese cabbage materials by RNA interference. The technical problems to be solved are not limited to the described technical themes, and those skilled in the art can clearly understand other technical themes not mentioned herein through the following description.

[0005] To solve the above technical problems, the present invention provides the following technical solutions: The present invention provides a method for preparing self-compatible Chinese cabbage, which down-regulates or inhibits or reduces the expression of the coding gene of BrTHE1 protein in the recipient Chinese cabbage or regulates the activity and / or content of the protein, so as to obtain self-compatible Chinese cabbage, and the recipient Chinese cabbage is self-incompatible; the BrTHE1 protein is any one of the following: A1) a protein with an amino acid sequence of SEQ ID NO:1; A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID No.1, having more than 80% identity with the protein shown in A1) and having the same function; A3) a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0006] In the above method, the recipient Chinese cabbage contains the coding gene.

[0007] The present invention also provides a method for improving the self-compatibility of Chinese cabbage, which down-regulates, inhibits or reduces the expression of the coding gene of BrTHE1 protein in the target Chinese cabbage or regulates the activity and / or content of the protein, so that the target Chinese cabbage changes from a self-incompatible plant to a self-compatible plant. The BrTHE1 protein is any one of the following: A1) a protein with an amino acid sequence of SEQ ID NO:1; A2) a protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID No.1, having more than 80% identity with the protein shown in A1) and having the same function; A3) a fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

[0008] In the above method, the target Chinese cabbage contains the coding gene.

[0009] The tag proteins described herein include but are not limited to: GST (glutathione S-transferase) tag protein, His6 tag protein (His-tag), MBP (maltose binding protein) tag protein, Flag tag protein, SUMO tag protein, HA tag protein, Myc tag protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag tag protein.

[0010] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the above protein of the present invention by using known methods, such as directed evolution or point mutation. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the above protein isolated from the present invention, as long as they encode the above protein and have the function of the above protein, are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0011] The above 75% or more identity can be 80%, 85%, 90% or 95% or more identity.

[0012] In this article, identity refers to the identity of amino acid sequences or nucleotide sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, and setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search, the identity value (%) of the amino acid sequence can be calculated.

[0013] In this article, the identity of more than 80% can be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0014] In the above method, down-regulating or inhibiting or reducing the expression of the coding gene of the protein in the receptor Chinese cabbage or regulating the activity and / or content of the protein includes introducing a substance that down-regulates or inhibits or reduces the expression level of the coding gene of BrTHE1 protein into the receptor Chinese cabbage.

[0015] In the above method, the substance is any one of the following: B1), an RNA molecule that inhibits or reduces or down-regulates the expression of the coding gene of the protein or an RNA molecule that inhibits or reduces or down-regulates the activity or content of the protein; B2), the coding gene expressing the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

[0016] In the above method, the RNA molecule described in B1) targets the mRNA transcribed from the coding gene of the aforementioned protein.

[0017] In the above method, the nucleotide sequence of the coding gene of the RNA molecule is nucleotides 1398 to 1647 of SEQ ID NO:2.

[0018] The present invention also provides a biological material related to the aforementioned protein, which is any one of the following: B1), an RNA molecule that inhibits, reduces or down-regulates the expression of the coding gene of the protein, or an RNA molecule that inhibits, reduces or down-regulates the activity or content of the protein; B2), the coding gene that expresses the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), a transgenic plant tissue containing the gene described in B2), or a transgenic plant tissue containing the expression cassette described in B3), or a transgenic plant tissue containing the recombinant vector described in B4); B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

[0019] Among the above-described methods or the above-described biological materials, the expression cassette containing a nucleic acid molecule as described in B3) refers to DNA that can express the above-described RNA molecule in a host cell. The expression cassette may further include a single-stranded or double-stranded nucleic acid molecule of all regulatory sequences necessary for expressing the nucleic acid molecule of any one of the above-described proteins or the DNA of the above-described RNA molecule. The regulatory sequences can direct the coding sequence to express any one of the above-described proteins or the DNA of the above-described RNA molecule in a suitable host cell under their compatible conditions. The regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences should include a promoter and transcription and translation termination signals. To introduce specific restriction enzyme sites into the vector for ligating the regulatory sequences to the coding region of the nucleic acid sequence encoding a protein or the DNA of the above-described RNA molecule, regulatory sequences with linkers can be provided. The regulatory sequence can be a suitable promoter sequence, i.e., a nucleic acid sequence recognizable by the host cell expressing the nucleic acid sequence. The promoter sequence contains transcriptional regulatory sequences that mediate the expression of the protein or the DNA of the above-described RNA molecule. The promoter can be any nucleic acid sequence having transcriptional activity in the selected host cell, including mutated, truncated, and hybrid promoters, and can be derived from genes encoding extracellular or intracellular proteins homologous or heterologous to the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3'-end of the nucleic acid sequence encoding a protein or the DNA of the above-described RNA molecule. Any terminator that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a suitable leader sequence, i.e., the untranslated region of mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5'-end of the nucleic acid sequence encoding a protein or the DNA of the above-described RNA molecule. Any leader sequence that can function in the selected host cell can be used in the present invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence linked to the amino terminus of the protein and can direct the protein or the DNA of the above-described RNA molecule into the cell secretion pathway. Any signal peptide coding region that can direct the expressed protein or the DNA of the above-described RNA molecule into the secretion pathway of the host cell used can be used in the present invention. It may also be necessary to add regulatory sequences that can regulate the expression of the protein or the DNA of the above-described RNA molecule according to the growth of the host cell. Examples of regulatory sequences are those systems that can respond to chemical or physical stimulants (including in the presence of regulatory compounds) to turn on or off gene expression. Other examples of regulatory sequences are those that can amplify genes.

[0020] Among the above-described methods or the above-described biological materials, the vector can be a plasmid, cosmid, phage, or viral vector.

[0021] In the above method or the above biological material, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0022] In the above method or the above biological material, the transgenic plant cell line does not include any propagation material.

[0023] In the above method or the above biological material, the transgenic plant organ in B8) can specifically be the stigma of cabbage.

[0024] In the above biological material, B1) the RNA molecule targets the mRNA transcribed from the gene encoding the above protein.

[0025] The present invention also provides an application, comprising any of the following applications of the aforementioned biomaterial: D1) Change the plant from a self-incompatible plant to a self-compatible plant; D2) preparing a product for converting a self-incompatible plant into a self-compatible plant; D3) Cultivate self-compatible plants; D4) preparing products for breeding self-compatible plants; D5) Plant breeding.

[0026] In the above application, the purpose of plant breeding is to break the self-incompatibility barrier of plants and transform self-incompatible plants into self-compatible plants.

[0027] In the above application, the plant is any one of the following: M1) dicots or monocots; M2) Cruciferae; M3) Cruciferae; M4) Brassica plants; M5) Cabbage.

[0028] In certain specific embodiments, the Brassica rapa is the inbred line 14CR.

[0029] The advantage of the present invention is that the present invention reveals for the first time the biological function of the cabbage BrTHE1 gene and silences / downregulates the cabbage gene through RNA interference technology. BrTHE1 The invention also aims to develop a new method for the self-incompatibility of Chinese cabbage, which can be used to generate transgenic materials containing the target gene. The invention also aims to develop self-compatible Chinese cabbage materials that can stably overcome the self-incompatibility of Chinese cabbage, broadening the scope for the propagation of self-incompatible lines and thus providing important breeding value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 For different RNAi strains BrTHE1 Statistical results of relative gene expression.

[0031] Figure 2 The growth of self-pollen tubes observed by aniline blue staining and the number of pollen tubes passing through the stigma. The left figure is a diagram showing the aniline blue staining of the stigma, and the right figure is a statistical chart of the number of self-pollen tubes passing through each stigma.

[0032] Figure 3 The number of developing seeds obtained in each silique of Chinese cabbage. The left figure is a diagram showing the seeds, and the right figure is a statistical chart of the number of seeds in each silique.

[0033] Figure 4 It is the map of the pBWA(V)HII-SLR-THE_rnai-TNOS vector. Specific Embodiments

[0034] The present invention will be further described in detail below in conjunction with specific embodiments. The examples given are only for clarifying the present invention and not for limiting the scope of the present invention. The following examples can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0035] The experimental methods in the following examples are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified.

[0036] In the following examples, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0037] The following examples use GraphPad Prism 8.0.1 statistical software to process the data. The experimental results are expressed as mean ± standard error. A two-tailed t test is used. 0.01 < P < 0.05 (*) indicates a difference, and P < 0.01 (**) indicates a significant difference.

[0038] Table 1. Instrument and Equipment

[0039] Table 2. Experimental Reagents

[0040] Example 1. Obtaining of Transgenic Chinese Cabbage I. RNA Interference Plasmid The recombinant vector pBWA(V)HII-SLR-THE_rnai-TNOS (the vector map is as Figure 4As shown in the figure, a recombinant vector was obtained by inserting a DNA fragment with a nucleotide sequence of SEQ ID NO:3 between the SLR promoter and the Tnos terminator of the pBWA(V)HII-SLR-TNOS backbone vector while keeping other nucleotides unchanged. The nucleotide sequence of the recombinant vector pBWA(V)HII-SLR-THE_rnai-TNOS is shown in the following "Nucleotide sequence of pBWA(V)HII-SLR-THE_rnai-TNOS". Among them, nucleotides 2353 to 3863 are the SLR promoter, and nucleotides 3864 to 4563 are SEQ ID NO:3. In SEQ ID NO:3, nucleotides 1 to 250 (forward sequence) target nucleotides 1398 to 1647 of SEQ ID NO:2, nucleotides 451 to 700 (reverse sequence) are the sequence reverse complementary to the aforementioned forward sequence, and nucleotides 251 to 450 are the loop connecting the aforementioned forward sequence and reverse sequence.

[0041] II. Preparation of transgenic Chinese cabbage 1. Preparation of Agrobacterium 1.1 Plasmid transformation Take 1 μL of plasmid (recombinant vector pBWA(V)HII-SLR-THE_rnai-TNOS) and add it to 50 μL of GV3101 Agrobacterium competent cells. After thorough mixing, transfer it to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix well, and transfer it to a 1.5 mL centrifuge tube. Incubate it on a shaker at 30 °C and 180 rpm for 30 min. Pipette 50 μL of the activated Agrobacterium liquid and inoculate it on an LB solid medium, and incubate it in the dark at 30 °C for 48 h.

[0042] 1.2 Detection of Agrobacterium 1.2.1 Synthesis of detection primers: HG-F: 5'-gctccaccatgttggcaagc-3' (2292 bp); E1427(553C): 5'-gcactcaatcatgagaagaagtgaaat-3' (2996 bp); 1.2.2 As shown in the following table, prepare the PCR amplification system. After completion, mix well thoroughly and use a PCR instrument for amplification. The amplification program is set accordingly according to primer information, etc.

[0043] Table 3. PCR amplification system

[0044] 1.2.3 Gel electrophoresis detection: Prepare 1% agarose gel (weigh 1.5 g of agarose powder, dissolve it in 150 mL of 1×TAE buffer, heat it in a microwave oven for about 3 minutes until the liquid becomes transparent. Add EB to the gel-making plate, pour the dissolved agarose liquid into the gel-making plate, mix well, insert a comb, and let it stand for 40 minutes until the gel turns milky white), load the samples, and complete the electrophoresis process; 1.2.4 View the PCR amplification results. When the electrophoresis bands of the positive control and the samples are clear, the sizes are correct, and there are no bands in the negative control, it indicates that the samples can enter the next step. Name the Agrobacterium with a positive plasmid PCR amplification result as GV3101 / pBWA(V)HII-SLR-THE_rnai-TNOS.

[0045] 2. Genetic transformation of Chinese cabbage Unless otherwise specified, the following reagents are all conventional reagents.

[0046] 2.1 Seed cleaning and germination Self-crossed line 14CR Chinese cabbage seeds: Recorded in the non-patent literature "FERONIA receptor kinase-regulated reactive oxygen species mediate self-incompatibility in Brassica rapa.", which can be obtained from Shandong Agricultural University by the public. This biological material is only used for repeating the relevant experiments of this invention and cannot be used for other purposes.

[0047] Disinfect with 75% ethanol for 30 - 60 s, wash once with sterile water for 1 min each time; disinfect with 0.15% mercuric chloride for 10 min, wash twice with sterile water for 1 min each time; wash with sterile water for 30 min, and inoculate on sterile filter paper to dry. Inoculate the Chinese cabbage seeds into a germination culture bottle and incubate in the dark at 23°C for 5 - 6 d; 2.2 Pre-culture Cut the hypocotyls of the Chinese cabbage seedlings germinated in 2.1 into segments 0.4 - 0.6 cm long, inoculate them on the pre-culture medium, and culture them under light at 23°C for 2 - 3 d to obtain explants.

[0048] 2.3 Agrobacterium infection and co-culture Pick Agrobacterium GV3101 / pBWA(V)HII-SLR-THE_rnai-TNOS into the infection solution to prepare an Agrobacterium resuspension with an OD 600nm = 0.2. Inoculate the explants into the Agrobacterium suspension for 10 min. Dry the infected explants on sterile filter paper and inoculate them on the co-culture medium, and incubate in the dark at 23°C for 48 - 72 h.

[0049] 2.4 Screening / differentiation Inoculate the explants on the co-culture medium onto the screening / differentiation medium, with 30 explants per dish, and culture them under light at 23°C, changing the plate every 15 days; 2.5 Rooting culture Inoculate the buds differentiated in 2.4 onto the rooting medium, and culture them under light at 23°C until roots grow, obtaining T0 generation plants.

[0050] 2.6 Detection of positive plants Extract the genomic DNA of T0 generation plants by the CTAB method and perform PCR detection. The detection method is shown in the Agrobacterium detection.

[0051] Example 2. In RNAi transgenic plants BrTHE1 Expression level detection Detection method: Extract the mRNA of T0 generation RNAi transgenic plants and reverse-transcribe it into cDNA. Use BrTHE1-F (5'-GAGAATTTAGTTGCGAAGGTCG-3') and BrTHE1-R (5'-CTTTAACCGCAGTACTCACATG-3') as amplification primers to detect the expression level of BrTHE1 in RNAi transgenic plants. PCR amplification system: 2× ChamQ Blue Universal SYBR qPCR Master Mix 10 µL, forward primer (10µM) 0.4 µL, reverse primer (10µM) 0.4 µL, template cDNA 2µL, ddH2O 7.2 µL. PCR amplification program: 95 °C for 30 s; 95 °C for 10 s, 62 °C for 30 s, 40 cycles; melting curve, 95 °C for 15 s, 60 °C for 1 min, 95 °C for 1 s; 4 °C, cooling.

[0052] The identification results of the expression level of BrTHE1 in BrTHE1 RNAi transgenic plants are as Figure 1 shown. The stigma BrTHE1 gene expression level of BrTHE1 RNAi-1 plants is lower than that of wild-type Chinese cabbage (inbred line 14CR), and the stigma BrTHE1 gene expression level of BrTHE1 RNAi-3 plants is significantly lower than that of wild-type Chinese cabbage (inbred line 14CR).

[0053] Example 3. Observe the number of pollen tubes penetrating the stigma of RNAi transgenic plants Experimental method: 1. Pollination: Use forceps to pick the stigma of the flower that blooms on the same day and has a cruciform petal, and insert it into the PGM medium (5 mM CaCl2, 5 mM KCl, 0.01% H3BO3, 1 mM MgSO4·7H2O, 10% sucrose, 0.8% agarose, pH 7.5), and perform artificial self-pollination with the self-pollen of the flower that blooms on the same day; 2. Aniline blue staining: a: 12 hours after pollination, fix the stigma in Carnoy's reagent (absolute ethanol: glacial acetic acid = 3:1) until the stigma is completely decolorized.

[0054] b: Wash the decolorized stigma 3 times with double-distilled water, and then soften the stigma in a 10 M NaOH solution in a water bath at 42°C for about 40 minutes. c: After washing the sodium hydroxide on the stigma with double-distilled water, place the softened stigma in a 0.1% aniline blue solution and stain it in the dark for more than 30 minutes.

[0055] d: After the staining is completed, use forceps to pick out the stigma onto a glass slide, cover it with a cover slip, and observe the number of pollen tubes passing through the stigma under the DAPI channel of an upright fluorescence microscope.

[0056] The results are as Figure 2 shown. The number of self-pollen tubes passing through the stigma of BrTHE1 RNAi-1 plants is more than that of wild-type Chinese cabbage (inbred line 14CR), and the number of self-pollen tubes passing through the stigma of BrTHE1 RNAi-3 plants is significantly more than that of wild-type Chinese cabbage (inbred line 14CR).

[0057] Example 4. Observe the number of seeds after self-pollination of RNAi transgenic plants Plant the T0 generation seeds of BrTHE1 RNAi-1 and BrTHE1 RNAi-3 in a solar greenhouse (temperature 25°C / 15°C, 16 h light / 8 h dark), and perform the following operations on the plants to be flowered: 1. Obtain seeds On a sunny day, perform self-pollination on the flowers that bloom on the same day and have a cruciform petal, and cover them with a parchment paper bag after pollination; 2. Seed observation: a: 12 days after pollination, retrieve the pollinated pods from the plants and bring them back to the laboratory.

[0058] b: Stick double-sided tape on a glass slide and fix the pod on the double-sided tape of the glass slide, with any one edge of the dorsal-ventral line of the pod facing down.

[0059] c: Use a dissecting needle to cut along the edge on the top of the pod and open the pod to both sides.

[0060] d: Observe the number of developing seeds in the fruit pods using a stereomicroscope and take pictures with a camera.

[0061] The results are as Figure 3 shown. The number of developing seeds obtained from BrTHE1 RNAi-1 plants is more than that of wild-type Chinese cabbage (inbred line 14CR), and the number of developing seeds obtained from BrTHE1 RNAi-3 plants is significantly more than that of wild-type Chinese cabbage (inbred line 14CR).

[0062] SEQ ID NO:1 (Amino acid sequence of BrTHE1) MGGPLLTPENDTLGRRWENDAEYLHVNSSVLVVTANPSSIKYSVSVTQETAPNMVYATADMMGEDANVAAPSFNLTWVLPVDPSFSYFVRVHFCDIVSQAMMNTLVFNLYVNDDLAHKSLDLSSLTNGLRVPYFDDYVAHASGESLTVSVGPDSLADITNATMSGLEVLKISNGAKSLSGVSPVKSLLFPGGGFNKKVVFFGSAVVAVTSVLLIAVCCYCCLAASRKKSPQKGGNGNGNGHPWLPLPLYGLSQTSHKSNTASCISLASTHLGRCFMFQEIMEATNKFDECSLLGVGGFGRVYKGTLEDGTKVAVKRGNPSSEQGMAEFRTEIEMLSKLRHRHLVSLIGYCDERSEMILVYEYMANGPLRSHLYGGELPPLSWKQRLEVCIGAARGLHYLHTGASLGIIHRDVKTTNILLDENLVAKVADFGLSKTGPSLDQTHVSTAVKGSFGYLDPEYFRRQQLTEKSDVYSFGVVLMEVLCCRPALNPVLPREQVNIAEWAMAWQKKGLLDQIMDGNLTGKVNTASLKKFGETAEKCLEEYGVDRPSMGDVLWNLEYALQLEETSSALMEADDNSTNHIHGIPMAPMESFDNSVSIDIVDRGGVNLGTGTDDDATTSAVFSQLVHPRGR.

[0063] SEQ ID NO:2 ( BrTHE1 CDS sequence)

[0064] SEQ ID NO:3 (tgtF-loop-tgtR) 5'-cgagaagtctgacgtgtactcgttcggagttgttctgatggaagtgctctgttgtagaccggctttaaacccggttttgccgagggagcaagtgaacatagcggaatgggccatggcgtggcagaagaagggtcttcttgatcagatcatggacggtaacttaaccgggaaagtgaacactgcgtcgcttaagaagttcggtgagacggctgagaagtgtttagaggagtatggagtggataggccttcgcctgcaggtctagtttttctccttcattttcttggttaggacccttttctctttttatttttttgagctttgatctttctttaaactgatctattttttaattgattggttatggtgtaaatattacatagctttaactgataatctgattactttatttcgtgtgtctatgatgatgatgatagttacagagcccgggcCGAAGGCCTATCCACTCCATACTCCTCTAAACACTTCTCAGCCGTCTCACCGAACTTCTTAAGCGACGCAGTGTTCACTTTCCCGGTTAAGTTACCGTCCATGATCTGATCAAGAAGACCCTTCTTCTGCCACGCCATGGCCCATTCCGCTATGTTCACTTGCTCCCTCGGCAAAACCGGGTTTAAAGCCGGTCTACAACAGAGCACTTCCATCAGAACAACTCCGAACGAGTACACGTCAGACTTCTCG-3'.

[0065] Nucleotide sequence of pBWA(V)HII-SLR-THE_rnai-TNOS

[0066] The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention and without the need for unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application is intended to cover any modifications, uses or improvements of the present invention, including changes made using conventional techniques known in the art that depart from the scope disclosed in this application.

Claims

1. A method for preparing self-compatible Chinese cabbage, which down-regulates or inhibits or reduces the expression of the coding gene of BrTHE1 protein in the recipient Chinese cabbage or regulates the activity and / or content of the protein, to obtain self-compatible Chinese cabbage, and the recipient Chinese cabbage is self-incompatible; the BrTHE1 protein is any one of the following: A1) A protein with the amino acid sequence of SEQ ID NO:1; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID NO:1, having more than 80% identity with the protein shown in A1) and having the same function; A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

2. A method for improving the self-compatibility of Chinese cabbage, which down-regulates or inhibits or reduces the expression of the coding gene of BrTHE1 protein in the target Chinese cabbage or regulates the activity and / or content of the protein, so that the target Chinese cabbage changes from a self-incompatible plant to a self-compatible plant, and the BrTHE1 protein is any one of the following: A1) A protein with the amino acid sequence of SEQ ID NO:1; A2) A protein obtained by substituting and / or deleting and / or adding amino acid residues to the amino acid sequence shown in SEQ ID NO:1, having more than 80% identity with the protein shown in A1) and having the same function; A3) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of A1) or A2).

3. The method according to claim 1 or 2, characterized in that, The down-regulation or inhibition or reduction of the expression of the coding gene of the protein in the recipient Chinese cabbage or the regulation of the activity and / or content of the protein includes introducing a substance that down-regulates or inhibits or reduces the expression level of the coding gene of BrTHE1 protein into the recipient Chinese cabbage.

4. The method according to claim 3, characterized in that, The substance is any one of the following: B1), an RNA molecule that inhibits or reduces or down-regulates the expression of the coding gene of the protein or an RNA molecule that inhibits or reduces or down-regulates the activity or content of the protein; B2), the coding gene expressing the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4).

5. The method according to claim 4, wherein The RNA molecule described in B1) targets the mRNA transcribed from the coding gene of the protein described in claim 3.

6. The method according to claim 5, wherein The nucleotide sequence of the coding gene of the RNA molecule is the nucleotide positions from 1398 to 1647 of SEQ ID NO:

2.

7. A biological material related to the protein described in claim 1, which is any one of the following: B1), an RNA molecule that inhibits or reduces or down-regulates the expression of the coding gene of the protein or an RNA molecule that inhibits or reduces or down-regulates the activity or content of the protein; B2), the coding gene expressing the RNA molecule described in B1); B3), an expression cassette containing the gene described in B2); B4), a recombinant vector containing the gene described in B2), or a recombinant vector containing the expression cassette described in B3); B5), a recombinant microorganism containing the gene described in B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4); B6), a transgenic plant cell line containing the gene described in B2), or a transgenic plant cell line containing the expression cassette described in B3), or a transgenic plant cell line containing the recombinant vector described in B4); B7), a transgenic plant tissue containing the gene described in B2), or a transgenic plant tissue containing the expression cassette described in B3), or a transgenic plant tissue containing the recombinant vector described in B4); B8), a transgenic plant organ containing the gene described in B2), or a transgenic plant organ containing the expression cassette described in B3), or a transgenic plant organ containing the recombinant vector described in B4).

8. The biomaterial according to claim 7, wherein The RNA molecule described in B1) targets the mRNA transcribed from the coding gene of the protein described in claim 1.

9. Application, characterized in that, Any of the following applications of the biological material described in claim 7 or 8: D1) converting a self-incompatible plant into a self-compatible plant; D2) preparing a product for converting a self-incompatible plant into a self-compatible plant; D3) cultivating a self-compatible plant; D4) preparing a product for cultivating a self-compatible plant; D5) plant breeding.

10. The application according to claim 9, wherein The plant is any of the following: M1) a dicotyledonous plant; M2) a plant of the order Brassicales; M3) a plant of the family Brassicaceae; M4) a plant of the genus Brassica; M5) Chinese cabbage.

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