Relevant gene of rosette leaf included angle as well as biological material, molecular marker and application thereof

Abstract

The invention discloses a rosette leaf included angle related gene as well as a biological material, a molecular marker and application thereof. The technical problem to be solved by the invention is to regulate the rosette leaf included angle of the plant. Specifically, the invention discloses any one of the following applications of the protein, a substance for regulating and controlling the expression of a coding gene of the protein or a substance for regulating and controlling the activity or content of the protein: A1) application in regulating and controlling the included angle of the rosette leaf of a plant; a2) application in preparation of products for regulating and controlling plant rosette leaf included angles; the plant protein is any one of the following proteins: B1) a protein with an amino acid sequence as shown in a sequence 2, or a protein which is obtained through substitution and / or deletion and / or addition of amino acid residues, has 80% or more of identity with the protein as shown in B1) and has the same function as the protein, or a protein with an amino acid residue as shown in SEQ ID NO: 1 and a protein with an amino acid residue as shown in SEQ ID NO: 2, or a protein with an amino acid residue as shown in SEQ ID NO: 1. The N terminal or / and the C terminal is / are connected with a protein tag to obtain the fusion protein. Compared with a wild type, a transgenic plant which overexpresses or inhibits the protein coding gene can increase the rosette leaf included angle of the plant, and contributes to regulation and improvement of the rosette leaf included angle.

Description

Technical Field

[0001] The present invention specifically relates to a gene related to the rosette leaf angle, a biological material, a molecular marker and an application thereof. Background Art

[0002] Chinese cabbage (Brassica rapa L. ssp. pekinensis) originated in China and is a variant of the Brassica campestris L. ssp. pekinensis in the genus Brassica of the Brassicaceae family that can form leaf heads, and is an annual or biennial herb. Chinese cabbage has a long cultivation history and is one of the important vegetables in China. Chinese cabbage is widely cultivated in various regions of China, and its cultivation area and consumption volume are among the top. Especially in the northern regions, Chinese cabbage is an essential vegetable for citizens in winter and spring. Therefore, as an important edible vegetable for the Chinese people, increasing its yield has always been the focus of research.

[0003] With the improvement of people's living quality and the reduction of vegetable areas around big cities, the demand for small-plant-type varieties suitable for high-density planting has increased sharply. At the same time, with the increase in labor costs, machine planting and harvesting have become new modes and directions for vegetable cultivation, and upright Chinese cabbage varieties are more conducive to machine harvesting. Therefore, small-angle (upright-type) varieties suitable for high-density planting have gradually become the breeding direction. Leaf angle is the inclination angle between a leaf and its own stem, and it is an important agronomic trait of Chinese cabbage, which is of great significance for the planting density and photosynthesis of Chinese cabbage. Leaf angle is a description of the spatial distribution state of plant leaves. The leaves of plants with small leaf angles are upright and compact, while the leaves of plants with large leaf angles are loose and flat. Plants with different leaf angles have different planting densities and light energy utilization rates, which will also lead to differences in water and fertilizer utilization efficiency, and ultimately result in yield differences.

[0004] By mining genes related to regulating the leaf angle of Chinese cabbage and analyzing the molecular mechanism of leaf angle formation, effective improvement can be carried out for the leaf angle. It can not only reduce the land area occupied by a single plant but also improve the light energy utilization rate of a single plant, thereby increasing the yield of Chinese cabbage. At present, research on leaf angle mainly focuses on gramineous crops such as maize, while research on leaf angle of Brassica crops such as Chinese cabbage is less, and the inheritance of Chinese cabbage leaf angle traits, leaf angle gene mapping and molecular regulation mechanism are still unclear.

[0005] Therefore, it is urgent to carry out research on the genetic law, gene mapping and functions of Chinese cabbage leaf angle genes, which is of great significance for improving the Chinese cabbage planting mode, guiding the breeding of excellent Chinese cabbage varieties, and increasing the yield and quality of Chinese cabbage. Summary of the Invention

[0006] The technical problem solved by the present invention is to regulate and / or predict the rosette leaf angle of plants.

[0007] To solve the above problems, the present invention provides the following applications.

[0008] Use of a protein, a substance that regulates the expression of the coding gene of the protein, or a substance that regulates the activity or content of the protein in any one of the following:

[0009] A1) Application in increasing or enhancing the rosette leaf angle of cruciferous plants;

[0010] A2) Application in preparing a product for increasing or enhancing the rosette leaf angle of cruciferous plants;

[0011] The protein is any one of the following:

[0012] B1) A protein with an amino acid sequence shown in Sequence 2;

[0013] B2) A protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in B1), having more than 80% identity with the protein described in B1) and having the same function as the protein;

[0014] B3) A fusion protein obtained by connecting a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0015] In the above proteins, the protein-tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using DNA in vitro recombination technology, in order to facilitate the expression, detection, tracing, and / or purification of the target protein. The protein tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0016] In the above proteins, identity refers to the identity of amino acid sequences. The identity of amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web page of 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 to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0017] Among the above-mentioned proteins, the identity of more than 80% may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

[0018] Among the above-mentioned proteins, Sequence 2 (SEQ ID No.2) consists of 502 amino acid residues.

[0019] In the present application, the regulation may be knockout or inhibition or reduction or down-regulation. The regulation may also be enhancement or increase or up-regulation.

[0020] In the above, knocking out or inhibiting or reducing or down-regulating the expression of the coding gene of the above-mentioned protein or the activity or content of the above-mentioned protein may increase the rosette leaf angle of the plant. The plant may be a dicotyledonous plant. The dicotyledonous plant may be a cruciferous plant. The cruciferous plant may be a Brassica plant. The Brassica plant may be Chinese cabbage. The Chinese cabbage may be Chinese cabbage variety Baiyang.

[0021] In the above, enhancing or increasing or up-regulating the expression of the coding gene of the above-mentioned protein or the activity or content of the above-mentioned protein may increase the rosette leaf angle of the plant. The plant may be a dicotyledonous plant. The dicotyledonous plant may be a cruciferous plant. The cruciferous plant may be an Arabidopsis plant. The Arabidopsis plant may be Arabidopsis thaliana. The Arabidopsis thaliana may be Arabidopsis thaliana variety Columbia (Col-0).

[0022] In the above-mentioned use, the protein is derived from Chinese cabbage. The Chinese cabbage may be Chinese cabbage 21M-193 or Chinese cabbage Baiyang.

[0023] In the above, the substance for regulating gene expression may be a substance that performs at least one of the following 6 regulations: 1) regulation at the gene transcription level; 2) regulation after gene transcription (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (that is, regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the mRNA degradation of the gene; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated by the gene).

[0024] In the above-mentioned use, the substance for regulating the expression of the coding gene of the above-mentioned protein is any of the following:

[0025] D1) A nucleic acid molecule that inhibits or reduces or down-regulates the expression of the coding gene of the above-mentioned protein;

[0026] D2) The coding gene expressing the nucleic acid molecule described in D1);

[0027] D3) An expression cassette containing the gene described in D2);

[0028] D4) A recombinant vector containing the gene described in D2), or a recombinant vector containing the expression cassette described in D3);

[0029] D5) A recombinant microorganism containing the gene described in D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4);

[0030] D6) A transgenic plant cell line containing the gene described in D2), or a transgenic plant cell line containing the expression cassette described in D3), or a transgenic plant cell line containing the recombinant vector described in D4);

[0031] D7) A transgenic plant tissue containing the gene described in D2), or a transgenic plant tissue containing the expression cassette described in D3), or a transgenic plant tissue containing the recombinant vector described in D4);

[0032] D8) A transgenic plant organ containing the gene described in D2), or a transgenic plant organ containing the expression cassette described in D3), or a transgenic plant organ containing the recombinant vector described in D4);

[0033] D9) A nucleic acid molecule encoding the above protein;

[0034] D10) An expression cassette containing the nucleic acid molecule described in D9);

[0035] D11) A recombinant vector containing the nucleic acid molecule described in D9), or a recombinant vector containing the expression cassette described in D10);

[0036] D12) A recombinant microorganism containing the nucleic acid molecule described in D9), or a recombinant microorganism containing the expression cassette described in D10), or a recombinant microorganism containing the recombinant vector described in D11);

[0037] D13) A transgenic plant cell line containing the nucleic acid molecule described in D9), or a transgenic plant cell line containing the expression cassette described in D10), or a transgenic plant cell line containing the recombinant vector described in D11);

[0038] D14) A transgenic plant tissue containing the nucleic acid molecule described in D9), or a transgenic plant tissue containing the expression cassette described in D10), or a transgenic plant tissue containing the recombinant vector described in D11);

[0039] D15) A transgenic plant organ containing the nucleic acid molecule described in D9), or a transgenic plant organ containing the expression cassette described in D10), or a transgenic plant organ containing the recombinant vector described in D11).

[0040] D1) The nucleic acid molecule may be a VIGS gene silencing vector. The target of the VIGS gene silencing vector is the 619-658th position of the nucleic acid sequence of SEQ ID NO.2. The VIGS gene silencing vector may be a recombinant vector containing a DNA molecule of the 619-658th position of the nucleotide sequence of SEQ ID NO.2.

[0041] D2) In the nucleic acid molecule of D2) or D9), those of ordinary skill in the art can easily mutate the nucleotide sequence that inhibits or reduces or down-regulates and / or enhances or increases or up-regulates the expression of the protein BraRLA coding gene of the present invention by using known methods, such as directed evolution or point mutation methods. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence that inhibits or reduces or down-regulates the expression of the protein BraRLA coding gene isolated from the present invention and having the function of inhibiting or reducing or down-regulating the expression of the protein BraRLA coding gene are all derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0042] Among the above-mentioned biomaterials, the expression cassette described in D3) refers to DNA that can express the gene in a host cell. This DNA can not only include a promoter that initiates gene transcription but also a terminator that terminates gene transcription. Further, the expression cassette can also include enhancer sequences. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically inducible promoter from tobacco, pathogenesis-related 1 (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); the tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); the heat shock promoter (U.S. Patent 5,187,267); the tetracycline-inducible promoter (U.S. Patent 5,057,422); seed-specific promoters, such as the foxtail millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), promoters specific to seed storage proteins (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res., 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627).

[0043] In the above D3), a recombinant expression vector containing the gene expression cassette can be constructed using a plant expression vector. The plant expression vector can be a Gateway system vector, a binary Agrobacterium vector, etc., such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When constructing a recombinant expression vector using IbpPGM, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added before the transcription start nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters; in addition, when constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signals and the start codon are extensive and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0044] The above 80% or more identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0045] 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 of 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 to calculate the identity of amino acid sequences, and then the identity value (%) can be obtained.

[0046] In the above D4), the backbone vector of the recombinant vector can be a pCAMBIA 2300 vector or a PTY-S plasmid.

[0047] The microorganism described in D5) above may be Agrobacterium tumefaciens. The Agrobacterium tumefaciens is GV3103. The microorganism described in D5) above may be Escherichia coli. The Escherichia coli may be Super Stbl3.

[0048] In the above text, the nucleic acid molecule described in B1) is a DNA molecule with a coding sequence of Sequence 1.

[0049] To solve the above problems, the present invention also provides a method for cultivating Chinese cabbage with large leaf angles.

[0050] The method includes down-regulating, weakening or reducing, or up-regulating, enhancing or increasing the expression level of the coding gene of the above protein in the target Chinese cabbage, and / or obtaining Chinese cabbage with large leaf angles by the activity and / or content of the protein, and the rosette leaf angle of the Chinese cabbage with large leaf angles is greater than that of the target Chinese cabbage.

[0051] In the method, down-regulating, weakening or reducing the expression level of the coding gene of the above protein in the target Chinese cabbage is achieved by virus-induced gene silencing.

[0052] The virus-induced gene silencing includes introducing the above VIGS gene silencing vector into the target Chinese cabbage.

[0053] To solve the above problems, the present invention also provides a method for cultivating Arabidopsis thaliana with large leaf angles. The method includes up-regulating, enhancing or increasing, or down-regulating, weakening or reducing the expression level of the coding gene of the above protein in the target Arabidopsis thaliana, and / or obtaining Arabidopsis thaliana with large leaf angles by the activity and / or content of the protein, and the rosette leaf angle of the Arabidopsis thaliana with large leaf angles is greater than that of the target Arabidopsis thaliana.

[0054] In the above text, the rosette leaf angle may be the angle between the central axis of the plant and the fully extended rosette leaf. The rosette leaf may be the rosette leaf at the 6-leaf stage or the 8-leaf stage.

[0055] The above method is applied in plant breeding.

[0056] The purpose of the breeding includes cultivating or selecting plants with large rosette leaf angles.

[0057] In the above application and method, the plant is any one of the following:

[0058] J1) Dicotyledonous plants;

[0059] J2) Cruciferous plants;

[0060] J3) Brassica plants or Arabidopsis;

[0061] J4) Chinese cabbage or Arabidopsis thaliana.

[0062] In the above text, the Arabidopsis thaliana can be Col-0 (Arabidopsis thaliana variety Columbia), and the Chinese cabbage can be Baiyang and 21M-193.

[0063] Use of a substance for detecting a molecular marker linked to the gene of the above-mentioned protein in any of the following;

[0064] A1) Use in predicting and / or assisting in predicting the rosette leaf angle of a plant;

[0065] A2) Use in preparing a product for predicting and / or assisting in predicting the rosette leaf angle of a plant.

[0066] The molecular marker can be the A05_18777194 marker or the A05_18955845 marker. The A05_18777194 marker is a SNP in the Chinese cabbage genome, where the 313th position in Sequence 4 is A or T. The A05_18955845 marker is a SNP in the Chinese cabbage genome, where the 250th position in Sequence 5 is G or C.

[0067] The substance can be a product. The detection substance can include reagents, kits, and instruments for detecting the A05_18777194 marker or the A05_18955845 marker or their genotypes. Specifically, primers and other reagents and instruments required for in vitro nucleic acid amplification for detecting the above-mentioned A05_18777194 marker or A05_18955845 marker or their genotypes.

[0068] The substance for detecting the A05_18777194 marker can be a substance for detecting the genomic sequence of the A05_18777194 marker, which includes an intron sequence. In the actual detection process, the A05_18777194 marker can be detected and analyzed by detecting the mRNA transcribed from the A05_18777194 marker gene caused by the A05_18777194 marker, the nucleotide polymorphism of the cDNA reverse transcribed from the A05_18777194 marker mRNA, or the amino acid polymorphism of the A05_18777194 marker protein.

[0069] The substance for detecting the A05_18955845 marker can be a substance for detecting the genomic sequence of the A05_18955845 marker, which includes an intron sequence. In the actual detection process, the A05_18955845 marker can be detected and analyzed by detecting the mRNA transcribed from the A05_18955845 marker gene caused by the A05_18955845 marker, the nucleotide polymorphism of the cDNA reverse transcribed from the A05_18955845 marker mRNA, or the amino acid polymorphism of the A05_18955845 marker protein.

[0070] To solve the above problems, the present invention also provides a method for predicting and / or assisting in predicting the rosette leaf angle of plants.

[0071] The method includes detecting the genotype of the above molecular marker in the plant to be tested, and predicting and / or assisting in predicting the rosette leaf angle of the plant according to the genotype of the plant to be tested. The genotype of the molecular marker includes the genotype of the A05_18777194 marker or the genotype of the A05_18955845 marker:

[0072] The rosette leaf angle of Chinese cabbage with the homozygous genotype of the A05_18777194 marker having a T at position 313 of SEQ ID NO: 4 is greater than that of Chinese cabbage with the heterozygous genotype of the A05_18777194 marker having a T at position 313 of SEQ ID NO: 4 and an A at position 313 of SEQ ID NO: 4; the rosette leaf angle of Chinese cabbage with the homozygous genotype of the A05_18777194 marker having a T at position 313 of SEQ ID NO: 4 is greater than that of Chinese cabbage with the heterozygous genotype of the A05_18777194 marker having a T at position 313 of SEQ ID NO: 4 and an A at position 313 of SEQ ID NO: 4 and the homozygous genotype of the A05_18777194 marker having an A at position 313 of SEQ ID NO: 4;

[0073] and / or,

[0074] The rosette leaf angle of Chinese cabbage with the homozygous genotype of the A05_18955845 marker having a G at position 250 of SEQ ID NO: 5 is greater than that of Chinese cabbage with the heterozygous genotype of the A05_18955845 marker having a G at position 250 of SEQ ID NO: 5 and a C at position 250 of SEQ ID NO: 5; the rosette leaf angle of Chinese cabbage with the homozygous genotype of the A05_ marker having a G at position 250 of SEQ ID NO: 5 is greater than that of Chinese cabbage with the heterozygous genotype of the A05_18955845 marker having a G at position 250 of SEQ ID NO: 5 and a C at position 250 of SEQ ID NO: 5 and the homozygous genotype of the A05_18955845 marker having a C at position 250 of SEQ ID NO: 5.

[0075] In the above applications and methods, the plant is a plant pure line or inbred line.

[0076] The application of the above method in plant breeding.

[0077] In the above applications and methods, the A05_18777194 marker or the A05_18955845 marker, or the substance for detecting the genotype of the A05_18777194 marker or the A05_18955845 marker, is one of the following D1), D2), D3) or D4):

[0078] D1) An in vitro nucleic acid amplification primer containing specific amplification of the SNP;

[0079] D2) An in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in D1);

[0080] D3) A kit containing the in vitro nucleic acid amplification primer described in D1) or the in vitro nucleic acid amplification reagent described in D2);

[0081] D4) A detection instrument containing the in vitro nucleic acid amplification primer described in D1), the in vitro nucleic acid amplification reagent described in D2), or the kit described in D3);

[0082] The A05_18777194 marker is that the 313th position in Sequence 4 is A or T;

[0083] The A05_18955845 marker is that the 250th position in Sequence 5 is G or C.

[0084] The in vitro nucleic acid amplification technology can be polymerase chain reaction (PCR), strand displacement amplification (SDA), ligase chain reaction (LCR), nucleic acid sequence-based amplification (NASBA), rolling circle nucleic acid amplification (RCA), loop-mediated isothermal amplification (lamp), helicase-dependent isothermal amplification technology (HDA), or Qβ replication technology.

[0085] In this application, polymerase chain reaction (PCR) is used as the amplification means for polymorphism detection.

[0086] The specific amplification described in D1) can detect the nucleotide sequences of the A05_18777194 marker or the A05_18955845 marker by the presence or absence of the amplification product or by the presence or absence of the amplification product combined with auxiliary reagents such as probes.

[0087] In the above applications, methods, and products, the in vitro nucleic acid amplification primer can be labeled with a label or not. The label refers to any atom or molecule that can be used to provide a detectable effect and can be linked to nucleic acids. Labels include but are not limited to dyes; radioactive labels such as 32P; binding moieties such as biotin; haptens such as digoxin (DIG); luminescent, phosphorescent, or fluorescent moieties; and individual fluorescent dyes or fluorescent dyes combined with moieties that can inhibit or shift the emission spectrum through fluorescence resonance energy transfer (FRET). The label can provide a signal detectable by fluorescence, radioactivity, colorimetry, gravimetry, X-ray diffraction or absorption, magnetism, enzyme activity, etc. The label can be a charged moiety (positive or negative charge) or, optionally, can be charge-neutral. The label can include nucleic acid or protein sequences or combinations thereof, as long as the sequence containing the label is detectable. In some embodiments, nucleic acids are directly detected without a label.

[0088] The primers for amplifying the genotype of the A05_18777194 marker may include A05_18777194 primer X: GAAGGTGACCAAGTTCATGCTTTCTTGGCCTTATCAGACGGTCT, A05_18777194 primer Y: GAAGGTCGGAGTCAACGGATTTTCTTGGCCTTATCAGACGGTCA, A05_18777194 primer Z: GTCCGAGGGAAGTGCGTCCG.

[0089] The primers for amplifying the genotype of the A05_18955845 marker may include A05_18955845 primer X: GAAGGTGACCAAGTTCATGCTGCAGACCAAAGCTATTTTAACTGAC, A05_18955845 primer Y: GAAGGTCGGAGTCAACGGATTGCAGACCAAAGCTATTTTAACTGAG, A05_18955845 primer Z: GTGGCCTCCAGCAGTTGCTTTCT.

[0090] Beneficial effects

[0091] The present invention discloses a gene related to the rosette leaf angle, its biological materials, molecular markers and applications. By performing KASP analysis on 300 individuals of the F2 generation of 21M-193 and Baiyang, a gene related to the rosette leaf angle, namely the Brarla gene, and two SNP markers linked to the rosette leaf angle, namely the A05_18777194 marker and the A05_18955845 marker, were discovered.

[0092] By constructing an overexpression vector to transform Arabidopsis plants, Arabidopsis overexpressing Brarla was obtained. It was found that compared with wild-type Arabidopsis, the rosette leaf angle of Arabidopsis overexpressing Brarla was significantly increased. At the same time, a VIGS vector of Brarla was constructed, and Chinese cabbage with silenced Brarla was obtained. Compared with wild-type Chinese cabbage, the rosette leaf angle of Chinese cabbage with silenced Brarla was significantly increased. It can be seen that the Brarla gene can regulate the rosette leaf angle of plants.

[0093] Moreover, according to the genotypes of the above-mentioned A05_18777194 marker and A05_18955845 marker to judge the phenotype of the rosette leaf angle of plants, the accuracy rate of the small rosette leaf angle can reach 97.18%, and the accuracy rate of the large rosette leaf angle can reach 96.10%, which can be used for predicting the rosette leaf angle of plants. Description of the drawings

[0094] Figure 1 is the measurement method of the rosette leaf angle, Figure 1Middle a: 0-200mm digital angle ruler; Figure 1 Middle b: Leaf angle measurement method.

[0095] Figure 2 Relative expression levels of Brarla genes in Chinese cabbage 21M-193, Chinese cabbage Baiyang, empty vector and PTYs-Brarla positive strains; A1: wild type of Chinese cabbage 21M-193 with large leaf angle; A2: wild type of Chinese cabbage Baiyang with small leaf angle; PTYs: virus empty vector; VIGS is PTYs-Brarla positive strain of Chinese cabbage Baiyang with small leaf angle; ***: p<0.001, there are extremely significant differences between the data; ns: no difference between the data.

[0096] Figure 3 This is the PTYs-BraRLA silenced phenotype of Chinese cabbage Baiyang, BraRLA is the VIGS-positive strain phenotype; A2 is the wild type of the parent Baiyang; the upper picture is a side view, and the lower picture is a top view, scale: 1 cm.

[0097] Figure 4 is the relative expression level of Arabidopsis thaliana overexpressing OE-Brarla, WT: wild-type Arabidopsis thaliana plant; OE-Brarla: Arabidopsis thaliana overexpressing plant.

[0098] Figure 5 The average values ​​of OE-Brarla leaf angles of Arabidopsis overexpressing plants at different stages.

[0099] Figure 6 This is the Arabidopsis OE-Brarla overexpression phenotype. Figure 6 Middle a: Overexpressing plants at the six-leaf stage; Figure 6 Middle b: wild-type plants at the six-leaf stage; Figure 6 Middle c: Overexpressing plants at the eight-leaf stage; Figure 6 Middle d: wild-type plants at the eight-leaf stage; Figure 6 Middle e: Overexpressing plants at the eight-leaf stage; Figure 6 Middle f: wild-type plants at the eight-leaf stage.

[0100] Figure 7 The KASP typing test results of the A05_18777194 marker in the F2 population and the KASP typing test results of the A05_18955845 marker in the F2 population are shown in FIG. Figure 7 a in the figure: KASP typing test of marker A05_18777194 in the F2 population, T:A is a heterozygous genotype, T:T is a homozygous large angle genotype, and A:A is a homozygous small angle genotype; Figure 7b in China: KASP genotyping detection of the A05_18955845 marker in the F2 population. C:G is the heterozygous genotype, G:G is the homozygous large included angle genotype, and C:C is the homozygous small included angle genotype.

[0101] Figure 8 It is the location map of the BraRLA gene on the chromosome. Specific implementation manners

[0102] The present invention will be further described in detail below in conjunction with specific implementation manners. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments 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.

[0103] The experimental methods in the following embodiments 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 specifications. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0104] The following embodiments use Excel statistical software to process data. The experimental results are expressed as mean ± standard deviation, and are tested by Two-tailed Student’s t-test. P < 0.05 (*) indicates significant difference, P < 0.01 (**) indicates extremely significant difference, and P < 0.001 (***) indicates extremely significant difference.

[0105] Materials and methods

[0106] Test materials

[0107] The large included angle Chinese cabbage 21M-193 (hereinafter also referred to as 21M-193 or parental 21M-193) is the hybrid offspring of three parents of Chinese cabbage, red flowering Chinese cabbage and Guangdong flowering Chinese cabbage. Its main characteristics are: the leaves are flat and prostrate, the included angle of the rosette leaves is large, the leaves grow parallel to the ground, there is no ball-forming trait, and the leaf color is deep.

[0108] The characteristics of the small included angle Chinese cabbage Baiyang (hereinafter also referred to as Baiyang or parental Baiyang) are: the included angle of the rosette leaves is small, it can form a normal ball, is stacked, and the leaf color is light green.

[0109] The sources and origins of the Chinese cabbage 21M-193 and Chinese cabbage Baiyang used in this application can be found in the article (Zhang Deshuang, Xin Xiaoyun, Zhu Hongbin, Zhang Fenglan, Yu Shuancang, Su Tongbing, Yu Yangjun, Wang Weihong, Zhao Xiuyun, Li Peirong, Lu Guixiang. Creation and application of materials with large included angle of rosette leaves and multiple tillers in Chinese cabbage [J]. Vegetables, 2024, (09): 50-57.).

[0110] Example 1: Obtaining SNPs Related to Rosette Leaf Angle

[0111] Construction of Genetic Population:

[0112] Hybridize 21M-193 with Baiyang to construct six-generation populations A1, A2, F1, F2, BC1, and BC1'. Among them, the backcross population BC1 is F1×21M-193, and the backcross population BC1' is F1×Baiyang.

[0113] Investigation and Measurement Methods for Morphological Traits:

[0114] For Chinese cabbage at the rosette stage above, find the latest completely extended healthy rosette leaf, and use a digital display angle gauge with a range of 0 - 200 mm to measure the angle between this leaf and the central axis of the plant, which is recorded as the leaf angle of the plant. Measure the leaf angle of all materials in the six generations ( Figure 1 where a is the digital display angle gauge, Figure 1 and b is the angle between the central axis and the leaf).

[0115] Pooling and BSA Resequencing

[0116] Using BSA (Bulked Segregant Analysis), hybridize the parents 21M-193 and Baiyang to obtain the F1 generation, and then self-cross to obtain the F2 generation segregation population. Select 50 extremely large-angle single plants and 50 extremely small-angle single plants from the F2 segregation population respectively. After extracting DNA using the improved CTAB method, mix the DNA of 50 extremely large-angle single plants and 50 extremely small-angle single plants at equal concentrations respectively to construct a large-angle pool and a small-angle pool. Send the DNA samples of the two parents 21M-193 and Baiyang, the large-angle mixed pool, and the small-angle mixed pool to Beijing Boyun Huakang Gene Technology Co., Ltd. for BSA resequencing. After resequencing, filter the obtained Raw Reads to get Clean Reads. Use the Burrow-Wheeler aligner (BWA) software to align the obtained Clean Reads with the Chinese cabbage reference genome V3.0 data. According to the positioning results of Clean Reads in the reference genome, use the GATK software toolkit with default parameters for SNP and Indels detection and annotation. Then use the SNP-index method to find the significant differences in genotype frequencies between the pools, and use Δ(SNP-index) to statistically analyze the association results. The stronger the association between the marked SNP and the trait, the closer Δ(SNP-index) is to 1. Finally, annotate the SNPs and genes in the candidate regions.

[0117] KASP Marker Development and Fine Mapping

[0118] Based on the BSA-seq resequencing results, since the initial mapping interval is too large and contains too many genes, which is not conducive to accurate prediction of candidate genes, KASP (Kompetitive Allele-Specific PCR) primers were designed using the known SNP sites (shown in Table 1). The reaction conditions and systems are shown in Tables 2 and 3. The KASP genotyping technology was used in combination with the phenotypic data to further fine-map the candidate genes in the two parents (21M-193 and Baiyang) and the 1129 F2 population. According to the SNP sites in the candidate interval, they were aligned with the sequences in the NCBI (National Center for Biotechnology Information) database, and 80bp DNA sequences were selected upstream and downstream of the SNP sites to design specific primers. The KASP primers were designed according to the software provided by the Vegetable Research Institute of Beijing Academy of Agriculture and Forestry Sciences based on the LGC company's KASP detection technology platform. The primer design should meet the requirements that the Tm value of the forward primer is about 62.5°C, the temperature difference does not exceed 1°C, and the GC content does not exceed 55%; the Tm value of the reverse primer is 66°C, and the GC content does not exceed 60%. The primers were synthesized by Beijing Sangon Biotech Co., Ltd. The SNPviewer 2.0 software was used to export the data. According to the genotyping results and the corresponding leaf angle phenotypic statistical results, the exchange individuals were screened.

[0119] Table 1 SNP marker sequences

[0120]

[0121]

[0122] The underlined sequence in primer X is the FAM tag sequence, and the underlined sequence in primer Y is the HEX tag sequence.

[0123] The primers X and Z in A05_18777194 amplify the fragment with the SNP site A05_18777194 being T (the 313th position in Sequence 4 is T), and the fluorescence signal of the fluorophore bound to the FAM tag sequence in the PARMS master mix can be read using a microplate reader or a fluorescence quantitative PCR instrument; the primers Y and Z in A05_18777194 amplify the fragment with the SNP site A05_18777194 being A (the 313th position in Sequence 4 is A), and the fluorescence signal of the fluorophore bound to the HEX tag sequence in the PARMS master mix can be read using a microplate reader or a fluorescence quantitative PCR instrument.

[0124] The primer X and primer Z in A05_18955845 amplify the fragment where the SNP locus A05_18955845 is C (the 250th position in Sequence 5 is C). The fluorescence signal of the fluorophore combined with the FAM tag sequence in the PARMS master mix can be read using a microplate reader or a real-time fluorescence quantitative PCR instrument; the primer Y and primer Z in A05_18955845 amplify the fragment where the SNP locus A05_18955845 is G (the 250th position in Sequence 5 is G). The fluorescence signal of the fluorophore combined with the HEX tag sequence in the PARMS master mix can be read using a microplate reader or a real-time fluorescence quantitative PCR instrument.

[0125] Table 2 KASP reaction system

[0126] Component Volume (μL) KASP Mastermix 5 μL KASP Primer mix 0.14 μL <![CDATA[Template DNA (50 ng·μL -1 )]]> 5 μL

[0127] Table 3 KASP reaction program

[0128]

[0129]

[0130] The F2 population was expanded to 1129 plants to further finely map the leaf angle gene. Using the QTL IciMapping software, a major QTL was identified in the candidate region, located between two markers A05_18329535 and A05_18955845. The LOD value of this QTL locus was 14.1, and the phenotypic contribution rate was 15.7. According to the BSA resequencing results, non-synonymous mutation SNPs in the candidate interval were screened, and KASP molecular markers were continuously developed in this interval. A total of 23 pairs of SNP molecular markers were designed, and all individual plants in the F2 population were genotyped to screen for recombinant individuals. It was found that 13 pairs of molecular markers had polymorphism and good genotyping. Finally, the candidate gene was mapped between two markers A05_18777194 and A05_18955845, and the genetic distance between the two markers was 0.6 cM, and the physical distance was 178.65 kb( Figure 8 ). By aligning with the reference genome, a total of 14 genes were included in this interval, and they may all be candidate genes.

[0131] Using the Chinese cabbage genome database version 3.0 of BRAD (http: / / brassicadb.cn), based on the mapped candidate interval, and through gene function annotation, among the above 14 candidate genes, a gene possibly related to the rosette leaf angle was found through functional analysis, namely the BraA05g025190.3C gene (hereinafter also referred to as the Brarla gene or BraRLA gene), and specific primers were designed according to the CDS and promoter sequences of the candidate gene.

[0132] Analysis of SNP Loci in Example 2

[0133] According to the above KASP results, two markers linked to the rosette leaf angle were found, namely the A05_18777194 marker and the A05_18955845 marker (specifically shown in Table 1). Using the two SNP markers A05_18777194 and A05_18955845, 300 individual plants were randomly selected from the F2 population and the KASP detection of the rosette leaf angle phenotype was carried out using the SNP primers of A05_18777194 and A05_18955845 (specifically shown in Table 1). The results showed that there were three phenotypes among the F2 individual plants: large leaf angle, small leaf angle, and intermediate type.

[0134] The rosette leaf angle of Chinese cabbage with the TT genotype of the A05_18777194 marker (the TT genotype is also called the TT type or T:T type) is greater than that of Chinese cabbage with the TA genotype of the A05_18777194 marker (the TA genotype is also called the TA type or T:A type). The rosette leaf angle of Chinese cabbage with the TA genotype of the A05_18777194 marker (the TA genotype) is greater than the rosette leaf angle of Chinese cabbage with the AA genotype of the A05_18777194 marker (the AA genotype is also called the AA type or A:A type).

[0135] The TT genotype of the A05_18777194 marker is a homozygous type with T at the 313th position of Sequence 4.

[0136] The AA genotype of the A05_18777194 marker is a homozygous type with A at the 313th position of Sequence 4.

[0137] The TA genotype of the A05_18777194 marker is a heterozygous type with T at the 313th position of Sequence 4 and A at the 313th position of Sequence 4.

[0138] The rosette leaf angle of Chinese cabbage with the GG genotype of the A05_18955845 marker (the GG genotype is also called the GG type or G:G type) is greater than that of Chinese cabbage with the GC genotype of the A05_18955845 marker (the GC genotype is also called the GC type or G:C type). The rosette leaf angle of Chinese cabbage with the GC genotype of the A05_18955845 marker (the GC genotype) is greater than the rosette leaf angle of Chinese cabbage with the CC genotype of the A05_18955845 marker (the CC genotype is also called the CC type or C:C type).

[0139] The GG genotype of the A05_189558454 marker is a homozygous type with G at the 250th position of Sequence 5.

[0140] The CC genotype of the A05_18955845 marker is a homozygous type with C at the 250th position of Sequence 5.

[0141] The GC genotype marked by A05_18955845 is a heterozygous type with G at position 250 and C at position 250 in Sequence 5.

[0142] The marker A05_18777194 is an SNP in the Chinese cabbage genome, which is A or T at position 313 in Sequence 4. The specific Sequence 4 is as follows:

[0143] ATGAAAGTACCACTAAAGCCAAAGATAATATTCATCCCTTACCCTGCGCAAGGCCATGTCACTCCGATGCTCCACCTCGCGTCGGCCTTCCTCAGCCGTGGATTCTCCCCTGTCGTTATGACTCCCGGGTCTATCCACCGTAGGATCTCGACGACTAACGAGGATCTTGGGATCACGTTCTTGGCCTTATCAGACGGTCTAGAACGTCCGGACGCACTTCCCTCGGACTTCTTCTCCATAGAGAGGTCGATGGAGAACATCATGCCGTCTCAGCTCGAACGATTCCTACTAGACGAAGACGCGGGCGTGGCTwGTGTTGTGGTTGATTTGTTGGCTTCGTGGGCTATAAAAGTGGCTGATCGGTGTGGTGTTCCTGTTGCCGGATTCTGGCCGGTGATGCTCGCTGCTTACCGTATGATCGAATCCATACCAGAGCTAGTAAGGACAGGCATAGTTTCCCGAAAAGGTAATCTGATTCGTTTTAATACAACGGTTAATTCTCATATATCGTAA; where W is A or T.

[0144] The marker A05_18955845 is an SNP in the Chinese cabbage genome, which is G or C at position 250 in Sequence 5. The specific Sequence 5 is as follows:

[0145]

[0146] The seeds of the individual plants in the F2 population obtained in Example 1 (see the construction of the genetic population in Example 1) were planted. When the Chinese cabbage grew to the rosette stage, the latest fully expanded and healthy rosette leaf was found, and the angle between this leaf and the central axis of the plant was measured using a digital angle gauge with a range of 0 - 200 mm. This was recorded as the leaf angle of the plant. The specific results are shown in Table 4.

[0147] The genotypes of the F2 population were detected using the primers constructed above. Figure 7 a: KASP genotyping detection of the A05_18777194 marker in the F2 population. T: The T:A point is the heterozygous genotype, T:T point is the homozygous large - angle genotype, and A:A point is the homozygous small - angle genotype. Figure 7 b: KASP genotyping detection of the A05_18955845 marker in the F2 population. C: The C:G point is the heterozygous genotype, G:G point is the homozygous large - angle genotype, and C:C point is the homozygous small - angle genotype. The specific results are shown in Table 4. The accuracy rate of the small leaf angle of the rosette leaf can reach 97.18%, and the accuracy rate of the large leaf angle of the rosette leaf can reach 96.10%.

[0148] Table 4 Detection results of the genotypes of 300 individual plants in the F2 population using the constructed KASP primers

[0149]

[0150]

[0151]

[0152]

[0153]

[0154]

[0155]

[0156]

[0157]

[0158]

[0159]

[0160] Example 3 Verification of the function of the BraA05g025190.3C gene

[0161] 1. Construction and transformation of the over - expression vector

[0162] Extract the RNA of the rosette leaves of the large-leaf angle parent 21M-193, reverse transcribe the RNA into cDNA, and obtain the cDNA of 21M-193.

[0163] Construction of overexpression vector

[0164] (1) Use the CDS primers of BraA05g025190.3C in Table 5 to amplify the above-obtained 21M-193 cDNA, and recover the CDS amplification sequence of the BraA05g025190.3C gene.

[0165] Table 5 Specific primers for the CDS and promoter of the BraA05g025190.3C gene

[0166]

[0167] (2) In the enzyme digestion system of the pCAMBIA 2300 plasmid (purchased from YouBio, product number VT1383) (see Table 6), carry out the enzyme digestion reaction (at 37 °C) for 3-5 h, recover the large fragment, and obtain the enzyme digestion product of the pCAMBIA 2300 plasmid.

[0168] Table 6 Vector enzyme digestion system

[0169] Component Volume (μL) Cutsmart 10 XbaI 2 SalI 2 pCAMBIA2300 plasmid 1 <![CDATA[ddH 2 O]]> Make up to 20 μL

[0170] (3) Homologous recombination

[0171] Use the Novoprotein ClonExpressⅡ OneStep CloningKit kit to carry out homologous recombination ligation of the above CDS amplification sequence of the BraA05g025190.3C gene and the enzyme digestion product of the pCAMBIA 2300 plasmid. After reacting at 37 °C for 30 min, store at 4 °C to obtain the ligation reaction solution. The ligation system is shown in Table 7.

[0172] Table 7 Homologous recombination ligation system

[0173] Ingredient Volume (μL) CDS amplification sequence of BraA05g025190.3C gene 3 Digested product of pCAMBIA2300 plasmid 4 Exnase Ⅱ 1 5xCE Ⅱ buffer 2

[0174] (4) Transform Escherichia coli DH5α

[0175] Take 10 μL of the above ligation reaction solution and transform Escherichia coli DH5α (TransGen Biotech, Beijing). Extract the plasmid from the grown single colonies for sequencing. Name the recombinant plasmid with the correct sequence as pCAMBIA 2300-BraA05g025190.3C. The recombinant plasmid pCAMBIA 2300-BraA05g025190.3C is obtained by replacing the fragment between 5'-AACACGGGTACC-3' and 5'-GTCGACATGGTG-3' of the pCAMBIA 2300 plasmid with the DNA fragment 1 (Sequence 3) with the nucleotide sequence, while keeping the other sequences of the plasmid vector unchanged. The recombinant plasmid pCAMBIA 2300-BraA05g025190.3C is also called the recombinant vector pCAMBIA2300-BraA05g025190.3C or pCAMBIA 2300-BraA05g025190.3C. Name the Escherichia coli DH5α containing the recombinant vector pCAMBIA 2300-BraA05g025190.3C as DH5α / pCAMBIA 2300-BraA05g025190.3C.

[0176] Sequence 1 is as follows:

[0177]

[0178] Sequence 2 is the protein sequence encoded by Sequence 1, which is specifically as follows:

[0179] MESCSVLEVDINGEETIFLNKQIICKYSGTLRKLLGKSTCSSSGNLKVIFNDFPGGGESFELLSRFCYNNCEWTVMPSNV

[0180] VFLHCAAKFMEVTKVLAQTEKCMEEIRYWAWPEVLLSLKQCQELETSSEAESLAVKLMDALVEKLCLAIEASPSSAACSP

[0181] SPDSSLFRFSCDSKSTESFKNSSSRITWWFDEVLVLSPGLVKTFLKLMVLRKFDNVTITRFLFYYQKVKFCSASSNEKRE

[0182] ILETTVDTLYVLDRSCVPYKSLFGVLRLALGLNINKSVMNKLEVMIGQQLEQATLDNLLVPSPSKSSHLYYVNLVLRFAK

[0183] AFLDGGKRGSSQMKKVARLIDQYVAEVAPDPCLKPSKFLSLLTLVPDSARESHEDIYRAIDMYLEAHAGLTNGERLNLIR

[0184] ALSYEKLSVQSRAHMSRNTKFQEIETLDDEQQQEQKQLVLRVEKVETSGENEKLKEHIEGIQWRVMELERACLKMQTQMEVIKKRTKSTSRGSNRSLPKLCS。

[0185] Sequence 3 is the gene sequence of Sequence 1, which is specifically as follows:

[0186]

[0187] (5) Plasmid extraction

[0188] Use the Novoprotein FastPure Plasmid Mini Kit to extract the plasmid from DH5α / pCAMBIA 2300 - BraA05g025190.3C to obtain the recombinant plasmid pCAMBIA 2300 - BraA05g025190.3C. The specific steps are shown in the instruction manual.

[0189] (6) Transformation of Agrobacterium tumefaciens GV3101

[0190] 1) Take out the Agrobacterium tumefaciens GV3101 competent cells (Bomed, Beijing) stored at -80 °C and thaw them on ice.

[0191] 2) Add 1 μg of the above recombinant plasmid pCAMBIA 2300 - BraA05g025190.3C to 50 μL of GV3101 competent cells. After gently flicking the bottom of the centrifuge tube to mix, incubate on ice for 5 min, freeze in liquid nitrogen for 5 min, incubate in a water bath at 37 °C for 5 min, and then incubate on ice for 5 min.

[0192] 3) Under sterile conditions, add 800 μL of antibiotic - free LB liquid medium and culture at 28 °C and 200 rpm for 2 h.

[0193] 4) Centrifuge at 5000 rpm for 5 min to collect the bacteria. Pour off part of the supernatant, leave 100 - 200 μL of supernatant, pipette and blow the bottom precipitate, and aspirate the suspended bacterial liquid and spread it on an LB solid medium containing Kan and Rif resistance. Invert the medium and place it in a temperature - controlled shaker at 28 °C for 2 d.

[0194] 5) Pick monoclonal colonies for plaque detection to obtain monoclonal Agrobacterium tumefaciens GV3101 / recombinant plasmid pCAMBIA 2300 - BraA05g025190.3C with clear and correct bands. Agrobacterium tumefaciens GV3101 / recombinant plasmid pCAMBIA 2300 - BraA05g025190.3C is Agrobacterium tumefaciens GV3101 containing the recombinant plasmid pCAMBIA 2300 - BraA05g025190.3C. Add an equal volume of 50% glycerol and mix, and store at -80 °C in the refrigerator for later use.

[0195] (7) Arabidopsis thaliana transformation method

[0196] 1) Aspirate 500 μL of the Agrobacterium tumefaciens GV3101 / recombinant plasmid pCAMBIA 2300 - BraA05g025190.3C solution and inoculate it into 50 mL of LB liquid medium containing Kan and Rif antibiotics. Incubate with shaking at 28 °C and 250 rpm for 18 - 24 h.

[0197] 2) After the cultivation, centrifuge at 5000 rpm for 15 min, discard the supernatant, add 1 / 2 MS liquid medium to resuspend the precipitated bacteria, adjust the OD600 to 0.8 using a UV spectrophotometer, and add 10 μL of Silwet L-77 after adjustment and mix well.

[0198] 3) Before infection, cut off the existing siliques of Col-0 Arabidopsis thaliana (Arabidopsis thaliana variety Columbia), and then immerse the inflorescence of Arabidopsis thaliana in the bacterial solution and take it out after 4 min;

[0199] 4) Place the infected Arabidopsis thaliana in the dark, light-proof and moist conditions for 16 h;

[0200] 5) Depending on the growth of Arabidopsis thaliana, infect again after one week.

[0201] (8) Screening of positive transgenic Arabidopsis thaliana plants

[0202] Sow the T 1 -generation seeds on 1 / 2 MS solid medium containing 25 mg / L Kan antibiotic, and screen for transgenic resistant seedlings by Kan. When the positive seedlings grow to 3-5 true leaves, transplant them into nutrient soil to grow, and harvest the T 2 -generation seeds individually. Then, continue to screen for individual progeny with a Kan resistance segregation ratio of 3:1 (resistant: non-resistant) from the T 2 -generation, and identify the T 2 -generation positive seedlings using the CDS amplification primers of BraA05g025190.3C (Table 5). Select the T 2 -generation positive seedlings with a 100% positive rate of individual progeny, and harvest their T 3 -generation seeds. They are homozygous plants transformed with pCAMBIA 2300-BraA05g025190.3C. Finally, sow the T 3 -generation seeds on 1 / 2 MS medium containing Kan for screening, and select the T 3 -generation seeds with all seedlings having Kan resistance for subsequent observation of overexpression phenotypes.

[0203] T 3 -generation Arabidopsis thaliana positive homozygous seedlings and Col-0 Arabidopsis thaliana seedlings are cultured at 21 °C under the condition of 16 h light / 8 h dark until the stage of 6 to 8 true leaves. Take the sixth rosette leaf of the T 3 -generation Arabidopsis thaliana positive homozygous seedlings and Col-0 Arabidopsis thaliana seedlings to extract total RNA, reverse transcribe to obtain cDNA, and use Real-Time to detect the T 3The expression level of the BraA05g025190.3C gene in homozygous positive Arabidopsis thaliana seedlings, with the internal reference gene being BrGAPDH (primers are shown in Table 8 below). The Real-Time reaction system and program are shown in Tables 9 and 10 below, and the results are as Figure 4 shown, where WT is Col-0 Arabidopsis thaliana seedlings, and OE-brarla is T 3 generation homozygous positive Arabidopsis thaliana seedlings, and the vertical axis represents the expression level of the BraA05g025190.3C gene.

[0204] Table 8 BraA05g025190.3C Real-Time primers

[0205]

[0206] Table 9 Real-Time reaction system

[0207] Component Volume (μL) AceQ qPCR SYBR Green MasterMix 5 μL Forward primer 0.5 μL Reverse primer 0.5 μL <![CDATA[Template cDNA (400 ng·μL -1 )]]> 1 μL <![CDATA[ddH 2 O]]> 2 μL

[0208] Table 10 Real-time reaction program

[0209] Step Temperature Time 1 94℃ 5 min 2 94℃ 15s 3 60℃ 15s 4 72℃ 15s 5 Go to step 2 40 cycles 6 72℃ 5 min

[0210] Meanwhile (when Arabidopsis thaliana grows to six and eight true leaves), select 30 T 3 generation homozygous positive Arabidopsis thaliana seedlings and 30 Col-0 Arabidopsis thaliana seedlings respectively, and use Image J software to measure the leaf angle (find the latest fully expanded healthy rosette leaf, measure the angle between this leaf and the plant's central axis, and record it as the leaf angle of this plant), record and take the average value. The change in the leaf angle of Arabidopsis thaliana is the most obvious, and the results are as Figure 5 shown, Figure 5 where WT is Col-0 Arabidopsis thaliana, and OE-brarla is T 3 generation homozygous positive Arabidopsis thaliana seedlings, and the six-leaf stage and eight-leaf stage are the six-leaf stage and eight-leaf stage of Arabidopsis thaliana respectively.

[0211] Moreover, take pictures to record the growth status, and the results are as Figure 6 shown, Figure 6 where a: T 3 generation homozygous positive Arabidopsis thaliana seedlings at the six-leaf stage; Figure 6 where b: Col-0 Arabidopsis thaliana seedlings at the six-leaf stage; Figure 6 where c: T 3 generation homozygous positive Arabidopsis thaliana seedlings at the eight-leaf stage; Figure 6 where d: Col-0 Arabidopsis thaliana seedlings at the eight-leaf stage; Figure 6 where e: T 3 generation homozygous positive

[0212] Arabidopsis thaliana seedlings;Figure 6 Col-0 Arabidopsis thaliana seedlings at the eight-leaf stage.

[0213] The results showed that Figure 6 In a and b, they are the comparisons between the T 3 generation of Arabidopsis thaliana positive homozygous seedlings and Col-0 Arabidopsis thaliana seedlings. The average leaf angle of the rosette leaves of the T 3 generation of Arabidopsis thaliana positive homozygous seedlings is 77°, and the average leaf angle of the rosette leaves of Col-0 Arabidopsis thaliana seedlings is 44°. Figure 6 In c, d, e, and f, they are the comparisons between the T 3 generation of Arabidopsis thaliana positive homozygous seedlings and Col-0 Arabidopsis thaliana seedlings at the eight-leaf stage. The leaf angles of the rosette leaves were measured in the same way, and it was found that the leaf angle of the rosette leaves of the T 3 generation of Arabidopsis thaliana positive homozygous seedlings increased by 5° compared with the six-leaf stage, and the average leaf angle was 82°. The average leaf angle of Col-0 Arabidopsis thaliana was 57°, and the leaf angle increased by 13° compared with the six-leaf stage.

[0214] 2. Construction of VIGS vector and transformation of strains

[0215] Method for constructing VIGS (Virus Induced Gene Silencing) vector: Download the CDS sequence of BraA05g025190.3C from the Chinese cabbage genome database website, select a 40bp specific fragment from it, design a palindromic DNA fragment with SnaBI restriction sites on both sides and 80bp, and add 15bp homologous sequences corresponding to both ends of the linearized PTY-S vector at both ends, a total of 110bp (CACGGACACTTCTACCTGATGGTGTTGAGGAAGTTCGATAATGTTACCATCACTATAGTGATGGTAACATTATCGAAC TTCCTCAACACCATCAGGTAAGTTCTCGATCT), which was synthesized by Nanjing Genscript Biotechnology Co., Ltd. and ligated with the vector pTY-S, and then transformed into Escherichia coli Stbl3.

[0216] PTY-S-BraA05g025190.3C-VIG is a VIGS gene silencing vector. The target site of PTY-S-BraA05g025190.3C-VIG is positions 207 - 220 of SEQ ID NO.2. PTY-S-BraA05g025190.3C-VIG is a recombinant vector containing a DNA molecule with the nucleotide sequence of positions 207 - 220 of SEQ ID NO.2.

[0217] PTY-S-BraA05g025190.3C-VIG is a recombinant plasmid obtained by inserting an 80-bp palindromic fragment containing a DNA molecule at positions 207-220 of SEQ ID NO.2 and 15-bp homologous sequences corresponding to both ends of the linearized PTY-S vector, a total of 110 bp (CACGGACACTTCTACCTGATGGTGTTGAGGAAGTTCGATAATGTTACCATCACTATAGTGATGGTAACATT ATCGAACTTCCTCAACACCATCAGGTAAGTTCTCGATCT) between 5’-GCACTCTCCGCTCAT-3’ and 5’-TTAAAATCGTTAGCT-3’ of the PTY-S plasmid (purchased from Nanjing Genscript Biotechnology Co., Ltd.), while keeping other sequences of the plasmid vector unchanged. The recombinant plasmid is named recombinant plasmid PTY-S-BraA05g025190.3C-VIG, also known as recombinant vector PTY-S-BraA05g025190.3C-VIG or PTY-S-BraA05g025190.3C-VIG. The obtained Agrobacterium tumefaciens GV3101 containing the recombinant vector PTY-S-BraA05g025190.3C-VIG is named GV3101 / PTY-S-BraA05g025190.3C-VIG. GV3101 / PTY-S is obtained in the same way. GV3101 / PTY-S is Agrobacterium tumefaciens GV 3101 containing the PTY-S plasmid.

[0218] The large-scale plasmid extraction was performed using the Giga kit (Omega, USA) as follows:

[0219] 1) Pick monoclonal colonies of PTY-S-BraA05g025190.3C-VIG and PTY-S empty vector (GV3101 / PTY-S-BraA05g025190.3C-VIG and GV3101 / PTY-S) on the medium and add them separately to 500 mL of LB liquid medium containing Amp resistance. Incubate in a temperature-controlled shaker at 30 °C and 225 rpm for 16 h.

[0220] 2) Centrifuge at 5000 rpm for 5 min at room temperature to collect the bacteria, and pour off the supernatant.

[0221] 3) Resuspend the bacteria with 125 mL of solutionⅠ / RNaseA.

[0222] 4) Add 125 mL of solutionⅡ and gently invert the mixture up and down 15-20 times. Let it stand at room temperature for 3 min.

[0223] 5) Add 125 mL of Neutralization Buffer and gently mix up and down until the solution is filled with white flocs;

[0224] 6) Centrifuge at 5000 rpm for 10 min and filter the liquid through a filter cloth into a new centrifuge bottle;

[0225] 7) Add 1 / 3 weight of PFC Binding Buffer and mix well;

[0226] 8) Pass the solution from step (7) through a HiBind DNA Giga column. First add 20 mL of DNA washing buffer and then add 40 mL to flow through the column;

[0227] 9) Keep the vacuum for 10 - 15 min to ensure that ethanol is completely removed;

[0228] 10) Add 3 - 5 mL of H 2 O or EB and incubate for 5 min, depending on the specific concentration;

[0229] 11) Insert the piston and slowly push it in to inject the plasmid into the tube;

[0230] 12) Use a NanoDrop2000 ultraviolet spectrophotometer to detect the plasmid concentration. Adjust the PTY - S plasmid and the PTY - S - BraA05g025190.3C - VIGS plasmid to 300 ng / μL to obtain the inoculation plasmid.

[0231] VIGS infects the small - leaf - angle Chinese cabbage Baiyang parent. The steps are as follows: Plant the small - leaf - angle parent Baiyang and the large - leaf - angle parent 21M - 193 in a temperature - controlled greenhouse at 25℃ / 18℃, 16 h during the day / 8 h at night. Randomly divide the small - leaf - angle parent into 3 groups, namely the non - infection control group, the infection empty - vector group, and the experimental group, with 30 plants in each group. Divide the large - leaf - angle parent 21M - 193 into 1 group, namely the large - leaf - angle parent 21M - 193 group, with 30 plants in each group.

[0232] When it grows to 3 - 4 true leaves, the large leaf angle parent 21M - 193 group and the non - infected control group with small leaf angle parent Baiyang are not treated. The infection empty - vector group and the experimental group are respectively infected with PTY - S plasmid and PTY - S - BraA05g025190.3C - VIG plasmid (one day before VIGS infection, Chinese cabbage is treated with darkness. When inoculating, gently rub the Chinese cabbage leaves with quartz sand until slight wounds are made). According to the leaf sizes of the infection empty - vector group and the experimental group, 6 - 10 μL of the above - mentioned PTY - S plasmid and PTY - S - BraA05g025190.3C - VIG plasmid are aspirated and smeared on the wounds. After 1 min, slowly rinse with distilled water and dry with a tissue. Cultivate under the conditions of 25℃ / 18℃, 16 h during the day / 8 h at night. One week later, depending on the growth of Chinese cabbage, when 3 new leaves grow, perform the above - mentioned infection step again. After continuous infection three times (the same steps as above), observe the phenotypes, measure the rosette leaf angle (find the latest completely extended healthy rosette leaf, measure the angle between this leaf and the central axis of the plant, and record it as the leaf angle of this plant), and take pictures. The results are as Figure 3 shown, Figure 3 in which, A2 is the non - infected control group (small leaf angle parent Baiyang), and BraRLA is the experimental group. After three infections are completed, collect the rosette leaves to extract total RNA, reverse - transcribe to obtain cDNA, and use Real - Time to perform qRT - PCR detection of the expression level of BraA05g025190.3C gene (the specific method refers to the steps in Example (7) Screening of positive transformed Arabidopsis plants) to identify VIGS positive seedlings. The results are as Figure 2 shown, Figure 2 in which, A1 is the large leaf angle parent 21M - 193 group (large leaf angle parent 21M - 193), A2 is the non - infected control group (small leaf angle parent Baiyang), PTYs is the infection empty - vector group, VIGS is the experimental group, and the vertical coordinate is the expression level of BraA05g025190.3C gene.

[0233] To sum up, VIGS belongs to post - transcriptional gene silencing. After a recombinant virus carrying a target gene cDNA fragment infects a plant, it induces phenotypic mutations due to target gene silencing. This method has characteristics such as simplicity, rapidity, and reliability, and is a practical method for studying plant gene functions. Currently, this technology has been applied to various cruciferous Brassica crops. Therefore, we used VIGS to silence the BraRLA gene of Chinese cabbage Baiyang with a small leaf angle to determine whether this gene has the function of regulating the leaf angle of Chinese cabbage. The research found that after the empty - vector PTY - s infected Chinese cabbage, the phenotype of Chinese cabbage did not change. When the PTYs - BraRLA virus plasmid infected Chinese cabbage with a small leaf angle, Baiyang showed a phenotype of increased leaf angle ( Figure 3 ).

[0234] To verify the function of PTYs - BraRLA at the transcriptional level, qRT - PCR analysis was performed on BraRLA of VIGS positive plants and CK to verify the difference in the expression level of this gene. The results showed that compared with the expression level of the BraRLA gene in the PTY - s empty vector, the expression level of the BraRLA gene in the VIGS positive plants was significantly down - regulated, and the relative expression level differed by nearly 2 - fold, with a highly significant difference. And compared with the expression level of the Brarla gene (BraA05g025190.3C gene) of the large - angle parent A 1 , the expression level of the BraRLA gene in the former was slightly higher than that of the Brarla gene in the latter ( Figure 2 ). This indicates that BraRLA is a key gene controlling the rosette leaf angle trait of Chinese cabbage.

[0235] The above has described the present invention in detail. For those skilled in the art, without departing from the purpose and scope of the present invention and without 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 intends to cover any modification, use or improvement of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the following appended claims.

Claims

1. Use of a protein, a substance for regulating the expression of a gene encoding the protein, or a substance for regulating the activity or content of the protein in any of the following: A1) Application in improving or increasing the angle of rosette leaves of cruciferous plants; A2) application in preparing a product for improving or increasing the angle of rosette leaves of cruciferous plants; The protein is any of the following: B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; B2) a protein obtained by substitution and / or deletion and / or addition of amino acid residues of the protein described in B1) which has an identity of more than 80% with the protein described in B1) and has the same function as the protein described in B1); B3) A fusion protein obtained by connecting the N-terminus and / or C-terminus of B1) or B2) to a protein tag.

2. The use according to claim 1, characterized in that The protein is derived from cabbage.

3. The use according to claim 1 or 2, characterized in that The substance that regulates the expression of the gene encoding the protein is any one of the following: D1) a nucleic acid molecule that inhibits, reduces or down-regulates the expression of a gene encoding a protein according to claim 1 or 2; D2) expressing the gene encoding the nucleic acid molecule described in D1); D3) an expression cassette containing the gene described in D2); D4) a recombinant vector containing the gene described in D2) or a recombinant vector containing the expression cassette described in D3); D5) a recombinant microorganism containing the gene described in D2), or a recombinant microorganism containing the expression cassette described in D3), or a recombinant microorganism containing the recombinant vector described in D4); D6) a transgenic plant cell line containing the gene described in D2), or a transgenic plant cell line containing the expression cassette described in D3), or a transgenic plant cell line containing the recombinant vector described in D4); D7) transgenic plant tissue containing the gene described in D2), or transgenic plant tissue containing the expression cassette described in D3), or transgenic plant tissue containing the recombinant vector described in D4); D8) a transgenic plant organ containing the gene described in D2), or a transgenic plant organ containing the expression cassette described in D3), or a transgenic plant organ containing the recombinant vector described in D4); D9) A nucleic acid molecule encoding the protein according to claim 1 or 2; D10) an expression cassette containing the nucleic acid molecule described in D9); D11) a recombinant vector containing the nucleic acid molecule described in D9), or a recombinant vector containing the expression cassette described in D10); D12) a recombinant microorganism containing the nucleic acid molecule described in D9), or a recombinant microorganism containing the expression cassette described in D10), or a recombinant microorganism containing the recombinant vector described in D11); D13) a transgenic plant cell line containing the nucleic acid molecule described in D9), or a transgenic plant cell line containing the expression cassette described in D10), or a transgenic plant cell line containing the recombinant vector described in D11); D14) transgenic plant tissue containing the nucleic acid molecule described in D9), or transgenic plant tissue containing the expression cassette described in D10), or transgenic plant tissue containing the recombinant vector described in D11); D15) A transgenic plant organ containing the nucleic acid molecule described in D9), or a transgenic plant organ containing the expression cassette described in D10), or a transgenic plant organ containing the recombinant vector described in D11).

4. A method for cultivating Chinese cabbage with a large leaf angle, characterized in that: The method comprises down-regulating, weakening or reducing, or up-regulating, enhancing or increasing the expression level of the gene encoding the protein in claim 1 or 2 in the target Chinese cabbage, and / or the activity and / or content of the protein to obtain Chinese cabbage with a large leaf angle, wherein the rosette leaf angle of the Chinese cabbage with a large leaf angle is greater than that of the target Chinese cabbage.

5. A method for cultivating Arabidopsis thaliana with a large leaf angle, characterized in that The invention comprises up-regulating, enhancing or increasing, or down-regulating, weakening or reducing the expression level of the gene encoding the protein described in claim 1 or 2 in the target Arabidopsis thaliana, and / or the activity and / or content of the protein to obtain Arabidopsis thaliana with a large leaf angle, wherein the rosette leaf angle of the Arabidopsis thaliana with a large leaf angle is greater than that of the target Arabidopsis thaliana.

6. Use of the method according to claim 4 or 5 in plant breeding.

7. The use according to any one of claims 1 to 3 and 6, and the method according to claims 4 to 5, characterized in that: The plant is any of the following: J1) dicotyledonous plants; J2) Cruciferae; J3) Brassica or Arabidopsis; J4) Brassica rapa or Arabidopsis thaliana.

8. Use of a substance for detecting a molecular marker linked to a gene of the protein according to claim 1 in any of the following; A1) Application in predicting and / or assisting in predicting the rosette leaf angle of a plant; A2) Application in the preparation of a product for predicting and / or assisting in predicting the angle of a plant rosette leaf.

9. A method for predicting and / or assisting in predicting the rosette leaf angle of a plant, comprising detecting the genotype of the molecular marker of claim 8 in a plant to be tested, and predicting and / or assisting in predicting the rosette leaf angle of the plant according to the genotype of the plant to be tested, wherein the genotype of the molecular marker comprises the genotype of the A05_18777194 marker and / or the genotype of the A05_18955845 marker: The rosette leaf angle of Chinese cabbage of which the genotype of marker A05_18777194 is homozygous for T at position 313 of sequence 4 is greater than the rosette leaf angle of Chinese cabbage of which the genotype of marker A05_18777194 is heterozygous for T at position 313 of sequence 4 and A at position 313 of sequence 4; the rosette leaf angle of Chinese cabbage of which the genotype of marker A05_18777194 is homozygous for T at position 313 of sequence 4 is greater than the rosette leaf angle of Chinese cabbage of which the genotype of marker A05_18777194 is heterozygous for T at position 313 of sequence 4 and A at position 313 of sequence 4 and the genotype of marker A05_18777194 is homozygous for A at position 313 of sequence 4; and / or, The rosette leaf angle of Chinese cabbage of the homozygous type with the genotype of marker A05_18955845 being G at position 250 of sequence 5 is greater than the rosette leaf angle of Chinese cabbage of the heterozygous type with the genotype of marker A05_18955845 being G at position 250 of sequence 5 and C at position 250 of sequence 5; the rosette leaf angle of Chinese cabbage of the homozygous type with the genotype of marker A05_18955845 being G at position 250 of sequence 5 is greater than the rosette leaf angle of Chinese cabbage of the heterozygous type with the genotype of marker A05_18955845 being G at position 250 of sequence 5 and the homozygous type with the genotype of marker A05_18955845 being C at position 250 of sequence 5.

10. The use according to claim 8 and the method according to claim 9, characterized in that: The A05_18777194 marker or the A05_18955845 marker, or the substance for detecting the genotype of the A05_18777194 marker or the A05_18955845 marker, is as follows D1), D2), D3) or D4): D1) containing in vitro nucleic acid amplification primers for specifically amplifying the SNP; D2) an in vitro nucleic acid amplification reagent containing the in vitro nucleic acid amplification primer described in D1); D3) a kit containing the in vitro nucleic acid amplification primers described in D1) or the in vitro nucleic acid amplification reagents described in D2); D4) A detection instrument containing the in vitro nucleic acid amplification primer described in D1), the in vitro nucleic acid amplification reagent described in D2) or the kit described in D3) The A05_18777194 marker is A or T at position 313 of sequence 4; The A05_18955845 marker is G or C at position 250 of sequence 5.

Images