Braamme cr1 gene and application thereof in regulating plant leaf size development

Abstract

The present application belongs to the technical field of plant breeding and genetic engineering, and particularly relates to BraAMMECR1 gene and its application in regulating leaf size development of Chinese cabbage. The present application uses two Chinese cabbage selfing lines Y2 (large leaf) and Y7 (small leaf) with extremely significant difference in leaf size to construct F2 population, and then uses BSA positioning and expression pattern analysis to screen a gene BraAMMECR1 for regulating leaf size development of Chinese cabbage, which is proved to effectively promote Chinese cabbage cell proliferation. The present application lays a foundation for analyzing the molecular regulation mechanism of Chinese cabbage leaf size development and improving Chinese cabbage yield through molecular breeding means, and therefore has good practical application value.

Description

Technical Field

[0001] This invention belongs to the field of plant breeding and genetic engineering technology, specifically involving the BraAMMECR1 gene and its application in regulating the size development of Chinese cabbage leaves. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Chinese cabbage (Brassica rapa L.ssp.pekinensis), often referred to as the "national vegetable," has an annual planting area of ​​approximately 30 million mu (about 2 million hectares) and a total yield of about 100 million tons, making it the largest-yielding vegetable crop in my country. Previous studies have shown a significant positive correlation between leaf size and yield, making it a key factor in yield traits and a focus of attention in Chinese cabbage breeding. However, the molecular mechanisms regulating leaf size development in Chinese cabbage remain a mystery. Therefore, identifying key genes regulating leaf size development and elucidating their regulatory mechanisms is crucial for rapidly innovating Chinese cabbage germplasm and cultivating superior new varieties through bio-breeding methods.

[0004] Extensive research in plants such as Arabidopsis thaliana has confirmed that leaf size is a complex trait controlled by multiple genes, and its regulation may involve a complex gene network with multiple components and pathways. At the cellular level, plant leaf growth can be divided into two coordinated processes: cell proliferation and cell expansion. In recent years, several genes controlling plant leaf size have been isolated from plants such as Arabidopsis thaliana and rice. For example, the ARL and TOR genes are involved in the positive regulation of cell expansion, while the AtKIN-13A and ARF8 genes are involved in the negative regulation of cell expansion. The regulatory process involved in cell proliferation is relatively more complex, involving two very important regulatory pathways: the ANT pathway and the TCP-GRF pathway. In the ANT pathway, when plants receive auxin signals, the expression of the ARGOS gene is induced. Then, by regulating the expression of the ANT gene, the level of the cyclin CYCD3 gene in cells is further regulated, ultimately regulating cell proliferation. Studies have confirmed that excessive or reduced expression of ARGOS and ANT genes in Arabidopsis thaliana can significantly increase or decrease the size of organs such as leaves, inflorescences, and flowers. In the TCP-GRF pathway, studies on some members of the Arabidopsis TCP transcription factor family (TCP2, TCP3, TCP4, TCP10, and TCP24) revealed that their transcriptional expression is regulated by miR319. The jaw-D mutant showed increased miR319 expression, which inhibited the expression of these TCP genes, leading to accelerated cell division at the leaf margins and a wavy leaf edge. This indicates that the aforementioned TCP transcription factors (Class II) are involved in the negative regulation of leaf size. Studies on GRF (growth-regulating factor) genes showed that their transcriptional expression is induced by gibberellin (GA), which then controls the number of cells in the leaf by regulating the expression of cell cycle genes, ultimately determining leaf size. Besides the ANT and TCP-GRF pathways, other genes in plants participate in regulating plant cell proliferation. For example, the Class I subfamily of TCP genes, as well as genes such as SWP and CYP78A7, participate in the positive regulation of cell proliferation, while genes such as DA, ROT4, and NGA participate in the negative regulation of cell proliferation.

[0005] Research on the development of Chinese cabbage leaf size has made some progress, but it is not in-depth. For example, the applicant studied the genes ANT1, GRF5, and GRF8 in Chinese cabbage using homologous cloning. They found that the transcriptional expression of ANT1 is induced by auxin, while the transcriptional expression of GRF5 and GRF8 is induced by gibberellin. Overexpression of these genes can promote the proliferation of transgenic plant cells and increase leaf size. Ge et al., Liu et al., Choi et al., and Wang Hongxia used QTL methods to locate traits controlling Chinese cabbage yield, such as gross weight, head weight, head height, and head width. However, these studies are still in the rough mapping stage and have not yet identified specific regulatory genes.

[0006] In summary, current research on the development of Chinese cabbage leaf size is significantly insufficient, and the answers provided are either incomplete or merely speculative. Using existing information to guide Chinese cabbage breeding remains inadequate. Therefore, there is an urgent need to use forward genetics to identify key genes regulating Chinese cabbage leaf size development and to elucidate their regulatory mechanisms, thereby better serving breeding practices. Summary of the Invention

[0007] To address the shortcomings of existing technologies, this invention aims to provide the BraAMMECR1 gene and its application in regulating the leaf size development of Chinese cabbage. Specifically, this invention utilizes two Chinese cabbage inbred lines, Y2 (large leaf) and Y7 (small leaf), with extremely significant differences in leaf size to construct an F2 population. Then, using BSA (bulked segregation analysis) localization and expression pattern analysis (RNA-seq), a gene named BraAMMECR1 that regulates the leaf size development of Chinese cabbage was screened, which can effectively promote Chinese cabbage cell proliferation. This invention lays the foundation for elucidating the molecular regulatory mechanism of Chinese cabbage leaf size development and improving Chinese cabbage yield through molecular breeding methods. Based on the above research results, this invention is thus completed.

[0008] To achieve the above-mentioned technical objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a gene named BraAMMECR1, said gene being selected from:

[0010] (a1) The nucleotide sequence shown in SEQ ID NO.1;

[0011] (a2) and (a1) encode proteins with the same amino acid sequence, but are different nucleotide sequences due to the degeneracy of the genetic code.

[0012] (a3) has ≥90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence identity) with the nucleotide sequence shown in (a1) or (a2) and encodes a nucleotide sequence having the same or similar functional protein;

[0013] A nucleotide sequence that is complementary to either (a4) or (a1)-(a3).

[0014] A second aspect of the present invention provides a protein encoded by the aforementioned gene BraAMMECR1. Specifically, the protein is selected from:

[0015] (b1) The amino acid sequence shown in SEQ ID NO.2;

[0016] (b2) A protein derived from the amino acid sequence shown in SEQ ID NO.2 by substitution and / or deletion and / or addition of one or more amino acid residues, and having the same function as the amino acid sequence shown in SEQ ID NO.2;

[0017] (b3) Other genes encoding proteins that have 90% or more (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (complete) sequence identity with the amino acid sequence shown in SEQ ID NO.2 and have the protein activity shown in SEQ ID NO.2.

[0018] In a third aspect, the present invention provides a recombinant expression vector containing the above-mentioned genes, a host bacterium, a transgenic cell, or a transgenic plant.

[0019] In one specific embodiment of the present invention, the recombinant expression vector is obtained by effectively linking the above-mentioned gene to the vector. The vector can be a cloning vector or an expression vector. The vector can be any one or more of a viral vector, plasmid, phage particle, or artificial chromosome, without specific limitations.

[0020] In this invention, the host bacterium refers to a host bacterium whose function has been altered by manipulating and modifying the genes of the target bacterium. This includes host bacteria obtained by introducing a foreign target gene or recombinant expression vector into the target bacterium, or host bacteria obtained by directly editing the endogenous genes of the target bacterium.

[0021] The target bacteria can be eukaryotic bacteria or prokaryotic bacteria.

[0022] The target bacteria can be bacteria, fungi, actinomycetes, etc. Specifically, the bacteria can be from genera such as *Escherichia*, *Agrobacterium*, *Flavobacterium*, *Alcaligenes*, *Pseudomonas*, and *Bacillus*, and more specifically, *Escherichia coli*, *Agrobacterium tumefaciens*, *Bacillus subtilis*, or *Bacillus pumilus*. The fungi can be yeasts. The fungi can be from genera such as *Fusarium*, *Verticillium*, *Penicillium*, *Aspergillus*, and *Cephalosporium*. The actinomycetes can be from genera such as *Streptomyces*, *Nocardia*, and *Micromonospora*.

[0023] In this invention, the transgenic cells can be isolated, ex vivo, cultured, or preferably part of a plant; wherein, the plant cells can be seed plant cells, specifically dicotyledonous plants, further cruciferous plants, and even more specifically Chinese cabbage.

[0024] A fourth aspect of the present invention provides the use of the BraAMMECR1 gene, protein, recombinant expression vector containing the BraAMMECR1 gene, host bacteria, or transgenic cells in any one or more of the following:

[0025] (c1) Regulates plant traits;

[0026] (c2) Improve and cultivate plants.

[0027] In this invention, the plant is any plant at any developmental stage. In particular, the plant can be a seed plant, further a dicotyledonous plant, and even further a cruciferous plant, with Chinese cabbage being the most preferred.

[0028] Specifically, (c1) regulates plant traits by regulating the size of Chinese cabbage leaves and plant structure.

[0029] The improvement and cultivation of plants (c2) refers to the improvement and cultivation of Chinese cabbage with larger leaves and a larger plant size, thereby obtaining high-yielding Chinese cabbage varieties. The leaf size can be assessed by the maximum leaf length and leaf width. This invention has demonstrated through experiments that Chinese cabbage overexpressing the BraAMMECR1-Y2 gene has longer and wider leaves than the wild-type control, resulting in larger leaves and a larger plant size.

[0030] A fifth aspect of the present invention provides a method for improving and breeding Chinese cabbage, the method comprising: increasing the expression level and / or activity of the BraAMMECR1 gene in Chinese cabbage, thereby obtaining Chinese cabbage with larger leaves and larger plant type traits, and thus obtaining a high-yielding Chinese cabbage variety.

[0031] In the above methods, increasing the expression level and / or activity of the BraAMMECR1 gene in Chinese cabbage can be achieved by means such as introducing a plasmid containing the BraAMMECR1 gene, operably linking a strong promoter to the BraAMMECR1 gene, and introducing an enhancer, etc., without specific limitations.

[0032] The beneficial technical effects of one or more of the above technical solutions are as follows:

[0033] The above technical solution screened a gene, BraAMMECR1, that regulates the size development of Chinese cabbage leaves. It can effectively promote the proliferation of Chinese cabbage cells, but does not change the size of transgenic Chinese cabbage cells. Ultimately, the leaves are longer and wider, resulting in larger leaves and larger Chinese cabbage plants. This is conducive to the analysis of the molecular regulatory mechanism of Chinese cabbage leaf size development and has broad application prospects and extremely high application value in Chinese cabbage molecular breeding. Attached Figure Description

[0034] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0035] Figure 1 The phenotypes of wild-type (WT) and overexpressing BraAMMECR1-Y2 (OE-Y2) plants were observed 40 days after sowing in this embodiment of the invention.

[0036] Figure 2 The maximum leaf length and leaf width of WT and OE-Y2 plants were statistically analyzed 40 days after sowing in this embodiment of the invention.

[0037] Figure 3 This is a scanning electron microscope image of the maximum leaf epidermis of WT and OE-Y2 plants 40 days after sowing, as shown in this embodiment of the invention.

[0038] Figure 4 This is a statistical representation of the number of epidermal cells per square millimeter of leaf in this embodiment of the invention. Detailed Implementation

[0039] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0040] It should be noted that the terminology used herein is for descriptive purposes only and is not intended to limit the exemplary embodiments according to this application. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof. This invention utilizes techniques and methods conventional in the fields of genetic engineering and molecular biology. Those skilled in the art can employ other conventional techniques, methods, and reagents in the art based on the embodiments provided in this invention, without being limited to the specific embodiments of this invention.

[0041] In this invention, the term "identity" or "consistency" refers to sequence similarity to an amino acid / nucleotide sequence. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0042] For sequence comparison, a sequence is typically used as a reference sequence and compared with the detection sequence. When using a sequence comparison algorithm, the detection and reference sequences are input into the computer, the coordinates of the subsequences are specified if necessary, and the parameters of the sequence algorithm program are specified. Then, based on the selected program parameters, the sequence comparison algorithm calculates the percentage sequence identity (consistency) of the detection sequence relative to the reference sequence.

[0043] Genes (nucleic acid molecules) can be DNA, such as cDNA, genomic DNA, or recombinant DNA, etc., without specific limitations.

[0044] Furthermore, a large number of transformation vectors available for plant transformation are known to those skilled in the art, and the nucleic acid molecules of this invention can be used in conjunction with any such vector. The choice of vector will depend on the preferred transformation technology and target plant species used for transformation.

[0045] Unless otherwise specified, all materials, reagents, strains, plasmids, enzymes, kits, etc. used in the following examples were obtained commercially.

[0046] Example

[0047] 1. Experimental Methods

[0048] 1.1 RNA extraction and reverse transcription

[0049] (1) Take 50-100mg of tissue and place it in a 1.5mL centrifuge tube. Add 1mL of Trizol and homogenize thoroughly. Let stand at room temperature for 5min.

[0050] (2) Add 0.2 mL of chloroform, shake for 15 s, and let stand for 2 min;

[0051] (3) Centrifuge at 4℃, 12,000g × 15min, and collect the supernatant;

[0052] (4) Add 0.5 mL of isopropanol, gently mix the liquid in the tube, and let it stand at room temperature for 10 min;

[0053] (5) Centrifuge at 4℃, 12,000g × 10min, and discard the supernatant;

[0054] (6) Add 1 mL of 75% ethanol, gently wash the precipitate, 4℃, 7500g×5min, and discard the supernatant;

[0055] (7) Dry, add an appropriate amount of DEPC H2O to dissolve (incubate at 65℃ for 10-15 min);

[0056] (8) Use the reverse transcription kit (TOYOBO Code: FSQ-101) to reverse transcribe RNA into cDNA.

[0057] 1.2 Cloning of gene fragments and construction of cloning vectors

[0058] Gene amplification primers were designed using Snapgene, and then PCR amplification was performed using KOD DNA polymerase with cDNA as a template. The PCR products obtained after agarose gel electrophoresis were ligated into the M5 HiPer pTOPO-Blunt vector and transformed into E. coli competent cells DH5α. After overnight culture, single clones were selected for PCR identification and sequencing.

[0059] 1.3 Construction and Identification of Overexpression Lines

[0060] (1) Expression vector construction: In this experiment, the overexpression vector used was pCAMBIA1307 plasmid. Primers were designed based on the expression vector map and the target gene sequence. The gene fragment with adapter was amplified using the cloning vector plasmid as a template and then ligated into the enzyme-digested pCAMBIA1307 vector.

[0061] (2) Agrobacterium transformation: Add 1 μL of recombinant plasmid to 50 μL of Agrobacterium competent cells, add 1 mL of YEP liquid medium, and incubate at 28℃ and 250 rpm for 3-5 h; spread on YEP solid medium with screening resistance, and incubate at 28℃ for 3 days; perform Agrobacterium PCR identification on single colonies, and store the correct strains at -20℃.

[0062] (3) Transformation of Chinese cabbage by inflorescence immersion method: Guangdong early-maturing Chinese cabbage was used as the experimental material. After the inflorescence was formed, the already formed pods were removed and set aside. 2 mL of activated Agrobacterium containing expression vector plasmid was added to 250 mL of YEP medium containing selection resistance and cultured overnight at 28°C. After collecting the bacterial cells, they were resuspended in the transformation medium and the OD600 was adjusted to 0.8. The inflorescence was immersed in the immersion solution for 30 seconds and cultured in the dark for one day. Then it was placed in a constant temperature incubator at 19°C.

[0063] (4) PCR identification of transgenic lines: Leaves of T3 generation transgenic Chinese cabbage and wild-type Chinese cabbage were taken with scissors and their DNA was extracted (extraction method refers to TaKaRa's plant DNA extraction kit). Using wild-type Chinese cabbage DNA as a negative control and the overexpressed plasmid as a positive control, the transgenic T3 generation plants were identified by PCR. The primers used were the universal primers M13-F / R on the pCAMBIA1307 vector.

[0064] Among them, primers for constructing the cloning vector:

[0065] 020370C-F:ATGGCGAACAGAGAGATGGCA(SEQ ID NO.3);

[0066] 020370C-R: TCAAGAAAAGGCCATGGCGG (SEQ ID NO. 4);

[0067] The primers for constructing the expression vector are as follows:

[0068] 20370xba1-F: cgacgagctctctagATGGCGAACAGAGAGATGGCA (SEQ ID NO.5);

[0069] 20370xba1-R:taccactagttctagTCAAGAAAGGCCATGGCGGG (SEQ ID NO. 6).

[0070] 2. Experimental Results

[0071] Using Chinese cabbage Y7 as material, the BraAMMECR1-Y2 gene was transformed. It was found that plants overexpressing the BraAMMECR1-Y2 gene in Y7 were significantly larger. Figure 1 and Figure 2 Furthermore, scanning electron microscopy revealed no significant difference in the number of epidermal cells within the same area between Y7 plants and plants overexpressing the BraAMMECR1-Y2 gene. Figure 3 and Figure 4In other words, overexpression of the BraAMMECR1-Y2 gene promoted the proliferation of transgenic plant cells but did not change the size of transgenic plant cells.

[0072] Nucleotide and amino acid sequence information used in this invention

[0073] BraAMMECR1 gene

[0074] (SEQ ID NO.1)

[0075] BraAMMECR1 protein

[0076] MANREMAVYCFDTLVSHYNNDETPPPAFDDANHPLFVTWKKIVNGGEPRLRGCIGTLEARRLINGFKDYALTSALRDRRFPPIQSKELPFLQCTVSVLTDYETADDYLDWEVGKHGIIIEFTEPVTNTKRSATYLPEVPAHEGWTKIEAIDSLVRKAGYNGEITESVRRRIRLTRYQSTLFSMHYSEYLSYVKATRGGVAPAINGTSKPAMAFS(SEQ IDNO.2)

[0077] It should be noted that the above examples are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the given examples, those skilled in the art can modify or make equivalent substitutions to the technical solutions of the present invention as needed, without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. Overexpression BraAMMECR1 The application of genes in promoting leaf and / or plant size increase in Chinese cabbage is characterized by, The BraAMMECR1 The gene has the nucleotide sequence shown in SEQ ID NO.

1.

2. A method for improving and cultivating Chinese cabbage, characterized in that, The method includes: improving the quality of Chinese cabbage BraAMMECR1 The expression level of genes is used to obtain Chinese cabbage with larger leaves and a larger plant shape, thereby obtaining high-yielding Chinese cabbage varieties; The BraAMMECR1 The gene has the nucleotide sequence shown in SEQ ID NO.1.

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