Tobacco NtD27 gene and its application

Abstract

This invention relates to the field of biotechnology, and more particularly to the tobacco NtD27 gene and its applications. Through research on the NtD27 gene, this application has discovered that this gene affects the development of tobacco axillary buds. Knockout mutations of the NtD27 gene can promote the growth of tobacco axillary buds, while overexpression of the NtD27 gene can effectively inhibit the growth of tobacco axillary buds. This provides potential germplasm resource support for molecular breeding of tobacco with fewer axillary buds and has significant practical value in the genetic improvement of important traits in tobacco and in basic botanical research.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the tobacco NtD27 gene and its applications. Background Technology

[0002] Tobacco is a widely cultivated and economically valuable crop. Proper topping and pruning are key agronomic measures for improving tobacco leaf quality. Although topping techniques are widely used, controlling the growth of axillary buds after topping remains a significant challenge in improving tobacco yield and quality. Existing pruning methods are mainly divided into manual and chemical pruning. The former is time-consuming and labor-intensive, and repeated operations can easily lead to diseases. The latter, while causing less damage to the tobacco plant, may affect leaf quality, pollute the environment, and is more costly. Effective axillary bud growth control is an important goal of green tobacco production. Therefore, identifying the regulatory genes that control tobacco axillary bud growth and using genetic engineering to obtain genetically improved tobacco plants has significant practical value. Summary of the Invention

[0003] In view of this, the technical problem to be solved by the present invention is to provide the tobacco NtD27 gene and its application.

[0004] In this invention, the tobacco NtD27 gene encodes a product that, according to bioinformatics analysis, is tobacco β-carotene isomerase.

[0005] This invention provides the application of tobacco β-carotene isomerase as a target in regulating the number of leaf buds and / or branches in plants.

[0006] Furthermore,

[0007] The amino acid sequence of the tobacco β-carotene isomerase is shown in SEQ ID NO:2.

[0008] The regulation includes raising and / or lowering.

[0009] The plants described in this invention include plants from the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, and / or Solanaceae families; further, the Solanaceae plants include tobacco.

[0010] This invention provides plant breeding or assisted breeding products, comprising at least one of the following: A) to C:

[0011] A) Amplification primers, detection primers, and / or targeting primers using the nucleic acid encoding the tobacco β-carotene isomerase as a template;

[0012] B) Targeting the interfering fragment of the tobacco β-carotene isomerase;

[0013] C) gRNA targeting the tobacco β-carotene isomerase;

[0014] D) Expression cassettes containing interfering fragments as described in B), or gRNAs as described in C);

[0015] E) A recombinant vector containing the interfering fragment as described in B), or the gRNA as described in C), or the interfering fragment as described in D);

[0016] F) Transformation or transfection of host cells with the recombinant vector described in E);

[0017] G), a mixture obtained by culturing host cells as described in F).

[0018] This invention provides a gRNA fragment, which is a fragment located approximately 20 bp before the recognition site (PAM site) of a CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a, and CRISPR / Cas14a) editing system targeting the target gene. The recognition site of the CRISPR / endonuclease system editing system varies depending on the endonuclease used; typically, the recognition site includes NGG (Cas9) and... / or TTN (Cpf1), etc.; In a specific embodiment of the present invention, the CRISPR / Cas9 system is used, and its recognition site is NGG, where N represents any one of the bases A, T, C, or G. The target site of the gRNA can be located at any position at the 5' end, middle, or 3' end of the nucleic acid encoding tobacco β-carotene isomerase, and the present invention does not limit this; In the present invention, tobacco β-carotene isomerase is used as the target, and gene editing is performed using the CRISPR / Cas9 system. Specifically, the nucleotide sequence of the target region of the gRNA fragment is shown in SEQ ID NO:7;

[0019] Furthermore, in this invention, tobacco β-carotene isomerase was obtained after gene editing using the CRISPR / Cas9 system. The base A at 271 bp of the nucleotide sequence shown in SEQ ID NO:1 was deleted, resulting in a frameshift mutation, which led to the loss and / or reduction of the function of tobacco β-carotene isomerase.

[0020] The expression cassette described in this invention refers to a DNA sequence from the start of the promoter to the end of the terminator. Regulatory fragments may also be included on either side of or between the promoter and terminator. These regulatory fragments may include promoters, enhancers, transcription termination signals, polyadenylation sequences, origins of replication, nucleic acid restriction sites, and homologous recombination sites operatively linked to the nucleic acid sequence, such as enhancers of promoters, poly(A) signals, etc.

[0021] The recombinant vector described in this invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule containing a desired coding sequence and suitable nucleic acid sequences or elements essential for the expression of an operablely linked coding gene in a specific host organism or for performing a desired operation.

[0022] In this specification, the terms "plasmid" and "vector" are sometimes used interchangeably because plasmids are currently the most commonly used form of vector. However, the present invention is intended to include other forms of expression vectors that perform equivalent functions and are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors, and / or simply potential genomic inserts.

[0023] The host cell described in this invention includes plant cells, and the plant includes plants of the Brassicaceae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Fabaceae, or Solanaceae families. Further, in a specific embodiment of this invention, the plant is a Solanaceae plant, and further, the Solanaceae plant is tobacco.

[0024] This invention provides a kit comprising the product and excipients described herein.

[0025] The excipients are used to preserve, bioreplicate, or assist the product in functioning; in this invention, the excipients include at least one of: DNA extraction reagent, dNTP, DNA reverse transcription reagent, culture medium, antibiotic, and / or buffer.

[0026] This invention provides the application of the product or kit described herein in plant breeding or assisted plant breeding.

[0027] This invention provides a method for plant breeding or assisted plant breeding, which includes plant breeding using the products or kits described in this invention.

[0028] This application, through research on the NtD27 gene, discovered that this gene affects the development of tobacco axillary buds. Knockout mutations of the NtD27 gene promote the growth of tobacco axillary buds, while overexpression of the NtD27 gene effectively inhibits its growth. This provides potential germplasm resource support for molecular breeding of tobacco with fewer axillary buds and has significant practical value in the genetic improvement of important traits in tobacco and in basic botanical research. Attached Figure Description

[0029] Figure 1 The electrophoresis diagram of the PCR amplification products of the NtD27 gene shows the amplification results of the NtD27 gene fragment;

[0030] Figure 2 The expression characteristics of the NtD27 gene in different tissues of tobacco are shown.

[0031] Figure 3 The results of NtD27 gene expression level analysis in NtD27 gene overexpression lines are shown.

[0032] Figure 4 The phenotypic diagram of NtD27 gene knockout editing is shown, and the phenotypic differences between the NtD27 gene knockout line (NtD27-KO) and the wild type are compared, especially the changes in lateral branch and axillary bud growth.

[0033] Figure 5 The phenotypic diagram of the NtD27 gene overexpression line is shown. The phenotypic differences between the NtD27 gene overexpression line (NtD27-OE) and the wild type were compared, especially the changes in lateral branch and axillary bud growth. Detailed Implementation

[0034] This invention provides the tobacco NtD27 gene and its applications. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired results. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to implement and apply the technology of this invention.

[0035] The NtD27 gene coding sequence: atggaagcaaatcttgttcaaccctaccatagcttttcaacagtatccatgagaaacagaaataagatcaatacttataaatacaaaaatataccatatccactatgctccatattaacgaagccacctgcaaataatcaaaatcataatgcagaaacaaccacaggcagttcttccaataatgtttacaaagataattggttcgatcgtctcgccattaatcatctctctcaaagtatacaagctaccacaggattaagaaacaataagagtggatatgaaggcttggcggaggctgcaaagatggtatataccaactttaactcaacgcgtcaagtaaatcttgttgttgaagctcttcaaaaggcattccctaagcctattctttccctgatgaagatggtactaccacaatctaagtgggcaagagagtattgcgctgctttcaccagagtcttcttctcttggctagtgggaccctgtgaggtgaaagagtcagaatttaacgggagaaaggaaaacaatgtggtccacatcaaaaaatgcaggtttctggaggaaaccaattgcgtgggaatgtgcactaacctctgcaagatgccttctcaattgttcatcaaggatacattaggaatgccagtgaacatggtaccaaactttgatgatatgagctgtgagatgatatttggacaggatgctcccccaccagatacagatccagcatttgtgcagccatgctacaaattatgcaaacttggcaataaacaccaacaggattgcaacagtcaaatgaagaagaaagatgtggagatttcataa (SEQ ID NO:1);

[0036] NtD27 amino acid sequence: MEANLVQPYHSFSTVSMRNRNKINTYKYKNIPYPLCSILTKPPANNQNHNAETTTGSSSNNVYKDNWFDRLAINHLSQSIQATTGLRNNKSGYEGLAEAAKMVYTNFNSTRQVNLVVEALQKAFPKPILSLM KMVLPQSKWAREYCAAFTRVFFSWLVGPCEVKESEFNGRKENNVVHIKKCRFLEETNCVGMCTNLCKMPSQLFIKDTLGMPVNMVPNFDDMSCEMIFGQDAPPPDTDPAFVQPCYKLCKLGNKHQQDCNSQMKKKDVEIS (SEQ IDNO:2);

[0037] The test materials used in this invention are all common commercially available products. The invention is further illustrated below with reference to embodiments:

[0038] Example 1 Cloning of the NtD27 gene

[0039] I. Experimental Materials, Reagents and Equipment

[0040] 1. Biomaterials

[0041] Tobacco variety: K326, a common cultivated tobacco variety.

[0042] Vectors: pKSE401 vector, pEASY-T1 Simple vector, PBI121 vector.

[0043] Competent cells: Trans1-T1 competent cells, DH5α chemocompetent cells.

[0044] Strain: LBA4404 Agrobacterium strain.

[0045] Primer synthesis and DNA sequencing were performed by Beijing BGI Genomics Co., Ltd.

[0046] 2. Experimental reagents

[0047] RNA extraction kit: SuperPure Plant polyRNA Kit, purchased from Zhengzhou Ansai Biotechnology Co., Ltd.

[0048] Quantitative real-time PCR enzyme: SYBR qPCR Kit, purchased from Zhengzhou Ansai Biotechnology Co., Ltd.

[0049] Reverse transcription kit: purchased from Takara Bio Engineering (Dalian) Co., Ltd.

[0050] Restriction endonucleases: BamHI and SacI enzymes, purchased from NEB.

[0051] DNA amplification enzyme: purchased from Beijing TransGen Biotech Co., Ltd.

[0052] Plant genome extraction and DNA purification kit: purchased from QIAGEN.

[0053] 3. Experimental Equipment

[0054] PCR synthesizer: Tprofessional Thermocycler, Biometra Corporation.

[0055] Quantitative PCR instrument: CFX96, Bio-Rad.

[0056] Ultraviolet gel imaging system: BioSpectrum, UVP Corporation.

[0057] II. Cloning of the NtD27 gene

[0058] 1. Prepare cDNA as a cloning template

[0059] 100 mg of tobacco (K326) leaves were thoroughly ground in liquid nitrogen, and total RNA was extracted according to the instructions of the RNA extraction kit. The extracted total RNA was then reverse transcribed into cDNA using a reverse transcription kit for later use.

[0060] 2. Design primers and perform PCR amplification.

[0061] Specific primers designed for amplifying the NtD27 gene are shown below:

[0062] NtD27-F: 5'-atggaagcaaatcttgttcaaccct-3' (SEQ ID NO: 3);

[0063] NtD27-R: 5'-ttatgaaatctccacatctttcttc-3' (SEQ ID NO:4);

[0064] Using the cDNA prepared in step 1 as a template, PCR amplification was performed using the primers described above. The PCR reaction conditions were: 94℃ pre-denaturation for 4 minutes; 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, and 72℃ extension for 40 seconds, for a total of 30 cycles; followed by a final extension at 72℃ for 10 minutes. The amplified products were stored at 4℃ for later use, or directly analyzed by electrophoresis (e.g., ...). Figure 1 (As shown).

[0065] 3. Purification of PCR amplification products and vector ligation

[0066] Purify the PCR amplification products according to the gel extraction kit instructions. The purified products are then ligated into the pEASY-T1 vector using the following ligation system:

[0067] DNA amplification product: 6 μL; pEASY-T1 vector: 1 μL;

[0068] After mixing the reaction mixture, it was connected at 25°C for 25 minutes.

[0069] 4. Transformation into competent cells

[0070] The ligation product was transformed into Trans1-T1 competent cells, and the specific steps are as follows:

[0071] (1) Thaw competent cells stored at -80℃ on ice.

[0072] (2) Add the ligation product to 50 μL of competent cells, mix gently, and incubate on ice for 30 minutes.

[0073] (3) Heat shock in a 42℃ water bath for 30 seconds, then immediately place on ice to cool for 2 minutes.

[0074] (4) Add 250 μL of LB medium (without antibiotics) equilibrated to room temperature and incubate at 37°C with shaking for 1 hour.

[0075] 5. Blue-white screening

[0076] On an LB agar plate (containing 60 μg / mL ampicillin), evenly spread 8 μL of a mixture of 500 mM IPTG and 40 μL of 20 mg / mL X-gal. Invert the plate and incubate overnight at 37°C. Pick white spots for amplification culture, extract plasmid DNA, and identify the recombinant plasmid by plasmid PCR.

[0077] 6. Sequencing and identification of the NtD27 gene

[0078] Positive clones were sent for sequencing to obtain the complete sequence of the NtD27 gene. The sequencing results showed that the coding region of the NtD27 gene is 819 bp in length, and its base sequence is shown in SEQ ID NO: 1. The amino acid sequence of the encoded NtD27 protein is shown in SEQ ID NO: 2.

[0079] Example 2: Analysis of NtD27 gene expression patterns

[0080] The expression pattern of the NtD27 gene was analyzed using real-time PCR technology by collecting samples from different tobacco tissues and organs. The specific experimental procedure is as follows:

[0081] 1. Sample collection and preservation

[0082] Tissue samples, including terminal buds, roots, petals, calyxes, stems, leaves, and leaf veins, were collected from tobacco plants at the budding stage. Samples were immediately flash-frozen in liquid nitrogen after collection and then stored at -80°C for later use.

[0083] 2. RNA extraction and cDNA synthesis

[0084] Total RNA was extracted from preserved tissue samples, and cDNA was synthesized using a reverse transcription kit. Extraction and reverse transcription were performed according to the kit instructions.

[0085] 3. Quantitative Real-Time PCR Analysis

[0086] In quantitative real-time PCR experiments, the NtL25 gene was used as an internal reference gene to standardize the expression level of the NtD27 gene. To detect the expression of the NtD27 gene, the following quantitative real-time PCR primers were designed:

[0087] NtD27-qF: 5'-gtggatatgaaggcttggcg-3' (SEQ ID NO:5);

[0088] NtD27-qR: 5'-ggtcccactagccaagagaa-3' (SEQ ID NO:6);

[0089] 4. Real-time PCR reaction conditions

[0090] The reaction conditions for real-time PCR are set as follows:

[0091] Step 1: Pre-denaturation at 95℃ for 10 seconds;

[0092] Step 2: PCR amplification reaction, 95℃ for 5 seconds, 60℃ for 30 seconds, for a total of 39 cycles;

[0093] Step 3: Dissolution curve analysis.

[0094] 5. Data Analysis

[0095] Each sample underwent three biological replicate experiments, using 2... -△△CT The relative gene expression levels were calculated. Results showed that the NtD27 gene was highly expressed in all tissues, including the terminal bud, root, petals, calyx, stem, leaf, and leaf veins. Specific analysis results are as follows: Figure 2 As shown.

[0096] The results of this experiment show that the NtD27 gene is widely expressed in different organs and tissues of tobacco, suggesting that it may play an important regulatory role in the growth and development of tobacco.

[0097] Example 3: Preparation of NtD27 knockout vector and expression vector plasmid

[0098] Editing vectors and expression vector plasmids for NtD27 gene knockout and overexpression were constructed. The specific experimental steps and operations are as follows:

[0099] 1. Construction of the knockout editing platform

[0100] To knock out the NtD27 gene, a PBI121 knockout vector based on the CRISPR / Cas9 system was constructed, and the steps are as follows:

[0101] (1) gRNA design and synthesis

[0102] Based on the NtD27 gene sequence, gRNAs targeting the NtD27 gene were designed using online gRNA design tools (such as CRISPR RGEN). GRNAs with high specificity and efficiency were screened, and two target sites located in the exon regions of the NtD27 gene were designed. The gRNA sequences are as follows:

[0103] gRNA1: 5'-ggattaagaaacaataagag-3' (SEQ ID NO:7, gRNA1 target sequence);

[0104] (2) Construction of gRNA vector

[0105] gRNA was inserted into the plant CRISPR / Cas9 gene editing vector (pKSE401). Specifically, the pKSE401 vector was digested with BsaI, and the linearized vector was recovered. Then, the synthesized gRNA was inserted into the linearized vector, and ligation was performed at 16°C for 2 hours using T4 DNA ligase.

[0106] (3) Vector construction and identification: Positive clones were identified by colony PCR and sequencing. The successfully constructed PBI121 knockout editing vector was used for subsequent NtD27 gene knockout experiments.

[0107] 2. Construction of overexpression vectors

[0108] To enable overexpression of the NtD27 gene in tobacco, an overexpression vector for NtD27 was constructed. The specific steps are as follows:

[0109] (1) Enzyme digestion and ligation of the vector

[0110] The PBI121 vector was double-digested with BamHI and SacI, and the linearized vector was recovered. The NtD27 gene fragment with enzyme-digested adapters obtained in Example 1 was ligated into the PBI121 vector. The ligation reaction was carried out at 25°C for 30 minutes, and the ligation system was as follows: NtD27 gene fragment: 6 μL; PBI121 vector: 1 μL; T4 DNA ligase buffer: 2 μL; T4 DNA ligase: 1 μL; total volume: 10 μL.

[0111] (2) Transformation of the carrier

[0112] The ligation product was transformed into *E. coli* DH5α competent cells. The specific transformation steps were as follows:

[0113] Step 1: Thaw competent cells on ice, add 2 μL of ligation product, mix gently, and incubate on ice for 30 minutes;

[0114] Step 2: Heat shock at 42℃ for 30 seconds, then immediately place on ice for 2 minutes;

[0115] Step 3: Add 500 μL of antibiotic-free LB medium and incubate at 37°C with shaking for 1 hour;

[0116] Step 4: Spread the mixture onto LB agar plates containing 50 μg / mL kanamycin and incubate at 37°C for 12–16 hours.

[0117] (3) Screening and identification of positive clones

[0118] Single colonies were selected, and positive clones were identified by colony PCR and double enzyme digestion. Subsequently, the positive clones were sequenced to ensure that the NtD27 gene fragment was correctly inserted into the PBI121 vector. The successfully constructed PBI121 overexpression vector was used for subsequent NtD27 gene knockout experiments.

[0119] Example 4: Construction of NtD27 gene knockout lines and overexpression transgenic lines

[0120] The specific experimental process for constructing NtD27 gene knockout edited lines and overexpression transgenic lines.

[0121] 1. Transformation, culture, and identification of Agrobacterium.

[0122] (1) Transformation of Agrobacterium: Add 1 μL of the NtD27 gene mutation vector and expression vector plasmid prepared in Example 3 to Agrobacterium competent cells, mix gently and transform;

[0123] (2) Culture: Add 600 μL of antibiotic-free LB liquid medium and culture at 28°C with shaking at 200 r / min for 1 hour. Then, spread 150 μL of bacterial culture onto LB culture dishes containing antibiotics (including kanamycin and rifampin) and culture at 28°C for 48-72 hours until single colonies are formed.

[0124] (3) Identification of positive clones: Select a single colony for amplification, extract the bacterial solution for PCR identification, confirm the positive clone strain, and ensure successful transformation.

[0125] 2. Transformation of tobacco plants

[0126] (1) Leaf treatment: Take leaves of sterile tobacco seedlings that have grown for about one month, and use a punch to treat the leaves into leaf discs with a diameter of 0.5 cm. Pre-culture the treated leaf discs on MS solid medium for 3 days.

[0127] (2) Agrobacterium infection: The transformed Agrobacterium engineered bacteria were cultured to OD. 600 =0.6, centrifuge at 4000 rpm for 5 minutes to collect the bacterial cells, and then suspend the bacterial cells in 20 mL of MS liquid medium. Place the pre-cultured leaf discs in the bacterial solution for 10 minutes to infect.

[0128] (3) Culture conditions: After the leaf discs were infected, excess bacterial solution around them was blotted dry with sterile filter paper. The solution was then transferred to MS solid medium containing 6-BA (2 mg / L) and NAA (0.5 mg / L) and cultured in the dark for 3 days. Afterward, the leaf discs were washed with sterile water containing Cef (cephalosporin) (400 mg / L), excess liquid was blotted off, and the solution was transferred to MS solid selection medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L), and Kan (50 mg / L) and cultured at 28°C under light.

[0129] (4) Rooting treatment: When the adventitious buds grow to 0.5 cm, they are transferred to MS solid medium containing Cef (200 mg / L) and Kan (50 mg / L) to root.

[0130] (5) Detection of gene-edited homozygous lines (NtD27 gene knockout line NtD27-KO) and transgenic positive lines (NtD27 gene overexpression line NtD27-OE): After the plants have grown for about one month, a small number of leaves were taken, and DNA was extracted according to the instructions of the plant genome extraction kit. The gene-edited homozygous lines and transgenic positive lines were detected by PCR amplification, cloning, and sequencing. The primer design is as follows:

[0131] NtD27-JF: 5'-ttcatttggagagaacacgggggac-3' (SEQ ID NO:8);

[0132] NtD27-JR: 5'-ttatgaaatctccacatctt-3' (SEQ ID NO:9);

[0133] The PCR conditions were: 94℃ pre-denaturation for 4 minutes; 94℃ denaturation for 30 seconds, 56℃ annealing for 30 seconds, 72℃ extension for 40 seconds, for a total of 25 cycles; and a final extension at 72℃ for 10 minutes. This method was used to identify gene-edited homozygous lines and transgenic positive lines.

[0134] NtD27 gene mutant lines were identified, and homozygous NtD27 gene editing lines were obtained. The mutation of NtD27 mutant 1 is the deletion of base A at 271 bp of the original gene sequence. Open reading frame prediction analysis shows that it is a loss-of-function mutation of NtD27.

[0135] (6) Detection of NtD27 expression level in transgenic positive lines (NtD27 gene overexpression lines)

[0136] A small number of leaves from the transgenic positive lines were collected, and RNA was extracted according to the instructions of the plant RNA extraction kit. cDNA was then reverse transcribed. The NtL25 gene was used as an internal control gene. The expression level of NtD27 in the transgenic positive lines was detected by real-time quantitative PCR. The primer design is as follows:

[0137] NtD27-qF: 5'-gtggatatgaaggcttggcg-3' (SEQ ID NO:5);

[0138] NtD27-qR: 5'-ggtcccactagccaagagaa-3' (SEQ ID NO:6);

[0139] The reaction conditions and analysis methods for quantitative real-time PCR are the same as in Example 2;

[0140] The results of NtD27 expression level detection in NtD27 overexpression positive lines are as follows: Figure 3 As shown, the expression level of NtD27 in the NtD27 overexpression positive lines was significantly higher than that in the wild type. Among them, WT was the wild type, and D27-1, D27-2, D27-4, D27-5, D27-8, D27-9, D27-11, D27-12, and D27-15 were all NtD27 overexpression positive lines. The different insertion positions of NtD27 led to the differences in expression levels.

[0141] 3. Phenotypic observation of gene-edited homozygous lines and transgenic positive lines

[0142] T0 generation plants from wild-type, gene-edited homozygous lines, and transgenic positive lines were transplanted into pots and cultured in a greenhouse for approximately 10 weeks. Observations showed that, compared to wild-type tobacco, the gene-edited homozygous lines exhibited a more pronounced branching phenotype and significant axillary bud growth (e.g., ...). Figure 4 As shown), the axillary bud growth of transgenic positive lines was significantly slowed (e.g. Figure 5 (As shown).

[0143] In summary, regulating the expression of the NtD27 gene can effectively control the proliferation of axillary buds and branches in tobacco, laying an applied foundation for tobacco plant architecture regulation and ultimately increasing tobacco yield. This provides important theoretical and experimental support for further breeding research and practice.

[0144] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of tobacco β-carotene isomerase as a target in inhibiting the number of tobacco leaf buds and / or branches; The amino acid sequence of the tobacco β-carotene isomerase is shown in SEQ ID NO:2; The inhibition of the number of tobacco leaf buds and / or branches is achieved by overexpressing tobacco β-carotene isomerase with the amino acid sequence shown in SEQ ID NO:2.

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