Tobacco NtD27 gene and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a tobacco NtD27 gene and application thereof. The research on the NtD27 gene finds that the gene affects the development of tobacco axillary buds. The growth of tobacco axillary buds can be promoted through knockout mutation of the NtD27 gene, and the growth of the tobacco axillary buds can be effectively inhibited through overexpression of the NtD27 gene; the method provides potential germplasm resource support for molecular breeding of few axillary buds of tobacco, and has important practical value in genetic improvement of important characters of tobacco and basic research of botany.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to tobacco NtD27 gene and application thereof. Background Art

[0002] Tobacco is a widely planted crop with high economic benefits. Reasonable topping and stubble removal is one of the key agronomic measures to improve the quality of tobacco leaves. Although topping technology has been widely used, controlling the growth of axillary buds after topping is still an important challenge to improve tobacco yield and quality. Existing stubble removal methods are mainly divided into manual and drug stubble removal. The former is time-consuming and labor-intensive, and repeated operations are prone to cause diseases. Although the latter causes less damage to tobacco plants, it may affect the quality of tobacco leaves, pollute the environment and has high costs. Effective means of controlling axillary bud growth is an important goal of green tobacco production. Therefore, it is of great practical value to find regulatory genes that control the growth of tobacco axillary buds and obtain genetically improved tobacco plants by genetic engineering. Summary of the invention

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

[0004] In the present invention, the tobacco NtD27 gene, the gene encoding product of which is analyzed by bioinformatics to be tobacco beta-carotene isomerase.

[0005] The invention provides the use of tobacco beta-carotene isomerase as a target in regulating the number of leaf buds and / or the number of branches of 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 increasing and / or decreasing.

[0009] The plants of the present invention include plants of the family Cruciferae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Leguminosae and / or Solanaceae; further, the Solanaceae plant includes tobacco.

[0010] The present invention provides a product for plant breeding or assisted breeding, which includes 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), an interfering fragment targeting the tobacco β-carotene isomerase;

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

[0014] D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C);

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

[0016] F), transforming or transfecting the host cell with the recombinant vector as described in E);

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

[0018] The present invention provides a gRNA fragment, which is a fragment targeting a target gene and located about 20 bp before the recognition site (PAM site) of the CRISPR / endonuclease system (such as CRISPR / Cas9, CRISPR / Cas12a, CRISPR / Cas12b, CRISPR / Cas13a and CRISPR / Cas14a) editing system; the recognition site of the CRISPR / endonuclease system editing system is different according to the different endonucleases. Generally, 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, wherein N represents any one of A, T, C or G base species, and the position of the target site of the gRNA can be at any position of the 5' end, the middle or the 3' end of the nucleic acid encoding tobacco β-carotene isomerase, and the present invention is not limited to this; In the present invention, tobacco β-carotene isomerase is used as a target, and gene editing is performed using the CRISPR / Cas9 system, and specifically, the nucleotide sequence of the target segment of the gRNA fragment is shown in SEQ ID NO: 7;

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

[0020] The expression cassette of the present invention refers to a DNA sequence starting from a promoter and ending at a terminator. A regulatory segment may also be included on both sides or between the promoter and the terminator, and the regulatory segment may include a promoter, an enhancer, a transcription termination signal, a polyadenylation sequence, a replication origin, a nucleic acid restriction site, and a homologous recombination site operably connected to the nucleic acid sequence, such as an enhancer of the promoter, a poly (A) signal, etc.

[0021] The recombinant vector of the present invention refers to a recombinant nucleic acid vector, which is a recombinant DNA molecule that contains a desired coding sequence and appropriate nucleic acid sequences or elements that are essential for the expression of an operably linked coding gene in a specific host organism or for the completion of a desired operation.

[0022] In this specification, "plasmid" and "vector" are sometimes used interchangeably, as plasmid is the most commonly used vector form. However, the present invention is intended to include such other forms of expression vectors that play an equivalent role, which are known or will become known in the art, including but not limited to: plasmids, phage particles, viral vectors and / or only potential genomic insertions.

[0023] The host cell of the present invention includes a plant cell, and the plant includes a plant of the family Cruciferae, Asteraceae, Chenopodiaceae, Rutaceae, Moraceae, Leguminosae or Solanaceae. Further, in a specific embodiment of the present invention, the plant is a plant of the family Solanaceae, and further, the plant of the family Solanaceae is tobacco.

[0024] The present invention provides a kit, which comprises the product of the present invention and auxiliary materials.

[0025] The auxiliary material is used to preserve or biologically replicate the product, or to assist the product in exerting its effects; in the present invention, the auxiliary material includes at least one of a DNA extraction reagent, a dNTP, a DNA reverse transcription reagent, a culture medium, an antibiotic and / or a buffer.

[0026] The present invention provides application of the product or the kit in plant breeding or assisted plant breeding.

[0027] The present invention provides a method for plant breeding or assisted plant breeding, which comprises using the product of the present invention or the kit of the present invention to carry out plant breeding.

[0028] Through the study of NtD27 gene, this application found that this gene affects the development of tobacco axillary buds. Through the knockout mutation of NtD27 gene, the growth of tobacco axillary buds can be promoted, and the overexpression of NtD27 gene can effectively inhibit the growth of tobacco axillary buds; it provides potential germplasm resource support for the molecular breeding of tobacco with few axillary buds, and has important practical value in the genetic improvement of important tobacco traits and basic botanical research. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The electrophoresis diagram of the PCR amplification product of NtD27 gene is shown, showing the amplification result of the NtD27 gene fragment;

[0030] Figure 2 Shows the expression characteristics of NtD27 gene in different tobacco tissues;

[0031] Figure 3 The results of the analysis of the expression level of NtD27 gene in the NtD27 gene overexpression strain are shown;

[0032] Figure 4 The figure shows the phenotype of NtD27 gene knockout editing, comparing the phenotypic differences between the NtD27 gene knockout strain (NtD27-KO) and the wild type, especially the changes in the growth of lateral branches and axillary buds;

[0033] Figure 5 The phenotype of the NtD27 gene overexpression line was shown, and the phenotypic differences between the NtD27 gene overexpression line (NtD27-OE) and the wild type were compared, especially in the changes in the growth of lateral branches and axillary buds. DETAILED DESCRIPTION

[0034] The present invention provides tobacco NtD27 gene and its application. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present 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 the present invention are all common commercial products and can be purchased on the market. The present invention is further described below in conjunction with the embodiments:

[0038] Example 1 Cloning of NtD27 gene

[0039] 1. Experimental Materials, Reagents and Equipment

[0040] 1. Biomaterials

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

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

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

[0044] Strain: LBA4404 Agrobacterium strain.

[0045] Primer synthesis and DNA sequencing: completed by Beijing Liuhe BGI Genomics Co., Ltd.

[0046] 2. Experimental Reagents

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

[0048] Fluorescence quantitative PCR enzyme: SYBR qPCR Kit, purchased from Zhengzhou Ansai Biotechnology Co., Ltd.

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

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

[0051] DNA amplification enzyme: purchased from Beijing Quanshijin Biotechnology Co., Ltd.

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

[0053] 3. Experimental Equipment

[0054] PCR synthesizer: Tprofessional Thermocycler, Biometra.

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

[0056] UV gel imaging system: BioSpectrum, UVP company.

[0057] 2. Cloning of NtD27 gene

[0058] 1. Preparation of cDNA as cloning template

[0059] Take 100 mg of tobacco (K326) leaves, grind them thoroughly in liquid nitrogen, and extract total RNA according to the instructions of the RNA extraction kit. Then use a reverse transcription kit to reverse transcribe the extracted total RNA into cDNA for later use.

[0060] 2. Design primers and perform PCR amplification

[0061] Specific primers were designed for amplifying the NtD27 gene, and the sequences are as follows:

[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, perform PCR amplification using the above primers. The PCR reaction conditions are: pre-denaturation at 94°C for 4 minutes; denaturation at 94°C for 30 seconds, annealing at 56°C for 30 seconds, extension at 72°C for 40 seconds, for a total of 30 cycles; and final extension at 72°C for 10 minutes. The amplified product is stored at 4°C for later use, or directly subjected to electrophoresis analysis (such as Figure 1 as shown).

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

[0066] Purify the PCR amplification product according to the instructions of the gel recovery kit. Connect the purified product to the pEASY-T1 vector. The connection system is as follows:

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

[0068] After the reaction mixture was mixed, the ligation was carried out at 25°C for 25 minutes.

[0069] 4. Transformation of competent cells

[0070] Transform the ligation product into Trans1-T1 competent cells. The specific steps are as follows:

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

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

[0073] (3) Heat shock in a 42°C water bath for 30 seconds, then immediately cool on ice 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 a solid LB plate (containing 60 μg / mL ampicillin), evenly spread 8 μL of 500 mM IPTG and 40 μL of 20 mg / mL X-gal mixture, invert the culture dish, and culture at 37°C overnight. Pick the white spots for amplification culture, extract plasmid DNA, and identify the recombinant plasmid by plasmid PCR.

[0077] 6. Sequencing and identification of NtD27 gene

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

[0079] Example 2 Analysis of the expression pattern of NtD27 gene

[0080] By collecting samples of different tobacco tissues and organs, the expression pattern of the NtD27 gene was analyzed using fluorescent quantitative PCR technology. The specific experimental process is as follows:

[0081] 1. Sample collection and storage

[0082] The buds, roots, petals, calyx, stems, leaves, veins and other tissue samples were collected from tobacco plants at the budding stage. The samples were immediately frozen in liquid nitrogen after collection and then stored in a -80°C refrigerator for later use.

[0083] 2. RNA Extraction and cDNA Synthesis

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

[0085] 3. Fluorescence quantitative PCR analysis

[0086] In the fluorescence quantitative PCR experiment, the NtL25 gene was used as an internal reference gene to standardize the expression of the NtD27 gene. To detect the expression of the NtD27 gene, the following fluorescence quantitative 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. Fluorescence quantitative PCR reaction conditions

[0090] The reaction conditions of fluorescence quantitative PCR were set as follows:

[0091] Step 1: pre-denaturation at 95°C for 10 seconds;

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

[0093] Step 3: Melting curve analysis.

[0094] 5. Data Analysis

[0095] Three biological replicates were performed for each sample. -△△CT The results showed that the NtD27 gene was highly expressed in the apical bud, root, petal, calyx, stem, leaf, and vein tissues. Figure 2 shown.

[0096] The results of this experiment showed that the NtD27 gene was widely expressed in different organs and tissues of tobacco, indicating 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] The editing vector and expression vector plasmid for NtD27 gene knockout and overexpression were constructed. The specific experimental steps and operations are as follows:

[0099] 1. Construction of knockout editing vector

[0100] In order to knock out the NtD27 gene, a PBI121 knockout vector based on the CRISPR / Cas9 system was constructed. The steps are as follows:

[0101] (1) gRNA design and synthesis

[0102] According to the sequence of the NtD27 gene, an online gRNA design tool (such as CRISPR RGEN) was used to design gRNA targeting the NtD27 gene. The gRNA with high specificity and high efficiency was screened, and two targets were designed to be located in the exon region of the NtD27 gene. 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] Insert gRNA into the plant CRISPR / Cas9 gene editing vector (pKSE401). The specific operation is: pKSE401 vector is digested with BsaI to recover the linearized vector; then the synthesized gRNA is inserted into the linearized vector and connected by T4 DNA ligase at 16°C for 2 hours.

[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 vector

[0108] In order to overexpress the NtD27 gene in tobacco, an NtD27 overexpression vector was constructed. The specific steps are as follows:

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

[0110] The PBI121 vector was double-digested with BamHI and SacI, and the linearized vector was recovered; the NtD27 gene fragment with restriction endonuclease adapter amplified in Example 1 was connected to 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 vector

[0112] The ligation product was transformed into E. coli DH5α competent cells. The specific transformation steps are:

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

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

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

[0116] Step 4: Spread on LB plate containing 50 μg / mL kanamycin and culture at 37°C for 12-16 hours.

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

[0118] Single colonies were picked and positive clones were identified by colony PCR and double enzyme digestion. Subsequently, positive clones were sent for sequencing 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 strain and overexpression transgenic strain

[0120] The specific experimental process of constructing NtD27 gene knockout editing strain and overexpression transgenic strain.

[0121] 1. Transformation, cultivation 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 the Agrobacterium competent cells respectively, and mix gently for transformation;

[0123] (2) Cultivation: Add 600 μL of LB liquid culture medium without antibiotics and culture at 28°C with shaking at 200 r / min for 1 hour. Then, spread 150 μL of the bacterial solution on an LB culture dish containing antibiotics (including kanamycin and rifampicin) and culture at 28°C for 48-72 hours until a single colony is formed.

[0124] (3) Identification of positive clones: Pick a single colony for expansion, extract the bacterial solution for PCR identification, confirm the positive clone strain, and ensure the 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, use a hole puncher to process the leaves into leaf disks with a diameter of 0.5 cm, and pre-culture the treated leaf disks on MS solid culture medium for 3 days.

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

[0128] (3) Culture conditions: Use sterile filter paper to absorb excess bacterial liquid around the infected leaf disc, then transfer to MS solid medium containing 6-BA (2 mg / L) and NAA (0.5 mg / L), and culture in the dark for 3 days. Then wash the leaf disc with sterile water containing Cef (cephalosporin) (400 mg / L), remove excess liquid, and transfer to MS solid screening medium containing 6-BA (2 mg / L), NAA (0.5 mg / L), Cef (200 mg / L) and Kan (50 mg / L), and culture at 28°C under light.

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

[0130] (5) Detection of gene-edited homozygous lines (NtD27 gene knockout lines NtD27-KO) and transgenic positive lines (NtD27 gene overexpression lines NtD27-OE): After the plants have grown for about one month, take a small amount of leaves and extract DNA according to the instructions of the plant genome extraction kit. Detect the gene-edited homozygous lines and transgenic positive lines by PCR amplification, cloning and sequencing. The primers are designed 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°C pre-denaturation for 4 minutes; 94°C denaturation for 30 seconds, 56°C annealing for 30 seconds, 72°C extension for 40 seconds, for a total of 25 cycles; and finally extension at 72°C for 10 minutes. This method was used to identify gene-edited homozygous strains and transgenic positive strains;

[0134] The NtD27 gene mutant strain was identified and the NtD27 gene-edited homozygous strain was obtained. The mutation of NtD27 mutant 1 was the deletion of base A at 271bp of the original gene sequence. The open reading frame prediction analysis showed that it was a NtD27 loss-of-function mutation.

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

[0136] A small amount of leaves from the transgenic positive lines were taken, RNA was extracted according to the instructions of the plant RNA extraction kit, and cDNA was reverse transcribed. The NtL25 gene was used as an internal reference gene, and the expression level of NtD27 in the transgenic positive lines was detected by real-time fluorescence quantitative PCR amplification. The primers were designed 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 method of fluorescent quantitative PCR were the same as those in Example 2;

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

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

[0142] The T0 plants of wild-type, gene-edited homozygous lines and transgenic positive lines were transplanted into pots and cultured in the greenhouse for about 10 weeks. The observation results showed that compared with wild-type tobacco, the gene-edited homozygous lines had more significant branching phenotypes and obvious axillary bud growth (such as Figure 4 The growth of axillary buds of transgenic positive lines was significantly slowed down (as shown in Figure 5 as shown).

[0143] In summary, by regulating the expression of the NtD27 gene, the proliferation of axillary buds and branches of tobacco can be effectively controlled, laying an application foundation for the regulation of tobacco plant type and the ultimate increase in tobacco yield. This provides important theoretical and experimental support for further breeding research and practice.

[0144] The above are only preferred embodiments of the present invention. It should be pointed out that, for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Application of tobacco β-carotene isomerase as a target in regulating the number of leaf buds and / or branches of plants.

2. The use according to claim 1, characterized in that: The amino acid sequence of the tobacco β-carotene isomerase is shown in SEQ ID NO:

2.

3. The use according to claim 1, characterized in that: The regulation includes increasing and / or decreasing.

4. The use according to any one of claims 1 to 3, characterized in that: The plants include tobacco.

5. A plant breeding or plant breeding assisted product, characterized in that: Including at least one of the following A) to C: A), amplification primers, detection primers and / or targeting primers using the nucleic acid encoding the tobacco β-carotene isomerase as a template; B), an interfering fragment targeting the tobacco β-carotene isomerase; C), gRNA targeting the tobacco β-carotene isomerase; D), an expression cassette containing the interference fragment as described in B), or the gRNA as described in C); E), a recombinant vector containing the interference fragment as described in B), or the gRNA as described in C), or the interference fragment as described in D); F), transforming or transfecting the host cell with the recombinant vector as described in E); G), culturing the mixture obtained by the host cells described in F).

6. The product according to claim 5, characterized in that The nucleotide sequence of the target region of the gRNA is shown in SEQ ID NO:

7.

7. A kit, characterized in that Including the product and auxiliary materials described in claim 5 or 6.

8. The kit according to claim 7, characterized in that The auxiliary materials include: at least one of a DNA extraction reagent, dNTP, a DNA reverse transcription reagent, a culture medium, an antibiotic and / or a buffer.

9. Use of the product according to claim 5 or 6 or the kit according to claim 7 or 8 in plant breeding or assisted plant breeding.

10. A method for plant breeding or assisted plant breeding, characterized in that The method comprises using the product according to claim 5 or 6 or the kit according to claim 7 or 8 for plant breeding.

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