Method for inhibiting growth of axillary buds of tobacco after topping

Abstract

The invention provides a method for inhibiting the growth of axillary buds after tobacco topping, which utilizes an axillary bud specific promoter to drive the overexpression of NtMYB306 protein in tobacco, so that the growth of the axillary buds after tobacco topping is inhibited, the growth of tobacco main stem leaves is further promoted, and the growth quality and yield of the tobacco leaves are improved.

Description

Technical Field

[0001] The present disclosure relates to the field of genetic engineering technology, and specifically, to a method for inhibiting the growth of axillary buds in tobacco after topping. Background Art

[0002] In tobacco production, topping is usually carried out to concentrate the supply of nutrients for leaf growth in order to improve the yield and quality of tobacco leaves. After topping, each leaf axil of the tobacco plant can regenerate 2 - 3 or more axillary buds. If allowed to grow, they will consume a large amount of nutrients and affect the vegetative growth of the main stem leaves.

[0003] As a leaf crop, allowing tobacco to flower and bear fruit will consume a large amount of its water, reducing the yield and quality of tobacco leaves. In the cultivation of high-quality tobacco, "topping" is an important measure to regulate the nutrition of tobacco plants and the yield and quality of tobacco leaves. It is necessary to remove the top flower buds or inflorescences at the appropriate time to prevent the unnecessary consumption of nutrients in the tobacco plants, and promote the concentrated supply of nutrients for leaf growth, increasing the leaf area and the weight of single leaves. However, after topping, axillary buds grow in clusters, consuming nutrients and failing to achieve the purpose of improving the quality of tobacco leaves.

[0004] In order to increase the yield and quality of tobacco leaves, it is often necessary to remove axillary buds and inhibit their growth. The traditional manual bud removal is laborious and troublesome, and it is not timely and thorough, which is likely to cause the growth of tobacco shoots in clusters and flowers all over the field, and is also prone to infectious diseases. At present, in field production, sprout inhibitors are used to inhibit the occurrence of axillary buds in tobacco plants after topping. The use of sprout inhibitors not only increases the production cost but also causes environmental pollution.

[0005] Therefore, it is crucial to cultivate a method that can effectively inhibit axillary buds after topping, while avoiding environmental and tobacco leaf pollution, with safe application, and capable of reducing production costs and labor input. Summary of the Invention

[0006] In order to improve the yield and quality of tobacco leaves, the present disclosure provides a method for inhibiting the growth of axillary buds in tobacco after topping, the method comprising the following steps:

[0007] Overexpressing NtMYB306 protein in tobacco, the NtMYB306 protein comprising:

[0008] (1) a protein with an amino acid sequence as shown in SEQ ID NO.2;

[0009] (2) a protein obtained by substituting, deleting or adding n amino acids to the protein with an amino acid sequence as shown in SEQ ID NO.2 and still having the function of inhibiting the growth of axillary buds in tobacco after topping, wherein n is an integer between 1 and 146.

[0010] Optionally, the method comprises: overexpressing the NtMYB306 protein in the axillary buds of tobacco.

[0011] Optionally, the method includes: transferring a gene expression cassette overexpressing the NtMYB306 protein into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

[0012] Optionally, the gene expression cassette overexpressing the NtMYB306 protein is as shown in SEQ ID NO.1, wherein the gene coding sequence of the NtMYB306 protein is located at positions 253-1227.

[0013] Optionally, the method includes: transferring the gene expression cassette into a tobacco expression vector to construct a recombinant expression vector;

[0014] transferring the recombinant expression vector into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

[0015] Optionally, the tobacco expression vector includes the PCHF3 vector and / or the PBI121 vector.

[0016] Optionally, the method includes inserting the gene expression cassette into a host cell to construct a transformant;

[0017] transferring the transformant into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

[0018] Optionally, the host cell is a genetically engineered bacterium; the genetically engineered bacterium includes Escherichia coli and / or Agrobacterium.

[0019] Optionally, the expression cassette overexpressing the NtMYB306 protein is transferred into the tobacco by the Agrobacterium-mediated method.

[0020] Through the above technical solution, the present disclosure provides a method for inhibiting axillary bud growth after topping tobacco. This method inhibits the growth of axillary buds after topping tobacco by overexpressing the NtMYB306 protein in tobacco, thereby promoting the growth of the main stem leaves of tobacco and improving the growth quality and yield of tobacco leaves.

[0021] Other features and advantages of the present disclosure will be described in detail in the subsequent specific implementation section. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The drawings are used to provide a further understanding of the present disclosure and constitute a part of the specification. Together with the following specific implementation, they are used to explain the present disclosure, but do not constitute a limitation to the present disclosure. In the drawings:

[0023] Figure 1It is the PCR result of the NtMYB306 gene and the leaf staining result 7 days after injecting 35S::NtMYB306 into Nicotiana benthamiana;

[0024] Figure 2 It is the axillary bud growth condition at the first leaf position within 3 weeks after topping the pNtTFL1::NtMYB306 material and the control plants;

[0025] Figure 3 It is (a) the axillary bud length at the first leaf position, (b) the leaf length at the first leaf position, and (c) the width within 3 weeks after topping the pNtTFL1::NtMYB306 material and the control plants; Detailed implementation manners

[0026] The following details the specific implementation manners of the present disclosure. It should be understood that the specific implementation manners described herein are only for explaining and illustrating the present disclosure, and are not used to limit the present disclosure.

[0027] The present disclosure provides a method for inhibiting axillary bud growth after topping tobacco, and the method includes the following steps:

[0028] Overexpress the NtMYB306 protein in tobacco, and the NtMYB306 protein includes:

[0029] (1) A protein with the amino acid sequence shown in SEQ ID NO.2;

[0030] (2) A protein with the amino acid sequence shown in SEQ ID NO.2 that has been substituted, deleted, or added with n amino acids and still has the function of inhibiting axillary bud growth after topping tobacco, where n is an integer between 1 and 146.

[0031] The inventors of the present disclosure have found through a large number of experiments that the protein with the amino acid sequence shown in SEQ ID NO.2 and its variants (proteins with 1 - 146 amino acids substituted, deleted, or added and still having the function) may interact with other proteins, nucleic acids, or small molecule substances through specific domains in tobacco cells, thereby maintaining the ability to inhibit axillary bud growth.

[0032] Optionally, the method includes: overexpressing the NtMYB306 protein in tobacco.

[0033] Optionally, the method includes: transferring the gene expression cassette for overexpressing the NtMYB306 protein into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

[0034] Optionally, the gene expression cassette overexpressing the NtMYB306 protein is as shown in SEQ ID NO.1, wherein the nucleotide sequence from position 253 to 1227 is the gene coding sequence of the NtMYB306 protein.

[0035] Optionally, the method includes: transferring the gene expression cassette into a tobacco expression vector to construct a recombinant expression vector;

[0036] transferring the recombinant expression vector into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

[0037] Optionally, the tobacco expression vector includes the PCHF3 vector and / or the PBI121 vector.

[0038] Optionally, the method further includes inserting the gene expression cassette into a host cell to construct a transformant;

[0039] transferring the transformant into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

[0040] Optionally, the host cell is a genetically engineered bacterium; the genetically engineered bacterium includes Escherichia coli and / or Agrobacterium.

[0041] Optionally, the expression cassette overexpressing the NtMYB306 protein is transferred into the tobacco by the Agrobacterium-mediated method.

[0042] Hereinafter, the present invention will be further described in detail by examples.

[0043] Example 1

[0044] Materials and Reagents

[0045] Plant materials: Seeds of Nicotiana tabacum cv. Honghuadajinyuan are preserved by the Biotechnology Research Center of the Tobacco Research Institute, Chinese Academy of Agricultural Sciences;

[0046] Seedlings and plasmids: Escherichia coli DH5α, Agrobacterium EHA105 strain, overexpression vectors PCHF3 and PBI121 backbone plasmids are all preserved by the Biotechnology Research Center of the Tobacco Research Institute, Chinese Academy of Agricultural Sciences; Restriction enzymes are products of NEB Company, T4 ligase is a product of Takara Bio Inc., and other biochemical reagents are purchased from companies such as Saisang, Sains, Xiubairui, and Biobest.

[0047] NtMYB306 nucleic acid sequence:

[0048] >NtMYB306 cds SEQ ID NO.1

[0049]

[0050] The amino acid sequence of NtMYB306 is as follows:

[0051] >NtMYB306protein SEQ ID NO.2

[0052] MGRPPCCDKVGVKKGPWTPEEDIMLVSYVQEHGPGNWRAVPTKTGLRRCSKSCRLRWTNYLRPGIKRGSFTDQEEKMIIQLQALLGNKWAAIASYLPERTDNDIKNYWNTHLKKKMKNLQEKCSGDDDLFSVENGHNFSSSNSTSRGQWERTLQTDINMAKKALHNALSLENSTPYIKQETPAKVSTYASSTENIARLLQGWMRSSSSTNNSENSKTSSNNIAATTDSSSCDGTPSAESKGGVGIMEAFESLFGLETFESSSSDQLSQTASPEASKFQVEIKKEENNSQVPLSVMLENWLFDENTIQGKDDLTIFSFDETADPF

[0053] Methods and steps

[0054] Cloning of the NtMYB306 gene

[0055] Primer design: Designed according to the CDS sequence of NtMYB306. For the convenience of constructing the vector, two sets of restriction enzyme sites were designed: SacI and PstI, and BamHI and SacI, respectively. The details are as follows: NtMYB306-F(SacI): NtMYB306-R(PstI): NtMYB306-F(BamHI): NtMYB306-R(SacI):

[0056] PCR amplification: Add the following components to a 0.2 mL centrifuge tube respectively:

[0057] 2×Phanta Max Master Mix 10μL <![CDATA[ddH 2 O]]> 7.4μL F(10mM) 0.8μL R(10mM) 0.8μL CDS plasmid template 1.0μL

[0058] The total is 20 μL. After mixing, pre-denature at 94 °C for 3 min, and then perform the PCR reaction: The reaction parameters are denaturation at 94 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s. After 35 cycles, continue to extend at 72 °C for 5 min and store at 16 °C;

[0059] Recovery of PCR products: The TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0 was used for the recovery of PCR amplification products, and the operation was carried out according to the instructions:

[0060] (1) Perform 1.0% agarose gel electrophoresis on 10 μL of the PCR amplification reaction solution. Electrophorese for 20 min at a constant voltage of 2 V / cm, cut out the agarose block containing the target fragment under ultraviolet light, and transfer it to a 1.5 mL centrifuge tube.

[0061] (2) Add 3 volumes of the gel dissolution solution Buffer GM, mix evenly, and dissolve the gel block at room temperature (15 - 25 °C).

[0062] (3) Transfer the dissolution solution to a Spin Column centrifuge column.

[0063] (4) Centrifuge at 12000 rpm / min at 25 °C for 1 min.

[0064] (5) Remove the liquid in the centrifuge tube, and add 700 μL of Buffer WB to the Spin Column centrifuge column.

[0065] (6) Centrifuge at 12000 rpm / min at 25 °C for 30 s.

[0066] (7) Repeat steps (5) and (6).

[0067] (8) Remove the liquid in the centrifuge tube, and centrifuge at 12000 rpm / min at room temperature for 1 min.

[0068] (9) Place the Spin Column on a new 1.5 mL centrifuge tube, add 30 μl of sterilized distilled water to the center of the Spin Column membrane, and let it stand at room temperature for 1 min.

[0069] (10) Centrifuge at 12,000 rpm / min at 25 °C for 1 min.

[0070] (11) Take 1.0 μL of the recovered liquid collected by centrifugation and measure the concentration of the DNA fragment on a NanoDrop 2000c spectrophotometer.

[0071] Cloning of the target fragment: According to the method provided by Takara Bio Inc., ligate the PCR amplification fragment with the pUC19-T Vertor: Add the following to a 0.5 mL sterile centrifuge tube:

[0072] 10×T4 DNA ligase Buffer 2.0μL pUC19-T Vector DNA 1.0μL PCR recovery product 4.0μL T4DNA ligase (3U / μL) 1.0μL ddH2O 2.0μL

[0073] A total of 10.0 μL. After thorough mixing, centrifuge for a few seconds, collect the liquid droplets on the tube wall to the bottom of the tube, and incubate in a 16°C metal bath for 16 - 18 h;

[0074] Preparation of E. coli DH5a competent cells:

[0075] (1) Pick a single colony of E. coli DH5a from an LB agar plate and inoculate it into 10 mL of LB liquid medium without antibiotics. Incubate overnight at 37°C with shaking at 300 rpm / min. The next day, transfer it to fresh LB liquid medium at a ratio of 1% (V / V) and incubate with shaking at 37°C until the OD reaches between 0.3 - 0.4;

[0076] (2) Transfer 50 - 100 mL of the culture broth into two pre - cooled sterile centrifuge tubes and place them on ice for 30 min;

[0077] (3) Centrifuge at 4°C, 6000 rpm / min for 5 min and discard the supernatant;

[0078] (4) Add 10 mL of pre - cooled 0.1 mol / L CaCl 2 solution to each centrifuge tube to resuspend the cells and incubate on ice for 30 min;

[0079] (5) Centrifuge at 4°C, 6000 rpm / min for 5 min to discard the supernatant, and then resuspend the cells in 2 mL of pre - cooled 0.1 mol / L CaCl 2 solution. These are the competent cells. Quick - freeze them in liquid nitrogen and store them in a - 70°C refrigerator;

[0080] Transformation of the ligation product:

[0081] (1) Use a sterile pipette tip to transfer 50 μL of competent cells into a 1.5 mL pre - cooled sterile centrifuge tube, add 10 μL of the ligation reaction solution, mix gently, and immediately place on ice for 30 min;

[0082] (2) Place the centrifuge tube in a 42°C water bath for heat shock for 60 s;

[0083] (3) Return it to ice for 3 - 5 min;

[0084] (4) Add 500 μL of LB liquid medium without additional antibiotics, mix well, and incubate at 37°C with shaking at 300 rpm / min for 60 min;

[0085] (5) Prepare an LB solid plate supplemented with 100 mg / mL ampicillin;

[0086] (6) Pipette 100 μL of the bacterial suspension onto the LB plate, and then use a sterile triangular - headed glass rod to spread the bacterial suspension evenly over the entire surface of the plate;

[0087] (7) Place the plate upright at 37 °C until the liquid is absorbed, then invert the petri dish and incubate at 37 °C for 16 h;

[0088] LB medium: Yeast Extract 5 g / L, Tryptone 10 g / L, NaCl 10 g / L. Dissolve in 1000 mL of distilled water, adjust the pH value to 7.0 with NaOH, and sterilize at 121 °C for 20 min;

[0089] Small-scale extraction of plasmid DNA:

[0090] (1) Use a sterile pipette tip to pick a white single colony from the LB plate and inoculate it into 5 mL of LB medium containing Amp (100 mg / mL) respectively;

[0091] (2) Incubate with continuous shaking at 37 °C and 300 rpm / min for 8 h;

[0092] (3) Centrifuge at 12,000 rpm / min at room temperature for 3 min, and aspirate and discard the supernatant as much as possible;

[0093] (4) Add 250 μL of Buffer P1 to the centrifuge tube containing the bacterial pellet, and use a vortex oscillator to completely suspend the bacterial pellet;

[0094] (5) Add 250 μL of Buffer P2 to the centrifuge tube, gently invert the tube up and down 4 - 6 times to fully lyse the bacteria;

[0095] (6) Add 350 μL of Buffer N3 to the centrifuge tube and immediately gently invert the tube up and down 4 - 6 times;

[0096] (7) Centrifuge at 12,000 rpm / min for 10 minutes, aspirate the supernatant, and add it to the SpinColumn CM placed in the Collection Tube;

[0097] (8) Centrifuge at 12,000 rpm / min for 60 seconds, pour out the waste liquid in the Collection Tube, and place the Spin Column CM back into the Collection Tube;

[0098] (9) Add 700 μL of Buffer PW to the Spin Column CM, centrifuge at 12,000 rpm / min for 60 seconds, pour out the waste liquid in the Collection Tube, and place the Spin Column CM back into the Collection Tube;

[0099] (10) Add 500 μL of Buffer PW to the Spin Column CM, centrifuge at 12,000 rpm for 60 seconds, pour out the waste liquid in the Collection Tube, and place the Spin Column CM back into the Collection Tube.

[0100] (11) Centrifuge at 12,000 rpm for 60 seconds, pour out the waste liquid, and leave the Spin Column CM with the lid open at room temperature for several minutes to thoroughly dry the residual Buffer PW on the adsorption membrane.

[0101] (12) Place the Spin Column CM in a new centrifuge tube, suspend and add 50 μl of Buffer EB dropwise to the middle part of the adsorption membrane, let it stand at room temperature for 2 minutes, centrifuge at 12,000 rpm for 1 minute, and collect the plasmid solution into the centrifuge tube. Store the plasmid at -20 °C.

[0102] Enzyme digestion identification of the vector

[0103] (1) In a 0.5 mL centrifuge tube, mix the following components in sequence:

[0104] 10×Buffer M 5.0μL Plasmid DNA 5.0μL ddH20 38μL Sacl 1μL PstI 1μL

[0105] The total is 50.0 μL;

[0106] (2) After mixing, centrifuge slightly, incubate at 37 °C for 3 hours, then take 5.0 μL of the enzyme digestion reaction solution for 1.0% agarose gel electrophoresis. After electrophoresis for 20 min under a constant voltage of 2 V / cm, observe under ultraviolet light and cut the gel for recovery;

[0107] DNA sequence determination and analysis: Pick positive clones for DNA sequence determination, and the sequencing is completed by Beijing Liuhe Huada.

[0108] Construction of PCHF3::NtMYB306 and pNTTFL1::NtMYB306 recombinant vectors:

[0109] Digestion and recovery of the target fragment: NtMYB306 was cut from the cloning vector using double digestion with SacI and PstI, and inserted into the similarly digested recombinant vector PCHF3 to construct the recombinant vector PCHF3::NtMYB306 carrying the NtMYB306 gene; NtMYB306 was cut from the cloning vector using double digestion with SacI and BamHI, and inserted into the similarly digested recombinant vector pNTTFL1::GUS to construct the recombinant expression vector pNTTFL1::NtMYB306 carrying the NtMYB306 gene; the digestion reaction system and steps, as well as the recovery steps of the digestion products, were the same as above; for the ligation reaction, Takara's T4 DNA ligase was used, and the system was as follows:

[0110] NtMYB306 fragment 1μL Vector linear fragment 4μL T4 DNA ligase 1μL 10×Buffer 2μL <![CDATA[ddH 2 O]]> 2μL

[0111] The total volume was 10 μL. After thorough mixing, it was centrifuged for a few seconds to collect the liquid droplets on the tube wall to the bottom of the tube, and then incubated in a water bath at 16 °C for 16 - 18 h;

[0112] Identification of the recombinant vectors PCHF3::NtMYB306 and pNTTFL1::NtMYB306: The above ligation products were transformed into Escherichia coli, and positive plaques were screened using PCR. At the same time, the positive plaques were sent for sequencing, and successful construction was achieved when the sequencing results were correct. Figure 1 a shows the PCR electrophoresis results of the recombinant vector pNTTFL1::NtMYB306; Figure 1 b shows the leaf staining results before and after injecting 35S::NtMYB306 into Nicotiana benthamiana.

[0113] PCR primers for verifying the PCHF3::NtMYB306 vector:

[0114] PCHF3-F: 5’-TTGAAGATGCCTCTGCCGAC-3’ SEQ ID NO.7,

[0115] NtMYB306-R: 5’-CTAAAAAGGGTCAGCAGTTTCATC-3’ SEQ ID NO.8;

[0116] PCR primers for verifying the pNTTFL1::NtMYB306 vector:

[0117] pNTTFL1-F: 5’-CTCATTGGTACCACGACGAG-3’ SEQ ID NO.9,

[0118] NtMYB306-R: 5’-CTAAAAAGGGTCAGCAGTTTCATC-3’ SEQ ID NO.10;

[0119] Preparation of Agrobacterium competent cells:

[0120] (1) Pick a single colony of Agrobacterium tumefaciens (EHA105) from the YEP plate (containing 50 μg / mL rifampicin), inoculate it into the YEP liquid medium containing 50 μg / mL rifampicin, and culture it at 28 °C with a rotation speed of 200 rpm / min for about 36 h;

[0121] (2) Take 2 mL of the bacterium solution after the first activation and inoculate it into 50 mL of YEP liquid medium containing the same antibiotic, and culture it under the same conditions until the OD600 reaches 0.5;

[0122] (3) Transfer the bacterium solution to a 50 mL sterile centrifuge tube and ice-bath for 30 min;

[0123] (4) Centrifuge at 5000 rpm / min for 10 min at 4 °C to collect the bacterial cells;

[0124] (5) Remove the supernatant, resuspend the bacterial cells in 10 mL of ice-bathed 0.15 M NaCl solution, and centrifuge to collect the bacterial cells;

[0125] (6) Resuspend again in 1 mL of ice-precooled 20 mM CaCl 2 solution, aliquot the bacterium solution into 1.5 mL sterile Eppendorf tubes at 50 μL / tube, quickly freeze it in liquid nitrogen for 1 min, and store it at -80 °C for later use. YEP medium: Beef extract 10 g / L, yeast extract 10 g / L, NaCl 5 g / L, pH 7.0;

[0126] Transformation of Agrobacterium with the recombinant vector

[0127] (1) Insert 0.1 - 1 μg (5 - 10 μL) of plasmid DNA into 50 μL of Agrobacterium competent cells, and then ice-bath for 30 min;

[0128] (2) Place it in liquid nitrogen for 1 min, and then immediately place it in a water bath at 37 °C for 5 min;

[0129] (3) Take out the centrifuge tube, add 0.5 mL of LB, and culture it with shaking at 28 °C and 220 rpm / min for 3 - 5 hours;

[0130] (4) Take out the bacterium solution, spread it on the LB plate containing spectinomycin (100 mg / mL) and rifampicin (20 μg / mL), and culture it upside down in an incubator at 28 °C. Colonies can be seen in about 2 days;

[0131] PCR verification of Agrobacterium monoclonal colonies:

[0132] Pick a single colony of PCHF3::NtMYB306 and inoculate it into a 2 mL centrifuge tube containing spectinomycin (100 mg / mL) and rifampicin (20 μg / mL). Pick a single colony of pNTTFL1::NtMYB306 and inoculate it into a 2 mL centrifuge tube containing kanamycin (100 mg / mL) and rifampicin (20 μg / mL). Incubate at 200 rpm / min and 28 °C for about 18 h.

[0133] PCR reaction system:

[0134] 2×Phanta Max Master Mix 10μL <![CDATA[ddH 2 O]]> 7.4μL F(10mM) 0.8μL R(10mM) 0.8μL Monoclonal bacterial solution 1μL

[0135] The total volume is 20 μL. After mixing, pre-denature at 94 °C for 3 min, and then perform PCR reaction: The reaction parameters are denaturation at 94 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s. After 35 cycles, continue to extend at 72 °C for 5 min and store at 16 °C. Pipette 10 μL of the PCR amplification reaction solution for 1.0% agarose gel electrophoresis, and electrophorese at a constant voltage of 2 V / cm for 20 min. Take a picture with a gel imager.

[0136] Nicotiana benthamiana injection process:

[0137] Pick Agrobacterium tumefaciens of PCHF3::NtMYB306 into 1 mL of LB medium (added with 100 mg / mL kanamycin and 20 μg / mL rifampicin), shake culture at 250 rpm and 28 °C for 16 h, then transfer the bacterial solution to fresh LB medium (added with 100 mg / mL kanamycin and 20 μg / mL rifampicin), shake culture at 250 rpm and 28 °C for 16 h. Centrifuge the bacterial solution at 4000 rpm for 10 min, discard the supernatant, and resuspend the cells with resuspension solution (10 mM MES, 150 μM AS, 10 mM MgCl 2 ) to adjust the resuspension solution to OD 600 = 1. Let the resuspension solution stand at 28 °C for 3 h. Select Nicotiana benthamiana at the 8 - 10 leaf stage, make a small hole on the back of the leaf with a needle, align the syringe without the needle with the small hole, and use your finger to block the syringe and the small hole through the leaf with the other hand. Push the piston to inject the bacterial solution into the leaf until obvious water stains appear. Observe for 7 days after injection and take pictures of the leaves.

[0138] Trypan blue staining process:

[0139] Mix 10 mL of glycerol, 10 mL of lactic acid, and 10 mL of sterile water evenly, then add 10 mg of trypan blue powder and 10 g of phenol to prepare a trypan blue solution. Immerse the Nicotiana benthamiana leaves injected for 7 days in the pre-boiled and preheated trypan blue staining solution, boil for 2 min, carefully pick out the leaves with forceps, rinse them thoroughly with sterile water, add an appropriate amount of chloral hydrate at 1.25 g / mL (dissolve 250 g of chloral hydrate in 40 mL of distilled water and make up the volume to 200 mL), gently rotate on a shaker for overnight decolorization, observe the cell death situation and take pictures for record.

[0140] Agrobacterium-mediated genetic transformation of tobacco:

[0141] Culture of tobacco sterile seedlings:

[0142] Soak the seeds of Nicotiana tabacum cv. Honghuadajinyuan in 15% sodium hypochlorite for 10 minutes, then rinse them 3 times with sterile water, sow the seeds on MS medium, transfer them to a plant light culture room for cultivation, and take the leaves for infection after one month;

[0143] Preparation of Agrobacterium infection solution:

[0144] Using the preserved original bacterial solution (-80 °C), take 100 μL and add it to YEB liquid (using Erlenmeyer flasks, 200 mL per flask + 100 mg / mL spectinomycin / kanamycin + 20 μg / mL rifampicin), culture at 28 °C, 200 rpm / min, in the dark for about 18 h until OD = 0.6 - 0.8. Centrifuge the bacterial solution at 4000 rpm / min for 5 min to collect the bacteria, and dilute it with MS 0 to OD600 = 0.8, then add 20 μg / mL of acetosyringone;

[0145] YEB medium: Contains 5 g of Tryptone, 1 g of yeast extract, 0.5 g of magnesium sulfate, 5 g of beef extract, 5 g of sucrose per liter, pH 7.0;

[0146] Process of genetic transformation

[0147] Cut off 0.5 cm of sterile leaves, immerse them in the bacterial solution for 5 min, blot them dry with sterile filter paper, place them with the veins facing up, spread them on the co-culture medium, culture in the dark at 22 °C for 3 days, and then inoculate the co-cultured leaves with the veins facing down onto the S 1 medium, place it in an artificial climate chamber, culture at about 25 °C for 3 weeks, and see adventitious buds growing at the leaf margins, with the bud length being 0.1 - 0.5 cm. Transfer the adventitious buds on the S 1 to the S 2 medium, culture under light for 2 weeks, and the adventitious buds grow into young seedlings. Break off the young seedlings on the S 2 onto the S 3 medium, culture under light for 2 weeks, and the seedlings gradually become strong. Transfer the seedlings on the S 3Select robust seedlings, remove the swollen part at the bottom and the yellowed leaves at the lower part, inoculate them onto the rooting medium, culture them under light for 2 weeks, and transplant them into nutrient pots after rooting. Extract DNA from each transgenic seedling for PCR detection.

[0148] Formulation of each step of the medium (1L), pH 5.8

[0149]

[0150] Positive detection of transgenic plants:

[0151] DNA extraction of transgenic plants:

[0152] (1) Take about 100 mg of fresh plant tissue, add liquid nitrogen and grind thoroughly;

[0153] (2) Collect the ground powder into a 1.5 mL centrifuge tube, add 400 μL of Buffer LP1 and 6 μL of RNase A (10 mg / mL), vortex for 1 minute, and let it stand at room temperature for 10 minutes to fully lyse;

[0154] (3) Centrifuge at 12000 rpm / min for 5 minutes, transfer the supernatant to a new 1.5 mL centrifuge tube;

[0155] (4) Add 1.5 times the volume of Buffer LP3 and mix well;

[0156] (5) Add all the solution and precipitate obtained in the previous step to Spin Columns DM, centrifuge at 12000 rpm / min for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0157] (6) Add 500 μL of Buffer GW2 to the adsorption column, centrifuge at 12000 rpm / min for 1 minute, pour out the waste liquid in the collection tube, and put the adsorption column back into the collection tube;

[0158] (7) Repeat step (6);

[0159] (8) Centrifuge at 12000 rpm / min for 2 minutes, pour out the waste liquid in the collection tube, and place the adsorption column at room temperature for several minutes to dry thoroughly;

[0160] (9) Place the adsorption column into a new 1.5 mL centrifuge tube, suspend and drop 50 μL of sterilized water onto the middle part of the adsorption membrane, let it stand at room temperature for 2 - 5 minutes, centrifuge at 12000 rpm / min for 1 minute, collect the DNA solution, and store the DNA at -20 °C.

[0161] Topping experiment on transgenic positive plants and control plants:

[0162] The transgenic positive plants and control plants were topped, and the growth of axillary buds at the first leaf position within 3 weeks after topping was observed. The results are as follows Figure 2 and 3 shown. Compared with the control material, the growth of axillary buds at the first leaf position of the pNTTFL1::NtMYB306 transgenic positive material was inhibited after topping, while the length and width of the first leaf were not affected. It can be seen that using the axillary bud-specific promoter to drive the NtMYB306 protein in tobacco can effectively inhibit the growth of axillary buds after topping, thereby promoting the transport of nutrients to the main stem leaves of tobacco and improving the growth quality and yield of tobacco leaves.

[0163] The preferred embodiments of the present disclosure have been described in detail above. However, the present disclosure is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0164] In addition, it should be noted that, in the case of no contradiction, the various specific technical features described in the above specific embodiments can be combined in any appropriate manner. To avoid unnecessary repetition, the present disclosure will not separately describe various possible combination methods.

[0165] Furthermore, any combination can be made between different embodiments of the present disclosure, as long as it does not violate the idea of the present disclosure, and it should also be regarded as the content disclosed by the present disclosure.

Claims

1. A method for inhibiting the growth of axillary buds of tobacco after topping, characterized in that: The method comprises the following steps: Overexpression of NtMYB306 protein in tobacco, wherein the NtMYB306 protein comprises: (1) a protein with an amino acid sequence as shown in SEQ ID NO.2; (2) A protein having an amino acid sequence as shown in SEQ ID NO. 2, with n amino acids substituted, deleted or added, and still having the function of inhibiting the growth of axillary buds after tobacco topping, wherein n is an integer between 1 and 146.

2. The method according to claim 1, wherein: The method comprises: overexpressing the NtMYB306 protein in axillary buds of tobacco.

3. The method according to claim 1, wherein: The method comprises: transferring a gene expression box for overexpressing NtMYB306 protein into tobacco to prepare transgenic tobacco, and planting and topping the transgenic tobacco.

4. The method according to claim 3, wherein: The gene expression cassette for overexpressing the NtMYB306 protein is shown in SEQ ID NO.1, wherein positions 253-1227 are the gene coding sequence of the NtMYB306 protein.

5. The method according to claim 3, wherein: The method comprises: transferring the gene expression cassette into a tobacco expression vector to construct a recombinant expression vector; The recombinant expression vector is transferred into tobacco to prepare transgenic tobacco, and the transgenic tobacco is planted and toppled.

6. The method according to claim 5, wherein: The tobacco expression vector includes a PCHF3 vector and / or a PBI121 vector.

7. The method according to claim 3, wherein: The method further comprises inserting the gene expression cassette into a host cell to construct a transformant; The transformant is transferred into tobacco to prepare transgenic tobacco, and the transgenic tobacco is planted and toppled.

8. The method according to claim 7, wherein: The host cell is a genetically engineered bacterium.

9. The method according to claim 8, wherein: The genetically engineered bacteria include Escherichia coli and / or Agrobacterium.

10. The method according to claim 3, wherein: The expression cassette for overexpressing the NtMYB306 protein is transferred into the tobacco by Agrobacterium-mediated method.

Images