CN115521936A - Method and material for delaying lateral branch growth of tobacco after topping - Google Patents

Abstract

The invention provides a method and a material for delaying the growth of lateral branches of tobacco after topping, wherein the method comprises the following steps: sowing, seedling raising and transplanting the transgenic tobacco, and topping; exogenous nucleic acid is inserted into the genome of the transgenic tobacco; the exogenous nucleic acid comprises an axillary bud specific promoter and a lethal gene driven by the axillary bud specific promoter; the axillary bud specific promoter comprises at least one of an NtTF1 (CEN-like protein 4) promoter, a CEN-like protein 2 promoter and a CEN-like protein 1 promoter; the lethal gene is at least one selected from diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene.

Description

Method and material for delaying lateral branch growth of tobacco after topping

Technical Field

The disclosure relates to the technical field of bioengineering, in particular to a method for delaying the growth of lateral branches of tobacco after topping and a recombinant vector.

Background

In tobacco production, topping is typically performed to concentrate the growth of the nutrient supply lamina in order to improve the yield and quality of the tobacco leaf. 2-3 or more axillary buds can be regenerated per 1 axillary leaf of the tobacco plant after topping. If the plants are allowed to grow, a large amount of nutrients are consumed, and the vegetative growth of main stem leaves is influenced.

Tobacco, as a leaf crop, is left to flower and bear fruits, which consumes a large amount of water, reducing the yield and quality of the tobacco leaves. In the planting of high-quality tobacco, "topping" is an important measure for regulating and controlling the nutrition of tobacco plants and the yield and quality of tobacco leaves, and buds or flower sequences at the top of the tobacco plants must be picked off at a proper time, so that the unnecessary consumption of nutrient substances in the tobacco plants is avoided, the nutrient substances are promoted to be intensively supplied to the leaves for growth, and the area and the weight of each leaf are increased. However, after topping, axillary buds grow clumpy and consume nutrients, and the aim of improving the quality of tobacco leaves cannot be fulfilled.

In order to improve the yield and quality of tobacco leaves, it is often necessary to remove axillary buds and inhibit their growth. The traditional manual bud picking is labor-consuming and troublesome, is not timely and thorough, is easy to cause tobacco branches and clusters, leads fireworks to fill the field, and is also easy to infect diseases. At present, the bud inhibitor is used in field production to inhibit the occurrence of axillary buds of tobacco plants after topping, and the use of the bud inhibitor not only increases the production cost, but also causes pollution to the environment, so that the cultivation of a method which can effectively inhibit the axillary buds after topping, simultaneously avoids the pollution to the environment and tobacco leaves, is safe to apply, and is vital in reducing the production cost and labor input.

Disclosure of Invention

In order to improve the yield and quality of tobacco leaves, the disclosure provides a method for delaying the growth of lateral branches of tobacco leaves after topping and a recombinant vector.

In one aspect, the present disclosure provides a method for delaying lateral shoot growth of tobacco after topping, the method comprising the steps of:

sowing, seedling raising and transplanting the transgenic tobacco, and topping; exogenous nucleic acid is inserted into the genome of the transgenic tobacco; the exogenous nucleic acid comprises an axillary bud specific promoter and a lethal gene driven by the axillary bud specific promoter;

the axillary bud-specific promoter comprises at least one of an NtTF1 (CEN-like protein 4) promoter, a CEN-like protein 2 promoter and a CEN-like protein 1 promoter;

the lethal gene is at least one selected from diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene.

According to the disclosure, the nucleotide sequence of diphtheria toxin A chain gene is shown in SEQ ID NO.1, the nucleotide sequence of Aspergillus oryzae gene RNase-T1 is shown in SEQ ID NO.2, and the nucleotide sequence of Barnase gene is shown in SEQ ID NO. 3.

According to the disclosure, the nucleotide sequence of the NtTF1 promoter is shown in SEQ ID No. 4.

According to the present disclosure, the exogenous nucleic acid is set forth in SEQ ID No. 5.

According to the present disclosure, the method further comprises: the exogenous nucleic acid shown as SEQ ID NO.5 is inserted into the genome of tobacco to prepare transgenic tobacco.

In another aspect, the present disclosure provides a recombinant vector into which the above-described foreign nucleic acid is inserted.

According to the present disclosure, the exogenous nucleic acid comprises an axillary bud-specific promoter and a lethal gene driven by the axillary bud-specific promoter;

the lethal gene is at least one of diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene; the axillary bud-specific promoter is at least one selected from the group consisting of an NtTF1 (CEN-like protein 4) promoter, a CEN-like protein 2 promoter and a CEN-like protein 1 promoter.

According to the present disclosure, the exogenous nucleic acid is set forth in SEQ ID No. 5.

Through the technical scheme, the method constructs the axillary bud special-shaped promoter and lethal gene recombinant vector, the axillary bud special-shaped promoter is used for driving expression of the lethal gene, the tobacco is infected by the recombinant vector to obtain a positive plant, and the growth of the tobacco axillary bud at the first leaf position of the topped positive plant is delayed for 1-2 weeks compared with that of a control, so that the yield and the quality of the tobacco are improved.

Additional features and advantages of the present disclosure will be set forth in the detailed description which follows.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure and are incorporated in and constitute a part of this specification, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure without limiting the disclosure. In the drawings:

FIG. 1 shows NtTF1 promoter amplification (left) with P _NtTF1 The DTA recombinant vector construction (right) is shown in the drawing.

FIG. 2 is P _NtTF1 The PCR detection result of DTA transgenic seedlings.

FIG. 3 shows the in-growth of the axillary buds at the first leaf position after topping of transgenic T1 plants and controls.

Detailed Description

The following describes in detail specific embodiments of the present disclosure. It should be understood that the detailed description and specific examples, while indicating the present disclosure, are given by way of illustration and explanation only, not limitation.

In one aspect, the present disclosure provides a method for delaying lateral shoot growth of tobacco after topping, the method comprising the steps of:

sowing, seedling raising and transplanting the transgenic tobacco, and topping; exogenous nucleic acid is inserted into the genome of the transgenic tobacco; the exogenous nucleic acid comprises an axillary bud specific promoter and a lethal gene driven by the axillary bud specific promoter;

the axillary bud specific promoter is at least one selected from the group consisting of an NtTF1 promoter, a CEN-like protein 2 promoter and a CEN-like protein 1 promoter;

the lethal gene is at least one selected from diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene.

Optionally, the diphtheria toxin A chain gene has a nucleotide sequence shown in SEQ ID No.1, the Aspergillus oryzae gene RNase-T1 has a nucleotide sequence shown in SEQ ID No.2, and the Barnase gene has a nucleotide sequence shown in SEQ ID No. 3.

Wherein the diphtheria toxin can not only delay the growth of the axillary bud of the transgenic tobacco after topping but also does not influence the growth of other organ tissues of the tobacco when the axillary bud specific promoter is applied.

Alternatively, the nucleotide sequence of the NtTF1 promoter is shown in SEQ ID NO. 4.

Optionally, the exogenous nucleic acid is as shown in SEQ ID NO. 5.

Optionally, the method further comprises: the exogenous nucleic acid shown as SEQ ID NO.5 is inserted into the genome of tobacco to prepare transgenic tobacco.

Alternatively, the exogenous nucleic acid may be obtained by ligating the nucleotide sequence of the axillary bud specific promoter with the sequence of the lethal gene, whereby the axillary bud specific promoter initiates expression of the lethal gene after insertion of the exogenous nucleic acid into the tobacco genome to form a transgenic tobacco plant, thereby delaying growth of tobacco axillary buds.

In another aspect, the present disclosure also provides a recombinant vector into which the above-described foreign nucleic acid is inserted.

Optionally, wherein the exogenous nucleic acid comprises an axillary bud specific promoter and a lethal gene driven by the axillary bud specific promoter; the lethal gene is at least one of diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene; the axillary bud specific promoter includes at least one of an NtTF1 (CEN-like protein 4) promoter, a CEN-like protein 2 promoter, and a CEN-like protein 1 promoter.

Preferably, the exogenous nucleic acid is shown as SEQ ID NO. 5.

The present invention will be described in further detail below with reference to examples.

Example 1

Materials: an upstream 246bp promoter sequence of NtTF1 (CEN-like protein 4), a PBI121 vector, a lethal gene DTA and a sequence thereof, and a tobacco variety;

the sequence of the upstream 246bp promoter of the NtTF1 is shown in SEQ ID NO. 4; the diphtheria toxin A chain (DTA) coding sequence is shown in SEQ ID NO. 1;

the method comprises the following steps:

vector construction process and genetic transformation process

Enzyme digestion identification of PBI121 vector

(1) In a 0.2mL centrifuge tube, the following ingredients were mixed in order: 10 Xbuffer M (5.0. Mu.L), plasmid DNA (5.0. Mu.L), ddH ₂ O (38. Mu.L), bamHI (1. Mu.L)/SacI (1. Mu.L) and HindIII (1. Mu.L)/BamHI (1. Mu.L) in a total reaction volume of 50. Mu.L;

(2) Mixing, centrifuging slightly, incubating at 37 deg.C for 3 hr, performing 1.0% agarose gel electrophoresis on 5.0 μ L enzyme digestion reaction solution, performing electrophoresis under 2V/cm constant pressure for 20min, observing under ultraviolet lamp, cutting, and recovering gel;

designing a primer according to an upstream 246bp promoter sequence of NtTF1, and constructing PBI121-P by adopting a homologous recombination method _NtTF1 The DTA recombinant vector designs two enzyme cutting sites for the convenience of constructing the vector: hindIII and BamHI;

P _NtTF1 -F:5’-TGATTACGCCAAGCTTCTCATTGGTACCACGACGAGT-3’SEQ ID NO.6 HindIII

P _NtTF1 -R:5’-GACCACCCGGGGATCCGGATCAGACATTTTTGAACCCA-3’SEQ ID NO.7 BamHI

designing a primer according to CDS sequence of DTA, and constructing PBI121-P by adopting a homologous recombination method _NtTF1 The DTA recombinant vector designs two enzyme cutting sites for the convenience of constructing the vector: bamHI and SacI

DTA-F：5’-GGACTCTAGAGGATCCATGGATCCTGATGATGTTGTTGA-3’SEQ ID NO.8 BamHI

DTA-R：5’-GATCGGGGAAATTCGAGCTCTTAGAGCTTTAAATCTCTGTAGGT-3’SEQ ID NO.9 SacI

And (3) PCR amplification: the following ingredients were added to a 0.2mL centrifuge tube: 2X Phanta Max Master Mix(10μL)、ddH ₂ O (8. Mu.L), F (10 mM)) (0.8. Mu.L), R (10 mM) (0.8. Mu.L), CDS plasmid template (0.4. Mu.L); the total volume of the reaction is 20 mu L; mixing the materials, performing pre-denaturation at 94 ℃ for 2min, and performing PCR: the reaction parameters are 94 ℃ denaturation 20s,60 ℃ annealing 20s,72 ℃ extension 30s, after 35 cycles, continuing extension for 5min at 72 ℃, and storing at 16 ℃;

and (3) recovering a PCR product: the recovery of PCR amplification product adopts TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0, the operation is carried out according to the instruction:

(1) Carrying out 1.0% agarose gel electrophoresis on 10 mu L of PCR amplification reaction solution, carrying out electrophoresis for 20min under the condition of constant voltage of 2V/cm, cutting an agarose block containing a target fragment under an ultraviolet lamp, and transferring the agarose block into a 1.5mL centrifuge tube;

(2) Adding 3 times of the volume of the gum block dissolving solution Buffer GM, uniformly mixing, and dissolving the gum blocks at room temperature of 15-25 ℃;

(3) Transferring the dissolved solution to a Spin Column centrifuge Column;

(4) Centrifuging at 12000rpm/min at 25 deg.C for 1min;

(5) Removing liquid in the centrifuge tube, and adding 700 mu L of Buffer WB to Spin Column centrifuge;

(6) Centrifuging at 12000rpm/min at 25 ℃ for 30s;

(7) Repeating the operation steps (5) and (6);

(8) Removing liquid in the centrifuge tube, and centrifuging at 12000rpm/min for 1min at room temperature;

(9) Placing Spin Column on new 1.5mL centrifuge tube, adding 30 μ L sterilized distilled water at center of Spin Column membrane, and standing at room temperature for 1min;

(10) Centrifuging at 25 deg.C for 1min at 12,000rpm/min;

(11) Taking 1.0 μ l of the recovery liquid collected by centrifugation, and carrying out DNA fragment concentration determination on a NanoDrop 2000c spectrophotometer;

cloning of the fragment of interest: according to the method provided by Baozhi corporation, the PCR amplified fragment is connected with the linear vector recovered by enzyme digestion: to a 0.2mL sterile centrifuge tube was added: 2.0. Mu.L of 5X In-Fusion HD Enzyme Premix, 4.0. Mu.L of linear vector, 1.0. Mu.L of PCR-recovered product, 3.0. Mu.L of ddH ₂ O, adding to the wholeThe product is 10.0 mu L, the mixture is fully and evenly mixed and then centrifuged for a plurality of seconds, the liquid on the tube wall is dripped to the tube bottom, and the reaction is carried out for 15min at 50 ℃;

preparation of coli DH5a competent cells:

(1) E.coli DH5a single colonies were picked from LB agar plates, inoculated into 10mL of LB liquid medium containing no antibiotics, and cultured overnight at 37 ℃ with shaking at 300 rpm/min. Transferring into fresh LB liquid culture medium according to the amount of 1% (V/V) the next day, and performing shake culture at 37 deg.C to 0.3-0.4;

(2) Transferring 50-100mL of culture solution into two pre-cooled sterile centrifuge tubes, and placing on ice for 30min;

(3) Centrifuging at 4 deg.C and 6000rpm/min for 5min, and removing supernatant;

(4) 10mL of precooled 0.1mol/L CaCl2 solution is added into each centrifuge tube respectively to resuspend the thalli, and ice bath is carried out for 30min;

(5) Centrifuging at 4 deg.C and 6000rpm/min for 5min to remove supernatant, and suspending thallus in 2mL precooled 0.1mol/CaCl ₂ And (4) obtaining competent cells in the solution. Quick freezing with liquid nitrogen, and storing in refrigerator at-70 deg.C;

conversion of ligation product:

(1) Taking 50 mu L of competent cells by using a sterile suction head, placing the competent cells in a 1.5mL precooled sterile centrifuge tube, adding 2.5 mu L of connection reaction liquid, gently mixing the competent cells and the connection reaction liquid uniformly, and immediately placing the mixture on ice for 30min;

(2) Placing the centrifugal tube in a constant-temperature water bath at 42 ℃ for heat shock for 30s;

(3) Putting back on ice for 3-5min;

(4) Adding 500 μ L LB liquid culture medium without additional antibiotics, mixing, shaking at 37 deg.C at 300rpm/min, and culturing for 60min;

(5) Preparing a solid plate of LB plus 100mg/ml kanamycin;

(6) Sucking 100 mu L of bacterial liquid, transferring the bacterial liquid to an LB flat plate, and uniformly coating the whole surface of the flat plate with the bacterial liquid by using a sterile triangular head glass rod;

(7) Placing the plate at 37 deg.C in forward direction until the liquid is absorbed, then inverting the plate, and culturing at 37 deg.C for 12-16h;

LB culture medium: adding 1000mL of distilled water into Yeast Extract 5g/L, peptone 10g/L and NaCl10g/L for dissolving, adjusting the pH value to 7.0 by using NaOH, and sterilizing at 121 ℃ for 20min;

small extraction of plasmid DNA:

(1) Picking white single colonies from the LB plate by using a sterile gun head and respectively inoculating the white single colonies into 5ml of LB culture medium containing Amp (100 mg/ml);

(2) Continuously oscillating and culturing for 8-10 h at 37 ℃ and 300 rpm/min;

(3) Centrifuging at room temperature for 3min at 12,000rpm/min, and discarding the supernatant as much as possible;

(4) Adding 250 mu L of BufferP1 into the centrifuge tube with the bacterial sediment, and completely suspending the bacterial sediment by using a vortex oscillator;

(5) Adding 250 mu L of BufferP2 into the centrifuge tube, and gently turning upside down and uniformly mixing for 4-6 times to ensure that the thalli are fully cracked;

(6) Adding 350 mu L of BufferN3 into the centrifuge tube, immediately and gently turning upside down and uniformly mixing for 4-6 times;

(7) Centrifuging at 12000rpm/min for 10min, sucking supernatant, and adding into Spin Column CM filled with Collection Tube;

(8) Centrifuging at 12000rpm/min for 60s, pouring out waste liquid in the Collection Tube, and putting Spin Column CM back into the Collection Tube;

(9) Adding 700 mu L Buffer PW into Spin Column CM, centrifuging at 12000rpm/min for 60s, pouring off waste liquid in the Collection Tube, and putting the Spin Column CM back into the Collection Tube;

(10) Adding 500 μ L Buffer PW into Spin Column CM, centrifuging at 12000rpm/min for 60s, pouring out waste liquid of Collection Tube, and placing Spin Column CM back into Collection Tube;

(11) Centrifuging at 12000rpm/min for 60s, and pouring out waste liquid. Uncovering the Spin Column CM, and placing the Spin Column CM at room temperature for a plurality of minutes to completely dry the residual Buffer PW on the adsorption film;

(12) The Spin Column CM was placed in a new centrifuge tube, 50. Mu.l of Buffer EB was added to the middle of the adsorption membrane in suspension, the tube was left at room temperature for 2min, centrifuged at 12000rpm/min for 1min, the solution was collected in the centrifuge tube, and the plasmid was stored at-20 ℃.

DNA sequencing and analysis: the selected positive clone is subjected to DNA sequence determination, and sequencing is completed by Beijing Liu-Hei-Huada;

PBI121-P _NtTF1 constructing a DTA recombinant expression vector: enzyme digestion and recovery of target fragments: inserting the NtTF1 promoter into a vector PBI121 at HindIII and BamHI sites, and inserting DTA into PBI121-P _NtTF1 In GUS recombinant vector, enzyme cutting sites are BamHI and SacI, and expression vector PBI121-P with DTA gene is constructed _NtTF1 DTA, as shown in FIG. 1, ntTF1 promoter amplification (left) with P _NtTF1 Constructing a DTA recombinant vector (right);

PBI121-P _NtTF1 identification of DTA recombinant expression vector: and (3) transforming the ligation product into escherichia coli, screening positive bacterial plaques by using PCR, detecting the positive bacterial plaques, and establishing successfully if the sequencing result is correct.

And (3) carrying out sensitive preparation on agrobacterium rhizogenes:

(1) A single colony of Agrobacterium tumefaciens (EHA 105) was picked from a YEP plate (containing 50. Mu.g/mL of rifampicin), inoculated in a YEP liquid medium containing 50. Mu.g/mL of rifampicin, cultured at 200rpm/min at 28 ℃ for about 36 hours;

(2) Inoculating 2mL of the first activated bacterial liquid into 50mL of YEP liquid culture medium containing the same antibiotics, and culturing under the same conditions until OD600 reaches 0.5;

(3) Transferring the bacterial liquid to a 50mL sterile centrifuge tube, and carrying out ice bath for 30min;

(4) Centrifuging at 4 deg.C and 5000rpm/min for 10min, and collecting thallus;

(5) Removing supernatant, suspending the thalli in 10mL of 0.15M NaCl solution in ice bath, and centrifugally collecting the thalli;

(6) Resuspended in 1mL of 20mM ice-chilled CaCl ₂ In the solution, the bacterial solution was dispensed into 1.5mL sterile Eppendorf tubes at 50. Mu.L/tube, frozen in liquid nitrogen for 1min, and stored at-80 ℃ for further use.

YEP medium: contains per liter: 10g of beef extract, 10g of yeast extract, 5g of NaCl and 7.0 of pH;

transforming agrobacterium with the recombinant vector:

(1) Inserting 0.1-1 mug (5-10 mug) of plasmid DNA into 50 muL of agrobacterium tumefaciens competent cells, and then carrying out ice bath for 30min;

(2) Placing into liquid nitrogen for 1min, immediately placing into 37 deg.C water bath kettle, and water-bathing for 5min;

(3) Taking out the centrifuge tube, adding 0.5mL LB, oscillating and culturing at 28 ℃ and 220rpm/min for 3-5 hours;

(4) Taking out bacterial liquid, coating on LB plate containing kanamycin (100 mg/mL) and rifampicin (20 mug/mL), inversely culturing in incubator at 28 deg.C, the bacterial colony can be seen in about 2 days;

PCR verification of agrobacterium monoclonal colonies: single colonies were picked and inoculated into 2mL centrifuge tubes containing kanamycin (100 mg/mL) and rifampicin (20. Mu.g/mL) at 200rpm/min for about 18h at 28 ℃.

Agrobacterium-mediated genetic transformation of tobacco:

culturing the tobacco aseptic seedlings: soaking safflower Honghua Dajinyuan tobacco seeds in 15% sodium hypochlorite for 10min, washing with sterile water for 3 times, dibbling the seeds on an MS culture medium, wherein the components of the culture medium are shown in table 1 (sprouting), transferring to a plant illumination culture chamber for culture, and taking leaves for infection after one month;

preparing an agrobacterium infection solution: using the preserved original strain solution (-80 ℃), 100. Mu.L of the stock strain solution was added to YEB (using a triangular flask, 200 mL/vial +100mg/mL spectinomycin + 20. Mu.g/mL rifampicin), and cultured at 28 ℃ at 200rpm/min in the dark for about 18 hours until OD = 0.6-0.8. Centrifuging at 4000rpm/min for 5min to collect bacterial liquid, diluting to OD600=0.8 by using MS0, and then adding 20 mug/mL acetosyringone;

YEB Medium: contains per liter: 5g of Tryptone, 1g of yeast extract, 0.5g of magnesium sulfate, 5g of beef extract, 5g of cane sugar and 7.0 of pH;

the process of genetic transformation: cutting off 0.5cm sterile leaves, immersing in bacteria solution for 5min, drying with sterile filter paper, spreading on co-culture medium (G), culturing in dark at 22 deg.C for 3 days, inoculating the co-cultured leaf vein downwards onto S1 culture medium, culturing in artificial climate room at 25 deg.C for 2-3 weeks until cluster buds grow at the edge of the leaf, wherein the bud length is 0.1-0.5cm, transferring the cluster buds on S1 culture medium onto S2 culture medium, culturing in light for 1-2 weeks, and allowing the cluster buds to grow into young plantlets. And (3) breaking the young plantlets on the S2 culture medium onto the S3 culture medium, and culturing for 1-2 weeks under the light, wherein the plantlets are gradually robust. Removing the expanded part at the bottom and the yellow leaves at the lower part of the robust seedling on the S3 culture medium, inoculating the seedling on a rooting culture medium (R), culturing for 1-2 weeks under the light, and transplanting the seedling into a nutrition pot after rooting.

TABLE 1 culture media used in the various steps of the tobacco genetic transformation procedure

The result of PCR detection of DNA extracted from each transgenic shoot is shown in FIG. 2. In FIG. 2, the samples from left to right of each lane are transgenic plants 1-5, and the results show that transgenic plants 1, 3, 4, 5 are positive plants.

The process of DNA extraction and PCR detection of transgenic plants is as follows:

(1) Taking about 100mg of fresh plant tissues, adding liquid nitrogen, and fully grinding;

(2) Collecting the ground powder into a 1.5mL centrifuge tube, adding 400 μ L Buffer LP1 and 6 μ L RNase A (10 mg/mL), vortexing and shaking for 1min, and standing at room temperature for 10min to allow full lysis;

(3) Centrifuging at 12000rpm/min for 5min, and transferring the supernatant into a new 1.5mL centrifuge tube;

(4) Adding 1.5 times volume of Buffer LP3, and fully and uniformly mixing;

(5) Adding the solution and the precipitate obtained in the previous step into Spin Columns DM, centrifuging at 12000rpm/min for 1 minute, pouring the waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube again;

(6) Adding 500 mu L of Buffer GW2 into the adsorption column, centrifuging at 12000rpm/min for 1 minute, pouring the waste liquid in the collecting tube, and putting the adsorption column back into the collecting tube again;

(7) Repeating the step (6);

(8) Centrifuging at 12000rpm/min for 2min, and pouring off waste liquid in the collecting pipe. Placing the adsorption column at room temperature for several minutes to completely dry;

(9) Placing the adsorption column into a new 1.5mL centrifuge tube, suspending and dropwise adding 50 mu L of sterilized water to the middle part of the adsorption membrane, standing at room temperature for 2-5 minutes, centrifuging at 12000rpm/min for 1 minute, collecting DNA solution, and storing DNA at-20 ℃;

transgenic plant positive verification PCR primer:

P _NtTF1 -F：5’-CTCATTGGTACCACGACGAG-3’SEQ ID NO.10，

DTA-R：5’-TTAGAGCTTTAAATCTCTGTAGGT-3’SEQ ID NO.11；

topping experiment and phenotype observation of transgenic positive plants and control plants:

topping the transgenic T1 generation plant and the control plant, and observing the growth condition of axillary buds in the four weeks, as shown in figure 3; in fig. 3, f to j are control plants, and a to e are transgenic positive plants, it can be seen that the axillary buds of the control plants grow normally, while the axillary buds of the transgenic plants obtained in the present disclosure start to be delayed in the first week compared to the control plants, and grow in the same manner as the control plants from the second week.

According to the technical scheme, the lethal gene expression is driven by the axillary bud specific promoter, the axillary bud specific promoter and lethal gene recombinant vector is constructed, and the growth of tobacco axillary buds can be effectively delayed by using the recombinant vector.

The preferred embodiments of the present disclosure have been described in detail above, however, the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present disclosure within the technical idea of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

It should be noted that, in the foregoing embodiments, various features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various combinations that are possible in the present disclosure are not described again.

In addition, any combination of various embodiments of the present disclosure may be made, and the same should be considered as the disclosure of the present disclosure as long as it does not depart from the gist of the present disclosure.

Sequence listing

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<211> 246

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctcattggta ccacgacgag taggagaata aactttttgg gcttttcttt tcttttcttt 60

ggttcccatt ttttgaacta tcaatatttt agtccaaaca cacctgactc tacagtgatc 120

tgatggccac tataaatatt ggctttttgc agctccaaaa tacaaatcgg tcgaactctt 180

catatatatt actcttaact ttaaataaat agatttctta tatatgggtt caaaaatgtc 240

tgatcc 246

<210> 5

<211> 14758

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

tgagcgtcgc aaaggcgctc ggtcttgcct tgctcgtcgg tgatgtactt caccagctcc 60

gcgaagtcgc tcttcttgat ggagcgcatg gggacgtgct tggcaatcac gcgcaccccc 120

cggccgtttt agcggctaaa aaagtcatgg ctctgccctc gggcggacca cgcccatcat 180

gaccttgcca agctcgtcct gcttctcttc gatcttcgcc agcagggcga ggatcgtggc 240

atcaccgaac cgcgccgtgc gcgggtcgtc ggtgagccag agtttcagca ggccgcccag 300

gcggcccagg tcgccattga tgcgggccag ctcgcggacg tgctcatagt ccacgacgcc 360

cgtgattttg tagccctggc cgacggccag caggtaggcc gacaggctca tgccggccgc 420

cgccgccttt tcctcaatcg ctcttcgttc gtctggaagg cagtacacct tgataggtgg 480

gctgcccttc ctggttggct tggtttcatc agccatccgc ttgccctcat ctgttacgcc 540

ggcggtagcc ggccagcctc gcagagcagg attcccgttg agcaccgcca ggtgcgaata 600

agggacagtg aagaaggaac acccgctcgc gggtgggcct acttcaccta tcctgcccgg 660

ctgacgccgt tggatacacc aaggaaagtc tacacgaacc ctttggcaaa atcctgtata 720

tcgtgcgaaa aaggatggat ataccgaaaa aatcgctata atgaccccga agcagggtta 780

tgcagcggaa aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg 840

gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt 900

atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag 960

gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt 1020

gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta 1080

ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt 1140

cagtgagcga ggaagcggaa gagcgccaga aggccgccag agaggccgag cgcggccgtg 1200

aggcttggac gctagggcag ggcatgaaaa agcccgtagc gggctgctac gggcgtctga 1260

cgcggtggaa agggggaggg gatgttgtct acatggctct gctgtagtga gtgggttgcg 1320

ctccggcagc ggtcctgatc aatcgtcacc ctttctcggt ccttcaacgt tcctgacaac 1380

gagcctcctt ttcgccaatc catcgacaat caccgcgagt ccctgctcga acgctgcgtc 1440

cggaccggct tcgtcgaagg cgtctatcgc ggcccgcaac agcggcgaga gcggagcctg 1500

ttcaacggtg ccgccgcgct cgccggcatc gctgtcgccg gcctgctcct caagcacggc 1560

cccaacagtg aagtagctga ttgtcatcag cgcattgacg gcgtccccgg ccgaaaaacc 1620

cgcctcgcag aggaagcgaa gctgcgcgtc ggccgtttcc atctgcggtg cgcccggtcg 1680

cgtgccggca tggatgcgcg cgccatcgcg gtaggcgagc agcgcctgcc tgaagctgcg 1740

ggcattcccg atcagaaatg agcgccagtc gtcgtcggct ctcggcaccg aatgcgtatg 1800

attctccgcc agcatggctt cggccagtgc gtcgagcagc gcccgcttgt tcctgaagtg 1860

ccagtaaagc gccggctgct gaacccccaa ccgttccgcc agtttgcgtg tcgtcagacc 1920

gtctacgccg acctcgttca acaggtccag ggcggcacgg atcactgtat tcggctgcaa 1980

ctttgtcatg cttgacactt tatcactgat aaacataata tgtccaccaa cttatcagtg 2040

ataaagaatc cgcgcgttca atcggaccag cggaggctgg tccggaggcc agacgtgaaa 2100

cccaacatac ccctgatcgt aattctgagc actgtcgcgc tcgacgctgt cggcatcggc 2160

ctgattatgc cggtgctgcc gggcctcctg cgcgatctgg ttcactcgaa cgacgtcacc 2220

gcccactatg gcattctgct ggcgctgtat gcgttggtgc aatttgcctg cgcacctgtg 2280

ctgggcgcgc tgtcggatcg tttcgggcgg cggccaatct tgctcgtctc gctggccggc 2340

gccagatctg gggaaccctg tggttggcat gcacatacaa atggacgaac ggataaacct 2400

tttcacgccc ttttaaatat ccgattattc taataaacgc tcttttctct taggtttacc 2460

cgccaatata tcctgtcaaa cactgatagt ttaaactgaa ggcgggaaac gacaatctga 2520

tcatgagcgg agaattaagg gagtcacgtt atgacccccg ccgatgacgc gggacaagcc 2580

gttttacgtt tggaactgac agaaccgcaa cgttgaagga gccactcagc cgcgggtttc 2640

tggagtttaa tgagctaagc acatacgtca gaaaccatta ttgcgcgttc aaaagtcgcc 2700

taaggtcact atcagctagc aaatatttct tgtcaaaaat gctccactga cgttccataa 2760

attcccctcg gtatccaatt agagtctcat attcactctc aatccaaata atctgcaccg 2820

gatctggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt 2880

gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg 2940

ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg 3000

gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc acgacgggcg 3060

ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg 3120

gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca 3180

tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc 3240

accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc 3300

aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca 3360

aggcgcgcat gcccgacggc gatgatctcg tcgtgaccca tggcgatgcc tgcttgccga 3420

atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg 3480

cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg 3540

aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg 3600

ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga 3660

ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag 3720

gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct 3780

catgctggag ttcttcgccc acgggatctc tgcggaacag gcggtcgaag gtgccgatat 3840

cattacgaca gcaacggccg acaagcacaa cgccacgatc ctgagcgaca atatgatcgg 3900

gcccggcgtc cacatcaacg gcgtcggcgg cgactgccca ggcaagaccg agatgcaccg 3960

cgatatcttg ctgcgttcgg atattttcgt ggagttcccg ccacagaccc ggatgatccc 4020

cgatcgttca aacatttggc aataaagttt cttaagattg aatcctgttg ccggtcttgc 4080

gatgattatc atataatttc tgttgaatta cgttaagcat gtaataatta acatgtaatg 4140

catgacgtta tttatgagat gggtttttat gattagagtc ccgcaattat acatttaata 4200

cgcgatagaa aacaaaatat agcgcgcaaa ctaggataaa ttatcgcgcg cggtgtcatc 4260

tatgttacta gatcgggcct cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg 4320

gcggctctga gggtggtggc tctgagggtg gcggttctga gggtggcggc tctgagggag 4380

gcggttccgg tggtggctct ggttccggtg attttgatta tgaaaagatg gcaaacgcta 4440

ataagggggc tatgaccgaa aatgccgatg aaaacgcgct acagtctgac gctaaaggca 4500

aacttgattc tgtcgctact gattacggtg ctgctatcga tggtttcatt ggtgacgttt 4560

ccggccttgc taatggtaat ggtgctactg gtgattttgc tggctctaat tcccaaatgg 4620

ctcaagtcgg tgacggtgat aattcacctt taatgaataa tttccgtcaa tatttacctt 4680

ccctccctca atcggttgaa tgtcgccctt ttgtctttgg cccaatacgc aaaccgcctc 4740

tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag 4800

cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt 4860

tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca 4920

caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc cccagattag 4980

ccttttcaat ttcagaaaga atgctaaccc acagatggtt agagaggctt acgcagcagg 5040

tctcatcaag acgatctacc cgagcaataa tctccaggaa atcaaatacc ttcccaagaa 5100

ggttaaagat gcagtcaaaa gattcaggac taactgcatc aagaacacag agaaagatat 5160

atttctcaag atcagaagta ctattccagt atggacgatt caaggcttgc ttcacaaacc 5220

aaggcaagta atagagattg gagtctctaa aaaggtagtt cccactgaat caaaggccat 5280

ggagtcaaag attcaaatag aggacctaac agaactcgcc gtaaagactg gcgaacagtt 5340

catacagagt ctcttacgac tcaatgacaa gaagaaaatc ttcgtcaaca tggtggagca 5400

cgacacactt gtctactcca aaaatatcaa agatacagtc tcagaagacc aaagggcaat 5460

tgagactttt caacaaaggg taatatccgg aaacctcctc ggattccatt gcccagctat 5520

ctgtcacttt attgtgaaga tagtggaaaa ggaaggtggc tcctacaaat gccatcattg 5580

cgataaagga aaggccatcg ttgaagatgc ctctgccgac agtggtccca aagatggacc 5640

cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 5700

ggattgatgt gatatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca 5760

agacccttcc tctatataag gaagttcatt tcatttggag agaacacggg ggactctaga 5820

ggatccccgg gtggtcagtc ccttatgtta cgtcctgtag aaaccccaac ccgtgaaatc 5880

aaaaaactcg acggcctgtg ggcattcagt ctggatcgcg aaaactgtgg aattgatcag 5940

cgttggtggg aaagcgcgtt acaagaaagc cgggcaattg ctgtgccagg cagttttaac 6000

gatcagttcg ccgatgcaga tattcgtaat tatgcgggca acgtctggta tcagcgcgaa 6060

gtctttatac cgaaaggttg ggcaggccag cgtatcgtgc tgcgtttcga tgcggtcact 6120

cattacggca aagtgtgggt caataatcag gaagtgatgg agcatcaggg cggctatacg 6180

ccatttgaag ccgatgtcac gccgtatgtt attgccggga aaagtgtacg tatcaccgtt 6240

tgtgtgaaca acgaactgaa ctggcagact atcccgccgg gaatggtgat taccgacgaa 6300

aacggcaaga aaaagcagtc ttacttccat gatttcttta actatgccgg aatccatcgc 6360

agcgtaatgc tctacaccac gccgaacacc tgggtggacg atatcaccgt ggtgacgcat 6420

gtcgcgcaag actgtaacca cgcgtctgtt gactggcagg tggtggccaa tggtgatgtc 6480

agcgttgaac tgcgtgatgc ggatcaacag gtggttgcaa ctggacaagg cactagcggg 6540

actttgcaag tggtgaatcc gcacctctgg caaccgggtg aaggttatct ctatgaactg 6600

tgcgtcacag ccaaaagcca gacagagtgt gatatctacc cgcttcgcgt cggcatccgg 6660

tcagtggcag tgaagggcga acagttcctg attaaccaca aaccgttcta ctttactggc 6720

tttggtcgtc atgaagatgc ggacttgcgt ggcaaaggat tcgataacgt gctgatggtg 6780

cacgaccacg cattaatgga ctggattggg gccaactcct accgtacctc gcattaccct 6840

tacgctgaag agatgctcga ctgggcagat gaacatggca tcgtggtgat tgatgaaact 6900

gctgctgtcg gctttaacct ctctttaggc attggtttcg aagcgggcaa caagccgaaa 6960

gaactgtaca gcgaagaggc agtcaacggg gaaactcagc aagcgcactt acaggcgatt 7020

aaagagctga tagcgcgtga caaaaaccac ccaagcgtgg tgatgtggag tattgccaac 7080

gaaccggata cccgtccgca aggtgcacgg gaatatttcg cgccactggc ggaagcaacg 7140

cgtaaactcg acccgacgcg tccgatcacc tgcgtcaatg taatgttctg cgacgctcac 7200

accgatacca tcagcgatct ctttgatgtg ctgtgcctga accgttatta cggatggtat 7260

gtccaaagcg gcgatttgga aacggcagag aaggtactgg aaaaagaact tctggcctgg 7320

caggagaaac tgcatcagcc gattatcatc accgaatacg gcgtggatac gttagccggg 7380

ctgcactcaa tgtacaccga catgtggagt gaagagtatc agtgtgcatg gctggatatg 7440

tatcaccgcg tctttgatcg cgtcagcgcc gtcgtcggtg aacaggtatg gaatttcgcc 7500

gattttgcga cctcgcaagg catattgcgc gttggcggta acaagaaagg gatcttcact 7560

cgcgaccgca aaccgaagtc ggcggctttt ctgctgcaaa aacgctggac tggcatgaac 7620

ttcggtgaaa aaccgcagca gggaggcaaa caatgaatca acaactctcc tggcgcacca 7680

tcgtcggcta cagcctcggg aattgctacc gagctcgaat ttccccgatc gttcaaacat 7740

ttggcaataa agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata 7800

atttctgttg aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat 7860

gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa 7920

aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg 7980

ggaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc gttacccaac 8040

ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa gaggcccgca 8100

ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttc 8160

ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct 8220

ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga tttgggtgat 8280

ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc 8340

acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcgggc 8400

tattcttttg atttataagg gattttgccg atttcggaac caccatcaaa caggattttc 8460

gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga 8520

agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccacccca gtacattaaa 8580

aacgtccgca atgtgttatt aagttgtcta agcgtcaatt tgtttacacc acaatatatc 8640

ctgccaccag ccagccaaca gctccccgac cggcagctcg gcacaaaatc accactcgat 8700

acaggcagcc catcagtccg ggacggcgtc agcgggagag ccgttgtaag gcggcagact 8760

ttgctcatgt taccgatgct attcggaaga acggcaacta agctgccggg tttgaaacac 8820

ggatgatctc gcggagggta gcatgttgat tgtaacgatg acagagcgtt gctgcctgtg 8880

atcaaatatc atctccctcg cagagatccg aattatcagc cttcttattc atttctcgct 8940

taaccgtgac aggctgtcga tcttgagaac tatgccgaca taataggaaa tcgctggata 9000

aagccgctga ggaagctgag tggcgctatt tctttagaag tgaacgttga cgatatcaac 9060

tcccctatcc attgctcacc gaatggtaca ggtcggggac ccgaagttcc gactgtcggc 9120

ctgatgcatc cccggctgat cgaccccaga tctggggctg agaaagccca gtaaggaaac 9180

aactgtaggt tcgagtcgcg agatcccccg gaaccaaagg aagtaggtta aacccgctcc 9240

gatcaggccg agccacgcca ggccgagaac attggttcct gtaggcatcg ggattggcgg 9300

atcaaacact aaagctactg gaacgagcag aagtcctccg gccgccagtt gccaggcggt 9360

aaaggtgagc agaggcacgg gaggttgcca cttgcgggtc agcacggttc cgaacgccat 9420

ggaaaccgcc cccgccaggc ccgctgcgac gccgacagga tctagcgctg cgtttggtgt 9480

caacaccaac agcgccacgc ccgcagttcc gcaaatagcc cccaggaccg ccatcaatcg 9540

tatcgggcta cctagcagag cggcagagat gaacacgacc atcagcggct gcacagcgcc 9600

taccgtcgcc gcgaccccgc ccggcaggcg gtagaccgaa ataaacaaca agctccagaa 9660

tagcgaaata ttaagtgcgc cgaggatgaa gatgcgcatc caccagattc ccgttggaat 9720

ctgtcggacg atcatcacga gcaataaacc cgccggcaac gcccgcagca gcataccggc 9780

gacccctcgg cctcgctgtt cgggctccac gaaaacgccg gacagatgcg ccttgtgagc 9840

gtccttgggg ccgtcctcct gtttgaagac cgacagccca atgatctcgc cgtcgatgta 9900

ggcgccgaat gccacggcat ctcgcaaccg ttcagcgaac gcctccatgg gctttttctc 9960

ctcgtgctcg taaacggacc cgaacatctc tggagctttc ttcagggccg acaatcggat 10020

ctcgcggaaa tcctgcacgt cggccgctcc aagccgtcga atctgagcct taatcacaat 10080

tgtcaatttt aatcctctgt ttatcggcag ttcgtagagc gcgccgtgcg tcccgagcga 10140

tactgagcga agcaagtgcg tcgagcagtg cccgcttgtt cctgaaatgc cagtaaagcg 10200

ctggctgctg aacccccagc cggaactgac cccacaaggc cctagcgttt gcaatgcacc 10260

aggtcatcat tgacccaggc gtgttccacc aggccgctgc ctcgcaactc ttcgcaggct 10320

tcgccgacct gctcgcgcca cttcttcacg cgggtggaat ccgatccgca catgaggcgg 10380

aaggtttcca gcttgagcgg gtacggctcc cggtgcgagc tgaaatagtc gaacatccgt 10440

cgggccgtcg gcgacagctt gcggtacttc tcccatatga atttcgtgta gtggtcgcca 10500

gcaaacagca cgacgatttc ctcgtcgatc aggacctggc aacgggacgt tttcttgcca 10560

cggtccagga cgcggaagcg gtgcagcagc gacaccgatt ccaggtgccc aacgcggtcg 10620

gacgtgaagc ccatcgccgt cgcctgtagg cgcgacaggc attcctcggc cttcgtgtaa 10680

taccggccat tgatcgacca gcccaggtcc tggcaaagct cgtagaacgt gaaggtgatc 10740

ggctcgccga taggggtgcg cttcgcgtac tccaacacct gctgccacac cagttcgtca 10800

tcgtcggccc gcagctcgac gccggtgtag gtgatcttca cgtccttgtt gacgtggaaa 10860

atgaccttgt tttgcagcgc ctcgcgcggg attttcttgt tgcgcgtggt gaacagggca 10920

gagcgggccg tgtcgtttgg catcgctcgc atcgtgtccg gccacggcgc aatatcgaac 10980

aaggaaagct gcatttcctt gatctgctgc ttcgtgtgtt tcagcaacgc ggcctgcttg 11040

gcctcgctga cctgttttgc caggtcctcg ccggcggttt ttcgcttctt ggtcgtcata 11100

gttcctcgcg tgtcgatggt catcgacttc gccaaacctg ccgcctcctg ttcgagacga 11160

cgcgaacgct ccacggcggc cgatggcgcg ggcagggcag ggggagccag ttgcacgctg 11220

tcgcgctcga tcttggccgt agcttgctgg accatcgagc cgacggactg gaaggtttcg 11280

cggggcgcac gcatgacggt gcggcttgcg atggtttcgg catcctcggc ggaaaacccc 11340

gcgtcgatca gttcttgcct gtatgccttc cggtcaaacg tccgattcat tcaccctcct 11400

tgcgggattg ccccgactca cgccggggca atgtgccctt attcctgatt tgacccgcct 11460

ggtgccttgg tgtccagata atccacctta tcggcaatga agtcggtccc gtagaccgtc 11520

tggccgtcct tctcgtactt ggtattccga atcttgccct gcacgaatac cagcgacccc 11580

ttgcccaaat acttgccgtg ggcctcggcc tgagagccaa aacacttgat gcggaagaag 11640

tcggtgcgct cctgcttgtc gccggcatcg ttgcgccaca tctaggtact aaaacaattc 11700

atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca gtaagtcaaa 11760

aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga cgcagaaggc 11820

aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc cacttacttt 11880

gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga caagttcctc 11940

ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa atggagtgtc 12000

ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt aatccaattc 12060

ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga tggagtgaaa 12120

gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt catcttcata 12180

ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc agccatcatg 12240

ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata gcatcatgtc 12300

cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca tttttaaata 12360

taggttttca ttttctccca ccagcttata taccttagca ggagacattc cttccgtatc 12420

ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc tcattttagc 12480

catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga agctaattat 12540

aacaagacga actccaattc actgttcctt gcattctaaa accttaaata ccagaaaaca 12600

gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga gccgattttg 12660

aaaccacaat tatgggtgat gctgccaact tactgattta gtgtatgatg gtgtttttga 12720

ggtgctccag tggcttctgt gtctatcagc tgtccctcct gttcagctac tgacggggtg 12780

gtgcgtaacg gcaaaagcac cgccggacat cagcgctatc tctgctctca ctgccgtaaa 12840

acatggcaac tgcagttcac ttacaccgct tctcaacccg gtacgcacca gaaaatcatt 12900

gatatggcca tgaatggcgt tggatgccgg gcaacagccc gcattatggg cgttggcctc 12960

aacacgattt tacgtcactt aaaaaactca ggccgcagtc ggtaacctcg cgcatacagc 13020

cgggcagtga cgtcatcgtc tgcgcggaaa tggacgaaca gtggggctat gtcggggcta 13080

aatcgcgcca gcgctggctg ttttacgcgt atgacagtct ccggaagacg gttgttgcgc 13140

acgtattcgg tgaacgcact atggcgacgc tggggcgtct tatgagcctg ctgtcaccct 13200

ttgacgtggt gatatggatg acggatggct ggccgctgta tgaatcccgc ctgaagggaa 13260

agctgcacgt aatcagcaag cgatatacgc agcgaattga gcggcataac ctgaatctga 13320

ggcagcacct ggcacggctg ggacggaagt cgctgtcgtt ctcaaaatcg gtggagctgc 13380

atgacaaagt catcgggcat tatctgaaca taaaacacta tcaataagtt ggagtcatta 13440

cccaattatg atagaattta caagctataa ggttattgtc ctgggtttca agcattagtc 13500

catgcaagtt tttatgcttt gcccattcta tagatatatt gataagcgcg ctgcctatgc 13560

cttgccccct gaaatcctta catacggcga tatcttctat ataaaagata tattatctta 13620

tcagtattgt caatatattc aaggcaatct gcctcctcat cctcttcatc ctcttcgtct 13680

tggtagcttt ttaaatatgg cgcttcatag agtaattctg taaaggtcca attctcgttt 13740

tcatacctcg gtataatctt acctatcacc tcaaatggtt cgctgggttt atcgcacccc 13800

cgaacacgag cacggcaccc gcgaccacta tgccaagaat gcccaaggta aaaattgccg 13860

gccccgccat gaagtccgtg aatgccccga cggccgaagt gaagggcagg ccgccaccca 13920

ggccgccgcc ctcactgccc ggcacctggt cgctgaatgt cgatgccagc acctgcggca 13980

cgtcaatgct tccgggcgtc gcgctcgggc tgatcgccca tcccgttact gccccgatcc 14040

cggcaatggc aaggactgcc agcgctgcca tttttggggt gaggccgttc gcggccgagg 14100

ggcgcagccc ctggggggat gggaggcccg cgttagcggg ccgggagggt tcgagaaggg 14160

ggggcacccc ccttcggcgt gcgcggtcac gcgcacaggg cgcagccctg gttaaaaaca 14220

aggtttataa atattggttt aaaagcaggt taaaagacag gttagcggtg gccgaaaaac 14280

gggcggaaac ccttgcaaat gctggatttt ctgcctgtgg acagcccctc aaatgtcaat 14340

aggtgcgccc ctcatctgtc agcactctgc ccctcaagtg tcaaggatcg cgcccctcat 14400

ctgtcagtag tcgcgcccct caagtgtcaa taccgcaggg cacttatccc caggcttgtc 14460

cacatcatct gtgggaaact cgcgtaaaat caggcgtttt cgccgatttg cgaggctggc 14520

cagctccacg tcgccggccg aaatcgagcc tgcccctcat ctgtcaacgc cgcgccgggt 14580

gagtcggccc ctcaagtgtc aacgtccgcc cctcatctgt cagtgagggc caagttttcc 14640

gcgaggtatc cacaacgccg gcggccgcgg tgtctcgcac acggcttcga cggcgtttct 14700

ggcgcgtttg cagggccata gacggccgcc agcccagcgg cgagggcaac cagcccgg 14758

<210> 6

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

tgattacgcc aagcttctca ttggtaccac gacgagt 37

<210> 7

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gaccacccgg ggatccggat cagacatttt tgaaccca 38

<210> 8

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

ggactctaga ggatccatgg atcctgatga tgttgttga 39

<210> 9

<211> 44

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gatcggggaa attcgagctc ttagagcttt aaatctctgt aggt 44

<210> 10

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

ctcattggta ccacgacgag 20

<210> 11

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ttagagcttt aaatctctgt aggt 24

Claims (8)

1. A method for delaying the growth of lateral branches of tobacco after topping, which is characterized by comprising the following steps:

sowing, seedling raising and transplanting the transgenic tobacco, and topping; exogenous nucleic acid is inserted into the genome of the transgenic tobacco; the exogenous nucleic acid comprises an axillary bud specific promoter and a lethal gene driven by the axillary bud specific promoter;

the axillary bud-specific promoter is at least one of NtTF1 promoter, CEN-like protein 2 promoter and CEN-like protein 1 promoter;

the lethal gene is at least one selected from diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene.

2. The method of claim 1, wherein the diphtheria toxin A chain gene has a nucleotide sequence shown in SEQ ID No.1, the Aspergillus oryzae gene RNase-T1 has a nucleotide sequence shown in SEQ ID No.2, and the Barnase gene has a nucleotide sequence shown in SEQ ID No. 3.

3. The method according to claim 1, wherein the nucleotide sequence of the NtTF1 promoter is represented by SEQ ID No. 4.

4. The method of claim 1, wherein the exogenous nucleic acid is set forth in SEQ ID No. 5.

5. The method of claim 1, wherein the method further comprises: inserting the exogenous nucleic acid shown as SEQ ID NO.5 into the genome of tobacco to prepare transgenic tobacco.

6. A recombinant vector having inserted therein the exogenous nucleic acid of claim 1.

7. The recombinant vector of claim 6, wherein said exogenous nucleic acid comprises an axillary bud-specific promoter and a lethal gene driven by said axillary bud-specific promoter;

the lethal gene is at least one selected from diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene; the axillary bud-specific promoter is at least one selected from the group consisting of an NtTF1 promoter, a CEN-like protein 2 promoter and a CEN-like protein 1 promoter.

8. The recombinant vector according to claim 6 or 7, wherein the exogenous nucleic acid is as shown in SEQ ID No. 5.

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