CN115521936B

CN115521936B - Method and material for delaying growth of lateral branches of tobacco after topping

Info

Publication number: CN115521936B
Application number: CN202210314795.8A
Authority: CN
Inventors: 吕婧; 代常波; 孙玉合; 李冰洁; 王李先秋
Original assignee: Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
Current assignee: Qingzhou Tobacco Research Institute of China National Tobacco Corp of Institute of Tobacco Research of CAAS
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2024-05-03
Anticipated expiration: 2042-03-28
Also published as: CN115521936A

Abstract

The invention provides a method and a material for delaying growth of lateral branches of tobacco after topping, wherein the method comprises the following steps: sowing, raising seedlings and transplanting transgenic tobacco, and topping; the genome of the transgenic tobacco is inserted with exogenous nucleic acid; 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 NtTF (CEN-like protein 4) promoter, CEN-like protein 2 promoter and CEN-likeprotein 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 growth of lateral branches of tobacco after topping

Technical Field

The present disclosure relates to the field of biotechnology, and in particular, to a method for delaying growth of tobacco side shoots after topping and a recombinant vector.

Background

In tobacco production, topping is typically performed to concentrate the growth of the nutrient supply blades in order to increase the yield and quality of the tobacco leaf. Each 1 leaf axil of the tobacco plant after topping can regenerate 2-3 or more axillary buds. If any of them grow, a great deal of nutrients are consumed, and the nutrition growth of the main stem leaves is affected.

Tobacco is used as leaf crop, and a large amount of water is consumed by flowering and fruiting of tobacco, so that the yield and quality of tobacco leaves are reduced. 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 the flower buds or inflorescences at the top of the tobacco plants must be picked in a proper period, so that unnecessary consumption of nutrients in the tobacco plants is avoided, the nutrients are intensively supplied for leaf growth, and the leaf area and the single leaf weight are increased. However, after topping, axillary buds are clustered, nutrient is consumed, and the purpose of improving tobacco quality is not achieved.

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 smoke fork clusters and fireworks Man Tian, and is easy to infect diseases. At present, bud inhibitors are used in field production to inhibit axillary buds of tobacco plants after topping, and the use of the bud inhibitors not only increases production cost, but also causes environmental pollution, so that the cultivation of the axillary buds after topping can be effectively inhibited, and meanwhile, the pollution to the environment and tobacco leaves is avoided, and the application is safe, so that the method for reducing production cost and labor input is of great importance.

Disclosure of Invention

In order to improve the yield and quality of tobacco leaves, the present 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 of delaying lateral branch growth of tobacco after topping thereof, the method comprising the steps of:

Sowing, raising seedlings and transplanting transgenic tobacco, and topping; the genome of the transgenic tobacco is inserted with exogenous nucleic acid; 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 NtTF (CEN-like protein 4) 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.

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

According to the present disclosure, the nucleotide sequence of NtTF a promoter is shown as SEQ ID NO. 4.

According to the present disclosure, the exogenous nucleic acid is shown as SEQ ID NO. 5.

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

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

According to the present disclosure, the exogenous nucleic acid includes an axillary bud specific promoter and a lethal gene driven by the 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 NtTF (CEN-like protein 4) promoter, CEN-like protein 2 promoter and CEN-like protein 1 promoter.

Through the technical scheme, the method constructs the recombinant vector of the axillary bud specific promoter and the lethal gene, drives the expression of the lethal gene by using the axillary bud specific promoter, infects tobacco by the recombinant vector to obtain a positive plant, and compared with a control, the growth of the axillary bud of the tobacco at the first leaf position of the positive plant after topping is delayed by 1-2 weeks, so that the yield and 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 are included to provide a further understanding of the disclosure, and are incorporated in and constitute a part of this specification, illustrate the disclosure and together with the description serve to explain, but do not limit the disclosure. In the drawings:

FIG. 1 is a schematic diagram of NtTF promoter amplification (left) and P _NtTF1:DTA recombinant vector construction (right).

FIG. 2 shows the PCR detection results of the DTA transgenic seedlings of P _NtTF1.

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

Detailed Description

The following describes specific embodiments of the present disclosure in detail. It should be understood that the detailed description and specific examples, while indicating and illustrating the disclosure, are not intended to limit the disclosure.

The axillary bud specific promoter is at least one selected from NtTF promoter, CEN-like protein 2 promoter and CEN-like protein 1 promoter;

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

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

Optionally, the nucleotide sequence of the NtTF1 promoter is shown as SEQ ID NO. 4.

Alternatively, the exogenous nucleic acid is set forth in SEQ ID NO. 5.

Optionally, the method further comprises: an exogenous nucleic acid as shown in SEQ ID NO.5 was 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 upon insertion of the exogenous nucleic acid into the tobacco genome to form a transgenic tobacco plant, the axillary bud specific promoter initiates expression of the lethal gene, thereby allowing delayed growth of the tobacco axillary buds.

In another aspect, the present disclosure also provides a recombinant vector into which the above exogenous 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 selected from diphtheria toxin A chain gene, aspergillus oryzae gene RNase-T1 and Barnase gene; the axillary bud specific promoter includes at least one of NtTF (CEN-like protein 4) promoter, CEN-like protein 2 promoter and CEN-like protein 1 promoter.

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

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

Example 1

Materials: ntTF1 (CEN-like protein 4) upstream 246bp promoter sequence, PBI121 vector, lethal gene DTA and sequence thereof, tobacco variety;

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

The method comprises the following steps:

Vector construction procedure and genetic transformation procedure

Enzyme digestion identification of PBI121 vector

(1) In a 0.2mL centrifuge tube, the following components were mixed in sequence: 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)/HindIII (1. Mu.L)/BamHI (1. Mu.L), and a total of 50. Mu.L of reaction volume;

(2) After uniform mixing, slightly centrifuging, incubating for 3 hours at 37 ℃, then taking 5.0 mu L of enzyme digestion reaction liquid to carry out 1.0% agarose gel electrophoresis, carrying out electrophoresis for 20 minutes under the constant pressure condition of 2V/cm, observing under an ultraviolet lamp, and cutting and recycling;

The primer is designed according to the upstream 246bp promoter sequence of NtTF1, a homologous recombination method is adopted to construct the PBI121-P _NtTF1, a DTA recombinant vector is provided, and two enzyme cutting sites are designed 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, constructing a PBI121-P _NtTF1 by adopting a homologous recombination method, wherein the DTA recombinant vector is provided with 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 (10. Mu.L), 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 was 20. Mu.L; mixing the materials, pre-denaturing at 94 ℃ for 2min, and performing PCR reaction: the reaction parameters are denaturation at 94 ℃ for 20s, annealing at 60 ℃ for 20s, extension at 72 ℃ for 30s, continuous extension at 72 ℃ for 5min after 35 cycles, and preservation at 16 ℃;

recovery of PCR products: recovery of PCR amplification products was performed using TaKaRa MiniBEST Agarose Gel DNA Extraction Kit Ver.4.0, following the instructions:

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

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

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

(4) 12000rpm/min, centrifuging at 25 ℃ for 1min;

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

(6) 12000rpm/min, and centrifuging at 25 ℃ for 30s;

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

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

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

(10) Centrifuging at 25 ℃ for 1min at 12,000 rpm/min;

(11) Taking 1.0 mu l of the centrifugally collected recovery liquid, and measuring the concentration of the DNA fragments on a NanoDrop 2000c spectrophotometer;

Cloning of the fragment of interest: the PCR amplified fragment was ligated with the linear vector recovered by digestion according to the method provided by Takara Bio Inc.: to a 0.2mL sterile centrifuge tube was added: 2.0 mu L of 5 XIn-Fusion HD Enzyme Premix, 4.0 mu L of linear carrier, 1.0 mu L of PCR recovery product and 3.0 mu L of ddH ₂ O are added into the mixture, the mixture is fully mixed and centrifuged for a plurality of seconds, and the liquid drops on the pipe wall are received at the pipe bottom and reacted for 15 minutes at 50 ℃;

preparation of the competent cells of the coll DH5 a:

(1) E.coli DH5a single colonies were picked from LB agar plates and inoculated into 10mL of antibiotic-free LB liquid medium, followed by shaking culture at 37℃and 300rpm/min overnight. Transferring into fresh LB liquid medium according to 1% (V/V) amount in the next day, and shake culturing at 37deg.C to 0.3-0.4;

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

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

(4) Adding 10mL of pre-cooled 0.1mol/L CaCl2 solution into each centrifuge tube to resuspend thalli, and carrying out ice bath for 30min;

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

Conversion of ligation products:

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

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

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

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

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

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

(7) The plate is placed forward at 37 ℃ until the liquid is absorbed, then the plate is inverted, and the plate is cultivated for 12-16 hours at 37 ℃;

LB medium: yeast Extract 5g/L, peptone Tryptone 10g/L, naCl g/L, adding 1000mL distilled water for dissolution, adjusting pH to 7.0 with NaOH, and sterilizing at 121deg.C for 20min;

small extraction of plasmid DNA:

(1) White single colonies were picked from LB plates with a sterile gun head and inoculated into 5ml LB medium containing Amp (100 mg/ml), respectively;

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

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

(4) Adding 250 mu L BufferP to a centrifuge tube with bacterial sediment, and thoroughly suspending the bacterial sediment by using a vortex oscillator;

(5) Adding 250 mu L BufferP to the centrifuge tube, gently reversing and uniformly mixing for 4-6 times to fully crack the thalli;

(6) Adding 350 mu L BufferN to the centrifuge tube, immediately and gently mixing the materials upside down for 4 to 6 times;

(7) Centrifuging at 12000rpm/min for 10min, sucking supernatant, and adding into Spin Column CM loaded 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 of Buffer PW to the Spin Column CM, centrifuging at 12000rpm/min for 60s, pouring out waste liquid in the Collection Tube, and putting the Spin Column CM back into the Collection Tube;

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

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

(12) Placing Spin Column CM in a new centrifuge tube, suspending and dripping 50 μl Buffer EB into the middle part of the adsorption film, standing at room temperature for 2min, centrifuging at 12000rpm/min for 1min, collecting the solution in the centrifuge tube, and preserving plasmid at-20deg.C.

DNA sequencing and analysis: the DNA sequence of the selected positive clone is determined, and the sequencing is completed by Beijing Liuhua Dada;

Construction of a recombinant expression vector of the PBI121-P _NtTF1: and (3) enzyme cutting and recovery of target fragments: inserting NtTF1 promoter into a vector PBI121, wherein the enzyme cutting sites are HindIII and BamHI, inserting DTA into a PBI121-P _NtTF1:: GUS recombinant vector, and constructing an expression vector PBI121-P _NtTF1:: DTA with a DTA gene, wherein the enzyme cutting sites are BamHI and SacI, as shown in figure 1, the NtTF1 promoter is amplified (left) and the P _NtTF1:: DTA recombinant vector is constructed (right);

Identification of the recombinant expression vector of the DTA of PBI121-P _NtTF1: and (3) converting the connection product into escherichia coli, screening positive bacterial plaques by using PCR, and carrying out sequencing on the positive bacterial plaques, wherein the sequencing result is correct, and the construction is successful.

Agrobacteria competent preparation:

(1) Single colonies of Agrobacterium tumefaciens (EHA 105) were picked from YEP plates (containing 50. Mu.g/mL rifampicin) and inoculated into YEP liquid medium containing 50. Mu.g/mL rifampicin at 200rpm/min for about 36h at 28 ℃;

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

(5) Removing the supernatant, re-suspending the bacterial cells in 10mL of 0.15M NaCl solution in an ice bath, and centrifugally collecting the bacterial cells;

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

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

Transforming agrobacterium with recombinant vector:

(1) 50. Mu.L of Agrobacterium competent cells were inserted with 0.1-1. Mu.g (5-10. Mu.L) of plasmid DNA and then ice-bathed for 30min;

(2) Placing into liquid nitrogen for 1min, and immediately placing into a water bath kettle at 37 ℃ for water bath for 5min;

(3) Taking out the centrifuge tube, adding 0.5mL LB, and culturing at 28deg.C under shaking at 220rpm/min for 3-5 hr;

(4) Taking out bacterial liquid, coating the bacterial liquid on an LB plate containing kanamycin (100 mg/mL) and rifampicin (20 mug/mL), and inversely culturing the bacterial liquid in an incubator at 28 ℃ for about 2 days to form visible bacterial colonies;

And (3) PCR verification of agrobacterium monoclonal colony: single colonies were picked and inoculated into 2mL centrifuge tubes containing kanamycin (100 mg/mL) and rifampicin (20. Mu.g/mL), incubated at 200rpm/min,28℃for about 18h.

Agrobacterium mediates genetic transformation of tobacco:

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

preparation of agrobacterium infection solution: using the stock solution (80 ℃ C.), 100. Mu.L of the stock solution was added to the YEB solution (200 mL/bottle+100 mg/mL spectinomycin+20. Mu.g/mL rifampin using a triangular flask), and the mixture was cultured at 28℃for about 200rpm/min under dark conditions until OD=0.6 to 0.8. Centrifuging at 4000rpm/min for 5min, collecting bacterial liquid, diluting to OD 600=0.8 by using MS0, and then adding 20 μg/mL of acetosyringone;

YEB medium: each liter contains: 5g of Tryptone, 1g of yeast extract, 0.5g of magnesium sulfate, 5g of beef extract, 5g of sucrose and pH 7.0;

Genetic transformation process: cutting off 0.5cm aseptic leaves, immersing in the bacterial liquid for 5min, sucking with aseptic filter paper, spreading on a co-culture medium (G) after She Maichao, culturing in darkness at 22 ℃ for 3 days, inoculating the co-cultured leaves veins downwards on an S1 culture medium, placing in a artificial climate chamber, culturing at about 25 ℃ for 2-3 weeks until cluster buds grow on the edges of the leaves, allowing the bud length to be 0.1-0.5cm, transferring the cluster buds on the S1 culture medium to an S2 culture medium, culturing under light for 1-2 weeks, and allowing the cluster buds to grow into young seedlings. And (3) picking young seedlings on the S2 culture medium onto the S3 culture medium, and culturing under light for 1-2 weeks, wherein the young seedlings are gradually strong. And (3) removing the bottom expanded part and the lower yellowing leaves of the strong seedlings on the S3 culture medium, inoculating the seedlings on a rooting culture medium (R), culturing for 1-2 weeks under light, and transplanting the seedlings into a nutrition pot after rooting.

TABLE 1 Medium used in various steps of the tobacco genetic transformation Process

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

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

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

(2) Collecting the ground powder into a 1.5mL centrifuge tube, adding 400 mu L Buffer LP1 and 6 mu L RNase A (10 mg/mL), shaking for 1 min by vortex, and standing at room temperature for 10 min to allow the powder to be fully cracked;

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

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

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

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

(7) Repeating step (6);

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

(9) Placing the adsorption column into a new 1.5mL centrifuge tube, suspending and dripping 50 μl of sterilized water into the middle part of the adsorption film, standing at room temperature for 2-5 min, centrifuging at 12000rpm/min for 1 min, collecting DNA solution, and preserving DNA at-20deg.C;

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 transgenic T1 generation plants and control plants, and observing the growth of axillary buds within four weeks, as shown in figure 3; in fig. 3, f to j are control plants, a to e are transgenic positive plants, and it can be seen that axillary buds of the control plants grow normally, whereas axillary buds of the transgenic plants obtained by the present disclosure start to delay in the first week compared to the control plants, and axillary bud growth is consistent with the control plants from the second week.

Through the technical scheme, the axillary bud specific promoter is utilized to drive the lethal gene to express, the recombinant vector of the axillary bud specific promoter and the lethal gene is constructed, and the growth of the axillary buds of tobacco can be effectively delayed by utilizing the recombinant vector.

The preferred embodiments of the present disclosure have been described in detail above, but the present disclosure is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solutions of the present disclosure within the scope of the technical concept of the present disclosure, and all the simple modifications belong to the protection scope of the present disclosure.

In addition, the specific features described in the foregoing embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, the present disclosure does not further describe various possible combinations.

Moreover, any combination between the various embodiments of the present disclosure is possible as long as it does not depart from the spirit of the present disclosure, which should also be construed as the disclosure of the present disclosure.

Sequence listing

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<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

1. A method for retarding the growth of tobacco side shoots after topping, said method comprising the steps of:

the axillary bud specific promoter is NtTF promoter;

the lethal gene is diphtheria toxin A chain gene;

the nucleotide sequence of the diphtheria toxin A chain gene is shown as SEQ ID NO. 1;

The nucleotide sequence of NtTF promoter is shown as SEQ ID NO. 4.

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

3. A recombinant vector for use in delaying growth of tobacco side shoots following topping, said recombinant vector being inserted with an exogenous nucleic acid according to claim 1.

4. The recombinant vector according to claim 3, wherein the exogenous nucleic acid is as shown in SEQ ID NO. 5.