CN113774070B - Method and material for inhibiting growth of lateral branches of tobacco after topping - Google Patents

Abstract

The invention provides a method for inhibiting growth of lateral branches of tobacco after topping, which comprises the following steps: s1, 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 includes a chemically inducible promoter and a lethal gene; s2, applying a chemical inducer on axillary buds of the transgenic tobacco after topping. In another aspect, the invention provides a diphtheria toxin and a gene encoding the diphtheria toxin. According to the technical scheme, the method disclosed by the invention is used for constructing the recombinant vector of the inducible promoter and the lethal gene, the promoter in the inducible expression system is used for driving the lethal gene to express, the recombinant vector is used for infecting tobacco to obtain a positive plant, the axillary buds of the positive plant after topping are subjected to chemical smearing treatment, and the growth of the treated tobacco axillary buds is inhibited compared with that of a control, so that the yield and quality of tobacco leaves are improved. Meanwhile, the use of the conventional chemical bud inhibitor is reduced, and the production cost and the environmental pollution are reduced.

Description

Method and material for inhibiting growth of lateral branches of tobacco after topping

Technical Field

The present disclosure relates to the field of bioengineering technology, and in particular, to a method for inhibiting growth of tobacco side branches after topping, a diphtheria toxin and a diphtheria toxin gene.

Background

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 tobacco 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 and material for inhibiting the growth of lateral branches of tobacco leaves after topping.

In one aspect, the invention provides a method for inhibiting growth of lateral branches of tobacco after topping, comprising the steps of:

s1, 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 includes a chemically inducible promoter and a lethal gene;

s2, applying a chemical inducer on axillary buds of the transgenic tobacco after topping.

On the other hand, the invention also provides a diphtheria toxin, wherein the diphtheria toxin is a diphtheria toxin with an improved amino acid sequence, and the amino acid sequence of the diphtheria toxin with the improved amino acid sequence is shown as SEQ ID NO. 2.

In still another aspect, the present invention also provides a diphtheria toxin gene, wherein the diphtheria toxin gene is a diphtheria toxin gene modified by coding amino acid sequences subjected to codon optimization, and the nucleotide sequence is shown in SEQ ID NO.1.

According to the technical scheme, the method disclosed by the invention is used for constructing the recombinant vector of the inducible promoter and the lethal gene, the promoter in the inducible expression system is used for driving the lethal gene to express, the recombinant vector is used for infecting tobacco to obtain a positive plant, the axillary buds of the positive plant after topping are subjected to chemical smearing treatment, and the growth of the treated tobacco axillary buds is inhibited compared with that of a control, so that the yield and quality of tobacco leaves 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 an electrophoresis image in the process of constructing a vector, wherein the left image PTA7002-DTA is converted into DH5A bacteria P (left) +enzyme digestion (right), and the right image PTA7002-DTA is converted into EHA105 bacteria P;

FIG. 2 is a positive assay for PTA7002-DTA transgenic seedlings;

FIG. 3 is CK (left) PTA7002-DTA (right) 12 days after the first leaf axillary buds of transgenic T1 generation plants and control are coated with 50. Mu.M DEX lanolin after topping;

FIG. 4 shows lanolin (left), lanolin+50. Mu.M DEX (right) after 5 days of application of different treatments of lanolin to axillary buds at the first leaf position after topping of transgenic T1-generation plants;

FIG. 5 shows lanolin +50μm DEX (left), lanolin (right) after 7 days of application of different treatment lanolin to axillary buds at the first leaf position after topping of transgenic T2-generation plants and control plants;

FIG. 6 shows the first leaf axillary buds (left), second leaf axillary buds (middle), third leaf axillary buds (right) 7 days after 50. Mu.M DEX lanolin is applied to the first leaf axillary buds after topping of transgenic T2-generation plants;

FIG. 7 shows the first leaf axillary buds (left), second leaf axillary buds (middle), third leaf axillary buds (right) after 50. Mu.M DEX lanolin is applied to the first leaf axillary buds after topping of transgenic T2-generation plants for 14 days;

FIG. 8 shows the first leaf axillary buds (left), second leaf axillary buds (middle), third leaf axillary buds (right) after the transgenic T2 generation plants are topped and smeared with 50. Mu.M DEX lanolin for 21 days;

FIG. 9 shows the first leaf axillary buds (left), second leaf axillary buds (middle) and third leaf axillary buds (right) after 50. Mu.M DEX lanolin is applied to the first leaf axillary buds 30 days after topping of transgenic T2 plants.

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.

In one aspect, the invention provides a method for inhibiting growth of lateral branches of tobacco after topping, comprising the steps of:

s1, 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 includes a chemically inducible promoter and a lethal gene; s2, applying a chemical inducer on axillary buds of the transgenic tobacco after topping.

Alternatively, wherein the lethal gene is the gene of the amino acid sequence engineered diphtheria toxin a chain; the amino acid sequence of the diphtheria toxin with the modified amino acid sequence is shown in SEQ ID NO. 2; the nucleotide sequence of the gene of the diphtheria toxin A chain modified by the coding amino acid sequence subjected to the codon optimization is shown as SEQ ID NO.1.

Wherein the amino acid sequence modified diphtheria toxin can stop the growth of axillary buds of the transgenic tobacco when a chemical inducer is applied, and does not influence the growth of other organs and tissues of the tobacco at other positions where the chemical inducer is not applied. The amino acid sequence of the diphtheria toxin is modified by changing the amino acid at the 2-3 positions from DP in the wild type to GA and deleting the amino acid at the 195-218 positions compared with the wild type diphtheria toxin.

Optionally, wherein the chemically inducible promoter is a dexamethasone inducible promoter, an ethanol inducible promoter, a tetracycline inducible promoter, a steroid inducible promoter; the inducer comprises at least one of dexamethasone, ethanol, tetracycline, and a steroid.

Optionally, the nucleotide sequence of the dexamethasone inducible promoter is shown in SEQ ID NO. 3; the nucleotide sequence of the tetracycline-inducible promoter is shown as SEQ ID NO. 4.

Alternatively, wherein the exogenous nucleic acid may be obtained by ligating the nucleotide sequence of the chemically inducible 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 chemically inducible promoter initiates expression of the lethal gene under the influence of the chemical inducer applied to the axillary buds, thereby stopping growth of the tobacco axillary buds, preferably the exogenous nucleic acid is as shown in SEQ ID NO. 5.

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

In one aspect, the invention also provides a diphtheria toxin gene, wherein the diphtheria toxin gene is a diphtheria toxin A chain gene modified by an amino acid sequence, and the nucleotide sequence of the diphtheria toxin A chain gene modified by the amino acid sequence is shown as SEQ ID NO.1.

On the other hand, the invention also provides a diphtheria toxin, wherein the diphtheria toxin is a diphtheria toxin with an improved amino acid sequence, and the amino acid sequence of the diphtheria toxin with the improved amino acid sequence is shown as SEQ ID NO. 2.

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

Example 1

The coding sequence of the diphtheria toxin A chain with optimized codons is shown as SEQ ID NO.1, and is synthesized by using a gene complete sequence synthesis mode; primer design: according to CDS sequence design of DTA, two enzyme cutting sites are designed for the convenience of constructing a vector: xhoI and SpeI

And (3) PCR amplification: the following ingredients were added to a 0.2mL centrifuge tube: 2X Phanta Max Master Mix (10 μl), ddH ₂ O (7.4. Mu.l), F (10 mM)) (0.8. Mu.l), R (10 mM) (0.8. Mu.l), CDS plasmid template (1.0. Mu.l); the total volume of the reaction was 20. Mu.l; mixing the materials, pre-denaturing at 94 ℃ for 3min, and performing PCR reaction: the reaction parameters are denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, continuous extension at 72 ℃ for 5min after 35 cycles, and preservation at 16 ℃; the total volume of the components is 20 mu l, after being uniformly mixed, the components are pre-denatured for 3min at 94 ℃, and then PCR reaction is carried out: the reaction parameters are denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, 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) 10 μl of the PCR amplification reaction solution was subjected to 1.0% agarose gel electrophoresis, electrophoresis was performed for 20min under a constant voltage of 2V/cm, agarose blocks containing the target fragment were cut off under an ultraviolet lamp, and transferred to 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 μl Buffer WB into a Spin Column;

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

(7) Repeating 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 μl 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 pUC19-T Vertor according to the method provided by Takara Bio Inc.: to a 0.5mL sterile centrifuge tube was added: 2.0. Mu.l 10 XT 4DNA ligase Buffer, 1.0. Mu.l pUC19-T Vector DNA, 4.0. Mu.l PCR recovery product, 1.0. Mu. l T4DNA ligase (3U/. Mu.l), 2.0. Mu.l ddH ₂ O, adding 10.0 mu l of total volume, fully and uniformly mixing, centrifuging for several seconds, dripping the pipe wall liquid into the pipe bottom, and carrying out metal bath at 16 ℃ for 16-18h;

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/CaCl2 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 1 competent cells by using a sterile suction head, placing the competent cells into a 1.5mL precooled sterile centrifuge tube, adding 10 mu 1 connecting 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 60s;

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

(4) Adding 500 mu 1 LB liquid culture medium without antibiotics, mixing, shaking culturing at 37deg.C at 300rpm/min for 60min;

(5) Preparation of LB-plus 100mg/mL ampicillin solid plates;

(6) Sucking 100 mu 1 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) 250 μl of BufferP1 was added to the centrifuge tube with the bacterial pellet left, and the bacterial pellet was thoroughly suspended using a vortex shaker;

(5) Adding 250 μl of BufferP2 into the centrifuge tube, gently mixing up and down for 4-6 times, and fully lysing the thalli;

(6) Adding 350 μl of BufferN3 into the centrifuge tube, and immediately and gently mixing the mixture upside down for 4-6 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 μl Buffer PW to Spin Column CM, centrifuging at 12000rpm/min for 60s, pouring out waste liquid in the Collection Tube, and putting 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.

Enzyme digestion identification of vectors

(1) In a 0.5mL centrifuge tube, the following components were mixed in sequence: 10 XBuffer M (5.0. Mu.1), plasmid DNA (5.0. Mu.1), ddH2O (38. Mu.l), xhol (1. Mu.l), speI (1. Mu.l), a total of 50. Mu.l reaction volume;

(2) After being evenly mixed, the mixture is slightly centrifuged, incubated for 3 hours at 37 ℃, then 5.0 mu l of enzyme digestion reaction liquid is taken for electrophoresis of 1.0% agarose gel, electrophoresis is carried out for 20 minutes under the constant pressure condition of 2V/cm, and then observation and gel cutting recovery are carried out under an ultraviolet lamp;

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 PTA7002-DTA plant expression vector.

Construction of PTA7002-DTA plant expression vector: and (3) enzyme cutting and recovery of target fragments: cutting DTA from the cloning vector by using Xhol and SpeI double enzyme cutting, inserting the DTA into a plant expression vector PTA7002 subjected to the same enzyme cutting, and constructing a plant expression vector PTA7002-DTA with a DTA gene; the enzyme digestion reaction system and the step, and the recovery step of enzyme digestion products are the same as the above;

ligation, T4DNA ligase from Takara Shuzo, was performed as follows: DTA fragment 1 mu 1, PTA7002 linear fragment 4 mu 1, T4DNA ligase 1 mu 1, 10 XBuffer 2 mu 1, ddH2O 2 mu 1, total 10 mu 1, after fully mixing, centrifuging for several seconds, dropping the tube wall liquid to the tube bottom, and water-bathing at 16 ℃ for 16-18h;

identification of PTA7002-DTA plant expression vector: transforming the ligation product into escherichia coli, screening positive bacterial plaques by a PCR method, and carrying out enzyme digestion identification by double enzyme digestion of Xhol and SpeI, wherein the results of the figures 1 and 2 show that the vector insert is correct in size; and meanwhile, the positive bacterial plaque is sent to be tested, and the sequencing result is correct, so that 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 CaCl2 solution, and 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: each liter contains: 10g of beef extract and 10g,NaCl 5g,pH7.0 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 0.5 mM LLB, 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 spectinomycin (100 mg/mL) and rifampicin (20 mug/mL), and inversely culturing the bacterial liquid in an incubator at 28 ℃ for about 2 days to obtain a bacterial colony;

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

PCR reaction system: 2X Phanta Max Master Mix. Mu.l, ddH2O 7.4. Mu.l, F (10 mM) 0.8. Mu.l, R (10 mM) 0.8. Mu.l, 1.0. Mu.l of a monoclonal bacterial liquid, 20. Mu.l in total, were mixed and then subjected to a PCR reaction after pre-denaturation at 94℃for 3 min: the reaction parameters are denaturation at 94 ℃ for 15s, annealing at 60 ℃ for 15s, extension at 72 ℃ for 30s, continuous extension at 72 ℃ for 5min after 35 cycles, and preservation at 16 ℃; sucking 10ul of PCR amplification reaction liquid to carry out 1.0% agarose gel electrophoresis, and carrying out electrophoresis for 20min under the constant voltage condition of 2V/cm, and photographing by a gel imager;

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, dibbling the seeds on MS culture medium, transferring to a plant illumination culture room for culture, and taking leaves for infection after one month;

preparation of agrobacterium infection solution: using the stock solution (-80 ℃) stored, 100. Mu.l was added to YEB solution (using a triangular flask, 200 mL/flask+100 mg/mL spectinomycin+20. Mu.g/mL rifampin), and cultured at 28℃at 200rpm/min under dark conditions for about 18 hours to OD=0.6-0.8. 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 7.0 of PH7;

genetic transformation process: cutting off 0.5cm aseptic leaves, immersing in the bacterial liquid for 5min, drying by using aseptic filter paper, spreading on a co-culture medium, culturing in darkness at 22 ℃ for 3 days, inoculating the co-cultured leaf veins downwards on an S1 culture medium, placing in a climatic chamber, culturing at 25 ℃ for 2-3 weeks until cluster buds grow on the edges of the leaves, allowing the buds to grow at 0.1-0.5cm, transferring the cluster buds on the S1 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 onto an 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, inoculating the seedlings on a rooting culture medium, culturing for 1-2 weeks under light, and transplanting the seedlings into a nutrition pot after rooting. The extracted DNA of each transgenic seedling was subjected to PCR detection, and the results are shown in Table 1.

TABLE 1

Transgenic plant positive detection, inducer DEX application experiment and surface form observation:

DNA extraction of transgenic plants:

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

(2) The ground powder was collected in a 1.5mL centrifuge tube, 400. Mu.l Buffer LP1 and 6. Mu.l RNase A (10 mg/mL) were added, vortexed for 1 minute, and left at room temperature for 10 minutes to allow full lysis;

(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 1min, pouring out the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe again;

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

(7) Repeating the 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 1min, collecting DNA solution, and preserving DNA at-20deg.C;

transgenic plant positive verification PCR primer:

PTA7002-F：5’-TTGCATGCCGGTCGACTCTA-3’

DTA-R：5’-GACTAGTTTATCTTCTAACTCTATTTCCAGCAC-3’

PCR reaction system: 2X Phanta Max Master Mix. Mu.l, ddH2O 7.4. Mu.l, F (10 mM) 0.8. Mu.l, R (10 mM) 0.8. Mu.l, DNA template 1.0. Mu.l, 20. Mu.l in total, were mixed and then pre-denatured at 94℃for 3min, followed by PCR reaction: the reaction parameters are 94 ℃, 15s denaturation, 15s annealing at 58 ℃,30 s extension at 72 ℃, 5min extension at 72 ℃ after 35 cycles, and 16 ℃ preservation; sucking 10 μl of PCR amplification reaction solution, performing 1.0% agarose gel electrophoresis, and performing electrophoresis under constant voltage of 2V/cm for 20min, and photographing by a gel imager;

inducer application experiments of transgenic positive plants and control plants:

to the melted lanolin was added 50. Mu.M of inducer DEX and cooled after the lanolin solidified. Topping transgenic T1 generation plants and control plants, immediately smearing lanolin containing DEX on axillary buds of the first leaf position of the topped plants, and observing the growth condition of the axillary buds, as shown in figure 3; the left graph is a control plant, the right graph is a transgenic positive plant, and the axillary bud growth of the control plant is normal, while the axillary bud growth of the transgenic plant is completely inhibited.

Topping transgenic T1 generation plants, immediately applying lanolin without DEX to axillary buds at the first leaf position of the plant after topping, comparing the lanolin with DEX to axillary buds at the first leaf position of the plant after topping, and observing the growth condition of the axillary buds, wherein the axillary buds of the transgenic plants are completely inhibited under the induction of DEX, as shown in figure 4, and the axillary buds of the transgenic plants grow normally under the condition that DEX is not applied.

Topping the transgenic T2 generation plant, immediately applying lanolin without DEX to axillary buds at the first leaf position of the plant after topping, comparing with the axillary buds at the first leaf position of the plant after topping, and observing the growth condition of the axillary buds, as shown in figure 5. The left panel of FIG. 5 shows a control, namely, transgenic negative seedlings (without DEX-induced promoter and diphtheria toxin gene inserted), after topping, the first leaf axillary buds will also grow out when applied with lanolin axillary buds containing DEX, and the right positive seedlings (with DEX-induced promoter and diphtheria toxin gene inserted) will still grow uninhibited when applied with pure lanolin (without DEX) after topping.

Topping transgenic T2 generation plants, and immediately applying lanolin containing DEX on axillary buds of the first leaf position, the second leaf position and the third leaf position of the topped plants, and observing axillary bud growth after 7 days, 14 days, 21 days and 30 days, as shown in figures 6, 7, 8 and 9. FIGS. 6 to 9 show that after the first leaf axillary buds are coated with DEX lanolin after topping of positive seedlings, the growth of the first leaf axillary buds is 1-3 in 1 month, and it can be seen that the treated first leaf axillary buds have no great change, i.e. the growth is inhibited, while the second and third leaf axillary buds below grow normally because the DEX-containing lanolin is not coated, thereby proving that the DEX-containing lanolin has an effect on the inhibition of the axillary buds of positive seedlings after topping.

Comparative example 1

A cultivation method for inhibiting growth of tobacco side branches after topping was provided by the same procedure as in example 1, except that the lethal gene used in the test was a gene encoding diphtheria toxin A chain which was not codon optimized and amino acid modified, the nucleotide sequence of the gene encoding diphtheria toxin A chain which was not amino acid modified was shown in SEQ ID NO.6, and the amino acid sequence was shown in SEQ ID NO. 7.

Constructing a recombinant vector by using the diphtheria toxin A chain gene which is not subjected to amino acid modification and an inducible promoter, acting on axillary buds of a first leaf position of a plant after topping, and observing the growth condition of the axillary buds. The result shows that the gene of diphtheria toxin A chain which is not subjected to amino acid modification is inserted into a vector to infect tobacco, and a transgenic plant cannot be obtained.

The inventor of the present disclosure has found through experiments that, when the genes encoding pseudomonas aeruginosa exotoxin, ricin A chain, aspergillus oryzae RNase-T1 and Bacillus amyloliquefaciens Barnase are combined with inducible promoters to construct recombinant vectors to infect tobacco, the growth of tobacco cells is inhibited without applying chemical inducer, so that transgenic plants cannot be obtained.

The sequences referred to in this application are as follows: SEQ ID NO.1 (nucleotide sequence of the gene of diphtheria toxin A chain); SEQ ID NO.2 (amino acid sequence of the amino acid sequence engineered diphtheria toxin A chain); SEQ ID NO.3 (nucleotide sequence of dexamethasone inducible promoter); SEQ ID NO.4 (nucleotide sequence of tetracycline-inducible promoter); SEQ ID NO.5 (foreign nucleic acid); SEQ ID NO.6 (nucleotide sequence of unmodified diphtheria toxin gene); SEQ ID NO.7 (unmodified diphtheria toxin amino acid sequence).

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

<120> method and Material for inhibiting growth of tobacco side branches after topping

<130> 17048CAAS-TRI-DCB

<160> 7

<170> SIPOSequenceListing 1.0

<210> 1

<211> 585

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 1

atgggagctg atgatgttgt tgattcttct aagtcttttg ttatggaaaa tttttcttct 60

tatcatggaa ctaagccagg atatgttgat tctattcaaa agggaattca aaagccaaag 120

tctggaactc aaggaaatta tgatgatgat tggaagggat tttattctac tgataataag 180

tatgatgctg ctggatattc tgttgataat gaaaatccac tttctggaaa ggctggagga 240

gttgttaagg ttacttatcc aggacttact aaggttcttg ctcttaaggt tgataatgct 300

gaaactatta agaaggaact tggactttct cttactgaac cacttatgga acaagttgga 360

actgaagaat ttattaagag atttggagat ggagcttcta gagttgttct ttctcttcca 420

tttgctgaag gatcttcttc tgttgaatat attaataatt gggaacaagc taaggctctt 480

tctgttgaac ttgaaattaa ttttgaaact agaggaaaga gaggacaaga tgctatgtat 540

gaatatatgg ctcaagcttg tgctggaaat agagttagaa gataa 585

<210> 2

<211> 194

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 2

Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu

1 5 10 15

Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile

20 25 30

Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp

35 40 45

Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala

50 55 60

Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys

85 90 95

Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr

100 105 110

Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe

115 120 125

Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly

130 135 140

Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

145 150 155 160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln

165 170 175

Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val

180 185 190

Arg Arg

<210> 3

<211> 3310

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60

agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120

gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180

cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240

ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300

caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat 360

gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420

atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480

cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540

ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600

tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660

atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720

tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg 780

cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840

ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900

gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960

tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gggatcttgg 1020

aggtgatgta acatgatcac aagctgatcc cccgaatttc cccgatcgtt caaacatttg 1080

gcaataaagt ttcttaagat tgaatcctgt tgccggtctt gcgatgatta tcatataatt 1140

tctgttgaat tacgttaagc atgtaataat taacatgtaa tgcatgacgt tatttatgag 1200

atgggttttt atgattagag tcccgcaatt atacatttaa tacgcgatag aaaacaaaat 1260

atagcgcgca aactaggata aattatcgcg cgcggtgtca tctatgttac tagatcggga 1320

attgatcccc cctcgacagc ttgcatgccg gtcgactcta gaggatccgg gtgacagccc 1380

tccgacgggt gacagccctc cgacgggtga cagccctccg aattctagag gatccgggtg 1440

acagccctcc gacgggtgac agccctccga cgggtgacag ccctccgaat tcgagctcgg 1500

tacccgggga tctgtcgacc tcgatcgaga tcttcgcaag acccttcctc tatataagga 1560

agttcatttc atttggagag gacacgctga agctagtcga ctctagcctc gagcggccgc 1620

cagtgtgatg gatatctgca gaattcggct tggatccagg atccaagccg aattccagca 1680

cactggcggc cgttactagt cgatccaggc ctcccagctt tcgtccgtat catcggtttc 1740

gacaacgttc gtcaagttca atgcatcagt ttcattgccc acacaccaga atcctactaa 1800

gtttgagtat tatggcattg gaaaagctgt tttcttctat catttgttct gcttgtaatt 1860

tactgtgttc tttcagtttt tgttttcgga catcaaaatg caaatggatg gataagagtt 1920

aataaatgat atggtccttt tgttcattct caaattatta ttatctgttg tttttacttt 1980

aatgggttga atttaagtaa gaaaggaact aacagtgtga tattaaggtg caatgttaga 2040

catataaaac agtctttcac ctctctttgg ttatgtcttg aattggtttg tttcttcact 2100

tatctgtgta atcaagttta ctatgagtct atgatcaagt aattatgcaa tcaagttaag 2160

tacagtatag gctttttgtg tcgagggggt accgagtcga ggaattcact ggccgtcgtt 2220

ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat 2280

ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag 2340

ttgcgcagcc tgaatggcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt 2400

cgccggcttt ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc 2460

tttacggcac ctcgacccca aaaaacttga tttgggtgat ggttcacgta gtgggccatc 2520

gccctgatag acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact 2580

cttgttccaa actggaacaa cactcaaccc tatctcgggc tattcttttg atttataagg 2640

gattttgccg atttcggaac caccatcaaa caggattttc gcctgctggg gcaaaccagc 2700

gtggaccgct tgctgcaact ctctcagggc caggcggtga agggcaatca gctgttgccc 2760

gtctcactgg tgaaaagaaa aaccacccca gtacattaaa aacgtccgca atgtgttatt 2820

aagttgtcta agcgtcaatt tgtttacacc acaatatatc ctgccaccag ccagccaaca 2880

gctccccgac cggcagctcg gcacaaaatc accactcgat acaggcagcc catcagtccg 2940

ggacggcgtc agcgggagag ccgttgtaag gcggcagact ttgctcatgt taccgatgct 3000

aattcggaag aacggcaact aagctgccgg ggtttgaaac acggatgatt ctcgcggagg 3060

gtagcatgtg attgtaacga tgacaggcgt tgctgcctgt gatcaaatat catcctccct 3120

ccgcagagag aatccgaatt atcagccttc ttattcattt ctcccgctta aaccggtgga 3180

acagcttttc gaatcttgag aactaatgcc gaacataata ggaaattcgc cttgataagg 3240

tcggctgaag gaagcctgat ggcctatttc ttagaaaggt gaaccgttgg accgaatatc 3300

aaatcttccc 3310

<210> 4

<211> 3480

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

tggaagggct aattcactcc caaagaagac aagatatcct tgatctgtgg atctaccaca 60

cacaaggcta cttccctgat tagcagaact acacaccagg gccaggggtc agatatccac 120

tgacctttgg atggtgctac aagctagtac cagttgagcc agataaggta gaagaggcca 180

ataaaggaga gaacaccagc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg 240

agagagaagt gttagagtgg aggtttgaca gccgcctagc atttcatcac gtggcccgag 300

agctgcatcc ggagtacttc aagaactgct gatatcgagc ttgctacaag ggactttccg 360

ctggggactt tccagggagg cgtggcctgg gcgggactgg ggagtggcga gccctcagat 420

cctgcatata agcagctgct ttttgcctgt actgggtctc tctggttaga ccagatctga 480

gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct 540

tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc 600

agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag 660

cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg 720

caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga 780

aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg 840

aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg 900

caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct 960

gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat 1020

cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca 1080

ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc 1140

aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag 1200

tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc 1260

aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg 1320

gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc 1380

cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc 1440

gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct 1500

ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa 1560

actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca 1620

gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt 1680

aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt 1740

ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta 1800

tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt 1860

actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct 1920

cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga 1980

cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgccttt aaaagaaaag 2040

gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca acagacatac 2100

aaactaaaga actacaaaaa caaattacaa aaattcaaaa ttttcgggtt tattacaggg 2160

acagcagaga tccagtttat cgacttaact tgtttattgc agcttataat ggttacaaat 2220

aaggcaatag catcacaaat ttcacaaata aggcattttt ttcactgcat tctagttttg 2280

gtttgtccaa actcatcaat gtatcttatc atgtctggat ctcaaatccc tcggaagctg 2340

cgcctgtctt aggttggagt gatacatttt tatcactttt acccgtcttt ggattaggca 2400

gtagctctga cggccctcct gtcttaggtt agtgaaaaat gtcactctct tacccgtcat 2460

tggctgtcca gcttagctcg caggggaggt ggtctggatc cgccggcacc ggtgtatacg 2520

ggaattcttt acgagggtag gaagtggtac ggaaagttgg tataagacaa aagtgttgtg 2580

gaattgaagt ttactcaaaa aatcagcact cttttatagg cgccctggtt tacataagca 2640

aagcttatac gttctctatc actgataggg agtaaactgg atatacgttc tctatcactg 2700

atagggagta aactgtagat acgttctcta tcactgatag ggagtaaact ggtcatacgt 2760

tctctatcac tgatagggag taaactcctt atacgttctc tatcactgat agggagtaaa 2820

gtctgcatac gttctctatc actgataggg agtaaactct tcatacgttc tctatcactg 2880

atagggagta aactcgaggt gataattcca cggggttggg gttgcgcctt ttccaaggca 2940

gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg 3000

ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg 3060

ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct 3120

tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg 3180

cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata 3240

gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg 3300

ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc 3360

ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc 3420

agggggatca tcgaattacc atgtctagac tggacaagag caaagtcata aactctgctc 3480

<210> 5

<211> 801

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

atattcccca aacaataccc aattataaac tttaccacag cgggtgccac tgtgcaaagc 60

tacacaaact ttatcagagc tgttcgcggt cgtttaacaa ctggagctga tgtgagacat 120

gaaataccag tgttgccaaa cagagttggt ttgcctataa accaacggtt tattttagtt 180

gaactctcaa atcatgcaga gctttctgtt acattagcgc tggatgtcac caatgcatat 240

gtggtcggct accgtgctgg aaatagcgca tatttctttc atcctgacaa tcaggaagat 300

gcagaagcaa tcactcatct tttcactgat gttcaaaatc gatatacatt cgcctttgga 360

ggtaattatg atagacttga acaacttgct ggtaatctga gagaaaatat cgagttggga 420

aatggtccac tagaggaggc tatctcagcg ctttattatt acagtactgg tggcactcag 480

cttccaactc tggctcgttc ctttataatt tgcatccaaa tgatttcaga agcagcaaga 540

ttccaatata ttgagggaga aatgcgcacg agaattaggt acaaccggag atctgcacca 600

gatcctagcg taattacact tgagaatagt tgggggagac tttccactgc aattcaagag 660

tctaaccaag gagcctttgc tagtccaatt caactgcaaa gacgtaatgg ttccaaattc 720

agtgtgtacg atgtgagtat attaatccct atcatagctc tcatggtgta tagatgcgca 780

cctccaccat cgtcacagtt t 801

<210> 6

<211> 657

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

atggatcctg atgatgttgt tgattcttct aaatcttttg tgatggaaaa cttttcttcg 60

taccacggga ctaaacctgg ttatgtagat tccattcaaa aaggtataca aaagccaaaa 120

tctggtacac aaggaaatta tgacgatgat tggaaagggt tttatagtac cgacaataaa 180

tacgacgctg cgggatactc tgtagataat gaaaacccgc tctctggaaa agctggaggc 240

gtggtcaaag tgacgtatcc aggactgacg aaggttctcg cactaaaagt ggataatgcc 300

gaaactatta agaaagagtt aggtttaagt ctcactgaac cgttgatgga gcaagtcgga 360

acggaagagt ttatcaaaag gttcggtgat ggtgcttcgc gtgtagtgct cagccttccc 420

ttcgctgagg ggagttctag cgttgaatat attaataact gggaacaggc gaaagcgtta 480

agcgtagaac ttgagattaa ttttgaaacc cgtggaaaac gtggccaaga tgcgatgtat 540

gagtatatgg ctcaagcctg tgcaggaaat cgtgtcaggc gatctctttg tgaaggaacc 600

ttacttctgt ggtgtgacat aattggacaa actacctaca gagatttaaa gctctaa 657

<210> 7

<211> 218

<212> PRT

<213> Artificial sequence (Artificial Sequence)

<400> 7

Met Asp Pro Asp Asp Val Val Asp Ser Ser Lys Ser Phe Val Met Glu

1 5 10 15

Asn Phe Ser Ser Tyr His Gly Thr Lys Pro Gly Tyr Val Asp Ser Ile

20 25 30

Gln Lys Gly Ile Gln Lys Pro Lys Ser Gly Thr Gln Gly Asn Tyr Asp

35 40 45

Asp Asp Trp Lys Gly Phe Tyr Ser Thr Asp Asn Lys Tyr Asp Ala Ala

50 55 60

Gly Tyr Ser Val Asp Asn Glu Asn Pro Leu Ser Gly Lys Ala Gly Gly

65 70 75 80

Val Val Lys Val Thr Tyr Pro Gly Leu Thr Lys Val Leu Ala Leu Lys

85 90 95

Val Asp Asn Ala Glu Thr Ile Lys Lys Glu Leu Gly Leu Ser Leu Thr

100 105 110

Glu Pro Leu Met Glu Gln Val Gly Thr Glu Glu Phe Ile Lys Arg Phe

115 120 125

Gly Asp Gly Ala Ser Arg Val Val Leu Ser Leu Pro Phe Ala Glu Gly

130 135 140

Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Gln Ala Lys Ala Leu

145 150 155 160

Ser Val Glu Leu Glu Ile Asn Phe Glu Thr Arg Gly Lys Arg Gly Gln

165 170 175

Asp Ala Met Tyr Glu Tyr Met Ala Gln Ala Cys Ala Gly Asn Arg Val

180 185 190

Arg Arg Ser Leu Cys Glu Gly Thr Leu Leu Leu Trp Cys Asp Ile Ile

195 200 205

Gly Gln Thr Thr Tyr Arg Asp Leu Lys Leu

210 215

Claims (4)

1. A method for inhibiting growth of lateral branches of tobacco after topping, said method comprising the steps of:

s1, 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 includes a chemically inducible promoter and a lethal gene;

s2, applying a chemical inducer on axillary buds of the transgenic tobacco after topping;

the lethal gene is a diphtheria toxin A chain gene modified by an amino acid sequence; the amino acid sequence of the diphtheria toxin A chain modified by the amino acid sequence is shown as SEQ ID NO. 2;

the genes of the diphtheria toxin A chain modified by the amino acid sequence are subjected to codon optimization;

the inducible promoter is a dexamethasone inducible promoter; the inducer is dexamethasone;

the nucleotide sequence of the dexamethasone inducible promoter is shown as SEQ ID NO. 3.

2. The method of claim 1, wherein the nucleotide sequence of the gene for the amino acid sequence engineered diphtheria toxin a chain is set forth in SEQ ID No.1.

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

4. The method as recited in claim 1, wherein 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.

Images