Tobacco NtEXT1 gene and application thereof in plant growth regulation

Abstract

The invention belongs to the technical field of tobacco genome analysis, and particularly relates to a tobacco NtEXT1 gene and application thereof in plant growth regulation. The CDS sequence of the tobacco NtEXT1 gene is as shown in SEQ ID No.1. The tobacco NtEXT1 gene is applied to multiple aspects of plant drought resistance, plant type regulation and control, root length regulation, salt resistance, cadmium resistance and the like. On the basis that the tobacco NtEXT1 gene is obtained through cloning, a preliminary experiment result shows that under the condition of overexpression of the gene, overexpression plants obviously improve the drought tolerance of tobacco seedlings by reducing stomatal conductance and transpiration rate of tobacco leaves. Based on the result, a certain technical foundation can be laid for tobacco drought resistance mechanism analysis and new variety cultivation of drought-resistant tobaccos.

Description

Technical Field

[0001] The present application belongs to the technical field of tobacco genome analysis, and specifically relates to a tobacco NtEXT1 gene and its application in plant growth regulation. Background Art

[0002] tobacco( Nicotiana tabacum L.) is mainly harvested for its leaves and is one of the most important economic crops not only in my country but also in the world. However, in recent years, due to factors such as climate change and extreme weather, seasonal drought has had a serious impact on tobacco planting, yield and quality during tobacco cultivation.

[0003] As one of the main environmental stresses in the process of plant growth, drought has a comprehensive impact on plant respiration, photosynthesis, stomatal movement, plant growth and other physiological aspects. When plants are subjected to drought stress, the stomata on the leaves will close, resulting in an increase in the intercellular carbon dioxide concentration, thereby reducing photosynthesis and transpiration, which ultimately affects plant growth and development. During the normal growth of tobacco, well-developed tobacco leaves have a higher water content and better leaf opening. As the main harvest object, tobacco leaves are extremely sensitive to the adverse environmental condition of drought. When facing a drought environment, not only will the water content of the leaves drop directly, but also indicators such as leaf size, weight, and content composition will be greatly affected. Therefore, a full study of the drought-resistant physiological mechanism and related regulatory genes in tobacco is of great technical significance for stabilizing tobacco quality and breeding new drought-resistant tobacco varieties. Summary of the invention

[0004] The purpose of this application is to provide a tobacco NtEXT1 gene and its application in plant growth regulation, thereby laying a certain foundation for the breeding of new varieties of related plants.

[0005] The specific technical solutions adopted in this application are as follows.

[0006] The tobacco NtEXT1 gene has a CDS of 1092 bp in length and a nucleotide sequence as shown in SEQ ID No. 1; specifically, it is as follows: The EXT1 protein encoded by the tobacco NtEXT1 gene contains 363 amino acid residues, has a molecular weight of 41.27 KDa, and an isoelectric point of 9.72; the specific amino acid sequence is as follows: MKSSGKLGQWSLIAVALAICLAASTVVADYSYGYASPLPSYNSKKHYKSPSLPKYPVLTPYYKSHAPSKNYYISPAPAKYYKFPTLAKYYKSPIPTKYYKSPAPTKYYKSPAPTKYYKSPAPAKYYKSPVPTKYYKSPAPAKYYKSPAPAKYNKSPVPAKYYKSPAPAKYYKSPSPVKYYKS SSPAKYYKSPSPTKYYKSHAPSKYYKSPSPIKYYKSPVYYKSPPPPAYYEKSPSYYKSPPPPPKYYEQSSYYKSPPPPPKYYEQSPSSYKSPPPPYYKESTPSYKSPPPPTKYYEQLLTYFSPPPPATYEKSPSYYSPPPPTNYYKQTPTYASPPPPEKYEQSATYASPPPPSASPPPTYY*.

[0007] The primer pair for preparing the tobacco NtEXT1 gene by PCR amplification, the specific primer pair sequence is designed as follows: NtEXT1- CDS-F: 5'-ATGAAAAGCTCAGGAAAGCTGGGGC-3', NtEXT1- CDS-R: 5'-TTAGTAGTAGGTTGGTGGAGGAGATGCTGA-3'.

[0008] The tobacco NtEXT1 gene is used in plant drought resistance. After the tobacco NtEXT1 gene is overexpressed, the drought resistance of the plant is improved by increasing the fresh weight, dry weight and water content of the aboveground and underground parts.

[0009] The tobacco NtEXT1 gene is used in plant drought resistance. After the tobacco NtEXT1 gene is overexpressed, the plant's drought resistance is improved by reducing the transpiration rate and the stomatal conductance index.

[0010] The tobacco NtEXT1 gene is used for regulating plant type, and after overexpressing the tobacco NtEXT1 gene, it is used to extend plant stem nodes.

[0011] The application of the tobacco NtEXT1 gene in regulating plant roots is to increase the length of plant roots after overexpressing the tobacco NtEXT1 gene.

[0012] The tobacco NtEXT1 gene is used in plant stress resistance regulation, and the gene is also related to the salt resistance and cadmium resistance functions of the plant.

[0013] The tobacco NtEXT1 gene is used in plant variety breeding to improve the drought resistance of plant varieties by overexpressing the NtEXT1 gene.

[0014] The method for breeding plant varieties using the tobacco NtEXT1 gene, wherein the plant is, for example, tobacco, and during breeding, the drought-resistant plant variety is obtained by overexpressing the NtEXT1 gene.

[0015] In this application, the inventor cloned a tobacco NtEXT1 gene. Preliminary experimental results showed that the gene was related to tobacco drought resistance. When the gene was overexpressed, the overexpressed plants significantly improved the drought resistance of tobacco seedlings by reducing the stomatal conductance and transpiration rate of tobacco leaves. Based on this result, a certain technical foundation can be laid for the analysis of tobacco drought resistance mechanism and the breeding of new drought-resistant tobacco varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The phenotypic differences between the NtEXT1 overexpression line and the wild type under control (upper panel A) and drought (lower panel B) conditions; Figure 2 The effect of drought stress on leaf number, stem node length and root length of NtEXT1 overexpression lines and wild type. Con.:Control, normal growth conditions; PEG: PEG treatment, drought stress conditions; Figure 3 The effect of drought stress on the growth of NtEXT1 overexpression lines and wild type; in the figure: (a) is the fresh weight (FW) of stem (aboveground part), (b) is the fresh weight (FW) of root (underground part); (c) is the dry weight (DW) of stem (aboveground part), (d) is the dry weight (DW) of root (underground part), (e) is the relative water content (RWC) of stem (aboveground part), and (f) is the relative water content (RWC) of root (underground part); Figure 4 The results of drought stress on the apparent transpiration rate and stomatal conductance of NtEXT1 overexpression lines and wild-type tobacco leaves; in the figure: (a) is the apparent transpiration rate; (b) is the stomatal conductance result; Figure 5The effect of drought stress on the superoxide anion and hydrogen peroxide content of NtEXT1 overexpression strain and wild type; in the figure: (a) is the MDA measurement result, (b) is the SOD measurement result, (c) is the CAT measurement result, and (d) is the POD measurement result; Figure 6 The effect of drought stress on the content of different hormones in NtEXT1 overexpression lines and wild type; in the figure: (a) is the result of hydrogen peroxide content, (b) is the result of superoxide anion; Figure 7 The effect of drought stress on the content of different hormones in NtEXT1 overexpression strains and wild type; in the figure: a is auxin IAA; b is cytokinin 2MeScZR; c is abscisic acid ABA; d is jasmonic acid 12-OH-JA; e is salicylic acid SA; f is gibberellin hormone GA20; Figure 8 The results of drought stress on the chemical composition of tobacco leaves of NtEXT1 overexpression strains and wild-type tobacco, in which: (a) is the result of total alkaloids, (b) is the result of total nitrogen, (c) is the result of reducing sugars, (d) is the result of total sugars, (e) is the result of potassium, (f) is the result of chlorine, (g) is the result of sugar-alkali ratio, (h) is the result of nitrogen-alkali ratio, and (i) is the result of potassium-chlorine ratio; Fig. 9 The expression changes of NtEXT1 gene under control, salt stress and cadmium stress were shown in the case of overexpression. DETAILED DESCRIPTION

[0017] The present application is further explained below in conjunction with the embodiments. Before introducing the specific embodiments, some experimental backgrounds in the following embodiments are briefly introduced as follows.

[0018] Biomaterials: Tobacco K326, a common and commonly used material in existing tobacco cultivation and research, can be obtained from public channels; The relevant primer synthesis and sequencing were provided by Wuhan Aidijing Biotechnology Co., Ltd.

[0019] Example 1 In the early stage of the research on whether exogenous selenium can alleviate the effect of drought on tobacco growth under drought stress, the inventor screened and obtained a gene with significant changes in expression under drought conditions based on the results of tobacco miRNA transcriptome sequencing analysis. NtEXT1Gene (this gene is a member of the Extensin protein family involved in cell wall changes), but whether this gene only responds to drought changes (can be used as a drought indicator gene) or has actual drought resistance function (functional gene) still needs further in-depth research. Therefore, in order to further clarify the actual function and application prospects of this gene, the inventors further cloned this gene and conducted relevant functional verification analysis. This example only briefly introduces the cloning process of this gene as follows.

[0020] 1. Extraction of total RNA and reverse transcription into cDNA Take K326 leaves as samples (three-leaf seedling leaves), extract total RNA and reverse transcribe into single-stranded cDNA. For related operations, refer to the instructions of the relevant kit, or refer to the following for details.

[0021] When extracting total RNA (using the RNA Rapid Extraction Kit, a product of EasyStud, product number DR0409050): Take 50 mg of leaf samples, add liquid nitrogen and grind into powder. After grinding, transfer to an RNase-free centrifuge tube (2 mL); Add 600 μL Buffer PR1(NT), vortex for 1 min, and let stand for 5 min (invert and mix twice during this period to fully lyse the plant tissue); After standing, centrifuge at room temperature and 12,000 rpm for 5 min. After centrifugation, transfer 400 μL of supernatant to a new RNase-free centrifuge tube, add an equal volume of anhydrous ethanol, mix well, and transfer all the solution to the RNA adsorption column placed in the collection tube (2 mL specification), and centrifuge at room temperature and 12,000 rpm for 30 s. Put the adsorption column back into the collection tube, add 500 μL Buffer PR2(NT) to the inner column, centrifuge at room temperature and 12000 rpm for 30 seconds, and discard the waste liquid (repeat washing once to ensure clean washing); Put the adsorption column back into the collection tube again, centrifuge at room temperature and 12000 rpm for 1 min, transfer the adsorption column to a clean RNase-free centrifuge tube (15 mL specification), add 30-60 μL RNA Elution Buffer, leave it at room temperature for 1 min, and centrifuge it at room temperature and 12000 rpm for 30 s. The eluate is the recovered solution containing the extracted total RNA.

[0022] When preparing cDNA by reverse transcription (using PrimeScript™ II 1st Strand cDNA Synthesis Kit, Takara product, catalog number: 6210A): First, prepare the reaction mixture according to the following ratio: Oligo dT Primer, 1 μL (50 μM); dNTP Mixture, 1 μL (10 mM each); Total RNA, 5 μg; RNase Free dH2O, add to 10 μL; After keeping at 65°C for 5 min, cool rapidly on ice; Subsequently, a reverse transcription reaction system was prepared based on the above reaction mixture: The above reaction mixture, 10 μL; 5×PrimeScript II Buffer, 4 μL; RNase Inhibitor, 0.5 μL (40 U / μl); PrimeScript II RTase (200 U / μl), 1 μL; RNase Free dH2O, add to 20 μL; After slowly mixing, react at 42°C for 60 min, keep at 95°C for 5 min, and then immediately cool on ice (the prepared result is cDNA).

[0023] 2. Primer design and PCR amplification Referring to the existing tobacco genome, the primer pairs for PCR amplification were designed as follows: NtEXT1- CDS-F: 5'-actagggtctcgcaccATGAAAAGCTCAGGAAAGCTGGGGC-3', NtEXT1- CDS-R: 5'-actagggtctctaccgTTAGTAGTAGGTTGGTGGAGGAGATGCTGA-3'; Subsequently, the cDNA prepared in step (i) was used as a template, KOD ONE high-fidelity enzyme (TOYOBO product) was used, and PCR amplification was performed using the above primers; the reference design of the 50 μl amplification system is as follows: KOD ONE, 25 μL; NtEXT1 -CDS-F, 1.5 μL; NtEXT1 -CDS-R, 1.5 μL; cDNA template, 2 μL; ddH2O, 20 μL; The PCR amplification program was as follows: 94°C, 2 min; 98°C, 10 sec, 56°C, 5 sec, 68°C, 5 sec, 30 cycles; 68°C, 5 min; after completion of amplification, the amplified product was detected by electrophoresis.

[0024] 3. Gene sequencing analysis After electrophoresis detection of the PCR amplification product in step (ii), the target band is recovered and purified, connected to the pMD18-T vector, transformed into E. coli DH5α, and the positive single clone is selected for sequencing. For related operations, refer to the relevant product manual or the conventional operation of existing molecular biology technology, or refer to the following.

[0025] When recovering and purifying the target band of the amplified product (using the Easy Gel Extraction & Clean-up Kit, a product of EasyStudy, catalog number: DR0101250): Cut out the gel block containing the target DNA band, place it in a centrifuge tube (1.5 mL), add 400 μL Buffer A1, and keep it at 65°C for 10 min (shake it every 2-3 min until the gel block is completely dissolved); The above solution was pipetted into a HiPure DNA adsorption column (placed in a 2 mL collection tube), centrifuged at room temperature and 12,000 rpm for 30 seconds, and the waste liquid was discarded; Place the adsorption column back into the collection tube, add 500 μL Buffer A2, centrifuge at room temperature and 12,000 rpm for 30 seconds, and discard the waste liquid (you can wash it again); Place the adsorption column back into the collection tube, centrifuge at 12,000 rpm at room temperature for 1 min, transfer the adsorption column into a centrifuge tube, add 30-50 μL of Elution 2.0, leave at room temperature for 2 min, and centrifuge at 12,000 rpm at room temperature for 1 min. The eluate is the solution containing the PCR amplification product (target DNA) after recovery and purification.

[0026] After the above-recovered and purified PCR amplification product (target DNA) was connected to the pMD18-T vector (product of Takara), the heat shock method was used to transform the E. coli DH5α competent cells. The specific transformation operation can be referred to as follows: Dissolve DH5α competent cells on ice in advance, add the target DNA (i.e., NtEXT1 gene) and 10 μL of pMD18-T vector ligation solution, mix well, place on ice for 5 min, heat shock at 42°C for 45 s, and then quickly place on ice for 2 min; Subsequently, 700 μL of LB liquid medium without antibiotics was added to the above transformation solution, mixed, and evenly spread on LB solid plate medium, and cultured inverted at 37°C overnight; After the culture was completed, a single clone was picked and inoculated into LB liquid culture medium (containing ampicillin antibiotics). After shaking on a shaker at 37°C and 200 rpm for 4 h, the bacterial solution was identified by PCR. The positive single clone identified correctly was further sequenced and analyzed to obtain NtEXT1 The actual sequence information of the gene.

[0027] The results of sequencing analysis showed that NtEXT1 The full length of the gene CDS is 1092bp (the sequence is shown in SEQ ID No. 1), encoding a total of 363 amino acids. The protein has a molecular weight of 41.27 KDa and an isoelectric point of 9.72.

[0028] Example 2 Based on the successful cloning of the EXT1 gene in Example 1, the inventors constructed a related recombinant overexpression plasmid vector to facilitate further research and clarification of the gene function. The specific construction process is briefly described as follows.

[0029] 1. Amplification of target gene NtEXT1 Gene Referring to Example 1, PCR amplification preparation NtEXT1 Gene; 2. Enzyme digestion-ligation reaction pEGOEP35S-H was used as the basic backbone vector, and it was linearized by Bsa I-HF restriction enzyme digestion. The linearized empty plasmid was ligated with T4 DNA ligase. NtEXT1 Genes are connected; The specific reference design of the 15μl enzyme digestion-ligation system is as follows: 10×CutSmart Buffer, 1.5μl; 10 mM ATP, 1.5 μl; pEGOEP35S-H empty plasmid, 100 ng; Target fragment ( NtEXT1 gene), 50 ng; Bsa I-HF, 10 U; T4 DNA ligase, 35 U; H2O, add to 15 μl; The reaction conditions were as follows (the golden gate ligation and recombination method in a one-step operation): first enzyme digestion at 37°C for 5 min; then ligation at 20°C for 5 min; 15 cycles.

[0030] 3. Plasmid transformation, screening and identification Referring to the operating instructions of the above-mentioned Example 1, the above-mentioned ligation system was transformed into DH5a Escherichia coli competent cells using the heat shock method, and further resistance (Kana resistance) screening and bacterial liquid PCR detection were performed; When testing bacterial liquid PCR, the 20μl amplification system is as follows: 2XTaq MIX, 10 μL; 35S-F primer, 0.5 μL; NOS-R primer, 0.5 μL; Bacterial liquid, appropriate amount; ddH2O, add to 20 μL; The reaction program was: 95°C, 5 min; 95°C, 30 sec, 55°C, 30 sec, 72°C, 90 sec, 25 cycles; 72°C, 5 min; The above 35S-F and NOS-R primer sequences are as follows: 35S-F:5'-CACGGGGGACTCTTGCCACC-3', NOS-R: 5'-ATCATCGCAAGACCGGCAAC-3'; The target band length is 1190 bp; The correct recombinant plasmid was identified by PCR of the bacterial solution, and the plasmid was further extracted and sequenced (Sanger sequencing) to ensure that the recombinant overexpression plasmid vector was constructed correctly.

[0031] When extracting plasmid, the specific operations can be referred to as follows: The identified single clone containing the correct recombinant plasmid was inoculated into LB liquid medium (50 μg / mL of kanamycin) and cultured at 37°C overnight; Take 4 mL of culture solution, centrifuge at room temperature and 10,000 rpm for 2 min, discard the supernatant, add 250 μL of Solution Ⅰ reagent (containing RNase A) to resuspend the bacterial pellet, add 250 μL of Solution Ⅱ reagent, gently invert several times until the bacteria are transparent (fully lysed); then add 350 μL of Solution Ⅲ reagent, invert several times until white compact flocs are formed; centrifuge at room temperature and 12,000 rpm for 10 min, and keep the supernatant; Add the above supernatant to the nucleic acid purification column (placed in the collection tube), centrifuge at room temperature and 12000 rpm for 1 min, and discard the filtrate; Take 500 μL of Buffer W1 and add it to the nucleic acid purification column. Centrifuge at room temperature and 12000 rpm for 30 seconds and discard the filtrate. Take 700 μL of Buffer W2 and add it to the nucleic acid purification column. Centrifuge at room temperature and 12000 rpm for 30 s, and discard the filtrate (this operation can be repeated once); Place the nucleic acid purification column in a collection tube, centrifuge at room temperature and 12000 rpm for 2 min, remove the residual liquid, transfer the nucleic acid purification column to an EP tube (1.5mL specification), add 50 μL of elution buffer for elution, add the elution buffer, let stand at room temperature for 2 min, centrifuge at room temperature and 12000 rpm for 2 min, and the remaining liquid is the recombinant plasmid solution (can be stored at -40℃ for future use) Example 3 Based on the overexpression recombinant plasmid vector constructed in Example 2, the inventors conducted further transformation and phenotypic verification. The relevant experimental process and results are briefly described as follows.

[0032] 1. Preparation of infection solution Take the recombinant overexpression plasmid extracted in Example 2, refer to the above operation instructions and conventional operations in the prior art, use the heat shock method to transform Agrobacterium, and further use YEB medium containing 50 mg / L Kan to screen the correct Agrobacterium strain for transformation; After amplification of the correctly transformed Agrobacterium strain (cultured at 30°C, 150 r / min), OD600 was adjusted to about 0.30 using AAM medium (product of Coolbo Company) containing 20 mg / L acetosyringone.

[0033] 2. Infection, transformation and screening Cut the leaves of K326 tobacco plants that have grown to the 3-4 leaf stage and have grown to 2-3 cm (about 4 weeks) into leaf discs of about 0.5 cm × 0.5 cm under sterile conditions using a scalpel and place them in the Agrobacterium suspension prepared in step (i) for infection for 15 minutes; After the immersion, the liquid on the leaf surface was dried with filter paper, and then the leaves were transferred to MS medium containing 0.5 mg / L NAA, 2.0 mg / L 6-BA and 20 mg / L acetosyringone and cultured in the dark for 48 hours. After dark culture, the cells were transferred to induction medium (MS medium containing 0.5 mg / L NAA, 2.0 mg / L 6-BA, 100 mg / L timentin, and 20 mg / L hygromycin) and cultured for about 10 days at 25°C, 50% humidity, and a photoperiod of 16 h light / 8 h dark (color temperature 4000K) to induce callus formation; The induced callus was further transferred to a differentiation medium (MS medium containing 0.5 mg / L NAA, 2.0 mg / L 6-BA, 100 mg / L timentin, and 30 mg / L hygromycin) and cultured for about 30 days (the medium was changed every 15 days) at 25°C, 50% humidity, and a photoperiod of 16 h light / 8 h dark (color temperature 4000K) to induce differentiation into tobacco seedlings; Finally, when green shoots grow out of the callus tissue (they begin to appear about 1 month after the leaf disc is placed in the callus differentiation medium), cut off the green shoots (1-2 cm high) and transfer the differentiated tobacco seedlings to the rooting medium (MS medium containing 100 mg / L timentin, 20 mg / L hygromycin and 100 mg / L Kan) and induce rooting under the culture conditions of 25°C, 50% humidity and a photoperiod of 16 h light / 8 h dark (color temperature 4000K).

[0034] 3. Simulated drought experiment Based on the screening in step (ii) above, the inventor successfully screened and obtained two NtEXT1 Based on the overexpression lines L6 and L7 (the lines were further cultured and the seeds were purified), the tobacco variety K326 (wild type, WT) was used as a control. NtEXT1 Further experimental studies were conducted on tobacco growth and stress (drought) resistance under overexpression conditions. The specific experimental results are briefly described as follows.

[0035] Experimental process: After the tobacco seeds are sterilized, they are placed in an artificial climate chamber for seed germination. After the tobacco leaves gradually take shape at the small cross stage, they are transferred to plug trays for soil culture. After about one month of cultivation, evenly growing and healthy tobacco seedlings are selected and transplanted to a hydroponic box (20L basic nutrient solution, pH 5.8) for further cultivation. Six days after transplanting, the treatment group was treated with 20% PEG (simulating drought) for 5 days. The experiment was designed randomly, with 3 plants as one replicate, and repeated three times. After the phenotypic changes were obvious, samples were collected for analysis of various indicators.

[0036] Based on the above experimental process, the specific experimental results are briefly introduced as follows.

[0037] (1) Comparison of growth phenotypes Intuitive growth phenotype comparison Figure 1 As shown in Figure 2, further measurement results of some growth indicators are shown in Figure 2. Figure 2 (number of leaves, length of stem nodes, length of roots), Figure 3 (fresh weight, dry weight, relative water content). Analysis shows that: Under normal growth conditions, the growth of overexpression lines L6 and L7 was not significantly different from that of the wild type; however, after 5 days of 20% PEG treatment (simulated drought), although there was no significant difference in the number of leaves, the length of the stem nodes increased by 38.10% and 19.05%, and the root length increased by 24.00% and 41.33%, respectively; that is, NtEXT1 Overexpression can affect phenotypic changes such as stem node length (affecting transpiration potential) and root length; In addition, after 5 days of 20% PEG treatment (simulated drought), the drought tolerance of the overexpression strain was significantly improved compared with the wild type, specifically: the aboveground fresh weight of L6 and L7 increased significantly by 39.09% and 43.98%, the aboveground dry weight increased significantly by 22.35% and 23.77%, the aboveground water content increased significantly by 2.57% and 2.80%, the underground fresh weight increased significantly by 56.26% and 84.35%, the underground dry weight increased significantly by 64.81% and 80.99%, and the underground water content increased significantly by 0.54% and 0.65%.

[0038] This result indicates that overexpression NtEXT1 The gene helps increase the water content in tobacco plants when faced with PEG treatment, thereby enhancing the plants' drought resistance.

[0039] (2) Transpiration rate and stomatal conductance Since stomatal conductance and transpiration rate are often directly related to the drought resistance of plants, the inventors further measured the stomatal conductance and transpiration rate of plants under different treatments (measured using a LI-600 measuring instrument, refer to its instructions for operation).

[0040] The results of transpiration rate and stomatal conductance are shown in Figure 4 The analysis showed that compared with the wild type, the apparent transpiration rate of the overexpression lines L6 and L7 was significantly reduced by 27.81% and 29.14%, and the stomatal conductance was significantly reduced by 28.52% and 25.16%.

[0041] This result indicates that when faced with drought stress, the NtEXT1 overexpressing strain improves the drought tolerance of tobacco plants by reducing the stomatal conductance and transpiration rate of tobacco leaves.

[0042] (3) Related physiological indicators Since various physiological agents and enzyme activities of plants will also undergo significant changes when facing adversity, the inventors used the underground parts of tobacco treated with 20% PEG for 5 days as samples to further determine metabolites and enzyme activities such as MDA (malondialdehyde), POD (peroxidase), SOD (superoxide dismutase), and CAT (catalase).

[0043] The results are as follows Figure 5 , Figure 6 As shown. It can be seen that compared with the wild type, the malondialdehyde (MDA) content of L6 and L7 was significantly reduced by 45.92% and 46.16%, the hydrogen peroxide content was significantly reduced by 59.40% and 60.13%, the superoxide anion production rate was significantly reduced by 59.78% and 57.17%, and the hydrogen peroxide content was significantly reduced by 50.6% and 51.4%. Compared with the wild type, the SOD activity of L6 and L7 increased significantly by 119.55% and 107.86%, the CAT activity increased significantly by 81.94% and 73.26%, and the POD activity increased significantly by 61.74% and 58.14%.

[0044] Taken together, these results indicate that overexpression of NtEXT1 can reduce drought-induced oxidative stress by affecting the activity of antioxidant enzymes.

[0045] During the determination of each indicator, the specific methods are as follows: After the tobacco underground samples were homogenized, they were centrifuged at 4°C and 12,000 g for 20 min, and the supernatant was taken for determination of various indicators; Malondialdehyde (MDA) determination was carried out according to the instructions of the test kit (product of Perriman, item number: BH-961153). For details, please refer to the following: Take 0.3 ml of detection reagent and 0.1 ml of sample to be tested, mix well, and keep warm at 95℃ for 30 min. After the end of the warming, quickly cool to about 25℃, centrifuge at 10000g for 10 min, and record the absorbance as A532 and A600 at wavelengths of 532 nm and 600 nm on a spectrophotometer. Peroxidase (POD) assay was performed with reference to the instructions of the Peroxidase (POD) Activity Test Kit (Periman Company, Catalog No.: BH-961149). The specifics are as follows: After the reagents in the kit are prepared into a working solution, the working solution is used to dilute the sample solution to be tested by an appropriate multiple, and the absorbance value A1 after the reaction is carried out for 1 minute and the absorbance value A2 after the reaction is carried out for 2 minutes are measured at a wavelength of 470 nm; Superoxide dismutase (SOD) assay: refer to the instructions of the superoxide dismutase (SOD) activity test kit (product of Perriman, catalog number: BH-961146) and use the WST-8 method for assay; Catalase (CAT) assay: refer to the instructions of the Catalase (CAT) activity test kit (product of Perriman, catalog number: BH-961148), the specific reference is as follows: 50 μL of the sample solution to be tested is mixed with 30 μL of reagent 1 and reacted at 25°C for 10 min, then reagents 2 and 3 (100 μL and 265 μL) are added, and the absorbance of the test sample and the control sample at 405 nm is measured; Determination of superoxide anion content: refer to the instructions of the superoxide anion content test kit (product of Perriman, product number: BH-961162); Determination of hydrogen peroxide (H2O2) content: refer to the instruction manual of the hydrogen peroxide (H2O2) content test kit (product of Perriman Company, item number: BH-961167).

[0046] (4) Hormone content Since hormones play a very important role in signal transduction and stress resistance regulation, the inventors measured the changes in some hormones in plants under different treatment conditions. The changes in endogenous hormone content in leaves of wild-type and overexpression strains L6 and L7 tobacco after 5 days of 20% PEG treatment are shown in Figure 2. Figure 7 It can be seen that compared with the wild type, under drought conditions, the contents of cytokinin 2MeScZR hormone, abscisic acid ABA, jasmonic acid hormone 12-OH-JA and gibberellin hormone GA20 in L6 and L7 plants decreased significantly by 37.18%, 26.02%, 58.04% and 28.27% (L6), and 37.04%, 39.94%, 68.88% and 38.42% (L7), respectively.

[0047] That is, when faced with drought stress, the NtEXT1 overexpressing strain enhanced the drought resistance of the plant by downregulating the levels of hormones such as cytokinin 2MeScZR, abscisic acid ABA, jasmonic acid hormone 12-OH-JA and gibberellin hormone GA20.

[0048] When determining the content of related hormones, the first unfolded leaf from top to bottom of tobacco treated with 20% PEG for 5 days was used as the sample, and the LC-MS / MS method was used to detect and determine the content of related hormones by Maiwei Metabolism Company in East Lake New Technology Development Zone of Wuhan.

[0049] (5) Chemical composition of tobacco leaves Since adverse conditions will have a certain impact on tobacco content and tobacco quality, during the experiment, the inventors further measured several chemical component indicators related to tobacco quality in tobacco under different treatment conditions. Figure 8 As shown. It can be seen that: Under drought conditions, compared with the wild type, the total alkaloids of L6 and L7 decreased significantly by 16.89% and 29.14%, and the potassium-chloride ratio decreased by 24.7% and 17.56%; the total sugar of L6 and L7 increased significantly by 31.05% and 30.08%, the chloride content increased by 38.07% and 21.71%, and the sugar-alkali ratio increased by 103.12% and 103.10%.

[0050] In the process of determining the above-mentioned chemical composition indicators, tobacco leaves were used as samples. After air-drying and grinding, the contents of total sugar and reducing sugar (YC / T159-2019), total nitrogen (YC / T161-2002), nicotine (YC / T468-2021), potassium (YC / T217-2007) and chlorine (YC / T162-2011) in tobacco leaves were determined respectively using a continuous flow analyzer (type AA3), and the sugar-alkali ratio, nitrogen-alkali ratio and potassium-chlorine ratio were calculated.

[0051] 4. Simulated salt stress and cadmium stress experiments Based on the screening in step (ii) above, two NtEXT1 Overexpression strains L6 and L7 were used as experimental materials (the strains were further cultured and purified seeds were harvested), and tobacco variety K326 (wild type, WT) was used as a control. NtEXT1 Further experimental studies were conducted on the growth of tobacco and its effects on stress resistance (salt stress, cadmium stress) under overexpression conditions. The specific experimental results are briefly described as follows.

[0052] Experimental process: K326 cultured to the three-leaf stage was transplanted into 1 / 2 Holgland nutrient solution. After 5 days of pre-culture, salt stress and cadmium stress were treated, which were divided into three treatments: control, 100 mM salt treatment (NaCl) and 5 μM cadmium treatment (CdCl2); Leaves were taken 24 hours after treatment, leaf RNA was extracted, reverse transcribed into cDNA, and detected by qRT-PCR technology. NtEXT1 Changes in gene expression. Referring to the existing tobacco genome, the primer pairs for PCR amplification were designed as follows: NtEXT1- qRT-F: 5'-AAAGCTCAGGAAAGCTGGGG-3', NtEXT1- qRT-R: 5'-TGCGAGGGTTGGAAATTGT-3'.

[0053] The results are as follows Fig. 9 It was shown that salt stress and cadmium stress significantly induced NtEXT1 The expression was upregulated. Compared with the control, the expression levels after salt stress and cadmium stress increased significantly by 4.0 and 5.3 times, respectively.

[0054] In other words, NtEXT1 Genes are also very important for the response to salt stress and cadmium stress, as well as the ability of plants to resist salt and cadmium.

Claims

1. A primer pair for preparing tobacco NtEXT1 gene by PCR amplification, characterized in that: The CDS sequence of the tobacco NtEXT1 gene is shown in SEQ ID No. 1; the specific primer pair sequence design is as follows: NtEXT1- CDS-F: 5’-ATGAAAAGCTCAGGAAAGCTGGGGC-3’, NtEXT1- CDS-R: 5'-TTAGTAGTAGGTTGGTGGAGGAGAGATGCTGA-3'.

2. Application of tobacco NtEXT1 gene in plant drought resistance, characterized in that: The CDS sequence of the tobacco NtEXT1 gene is shown in SEQ ID No.

1. After the tobacco NtEXT1 gene is overexpressed, the drought resistance of the plant is improved by increasing the fresh weight, dry weight and water content of the aboveground and underground parts; Alternatively: After overexpressing the tobacco NtEXT1 gene, the plant's drought resistance is improved by reducing the transpiration rate and stomatal conductance index.

3. The use of tobacco NtEXT1 gene in plant drought resistance as claimed in claim 2, characterized in that: The plant is tobacco.

4. Application of tobacco NtEXT1 gene in regulating plant type, characterized in that: Overexpression of the tobacco NtEXT1 gene was used to extend plant stem nodes.

5. The use of tobacco NtEXT1 gene in regulating plant type according to claim 4, characterized in that: The plant is tobacco.

6. Application of tobacco NtEXT1 gene in plant root regulation, characterized in that: Overexpression of the tobacco NtEXT1 gene was used to increase plant root length.

7. The use of tobacco NtEXT1 gene in plant root regulation according to claim 6, characterized in that: The plant is tobacco.

8. Application of tobacco NtEXT1 gene in plant variety breeding, characterized in that: Improving drought resistance in plant varieties by overexpressing the NtEXT1 gene.

9. A method for breeding plant varieties using tobacco NtEXT1 gene, characterized in that: The plant is tobacco, and during cultivation, a drought-resistant plant variety is obtained by over-expressing the NtEXT1 gene.

10. Application of tobacco NtEXT1 gene in plant resistance, characterized in that: The resistance is salt resistance and / or cadmium resistance.

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