Application of NtTCP7 gene in drought tolerance of tobacco

Abstract

The invention discloses application of an NtTCP7 gene in tobacco drought tolerance, and belongs to the technical field of molecular biology. According to the application of the NtTCP7 gene disclosed by the invention in the drought tolerance of the tobacco, the sequence of the NtTCP7 gene is as shown in SEQ ID NO. 1. The NtTCP7 gene negatively regulates the drought tolerance of the tobacco, and the drought tolerance of the tobacco is remarkably improved by knocking out the gene. Excavation and functional identification of the gene provide important gene resources and theoretical basis for drought-resistant breeding of tobacco.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and more particularly to the application of NtTCP7 gene in tobacco drought tolerance. Background Art

[0002] Tobacco (Nicotiana tabacum L.) originates in tropical regions with abundant rainfall and has a high water requirement. With its large leaves, tobacco plants require and consume a large amount of water. Water is crucial for the morphological composition and metabolic processes of tobacco plants and is essential for high-quality leaf production. During the growing season, if field water holding capacity falls below 50%, or if leaf moisture content decreases by 6%-8%, tobacco yield and quality are severely impacted, and the plants may wilt. Recent years of drought have severely impacted tobacco yield and quality, causing significant economic losses to farmers. Currently, the use of molecular biotechnology to identify drought-tolerant tobacco genes, develop molecular markers, and target improvements in major cultivated varieties to cultivate new drought-tolerant tobacco varieties is an important approach to addressing this issue.

[0003] TCP (TeosiNte branched 1 / Cycloidea / Proliferating cell factor) transcription factors are a family of plant-specific transcription factors. The proteins they encode possess a conserved TCP domain and belong to the class of bHLH transcription factors. They were first described by Cubas et al. in 1999. Based on differences within the TCP domain, TCP transcription factors can be divided into two subfamilies: Class I, also known as PCF or TCP-p, comprises the PCF1 and PCF2 subclasses, and Class II, also known as TCP-c, comprises the CYC / TB1 and CIN subclasses. The main differences between these two subclasses are that Class II proteins have four additional amino acid residues (CTAK) in the basic region compared to Class I proteins, and the helix II domain of Class II proteins is longer than that of Class I proteins. Furthermore, a few Class II proteins contain an arginine-rich R domain (KELRAKARERARERTKEK) and an ECE domain containing a glutamic acid-cysteine-glutamic acid sequence (SECEV). TCP transcription factors are involved in regulating plant growth and development, and play a vital role in the evolution of plant morphology and structure, such as embryonic growth, leaf development, floral organ morphogenesis, pollen development, seed germination and aging. They are also related to regulatory pathways related to cell proliferation, cell cycle regulation and hormone response.

[0004] Recent research has revealed the mechanisms of action of TCP transcription factors, demonstrating their crucial role in plant resistance to biotic and abiotic stresses. Research on the role of TCP transcription factors in plant responses to abiotic stresses started relatively late, resulting in limited reports on their involvement. Existing studies indicate that TCP family genes respond to stress by regulating cellular osmotic pressure, reducing cellular damage such as reactive oxygen species, and decreasing cellular sensitivity to hormones. At room temperature, the Arabidopsis AtTCP17 protein binds to the CRY1 (Cryptochrome 1) protein, inhibiting the dimerization of PIF4 (Phytochrome INteracting Factors 4) with AtTCP17. However, at elevated temperatures, CRY1 reduces AtTCP17 binding and promotes its interaction with PIF4, thereby increasing PIF4 transcriptional activity. This regulates thermomorphogenesis in Arabidopsis and protects against heat damage. Natural variation in the maize ZmTCP42 promoter is closely associated with drought resistance. Overexpression of the ZmTCP42 gene in Arabidopsis thaliana makes transgenic plants more sensitive to ABA and increases their tolerance to drought stress. Transcriptome analysis of upland cotton revealed that 41 TCP family genes respond to temperature, salt, and drought stress.

[0005] Drought stress severely impacts tobacco yield and quality. Using biological techniques to identify drought-tolerant genes and explore their functions has important theoretical and practical implications for the breeding and cultivation of drought-tolerant varieties. Therefore, providing a method for applying the NtTCP7 gene to tobacco drought tolerance is an urgent need for those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the use of the NtTCP7 gene in tobacco drought tolerance.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] Application of the NtTCP7 gene in tobacco drought tolerance, the NtTCP7 gene sequence is shown in SEQ ID NO.1.

[0009] Furthermore, the knockout of NtTCP7 gene is used in positively regulating tobacco drought tolerance. The NtTCP7 gene sequence is shown in SEQ ID NO.1.

[0010] Furthermore, the application of biological materials with NtTCP7 gene knockout in positively regulating tobacco drought tolerance, the NtTCP7 gene sequence is shown in SEQ ID NO.1;

[0011] The biological material is any one of the following:

[0012] A: an expression cassette capable of silencing the NtTCP7 gene whose nucleotide sequence is shown in SEQ ID NO.1;

[0013] B: recombinant vector containing the expression cassette described in A;

[0014] C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

[0015] Furthermore, the application of overexpressing the NtTCP7 gene in negatively regulating tobacco drought tolerance is shown in SEQ ID NO.1.

[0016] Furthermore, the application of biological materials overexpressing the NtTCP7 gene in negatively regulating tobacco drought tolerance, the NtTCP7 gene sequence is shown in SEQ ID NO.1;

[0017] The biological material is any one of the following:

[0018] A: an expression cassette capable of overexpressing the NtTCP7 gene whose nucleotide sequence is shown in SEQ ID NO.1;

[0019] B: recombinant vector containing the expression cassette described in A;

[0020] C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

[0021] Furthermore, the NtTCP7 gene is used in tobacco breeding, and the NtTCP7 gene sequence is shown in SEQ ID NO.1.

[0022] Furthermore, the NtTCP7 gene is used in breeding tobacco drought-resistant germplasm, and the NtTCP7 gene sequence is shown as SEQ ID NO.1.

[0023] Through the above technical solution, it can be seen that compared with the prior art, the present invention discloses the application of the NtTCP7 gene in tobacco drought tolerance. A drought-responsive gene NtTCP7 was isolated from tobacco. The length of the CDS sequence of this gene is 744bp, encoding 247 amino acids. Using the flue-cured tobacco variety K326 as the background, two knockout strains of this gene, tcp7-1-5 (ko#1) and tcp7-10-3 (ko#10), were obtained using CRISPR technology; by constructing an overexpression vector and transforming K326, two overexpression strains TCP7-OE#2 and TCP7-OE#5 were obtained. The phenotypic identification results showed that under drought stress, compared with K326 (WT), the two knockout strains had a lighter degree of wilting, and the two overexpression strains had a more severe degree of wilting. Physiological and chemical results showed that under drought stress, the relative electrical conductivity (EL), hydrogen peroxide (H2O2), and superoxide anion (OFR) contents in the leaves of the two knockout lines were significantly lower than those of the WT, while the opposite was true for the two overexpression lines. NBT and DAB staining revealed that under drought stress, the leaves of the two knockout lines had a smaller and lighter staining area than the WT, while the two overexpression lines had a larger and darker staining area than the WT. These results suggest that the NtTCP7 gene negatively regulates tobacco drought tolerance. The discovery and functional identification of this gene provide important genetic resources and theoretical foundations for tobacco drought tolerance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are merely embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying any creative work.

[0025] Figure 1 This is the phylogenetic tree of NtTCP7 of the present invention;

[0026] Figure 2 This is the sequence alignment of the NtTCP7 homologous proteins of the present invention;

[0027] Figure 3 This is the expression analysis of the NtTCP7 overexpression strain of the present invention;

[0028] Figure 4 Sequence alignment and peak analysis near the target site of the knockout strain of the present invention;

[0029] Figure 5 Comparison of NtTCP7 protein sequences between the knockout strains and WT of the present invention;

[0030] Figure 6This is the phenotype after 5 h of drought stress treatment simulated by 400 mM mannitol of the present invention;

[0031] Figure 7 This is the analysis of EL changes after drought treatment in the present invention;

[0032] Figure 8 Analysis of the changes in H2O2 (left) and OFR (right) contents after drought treatment according to the present invention;

[0033] Figure 9 These are the NBT and DAB staining results after drought treatment in the present invention. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0035] The NtTCP7 CDS sequence is shown in SEQ ID NO.1.

[0036] ATGTCGACGTCGGTAGAAGCTAACGGCGGCGCTACACTTACGCAGCAACACTCTTCTGATGCTGGCAATAGTGGTGCTTTGGTAGTGAAGAAGCCGCCGGCAAAGGACCGTCATAGCAAAGTCGATGGACGAGGAAGGCGTATAAGGATGCCAATTGTTTGTGCGGCTAGGGTTTTCCAGCTCACTCGCGAACTAGGTCATAAGTCCGACGGTCAAACCATTGAATGGCTGCTGCGTCAAGCTGAACCCTCGATTATCGCCGCCACTGGTACTGGTACTATCCCTGCTAGCTTTTCAACTCTCTCCGTTTCCCTACGGAATTCTTCGGTTTCTTCTTCTCTTTCCGCTCCGCTTGATTACAAGTCGTCATCACAGCCGTCGATTTCTCCCCCGCCGTTTTTGCTCGGGAAGCGTCTGCGTCCCGAGGATGACGACGGGAGTGGGGACAAGGATGACGCTAGTAACGGAGCTACGGTTGTGGGACCTACCGCTGGGTTCTGGGCTGTTCCAGCTAGGCCGGATTTTGGCCAGGTTTGGAGCTTTGCGCCGCCACCACCGCCGCCGGAGATGGTGGCGCCGACACATTCATCGGCAGCGAGATTTCTTCAGCAACAAATGGGAGAGGAGTCGGCTGCTAGATTAGGGAATTACTTTCCTATAGCTCAAGGGCACCTGAATTTGCTAGCTTCTCTGTCGGGCTCCGCACCGCCTTCTTCTG GGAGAAGAGACGATGACGGAAACTGA ; SEQ ID NO.1.

[0037] The NtTCP7 protein sequence is shown in SEQ ID NO.2.

[0038] MSTSVEANGGATLTQQHSSDAGNSGALVVKKPPAKDRHSKVDGRGRRIRMPIVCAARVFQLTRELGHKSDGQTIEWLLRQAEPSIIAATGTGTIPASFSTLSVSLRNSSVSSSLSAPLDYKSSSQ PSISPPPPFLLGKRLRPEDDDGSGDKDDASNGATVVGPTAGFWAVPARPDFGQVWSFAPPPPPPEMVAPTHSSAARFLQQQMGEESAARLGNYFPIAQGHLNLLASLSGSAPPSSGRRDDDGN; SEQ ID NO.2.

[0039] Example 1 Gene sequence feature analysis

[0040] The NtTCP7 protein sequence involved in the present invention (as shown in SEQ ID NO.2) was obtained from the National Center for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / BLAST / ). Gene homology was analyzed using MEGA12.0 software, a phylogenetic tree was constructed using the neighbor-joining method (NJ), and protein similarity was compared using DNAMAN software.

[0041] Phylogenetic tree analysis revealed that the NtTCP7 was most closely related to NtoTCP7 (XP 009594814.1) of Nicotiana velutina. Figure 1 Protein sequence homology analysis revealed that the protein sequence has a typical TCP domain and has a high homology with NtoTCP7 of Nicotiana tomentosa, NaTCP7L of Nicotiana attenuata, and NsTCP7L of Nicotiana sylvatica ( Figure 2 ).

[0042] Example 2 Overexpression vector construction

[0043] 1) cDNA acquisition

[0044] (1) RNA extraction

[0045] This experiment mainly uses the kit FastPure Universal PlaNt Total RNA Isolation Kit to extract plant total RNA (NoviZan, catalog number: RC411-01). For specific steps, please refer to the instruction manual.

[0046] (2) Reverse transcription

[0047] This experiment used the reverse transcription kit HiScript III 1st cDNA was synthesized using the Strand cDNA Synthesis Kit (+gDNAwiper) (Novagen, Cat. No.: R312-02). Specific steps were referred to the instruction manual.

[0048] 2) Synthetic primers

[0049] Synthesize the following target gene amplification primers:

[0050] Primer F1: AACACGGGGGACTTTGCAACatgtcgacgtcggtagaagctaacg;

[0051] SEQ ID NO.3;

[0052] Primer R1: TGAAGACAGAGCTAGTTACAtcagtttccgtcatcgtctcttctcc; SEQ ID NO.4.

[0053] 3) Amplify the target fragment

[0054] Perform the amplification reaction according to the following system and procedures:

[0055] PCR system: Nuclease-free Water 20μL, Biorun PfuPCRMix 25μL, Primer F12μL, Primer R12μL, cDNA 1μL, Total volume 50μL.

[0056] PCR program: 94°C for 5 min; 94°C for 30 sec, 50°C for 45 sec, 72°C for 106 sec, 30 cycles; 72°C for 10 min; 16°C for 30 min.

[0057] Then, perform 1% agarose gel electrophoresis at 5 v / cm for 20 minutes, cut out the electrophoretic fragment of Gname (744 bp) under ultraviolet light, and place it in a system for sol recovery. The recovery procedure is described in the instructions of the Novizan (Cat. No.: DC301-01) DNA recovery kit; dissolve the recovered DNA in a total volume of 40 μL of water (the recovered product is labeled: rDNAG1), and recombinant with the vector after testing.

[0058] 4) Recombination of target fragment and vector

[0059] (1) The target fragment obtained in step 3) and the linearized vector pBWA(V)HS-ccdB(D) purified by BsaI / EcoI digestion were added to the recombinant system (Novozyme, II One Step Cloning Kit) for reaction, and the specific method refers to the instruction manual.

[0060] (2) Take 1 μL of homologous recombination ligation product and add it to 20 μL of Escherichia coli chemically competent cells (Fast-T1 chemically competent cells, Novezan, catalog number: C505-03) for transformation, coat a 50 μg / mL Km resistance LB solid culture plate, and culture at 37°C overnight.

[0061] (3) Select positive clones

[0062] Pick a single clone for colony PCR, the primer sequences are as follows:

[0063] Primer F2: tTCATTTGGAGAGAACACGGGggac; SEQ ID NO.5;

[0064] Primer R2: gcataagctggtataggaaaaactg; SEQ ID NO. 6.

[0065] PCR system: Nuclease-free Water 9.5μL; Biorun Magic PCR Mix 12.5μL; PrimerF2 (100μM) 1μL; Primer R2 (100μM) 1μL; bacterial liquid 1μL; Total volume 25μL.

[0066] PCR program: 94°C for 5 min; 94°C for 30 sec, 50°C for 45 sec, 72°C for 100 sec, 30 cycles; 72°C for 10 min; 16°C for 30 min.

[0067] Next, use 1% agarose gel electrophoresis to identify positive clones. The target band is approximately 662 bp. Take 200 μL of the bacterial suspension corresponding to one to three positive bands and send it to a sequencing company for verification.

[0068] 5) Extract plasmid pBWA(V)HS-ccdB(D)-NtTCP7

[0069] Correctly sequenced, intact clones were added to 5-10 ml of LB liquid medium containing 50 μg / mL Km in a conical flask and cultured overnight at 37°C with shaking at 200 rpm. Add 500 μl of fresh culture medium to an equal volume of sterile 50% glycerol and store the E. coli culture at -80°C. Use the remaining culture medium to extract the plasmid using a plasmid extraction kit (FastPure EndoFree Plasmid Mini Kit-Box2, Cat. No. DC203-01).

[0070] Example 3 Gene Editing Vector Construction

[0071] 1) Target design

[0072] Using the online analysis tool http: / / crispor.tefor.net / , we designed targets on gene exons and obtained the following two specific targets:

[0073] Target1: ACGTCGGTAGAAGCTAACGGCGG; SEQ ID NO.7;

[0074] Target2: CAACTCTCTCCGTTTCCCTACGG; SEQ ID NO.8.

[0075] Then, primers for constructing the CRSIPR vector were designed, and their sequence information is as follows:

[0076] F1(+): cagtGGTCTCatgcaACGTCGGTAGAAGCTAACGG; SEQ ID NO.9;

[0077] R1(-): cgatGGTCTCaaaacTAGGGAAACGGAGAGAGTTG; SEQ ID NO. 10.

[0078] 2) PCR amplification

[0079] Prepare 50 μL of the system according to Table 1 and perform the amplification reaction according to the procedure in Table 2.

[0080] Table 1 PCR system

[0081]

[0082]

[0083] Table 2 PCR program

[0084]

[0085] The target fragment (about 250 bp) was recovered from the gel, and the recovered DNA was dissolved in a total volume of 30 μL of water. After testing, it was ligated with the vector.

[0086] 3) Enzyme digestion and ligation

[0087] The enzyme digestion and ligation system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0088] Table 3 Enzyme Digestion and Ligation System

[0089]

[0090] Table 4 Enzyme digestion and ligation reaction conditions

[0091]

[0092] 4) Transformation and identification

[0093] Transform 5-10 μL of the ligation product into competent E. coli cells (Fast-T1 chemically competent cells, Novezan, Cat. No. C505-03) according to the manufacturer's instructions. Transform the cells into LB plates coated with 50 μg / mL kanamycin-resistant solid culture medium, incubate at 37°C for 12 hours, and perform colony PCR analysis.

[0094] Primer information is as follows:

[0095] F3: gtaaaacgacggccagt; SEQ ID NO.11;

[0096] R3: ccagaaattgaacgccgaag; SEQ ID NO. 12.

[0097] Pick 10 single colonies for PCR identification. The identification system and reaction conditions refer to Tables 5 and 6.

[0098] Table 5 Colony PCR reaction system

[0099]

[0100] Table 6 PCR reaction program

[0101]

[0102] The target band is a fragment of about 800 bp.

[0103] Select the bacterial liquid corresponding to 1-3 positive bands, take 100 μL of it for sample sequencing, and inoculate the remaining 400 μL of bacterial liquid into 5-10 ml of LB containing kanamycin resistance. Shake the tube and wait for the sequencing results to come out. Take a tube corresponding to the correct sequencing to extract the plasmid. After extracting the plasmid, save the strain and plasmid.

[0104] Example 4 Tobacco genetic transformation

[0105] 1) Agrobacterium preparation

[0106] Add 1 μL of plasmid (overexpression vector or gene editing vector) to 50 μL of GV3101 competent Agrobacterium cells and transform the culture into LB plates coated with 50 μg / mL kanamycin. Incubate at 28°C for 48 hours, and perform colony PCR. The amplification primers, reaction system, and reaction procedure are the same as above. PCR products are detected by gel electrophoresis. If the electrophoresis results show clear and correct electrophoretic bands in the positive control (using the sequenced plasmid as described above as a template) and the sample, and no bands are observed in the negative control (using water as a template), the sample is suitable for the next step and can be used to infect tobacco.

[0107] Single colonies were selected and placed in liquid LB medium containing 25 μg / mL rifampicin and 100 μg / mL kanamycin (Km) antibiotics, and shaken at 28°C for 24 h. The shaken bacterial solution was centrifuged at 4000 r / min for 10 min, the supernatant was discarded, and the suspension was resuspended in infiltration buffer (containing 10 mM MgCl2, pH = 5.2, 10 mM 2-(N-morpholino)ethanesulfonic acid (MES) and 0.1 mM acetosyringone), and incubated at room temperature for more than 3 h until the OD 600 The value is about 0.6 and is used as the infection solution.

[0108] 2) Genetic transformation

[0109] Select plump, uniform "K326" tobacco seeds, sterilize them with a 10% sodium hypochlorite solution for 15 minutes, rinse five times with sterile water, and blot dry with sterile absorbent paper. Sow four seeds per bottle on a medium containing MS (30 g / L sucrose) and 8 g / L agar (pH 5.8). Place in a 25°C incubator with a constant light intensity of 1600 lx and a photoperiod of 16 h (light) / 8 h (dark) for 45 days. After the sterile seedlings develop four leaves, cut them into small pieces approximately 5 mm x 5 mm, and remove the leaf veins. After pre-incubation for two days in MS medium containing 2 mg / L 6-BA and 0.2 mg / L IAA, soak them in Agrobacterium infection solution. The infected explants are then incubated in MS medium containing 2 mg / L 6-BA and 0.2 mg / L IAA in the dark for two days. After co-cultivation, the culture medium was supplemented with 50 mg / L kanamycin and 500 mg / L carbenicillin for screening and induction of resistant callus. Culture conditions were the same as above, with subculture every 14 days. When resistant buds on the callus grew to 2 cm, they were transferred to rooting medium (MS + 50 mg / L kanamycin + 500 mg / L carbenicillin + 0.2 mg / L IAA). Rooting took about 7 days. When the seedlings grew to about 6 cm, the bottle caps were opened and hardened for 2 days. The seedlings were then transplanted into a high-temperature sterilized tobacco substrate (Hunan Tianliang Agricultural Technology Development Co., Ltd.), covered with plastic film to retain moisture, and cultured under light at 25-27°C for further seed harvesting.

[0110] Example 5 Positive seedling screening and identification

[0111] 1) Screening and identification of overexpression positive seedlings

[0112] (1) Harvest contemporary transgenic tobacco seeds and germinate them on a medium containing 150 μg / mL Hyg B. The entire process needs to be carried out in a sterile environment. First, soak the seeds in ddH2O for 12 hours; pour out the ddH2O in the EP tube, then add 1 mL of 75% alcohol and vortex for 30 seconds; pour out the alcohol and rinse three times with 1 mL of ddH2O; add 1 mL of NaClO solution and mix well. This process should not exceed 5 minutes; then rinse four times with sterilized ddH2O; spread the rinsed seeds on MS medium containing 150 μg / mL Hyg B. Finally, seal with parafilm and place in a tissue culture room for 7 days.

[0113] (2) Transfer the green seedlings grown on the culture medium to soil for further cultivation. Wait for the seeds to mature and divide them to collect the T1 generation seeds.

[0114] (3) Continue screening the collected T1 generation seeds according to the method in step (1). Place them in the tobacco tissue culture room for 7 days. A culture dish containing only green seedlings is considered a possible positive seedling.

[0115] (4) Ten possible positive seedlings were selected from each dish and transferred to soil for further culture. Meanwhile, the remaining small green seedlings in the dish were collected to extract RNA, reverse transcribed into cDNA, and quantitatively detected the expression level of the target gene.

[0116] (5) RNA was extracted from transgenic tobacco to verify the expression of the target gene. The methods of total RNA extraction and cDNA synthesis were the same as above, and the expression of the target gene NtTCP7 was detected using real-time quantitative PCR technology.

[0117] NtTCP7 fluorescent quantitative primers:

[0118] NtTCP7-qF:CCCTACGGAATTCTTCGGT; SEQ ID NO.13;

[0119] NtTCP7-qR: ACGCAGACGCTTCCCGAGCAAA; SEQ ID NO.14;

[0120] Tobacco internal reference gene ACTIN7 fluorescence quantitative primers:

[0121] NtACT7-qF: CCACACTGGTGTTATGGTTG; SEQ ID NO.15;

[0122] NtACT7-qR: AATACCGTGCTCAATTGGG; SEQ ID NO. 16.

[0123] The real-time quantitative PCR (kit Thermo Fisher Scientific, catalog number: A25742) amplification system and procedure are shown in Tables 7 and 8.

[0124] Table 7 Real-time quantitative PCR reaction system

[0125]

[0126] Table 8 Real-time quantitative PCR reaction program

[0127]

[0128]

[0129] (5) When the seeds of the current generation of plants mature, collect the seeds of the strains with high expression levels for subsequent phenotypic identification.

[0130] Results of screening of NtTCP7 homozygous overexpression strains: After 15 positive seedlings were harvested, T0 seeds were screened with 150 μg / mL hygromycin and two strains with high expression levels, TCP7-OE#2 (OE#2) and TCP7-OE#5 (OE#5), were identified through expression analysis. T1 generation seeds were further harvested and T2 generation seeds (pure lines) were obtained by treating T1 seeds with 150 μg / mL hygromycin ( Figure 3 ).

[0131] 2) Screening and identification of gene knockout positive seedlings

[0132] Cut the leaves of sterile rooted tissue culture seedlings and use DNA was extracted using the PlaNt DNA Isolation Mini Kit (Nanjing Novozymes, Cat. No. DC104-01). Detailed procedures were described in the manufacturer's instructions. PCR amplification of the knockout material was performed using the high-fidelity PrimeSTAR Max DNA Polymerase (from Bio-Rad Biotechnology (Beijing) Co., Ltd.).

[0133] The primer sequence information is as follows:

[0134] TCP7-crispr-F: CTACCCCCACCCAGACTA; SEQ ID NO.17;

[0135] TCP7-crispr-R: CAGAGAAGCTAGCAAATTCAGG; SEQ ID NO. 18.

[0136] Fragment length: 799bp.

[0137] The PCR reaction system is shown in Table 9; the PCR program is shown in Table 10.

[0138] Table 9 PCR reaction system

[0139]

[0140] Table 10 PCR program

[0141]

[0142]

[0143] Results of screening of NtTCP7 homozygous knockout strains: 2 successfully edited strains, tcp7-1-5 (ko#1) and tcp7-10-3 (ko#10), were screened through sequencing analysis of 30 positive seedlings. It was found that ko#1 had a base insertion at target site 1; ko#10 had a base insertion at target site 1 and a large base deletion at target site 2, and both had clean single peaks near the target sites, indicating that these two strains were homozygous mutations ( Figure 4 Protein sequence comparison revealed that NtTCP7 protein translation terminated prematurely in ko#1 and ko#10 strains ( Figure 5 ), indicating that the function of the NtTCP7 gene was disrupted in these two strains.

[0144] Example 6 Identification of tobacco drought-tolerant phenotypes

[0145] Seeds of knockout lines, overexpression lines, and wild-type plants with uniformly plump and large grains were selected, sterilized, and placed at 4°C for 48 hours. The seeds were then sown in small, well-watered boxes filled with tobacco-specific substrate, covered with a lid to maintain moisture, and incubated in a greenhouse (25°C, 75% humidity, 16 hours of light, 8 hours of darkness) for germination.

[0146] Mannitol-induced drought simulation experiment: 30 days after sowing (at the six-leaf, one-heart stage), tobacco seedlings of uniform growth were selected and infiltrated with 200 ml of 400 mM mannitol solution per plant. Phenotypic changes were observed and photographed after treatment. At 0 h and 7 days, the third leaf (counting from the top) was sampled for physiological and biochemical analysis. Five biological replicates were set up for each treatment for physiological and biochemical analysis. Each seedling was infused with 200 ml of H2O per plant as the control (CK).

[0147] like Figure 6As shown, under control conditions, the two overexpression and knockout lines showed no significant phenotype differences from the WT. After 5 hours of simulated drought stress treatment with 400 mM mannitol, the leaves of the two overexpression lines wilted more severely compared to the WT, while the leaves of the two knockout lines showed only slight drooping, with no significant changes from their pre-treatment levels. This result indicates that the two knockout lines have significantly enhanced drought tolerance compared to the WT, and that NtTCP7 negatively regulates drought tolerance in tobacco.

[0148] Example 7 Determination of physiological and biochemical indicators of knockout strains

[0149] The hydrogen peroxide (H2O2) content was determined using a hydrogen peroxide test kit (Nanjing Jiancheng, catalog number: A064-1-1); the superoxide anion capacity (OFR) was determined using an oxygen free radical (OFR) kit (Jiangsu Edison Biotechnology, catalog number: ADS-W-YH008); the specific method was carried out according to the kit instructions.

[0150] Relative conductivity: Use a circular borer to extract 0.2 g of fresh leaf tissue from the fourth true leaf of a seedling (counting downward from the top leaf). Place the 0.2 g fresh leaf tissue into a centrifuge tube containing 25 ml of ddH2O and shake at 37°C for 24 hours. Measure the first conductivity value, L1, of the exudate. Autoclave the EP tube at 120°C for 15 minutes. After cooling to room temperature, measure the second conductivity value, L2, of the exudate. Calculate relative conductivity (EL = L1 / L2 * 100%).

[0151] 3,3'-Diaminobenzidine (DAB) staining: Prepare 0.1 mg / mL DAB in 50 mM Tris-acetate buffer (pH 5.0). Soak leaves in the staining solution overnight at room temperature in the dark. Remove the staining solution, add anhydrous ethanol, and incubate in a boiling water bath for 10 minutes (longer if chlorophyll is difficult to dehydrate). Finally, transfer the leaves to anhydrous ethanol, photograph under a microscope or camera, and store.

[0152] Nitroblue tetrazolium chloride (NBT) staining: Prepare 1 mg / mL NBT staining solution in 10 mM PBS (pH 7.8). Place excised leaves in the NBT staining solution and stain under light for 1-2 hours. Decolorize when a color phenotype (blue leaves) develops. Remove the staining solution, add anhydrous ethanol, boil in boiling water for 10 minutes, store in 70% ethanol, and photograph under a microscope or camera.

[0153] Physiological and biochemical results showed that under control conditions, the EL (relative electrical conductivity) of the two overexpression and knockout lines had no significant difference from that of WT. After 7 days of drought stress treatment with 400 mM mannitol, the EL of the two overexpression lines was significantly higher than that of WT, the EL of ko#1 was lower than that of WT, and the EL of ko#10 was significantly lower than that of WT. Figure 7 ).

[0154] like Figure 8 As shown in the figure, there was no significant difference in the OFR (superoxide anion) and H2O2 (hydrogen peroxide) contents between the overexpression lines, the knockout lines and the WT before drought treatment; after mannitol-simulated drought stress treatment, the H2O2 contents in the two overexpression lines were higher than those in the WT, while the H2O2 contents in the two knockout lines were significantly lower than those in the WT ( Figure 8 Left); After mannitol-induced drought stress treatment, OFR in the two overexpression lines was higher than that in WT, while OFR in the two knockout lines was lower than that in WT ( Figure 8 right).

[0155] ROS histochemical staining was performed on the overexpression lines, knockout lines and wild type after 7 days of drought stress simulated by 400 mM mannitol. The results of NBT and DAB staining showed that ( Figure 9 Under normal conditions, leaves of both the overexpression and knockout lines showed slight blue and reddish-brown spots compared to WT leaves, but no significant differences were observed between the lines. After simulated drought treatment, leaves of the two overexpression lines showed larger and darker staining than those of the WT, indicating that the overexpression lines accumulated more OFR / H2O2 than the WT. However, leaves of the two knockout lines showed smaller and lighter staining than those of the WT, indicating that the knockout lines accumulated less OFR / H2O2 than the WT.

[0156] The above description of the disclosed embodiments is intended to enable one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be readily apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention is not limited to the embodiments shown herein but is intended to conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Application of NtTCP7 gene in tobacco drought tolerance, characterized in that, The NtTCP7 gene sequence is shown in SEQ ID NO.

1.

2. Application of knocking out the NtTCP7 gene in positively regulating tobacco drought tolerance, characterized in that: The NtTCP7 gene sequence is shown in SEQ ID NO.

1.

3. Application of biomaterials with NtTCP7 gene knockout in positively regulating tobacco drought tolerance, characterized in that: The NtTCP7 gene sequence is shown in SEQ ID NO.1; The biological material is any one of the following: A: an expression cassette capable of silencing the NtTCP7 gene whose nucleotide sequence is shown in SEQ ID NO.1; B: recombinant vector containing the expression cassette described in A; C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

4. Application of overexpression of NtTCP7 gene in negatively regulating tobacco drought tolerance, characterized in that: The NtTCP7 gene sequence is shown in SEQ ID NO.

1.

5. Application of biomaterials overexpressing the NtTCP7 gene in negatively regulating tobacco drought tolerance, characterized in that: The NtTCP7 gene sequence is shown in SEQ ID NO.1; The biological material is any one of the following: A: an expression cassette capable of overexpressing the NtTCP7 gene whose nucleotide sequence is shown in SEQ ID NO.1; B: recombinant vector containing the expression cassette described in A; C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

6. Application of NtTCP7 gene in tobacco breeding, characterized in that, The NtTCP7 gene sequence is shown in SEQ ID NO.

1.

7. Application of NtTCP7 gene in breeding tobacco drought-tolerant germplasm, characterized in that, The NtTCP7 gene sequence is shown in SEQ ID NO.1.

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