Application of ntcp7 gene in tobacco drought tolerance

Abstract

The application discloses application of an NtTCP7 gene in tobacco drought resistance and belongs to the technical field of molecular biology. The application discloses the application of the NtTCP7 gene in tobacco drought resistance, and the sequence of the NtTCP7 gene is shown as SEQ ID NO. 1. The NtTCP7 gene negatively regulates the tobacco drought resistance, and knocking out the gene leads to significant improvement of the tobacco drought resistance. The mining and function identification of the gene provide important gene resources and a theoretical basis for tobacco drought-resistant breeding.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of molecular biology, and more particularly to application of NtTCP7 gene in tobacco drought resistance. BACKGROUND

[0002] Tobacco (Nicotiana tabacum L.) originates from the tropics with abundant rainfall and has high requirements for water. Tobacco leaves are large, water consumption is high, and water participates in the morphological structure and metabolic process of tobacco, which is the basis for high-quality tobacco production. If the field water holding capacity is less than 50% or the leaf water content is reduced by 6%-8% during the growing season, the yield and quality of tobacco will be seriously affected, and the tobacco plants can appear wilting. In recent years, continuous drought has seriously affected the yield and quality of tobacco and has caused significant economic losses to tobacco farmers. At present, an important way to solve the problem is to use molecular biological technology to mine drought-resistant genes of tobacco, develop molecular markers, and conduct directional improvement on main tobacco varieties to cultivate new drought-resistant tobacco varieties.

[0003] TCP (Teosinte branched 1 / Cycloidea / Proliferating cell factor) transcription factor is a plant-specific transcription factor family, the encoded protein has a conserved TCP domain, belongs to a class of bHLH transcription factors, and was first reported by Cubas et al. in 1999. According to the differences in the TCP domain, the TCP transcription factor can be divided into two subfamilies: Class I, also known as PCF class or TCP-p class, including PCF1 subclass and PCF2 subclass, and Class II, also known as TCP-c class, including CYC / TB1 subclass and CIN subclass. The main difference between the two subclasses is that Class II has four more amino acid residues (CTAK) in the basic region than Class I, and the helix II domain of Class II protein is longer than that of Class I protein. Meanwhile, a few Class II proteins contain an arginine-rich (KELRAKARERARERTKEK) R domain and an ECE domain containing a glutamic acid-cysteine-glutamic acid sequence (SECEV). TCP transcription factors are involved in the regulation of plant growth and development, and play a crucial role in the evolution of plant morphology and structure, such as embryonic growth, leaf development, floral organ morphogenesis, pollen development, seed germination and senescence. At the same time, they are also related to the regulation pathways of cell proliferation, cell cycle regulation and hormone response.

[0004] In recent years, the mechanism of TCP transcription factors has been revealed, and it is found that TCP transcription factors play an important role in the process of plant resistance to biotic and abiotic stress. The research on the role of TCP transcription factors in plant response to abiotic stress started late, so there are not many reports on the involvement of TCP transcription factors in abiotic stress. Existing research shows that TCP family genes respond to adverse stress by regulating cell osmotic pressure, reducing damaged cell substances such as active oxygen, and reducing cell sensitivity to hormones. At normal temperature, the AtTCP17 protein of Arabidopsis thaliana binds with the CRY1 (Cryptochrome 1) protein to inhibit the formation of a dimer of PIF4 (Phytochrome INteracting Factors 4) and AtTCP17, but under high temperature conditions, CRY1 promotes the interaction of AtTCP17 and PIF4 by reducing the binding of AtTCP17, thereby increasing the transcriptional activity of PIF4 and regulating the heat morphogenesis of Arabidopsis thaliana to resist damage caused by high temperature. The natural variation of the maize ZmTCP42 promoter is closely related to drought resistance, and overexpression of the ZmTCP42 gene in Arabidopsis thaliana makes the transgenic plants more sensitive to ABA and increases the drought tolerance of the transgenic plants. There are studies on the transcriptome analysis of Gossypium hirsutum, and 41 TCP family genes respond to temperature, salt and drought stress.

[0005] Drought stress seriously affects the yield and quality of tobacco. It is of great theoretical and practical significance to use biological techniques to mine drought-resistant genes and explore the functions of the genes for breeding and cultivating drought-resistant varieties. Therefore, providing the application of NtTCP7 gene in tobacco drought resistance is a problem that those skilled in the art need to solve. SUMMARY

[0006] Therefore, the application provides the application of NtTCP7 gene in tobacco drought resistance.

[0007] In order to achieve the above purpose, the technical scheme adopted by the application is as follows:

[0008] The application of NtTCP7 gene in tobacco drought resistance, wherein the sequence of the NtTCP7 gene is shown as SEQ ID NO. 1.

[0009] Further, the application of knocking out NtTCP7 gene in the positive regulation of tobacco drought resistance, wherein the sequence of the NtTCP7 gene is shown as SEQ ID NO. 1.

[0010] Further, the application of biological material for knocking out NtTCP7 gene in the positive regulation of tobacco drought resistance, wherein the sequence of the NtTCP7 gene is shown as SEQ ID NO. 1.

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

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

[0013] B: a recombinant vector containing the expression cassette of A;

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

[0015] Further, the application discloses an application of overexpressing NtTCP7 gene in negative regulation of tobacco drought resistance, wherein the NtTCP7 gene sequence is shown as SEQ ID NO. 1.

[0016] Further, the application discloses an application of biological material of overexpressing NtTCP7 gene in negative regulation of tobacco drought resistance, wherein the NtTCP7 gene sequence is shown as SEQ ID NO. 1.

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

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

[0019] B: a recombinant vector containing the expression cassette of A;

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

[0021] Further, the application discloses an application of NtTCP7 gene in tobacco breeding, wherein the NtTCP7 gene sequence is shown as SEQ ID NO. 1.

[0022] Further, the application discloses an application of NtTCP7 gene in breeding tobacco drought-resistant germplasm, wherein the NtTCP7 gene sequence is shown as SEQ ID NO. 1.

[0023] Compared with the prior art, the application provides application of the NtTCP7 gene in tobacco drought resistance, a drought response gene NtTCP7 is separated in tobacco, the length of the CDS sequence of the gene is 744bp, and 247 amino acids are encoded. Taking a flue-cured tobacco variety K326 as background, two knockout strains tcp7-1-5 (ko#1) and tcp7-10-3 (ko#10) of the gene are obtained by using CRISPR technology; two overexpression strains TCP7-OE#2 and TCP7-OE#5 are obtained by transforming K326 by constructing an overexpression vector. Phenotype identification results show that, compared with K326 (WT), the wilting degree of the two knockout strains is lighter, and the wilting degree of the two overexpression strains is more serious under drought stress. Physiological and biochemical results show that, under drought stress, the relative conductivity (EL), hydrogen peroxide (H2O2) and superoxide radical anion (OFR) contents in the leaves of the two knockout strains are significantly lower than those of WT, and the two overexpression strains are contrary. NBT and DAB staining results show that, under drought stress, compared with WT, the staining area of the leaves of the two knockout strains is smaller, and the staining degree is lighter, and compared with WT, the staining area of the leaves of the two overexpression strains is larger, and the staining degree is deeper. The above results show that the NtTCP7 gene negatively regulates the drought resistance of tobacco. The mining and function identification of the gene provide important gene resources and theoretical basis for tobacco drought resistance breeding. BRIEF DESCRIPTION OF DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or the prior art description. Obviously, the drawings in the following description are only embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of the provided drawings.

[0025] Figure 1 Phylogenetic tree of NtTCP7 in the present application;

[0026] Figure 2 Sequence alignment of NtTCP7 homologous proteins in the present application;

[0027] Figure 3 Expression quantity analysis of NtTCP7 overexpression strains in the present application;

[0028] Figure 4 Sequence alignment and peak graph analysis of the knockout strain target site in the present application;

[0029] Figure 5 Sequence alignment of NtTCP7 protein in the knockout strain and WT in the present application;

[0030] Figure 6Phenotype of 400 mM mannitol drought stress treatment for 5 h of the application;

[0031] Figure 7 EL change analysis after drought treatment of the application;

[0032] Figure 8 H2O2(left) and OFR(right) content change analysis after drought treatment of the application;

[0033] Figure 9 NBT and DAB staining results after drought treatment of the application. DETAILED DESCRIPTION

[0034] The technical solutions in the embodiments of the application will be clearly and completely described in combination with the accompanying drawings of the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by those skilled in the art without creative labor fall within the protection scope of the application.

[0035] The NtTCP7 CDS sequence is shown as 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 this invention (as shown in SEQ ID NO.2) was obtained from the National CeNterfor Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / BLAST / ). Gene homology was analyzed using MEGA12.0 software and a phylogenetic tree was constructed using the neighbor-to-neighbor (NJ) method. Protein similarity was compared using DNAMAN software.

[0041] Phylogenetic analysis revealed that this NtTCP7 is most closely related to the tobacco velvet NtoTCP7 (XP 009594814.1). Figure 1 Homology analysis of the protein sequence revealed that the protein sequence possesses a typical TCP domain and shows high homology with NtoTCP7 from *Nicotiana flavescens*, NaTCP7L from *Nicotiana acuminata*, and NsTCP7L from *Nicotiana scabra*. Figure 2 ).

[0042] Example 2 Construction of overexpression vector

[0043] 1) cDNA Acquisition

[0044] (1) RNA extraction

[0045] This experiment mainly used the FastPure Universal PlaNt Total RNA Isolation Kit to extract total RNA from plants (Novozymes, catalog number: RC411-01). The specific steps were as described in the instruction manual.

[0046] (2) Reverse transcription

[0047] This experiment used the HiScript III reverse transcription kit.st Use the Strand cDNA SyNthesis Kit (+gDNAwiper) to synthesize cDNA (Novozymes, catalog number: R312-02). Refer to the instruction manual for specific steps.

[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] The amplification reaction was carried out according to the following system and procedure:

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

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

[0057] Then, the Gname (744bp) fragment was excised under UV light by 1% agarose gel electrophoresis at 5V / cm for 20 minutes and placed in a system for sol-gel recovery. The recovery procedure is as described in the Novizan (Catalog No.: DC301-01) DNA Recovery Kit instructions. The recovered DNA was dissolved in 40μL of water (the recovered product was labeled as: rDNAG1). After verification, it was recombined with the vector.

[0058] 4) Recombination of target fragment with vector

[0059] (1) The target fragment obtained in step 3) and the linearized vector pBWA(V)HS-ccdB(D) purified by BsaI / EcoI enzyme digestion were added to the recombinant system at a molar ratio of 2:1 (Novizan, The reaction is carried out using the IIOne StepCloning Kit. Please refer to the instruction manual for specific methods.

[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, Novizan, catalog number: C505-03) for transformation. Spread the transformation on 50 μg / mL Km resistant LB solid culture dishes and incubate overnight at 37°C.

[0061] (3) Selecting positive clones

[0062] Select single colonies for colony PCR. 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℃ for 5 min; 94℃ for 30 sec, 50℃ for 45 sec, 72℃ for 100 sec, 30 cycles; 72℃ for 10 min; 16℃ for 30 min.

[0067] Then, positive clones were identified using 1% agarose gel electrophoresis. The target band was a fragment of approximately 662 bp. 200 μL of bacterial culture corresponding to 1-3 positive bands was sent to a sequencing company for verification.

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

[0069] Add correctly sequenced and intact clones to 5-10 ml of LB liquid medium containing 50 μg / mL Km in an Erlenmeyer flask and incubate overnight at 37°C with a shaker at 200 rpm. Take 500 μl of fresh bacterial culture and add an equal volume of sterilized 50% glycerol, then store the E. coli culture at -80°C. Extract plasmids from the remaining bacterial culture using a plasmid extraction kit (FastPure EndoFree Plasmid Mini Kit-Box2, catalog number DC203-01).

[0070] Example 3: Construction of gene editing vector

[0071] 1) Target design

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

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

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

[0075] Then, primers for 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 a 50 μL 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 Procedure

[0084]

[0085] The target fragment (approximately 250 bp) was recovered by gel extraction. The recovered DNA was dissolved in 30 μL of water. After verification, it was ligated into 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, following the instructions (Fast-T1 chemocompetent cells, Novizan, catalog number: C505-03). Plate the transformed cells onto 50 μg / mL kanamycin-resistant LB agar plates and incubate at 37°C for 12 hours. Perform colony PCR identification.

[0094] Primer information is as follows:

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

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

[0097] Ten single colonies were selected for PCR identification. The identification system and reaction conditions are shown in Tables 5 and 6.

[0098] Table 5 Colony PCR Reaction System

[0099]

[0100] Table 6 PCR reaction procedures

[0101]

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

[0103] Select bacterial suspensions corresponding to 1-3 positive bands, take 100μL for sequencing, and inoculate the remaining 400μL of bacterial suspensions into 5-10ml of kanamycin-resistant LB. Shake the test tubes and wait for the sequencing results. Take the tube with the correct sequencing results to extract the plasmid. After extracting the plasmid, preserve the bacterial strain and plasmid.

[0104] Example 4 Tobacco Genetic Transformation

[0105] 1) Preparation of Agrobacterium

[0106] Add 1 μL of plasmid (overexpression vector or gene editing vector) to 50 μL of Agrobacterium GV3101 competent cells, transform and plate onto LB agar plates containing 50 μg / mL kanamycin resistance, incubate at 28°C for 48 h, and perform colony PCR identification. The amplification primers, reaction system, and reaction procedure are the same as above. Detect the PCR products by gel electrophoresis. If the electrophoretic bands of the positive control (using the correctly sequenced plasmid as a template) and the sample are clear and of the correct size, and the negative control (using water as a template) shows no band, it indicates that the sample can proceed to the next step and can be used to infect tobacco.

[0107] Single colonies were selected and cultured in liquid LB medium containing 25 μg / mL rifampicin and 100 μg / mL kanamycin. The culture was incubated at 28°C for 24 h with shaking. The culture was then centrifuged at 4000 rpm for 10 min, the supernatant was discarded, and the culture was resuspended in a immersion buffer (containing 10 mM MgCl2, pH 5.2, 10 mM 2-(N-morpholine)ethanesulfonic acid (MES), and 0.1 mM acetylsylphenone) and incubated at room temperature for at least 3 h until OD500 reached. 600 The value is approximately 0.6, which is used as a disinfectant.

[0108] 2) Genetic transformation

[0109] Select plump and uniform "K326" tobacco seeds, disinfect them with 10% sodium hypochlorite solution for 15 min, rinse them 5 times with sterile water, and slightly pat them dry with sterile absorbent paper. Sow 4 seeds / bottle on MS medium containing 30 g / L sucrose and 8 g / L agar (pH = 5.8). Incubate at 25℃ under constant temperature and light conditions of 1600 lx light intensity and 16 h (light) / 8 h (dark) photoperiod for 45 days. After the sterile seedlings have grown 4 leaves, cut them into small pieces of about 5 mm x 5 mm and remove the veins. Pre-culture them on MS medium containing 2 mg / L 6-BA and 0.2 mg / L IAA for 2 days, then soak them in Agrobacterium infection solution. The infected explants are then placed in MS medium containing 2 mg / L 6-BA and 0.2 mg / L IAA and cultured in the dark for 2 days. After co-culturing, 50 mg / L kanamycin and 500 mg / L carbenicillin were added to the culture medium for selection to induce the production of resistant callus. The culture conditions were the same as above, with subculturing every 14 days. When the resistant shoots on the callus reached 2 cm, they were transferred to rooting medium (MS + 50 mg / L kanamycin + 500 mg / L carbenicillin + 0.2 mg / L IAA). Rooting occurred in about 7 days. When the seedlings reached about 6 cm, the mouths of the culture bottles were opened for hardening off for 2 days. The seedlings were then transplanted into high-temperature sterilized tobacco-specific substrate (Hunan Tianliang Agricultural Technology Development Co., Ltd.), covered with plastic film to retain moisture, and cultured under light at 25–27℃ for further seed harvesting.

[0110] Example 5: Screening and Identification of Positive Seedlings

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

[0112] (1) Harvested contemporary transgenic tobacco seeds were germinated on a medium containing 150 μg / mL Hyg B. The entire process was carried out in a sterile environment. First, the seeds were soaked in ddH2O for 12 h; the ddH2O in the EP tube was poured out, and then 1 mL of 75% ethanol was added and vortexed for 30 sec; the ethanol was poured out, and 1 mL of ddH2O was added to rinse three times; 1 mL of NaClO solution was added and mixed well, and this process did not exceed 5 min; then the seeds were rinsed four times with sterile ddH2O; the rinsed seeds were spread evenly on MS medium containing 150 μg / mL Hyg B. Finally, the tubes were sealed with sealing film and cultured in a tissue culture room for 7 days.

[0113] (2) Transfer the green seedlings that grow on the culture medium into the soil for further cultivation. Wait for the seeds to mature, then divide the plants and harvest the T1 generation seeds.

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

[0115] (4) Select 10 potential positive seedlings from each dish and transfer them to soil for further cultivation. At the same time, collect the remaining small green seedlings in the dish, extract RNA, reverse transcribe it into cDNA, and quantitatively detect the expression level of the target gene.

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

[0117] NtTCP7 fluorescence quantitative primers:

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

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

[0120] Primers for quantitative real-time analysis of the tobacco internal reference gene ACTIN7:

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

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

[0123] The amplification system and procedure for real-time quantitative PCR (Thermo Fisher Scientific kit, catalog number: A25742) 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) Once the seeds of this generation of plants mature, collect seeds from lines with high expression levels for subsequent phenotypic identification.

[0130] Screening results of NtTCP7 homozygous overexpression lines: After harvesting seeds from 15 positive seedlings, T0 seeds were screened with 150 μg / mL hygromycin and two lines with high expression levels, TCP7-OE#2 (OE#2) and TCP7-OE#5 (OE#5), were identified through expression analysis. Further seed harvesting yielded T1 generation seeds, 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 leaves from sterile rooted tissue culture seedlings and use... DNA was extracted using the PlaNt DNA Isolation Mini Kit (Nanjing Novizan, catalog number: DC104-01) plant total DNA extraction kit, following the instructions. PCR amplification of the knockout material was performed using PrimeSTAR Max DNA Polymerase, a high-fidelity enzyme from Biotech (Beijing) Co., Ltd.

[0133] 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 procedure is shown in Table 10.

[0138] Table 9 PCR Reaction System

[0139]

[0140] Table 10 PCR Procedure

[0141]

[0142]

[0143] Screening results of NtTCP7 homozygous knockout lines: Sequencing analysis of 30 positive seedlings revealed two successfully edited lines, tcp7-1-5 (ko#1) and tcp7-10-3 (ko#10). Furthermore, ko#1 showed an insertion at target site 1; ko#10 showed an insertion at target site 1 and a large deletion at target site 2, with clean single peaks near the target sites, indicating that these two lines are homozygous mutants. Figure 4 Protein sequence alignment revealed that the translation of the NtTCP7 protein terminated prematurely in the ko#1 and ko#10 lines. Figure 5 This indicates that the NtTCP7 gene function was disrupted in both strains.

[0144] Example 6: Identification of drought-resistant phenotypes in tobacco

[0145] Seeds from knockout pure lines, overexpression pure lines, and wild-type plants with plump and uniform grain size were selected, sterilized, and then placed at 4℃ for 48 hours. They were then sown in small square boxes pre-filled with tobacco-specific substrate and fully watered, covered to keep warm and moist, and incubated in a greenhouse (temperature 25℃, humidity 75%, 16 hours light, 8 hours darkness) for germination.

[0146] Mannitol-simulated drought experiment: 30 days after sowing (six-leaf stage), tobacco seedlings with uniform growth were selected, and each seedling was irrigated with 200 ml of 400 mM mannitol solution. Phenotypic changes were observed and photographs were taken after treatment. At 0 h and 7 d, the third leaf (counting from the top) was taken as plant samples for physiological and biochemical index detection. Five biological replicates were set up for each treatment for physiological and biochemical detection. A control (CK) was established by irrigating each seedling with 200 ml of H2O.

[0147] like Figure 6As shown, under control conditions, the two overexpression and knockout lines showed no significant difference in phenotype from the WT line. After 5 hours of treatment with 400 mM mannitol simulating drought stress, compared with WT, the leaves of the two overexpression lines showed more severe wilting, while the leaves of the two knockout lines only showed slight drooping, with no significant change from before the treatment. These results indicate that compared with WT, the two knockout lines exhibited significantly enhanced drought tolerance, and NtTCP7 negatively regulates the drought tolerance of tobacco.

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

[0149] Hydrogen peroxide (H2O2) content was determined using a hydrogen peroxide test kit (Nanjing Jiancheng, catalog number: A064-1-1); superoxide anion capacity (OFR) was determined using a superoxide anion (Oxygen free radical, OFR) kit (Jiangsu Edison Biotechnology, catalog number: ADS-W-YH008); specific methods were performed according to the kit instructions.

[0150] Relative conductivity: Take 0.2g of fresh leaf tissue from the fourth true leaf of the seedling (counting downwards from the top leaf) using a round punch, place it in a centrifuge tube containing 25ml of ddH2O, and treat it on a shaker at 37℃ for 24h. Measure the conductivity L1 of the first extravasation. Then, place the EP tube in an autoclave at 120℃ for 15 minutes. After cooling to room temperature, measure the conductivity L2 of the second extravasation. Calculate the relative conductivity (EL = L1 / L2 * 100%).

[0151] 3,3'-Diaminobenzidine (DAB) staining: First, prepare 0.1 mg / mL DAB and dissolve it in 50 mM Tris-acetic acid buffer (pH 5.0). Immerse the 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 (if chlorophyll is difficult to dehydrate, the time can be longer). Finally, transfer the leaves to anhydrous ethanol, photograph and observe them under a microscope or camera, and preserve them.

[0152] Nitrotetrazole blue (NBT) staining: First, prepare an NBT staining solution of 1 mg / mL in 10 mM PBS (pH 7.8). Cut off leaves and place them in the NBT staining solution. Stain under light for 1-2 hours. Once a colorimetric phenotype (leaf turning blue) is observed, destain. Remove the staining solution, add anhydrous ethanol, boil in boiling water for 10 minutes, and store in 70% ethanol. Observe under a microscope or camera.

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

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

[0155] ROS histochemical staining was performed on overexpressing lines, knockout lines, and wild-type lines after 7 days of simulated drought stress with 400 mM mannitol. NBT and DAB staining results showed that ( Figure 9 Under normal conditions, both the overexpression and knockout lines showed slight blue / reddish-brown spots on their leaves, similar to the WT lines, but there was no significant difference between the lines. After simulated drought treatment, the leaves of the two overexpression lines showed larger and deeper staining areas than the WT lines, indicating that the overexpression lines accumulated more OFR / H2O2 than the WT plants; while the leaves of the two knockout lines showed smaller and lighter staining areas than the WT lines, indicating that the two knockout lines accumulated less OFR / H2O2 than the WT plants.

[0156] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those 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 invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Knockout NtTCP7 The application of genes in the positive regulation of drought resistance in tobacco is characterized by, The NtTCP7 The gene sequence is shown in SEQ ID NO.

1.

2. Knockout NtTCP7 The application of gene-based biomaterials in the positive regulation of drought resistance in tobacco is characterized by, The NtTCP7 The gene sequence is shown in SEQ ID NO.1; The biomaterial is any one of the following: A: Enables nucleotide sequences as shown in SEQ ID NO.1 NtTCP7 Gene silencing expression cassettes; B: A recombinant vector containing the expression cassette described in A; C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

3. Knockout NtTCP7 The application of genes in tobacco drought resistance breeding is characterized by, The NtTCP7 The gene sequence is shown in SEQ ID NO.

1.

4. Knockout NtTCP7 The application of genes in the breeding of drought-resistant tobacco germplasm is characterized by, The NtTCP7 The gene sequence is shown in SEQ ID NO.1.

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