Application of ntcp7 gene in tobacco cold tolerance

Abstract

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

[0002] Tobacco (Nicotiana tabacum L.) is an important economic crop originally from tropical and subtropical regions, which is extremely vulnerable to low temperature damage. Low temperature stress is one of the main environmental factors limiting the growth, quality and yield of tobacco. The low temperature stress on tobacco seedlings is more obvious. When the tobacco is subjected to 12℃ low temperature stress for about two weeks at the age of 6-7 leaves, the tobacco will bloom early, reduce the yield and quality of tobacco leaves, and thus reduce the income of tobacco farmers and cause great losses to the tobacco industry. At present, an important way to solve the problem is to use molecular biological technology to mine low temperature resistant genes of tobacco, develop molecular markers, and conduct directional improvement on main tobacco varieties to cultivate new low temperature 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 difference 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 R domain rich in arginine (KELRAKARERARERTKEK) and an ECE domain containing glutamic acid-cysteine-glutamic acid sequence (SECEV). The TCP transcription factor is involved in the regulation of plant growth and development, plays 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, and is also related to the regulation pathways of cell proliferation, cell cycle regulation and hormone response.

[0004] In recent years, the mechanism of TCP transcription factor has been revealed, and it plays an important role in the process of plant resistance to biotic and abiotic stress. The research on the role of TCP transcription factor in plant response to abiotic stress started late, so there are not many reports on TCP transcription factor involved in abiotic stress. Existing research shows that TCP family genes respond to adversity 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 combines 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 reduces the binding with AtTCP17 to promote the interaction between AtTCP17 and PIF4, thereby increasing the transcriptional activity of PIF4 and regulating the thermal morphogenesis of Arabidopsis thaliana to resist the 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 at the same time 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] Low temperature stress seriously affects the yield and quality of tobacco. It has important theoretical and practical significance to use biological technology to mine low temperature resistant genes and explore gene function for breeding and cultivating low temperature resistant varieties. Therefore, providing the application of NtTCP7 gene in tobacco cold tolerance 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 cold tolerance.

[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 cold tolerance, wherein the sequence of the NtTCP7 gene is shown as SEQ ID NO. 1.

[0009] Further, the application of knocking out NtTCP7 gene in regulating tobacco cold tolerance, 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 regulating tobacco cold tolerance, 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 cold tolerance, 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 cold tolerance, 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 screening of tobacco cold-tolerant 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 cold tolerance, a low-temperature response gene NtTCP7 is separated from tobacco, the length of the CDS sequence of the gene is 744 bp, 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 low-temperature stress. Physiological and biochemical results show that, under low-temperature 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 low-temperature 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 cold tolerance of tobacco. The mining and functional identification of the gene provide important gene resources and theoretical basis for tobacco low-temperature tolerance 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 only constitute the 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 of the target site and peak graph analysis of knockout strains in the present application;

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

[0030] Figure 6 Phenotype after low-temperature (4℃) treatment for 6.5 h in the present application;

[0031] Figure 7 Analysis of EL change after low temperature treatment of the application;

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

[0033] Figure 9 NBT and DAB staining results after low temperature 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 in 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] MSTSVEANGGATLTQQHSSDAGNSGALVVKKPPAKDRHSKVDGRGRRIRMPIVCAARVFQLTRELGHKSDGQTIEWLLRQAEPSIIAATGTGTIPASFSTLSVSLRNSSVSSSLSAPLDYKSSSQPSISPPPFLLGKRLRPEDDDGSGDKDDASNGATVVGPTAGFWAVPARPDFGQVWSFAPPPPPPEMVAPTHSSAARFLQQQMGEESAARLGNYFPIAQGHLNLLASLSGSAPPSSGRRDDDGN; SEQ ID NO. 2.

[0039] Example 1 Gene sequence feature analysis

[0040] The NtTCP7 protein sequence (as shown in SEQ ID NO. 2) involved in the present application is from National Center for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / BLAST / ), the gene homology is analyzed by using MEGA12.0 software, the phylogenetic tree is constructed by using NJ method, and the protein similarity alignment is performed by using DNAMAN software.

[0041] The phylogenetic tree analysis found that the NtTCP7 has the closest genetic relationship with NtoTCP7 (XP 009594814.1) of Nicotiana tomentosiformis ( Figure 1 ). The protein sequence homologous alignment analysis found that the protein sequence has a typical TCP domain, and has higher homology with NtoTCP7 of Nicotiana tomentosiformis, NaTCP7L of Nicotiana attenuata and NsTCP7L of Nicotiana sylvestris ( Figure 2 ).

[0042] Example 2 Construction of overexpression vector

[0043] 1) cDNA acquisition

[0044] (1) RNA extraction

[0045] In this experiment, the total RNA of plants was extracted by using the kit FastPure Universal Plant Total RNA Isolation Kit (Novizen, item number: RC411-01), and the specific steps were referred to the instruction manual.

[0046] (2) Reverse transcription

[0047] In this experiment, the reverse transcription kit HiScript III 1st Strand cDNA SyNthesis Kit(+gDNAwiper) for cDNA synthesis (Novagen, Cat No: R312-02), according to the instruction manual.

[0048] 2) Synthesis of primers

[0049] The following target gene amplification primers were synthesized:

[0050] Primer F1: AACACGGGGGACTTTGCAACatgtcgacgtcggtagaagctaacg;

[0051] SEQ ID NO. 3;

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

[0053] 3) Amplification of target fragments

[0054] The amplification reaction was performed according to the following system and procedure:

[0055] PCR system: Nuclease-free Water 20 μL, Biorun Pfu PCR Mix 25 μL, Primer F1 12 μL, Primer R1 12 μL, cDNA 1 μL, Total volume 50 μL.

[0056] PCR procedure: 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, 1% agarose gel electrophoresis was performed at 5 v / cm for 20 min, and the electrophoresis fragment of Gname (744 bp) was cut out under ultraviolet light and placed in a system for gel recovery, according to the instruction manual of the Novagen (Cat No: DC301-01) DNA recovery kit; the recovered DNA (recovery product labeled as: rDNA G1) was dissolved in a total volume of 40 μL of water, and after detection, it was recombined with the vector.

[0058] 4) Recombination of target fragments and vectors

[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 recombination system in a molar ratio of 2:1 (Novagen, Cat No: R312-02, Cat No: R400-01), according to the instruction manual.

[0060] IIOne Step Cloning Kit) according to the manufacturer's instructions.

[0061] (2) Take 1 μL of the homologous recombination ligation product and add it to 20 μL of E. coli chemically competent cells (Fast-Tl chemically competent cells, Novagen, Cat. No. C505-03) for transformation, and then spread on 50 μg / mL Km-resistant LB solid plates and incubate at 37°C overnight.

[0062] (3) Select positive clones

[0063] Take a single colony for colony PCR, and the primer sequences are as follows:

[0064] Primer F2: tTCATTTGGAGAGAACACGGGggac; SEQ ID NO. 5;

[0065] Primer R2: gcataagctggtataggaaaaactg; SEQ ID NO. 6.

[0066] PCR system: Nuclease-free Water 9.5 μL; Biorun Magic PCR Mix 12.5 μL; Primer F2 (100 μM) 1 μL; Primer R2 (100 μM) 1 μL; Bacterial solution 1 μL; Total volume 25 μL.

[0067] 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.

[0068] Then, use 1% agarose gel electrophoresis to detect and identify positive clones. The target band is a fragment of about 662 bp. Take 1-3 positive bands corresponding to the bacterial solution, and take 200 μL to send to a sequencing company for verification.

[0069] 5) Extract plasmid pBWA(V)HS-ccdB(D)-NtTCP7

[0070] The correct and complete clones were sequenced, and 5-10 ml of LB liquid medium containing 50 μg / mL Km was added to the triangular flask, 200 rpm, 37°C shaking bed overnight culture. 500 μl of fresh bacterial liquid was added to the same volume of sterilized 50% glycerol, and the E. coli bacterial liquid was stored at -80°C. The remaining bacterial liquid was extracted using a plasmid extraction kit (FastPure EndoFree Plasmid Mini Kit-Box2, item number DC203-01) to extract the plasmid.

[0071] Example 3 Gene editing vector construction

[0072] 1) Target design

[0073] Using the online analysis tool http: / / crispor.tefor.net / , target points were designed on the gene exons, and the following two specific targets were obtained through analysis:

[0074] Target1: ACGTCGGTAGAAGCTAACGGCGG; SEQ ID NO. 7;

[0075] Target2: CAACTCTCTCCGTTTCCCTACGG; SEQ ID NO. 8.

[0076] Further design of CRSIPR vector construction primers, the sequence information is as follows:

[0077] F1 (+): cagtGGTCTCatgcaACGTCGGTAGAAGCTAACGG; SEQ ID NO. 9;

[0078] R1 (-): cgatGGTCTCaaaacTAGGGAAACGGAGAGAGTTG; SEQ ID NO. 10.

[0079] 2) PCR amplification

[0080] According to Table 1, 50 μL system was prepared and the amplification reaction was carried out according to Table 2 procedure.

[0081] Table 1 PCR system

[0082]

[0083]

[0084] Table 2 PCR program

[0085]

[0086] The gel recovery target fragment (about 250 bp) was dissolved in a total volume of 30 μL water, and the recovered DNA was linked to the vector after detection.

[0087] 3) Enzymatic digestion and ligation

[0088] The enzyme digestion and ligation system is shown in Table 3, and the reaction conditions are shown in Table 4.

[0089] Table 3 Enzymatic digestion and ligation system

[0090]

[0091] Table 4 Enzymatic digestion and ligation reaction conditions

[0092]

[0093] 4) Transformation and identification

[0094] 5-10 μL of the ligation product was transformed into E. coli competent cells, and the specific method was referred to the instruction (Fast-T1 chemical competent cells, Novozyme, item number: C505-03). The transformation was coated with 50 μg / mL kanamycin-resistant LB solid culture dish, and incubated at 37°C for 12 hours, and then colony PCR identification was performed.

[0095] The primer information is as follows:

[0096] F3: gtaaaacgacggccagt; SEQ ID NO. 11;

[0097] R3: ccagaaattgaacgccgaag; SEQ ID NO. 12.

[0098] Ten single colonies were selected for PCR identification, and the identification system and reaction conditions were referred to Tables 5 and 6.

[0099] Table 5 Colony PCR reaction system

[0100]

[0101] Table 6 PCR reaction program

[0102]

[0103] The target band is a fragment of about 800 bp.

[0104] One to three positive band corresponding bacterial liquid was selected, 100 μL was taken for sequencing, and the remaining 400 μL bacterial liquid was inoculated into 5-10 ml kanamycin-resistant LB, and the test tube was shaken. After the sequencing result was obtained, one tube was taken to extract the plasmid corresponding to the correct sequencing, and the strain and plasmid were preserved after the plasmid was extracted.

[0105] Example 4 Tobacco genetic transformation

[0106] 1) Agrobacterium preparation

[0107] Take 1 μL plasmid (overexpression vector or gene editing vector) into 50 μL GV3101 Agrobacterium competent cells, transform LB plate containing 50 μg / mL kanamycin resistance, 28°C culture for 48h, colony PCR identification. Amplification primer, reaction system and reaction program are the same as above. The PCR product is detected by gel electrophoresis, and the electrophoresis results of positive control (using the above sequencing correct plasmid as template) and sample are clear, the size is correct, and the negative control (template is water) has no band. Under the condition that the sample can enter the next step and can be used for infection of tobacco.

[0108] Select single colony in liquid LB medium containing 25 μg / mL rifampicin and 100 μg / mL kanamycin (Km) antibiotics, 28°C shaking bacteria for 24h; centrifuge the shaken bacteria at 4000r / min for 10min, discard the supernatant, resuspend in infiltration buffer (containing 10mM MgCl2, pH=5.2 10mM 2-(N-morpholino) ethanesulfonic acid (MES) and 0.1mM acetyl-syringone), incubate at room temperature for more than 3h, until OD 600 value is about 0.6 as the infection solution for standby.

[0109] 2) Genetic transformation

[0110] Select full and consistent "K326" tobacco seeds with 10% sodium hypochlorite solution for 15 min, rinse with sterile water 5 times, and slightly absorb with sterile absorbent paper, 4 grains / bottle on MS + sucrose 30 g / L + agar 8 g / L (pH = 5.8) medium. Put into 25℃ light incubator, culture conditions are light intensity 1600lx, light cycle 16h(light) / 8h(dark) for 45d. After the aseptic seedling grows 4 leaves, cut into about 5mmx5mm small pieces, remove the veins. After 2d pre-culture in MS medium containing 2mg / L 6-BA and 0.2mg / L IAA, soak in Agrobacterium infection solution. The infected explants are placed in MS medium containing 2mg / L 6-BA and 0.2mg / L IAA for dark culture for 2d. After co-culture, 50mg / L kanamycin and 500mg / L carbenicillin are added to the medium for screening to induce the production of resistant callus, and the culture conditions are the same as above, subculture every 14d. When the resistant buds on the callus grow to 2cm, the buds are moved to rooting medium (MS + 50mg / L kanamycin + 500mg / L carbenicillin + 0.2mg / L IAA), and root in about 7d. When the seedlings grow to about 6cm, open the bottle mouth of the culture bottle, and harden for 2d. Then the seedlings are transplanted into tobacco special substrate (Hunan Tianliang Agricultural Technology Development Co., Ltd.) sterilized by high temperature, covered with plastic film for moisture, and cultured under light at 25-27℃. Further harvest seeds.

[0111] Example 5 Positive seedling screening and identification

[0112] 1) Overexpression positive seedling screening and identification

[0113] (1) The harvested transgenic tobacco seeds of the current generation are germinated on medium containing 150μg / mL Hyg B. The whole process needs to be carried out in a sterile environment. First, soak the seeds in ddH2O for 12h; pour out the ddH2O in the EP tube, then add 1mL 75% alcohol and vortex for 30sec; pour out the alcohol and add 1mL ddH2O for 3 times of rinsing; add 1mL NaClO solution and mix well, the process should not exceed 5min; then rinse with sterile ddH2O four times; spread the rinsed seeds on MS medium containing 150μg / mL Hyg B. Finally, seal with sealing film and put into the tissue culture room for 7 days.

[0114] (2) The green seedlings grown on the medium are transferred to the soil for further culture. Wait for the seeds to mature and collect T1 generation seeds.

[0115] (3) The collected T1 generation seeds are further screened according to the method of step (1). Put into the tobacco tissue culture room for 7 days. The culture dish with all green seedlings in the same dish is considered as possible positive seedlings.

[0116] (4)Select 10 possible positive seedlings per dish and transfer them into soil for further culture. At the same time, collect the remaining small green seedlings in the dish, extract RNA, reverse transcribe into cDNA, and quantitatively detect the expression amount of the target gene.

[0117] (5) Extract transgenic tobacco RNA for expression verification of the target gene. The total RNA extraction and cDNA synthesis method are the same as above. Real-time quantitative PCR technology is used to detect the expression of the target gene NtTCP7.

[0118] NtTCP7 fluorescent quantitative primer:

[0119] NtTCP7-q-F: CCCTACGGAATTCTTCGGT; SEQ ID NO. 13;

[0120] NtTCP7-q-R: ACGCAGACGCTTCCCGAGCAAA; SEQ ID NO. 14;

[0121] Tobacco internal reference gene ACTIN7 fluorescent quantitative primer:

[0122] NtACT7-q-F: CCACACTGGTGTTATGGTTG; SEQ ID NO. 15;

[0123] NtACT7-q-R: AATACCGTGCTCAATTGGG; SEQ ID NO. 16.

[0124] The amplification system and procedure of real-time quantitative PCR (kit Thermo Fisher Scientific, item number: A25742) are shown in Tables 7 and 8.

[0125] Table 7 Real-time quantitative PCR reaction system

[0126]

[0127] Table 8 Real-time quantitative PCR reaction procedure

[0128]

[0129]

[0130] (5) After the seeds of the current generation plants mature, collect the seeds of the high-expression strain for subsequent phenotype identification.

[0131] NtTCP7 homozygous overexpression strain material screening results: through the 15 positive seedlings, the T0 seed was screened with 150 μg / mL hygromycin and identified by expression analysis to 2 high expression strains TCP7-OE#2 (OE#2) and TCP7-OE#5 (OE#5), further seed collection obtained T1 generation seed, T1 seed was screened with 150 μg / mL hygromycin to obtain T2 generation seed (pure line) Figure 3

[0132] 2) Gene knockout positive seedling screening and identification

[0133] Cut sterile rooting tissue culture seedling leaves, use PlaNt DNA Isolation Mini Kit (Nanjing Nuowezan, item number: DC104-01) plant total DNA extraction kit to extract DNA, detailed method refers to the instruction. Use the high fidelity enzyme PrimeSTAR Max DNA Polymerase of Baorui Biotechnology (Beijing) Co., Ltd. to perform PCR amplification on the knockout material.

[0134] The primer sequence information is as follows:

[0135] TCP7-crispr-F: CTACCCCCAACCCAGACTA; SEQ ID NO. 17;

[0136] TCP7-crispr-R: CAGAGAAGCTAGCAAATTCAGG; SEQ ID NO. 18.

[0137] Fragment length: 799 bp.

[0138] The PCR reaction system is shown in Table 9; the PCR program is shown in Table 10.

[0139] Table 9 PCR reaction system

[0140]

[0141] Table 10 PCR program

[0142]

[0143]

[0144] ​NtTCP7 homozygous knockout line material screening results: through sequencing analysis of 30 positive seedlings, two successfully edited lines tcp7-1-5 (ko#1) and tcp7-10-3 (ko#10) were screened from them. It was found that ko#1 had a base insertion at the target site 1; ko#10 had a base insertion at the target site 1 and a large base deletion at the target site 2, and the target site was clean single peak, indicating that the two lines were homozygous mutation. Figure 4 Protein sequence alignment found that the NtTCP7 protein translation in ko#1 and ko#10 lines was terminated prematurely, Figure 5 , indicating that the function of NtTCP7 gene in the two lines was destroyed.

[0145] Example 6 Tobacco cold tolerance phenotype identification

[0146] Select two knockout pure lines, two overexpression pure lines and wild type plant seeds with full and uniform size, disinfect and sterilize, and then place them in 4℃ low temperature treatment for 48h. Then sow them in small boxes filled with tobacco special substrate and fully soaked, cover with lid to keep warm and moist, and incubate germination in greenhouse (temperature 25℃, humidity 75%, 16h light, 8h darkness).

[0147] Low temperature stress treatment: after 30d of sowing (six-leaf-one-heart stage), select tobacco seedlings with consistent growth and place them in a 4℃ incubator for low temperature treatment, observe the phenotype and take pictures. At 0h and 5d time points, take the third leaf (counted from the top) as plant samples for physiological and biochemical index detection, with 5 biological replicates for each treatment. After sampling, quickly wrap with tin paper and put into liquid nitrogen, store at -80℃.

[0148] As shown in Figure 6 , there was no obvious difference in phenotype between the two overexpression and knockout lines and K326 (WT) under control conditions (temperature 25℃). After 6.5h of 4℃ low temperature treatment, the leaf wilting of the two overexpression lines was more severe compared with WT, while the leaf of the two knockout lines only showed slight drooping, with no obvious change from before treatment. The results showed that the cold tolerance of the two knockout lines was significantly enhanced compared with WT, and NtTCP7 negatively regulated the cold tolerance of tobacco.

[0149] Example 7 Physiological and biochemical index determination of knockout lines

[0150] The content of hydrogen peroxide (H2O2) was determined by hydrogen peroxide test kit (Nanjing Jiancheng, item number: A064-1-1); the superoxide anion capacity (OFR) was determined by superoxide anion (OFR) kit (Jiangsu Aidisen Biological Technology, item number: ADS-W-YH008); the specific method was carried out according to the kit instructions.

[0151] Relative conductivity: Fresh leaves of the 4th leaf from the top of seedlings were taken with a circular punch, 0.2 g of fresh tissue leaves were placed in a centrifuge tube containing 25 ml of ddH2O, and treated at 37°C for 24 h on a shaker. The first exudate conductivity value L1 was measured, and the EP tube was then placed in an autoclave at 120°C for 15 min. After the temperature cooled to room temperature, the second exudate conductivity value L2 was measured. The relative conductivity (EL = L1 / L2*100%) was calculated.

[0152] 3,3'-diaminobenzidine (DAB) staining: First, prepare 0.1 mg / mL DAB in 50 mM Tris-acetate buffer (pH 5.0). Soak the leaves in the staining solution at room temperature in the dark overnight. Remove the staining solution and add anhydrous ethanol, and boil in a water bath for 10 min (if the chlorophyll is difficult to dehydrate, the time can be longer). Finally, transfer the leaves to anhydrous ethanol, and take photos under a microscope or camera for observation and preservation.

[0153] Nitro blue tetrazolium chloride (NBT) staining: First, prepare NBT staining solution 1 mg / mL in 10 mM PBS (pH 7.8). Cut the leaves and place them in the NBT staining solution, and stain under light for 1-2 h. When the color phenotype appears (the leaves are blue), decolorize. Remove the staining solution and add anhydrous ethanol, and boil in boiling water for 10 min. Store in 70% ethanol and take photos under a microscope or camera for observation.

[0154] The physiological and biochemical results show that under the control condition, the EL (relative conductivity) of the two overexpression and knockout lines has no significant difference from that of the WT. After low temperature stress treatment, the EL of the two overexpression lines is significantly higher than that of the WT, and the EL of the two knockout lines is lower than that of the WT. Figure 7 ).

[0155] As shown in Figure 8 , before low temperature treatment, the OFR (superoxide anion) and H2O2 (hydrogen peroxide) contents of the overexpression lines and the knockout lines have no significant difference from those of the WT. After low temperature stress, the OFR of the two overexpression lines is significantly higher than that of the WT, the OFR content of ko#1 is significantly lower than that of the WT, and the OFR content of ko#10 is lower than that of the WT Figure 8 ). The H2O2 content of the two overexpression lines is higher than that of the WT, the H2O2 content of ko#1 is significantly lower than that of the WT, and the H2O2 content of ko#10 is lower than that of the WT Figure 8 ).

[0156] ROS histochemical staining was performed on the overexpression lines, the knockout lines and the wild type after 4°C (5 d) low temperature treatment. The NBT and DAB staining results show that Figure 9), under normal conditions, the leaves of overexpression and knockout lines all have slight blue spots / red-brown spots as compared with WT leaves, but there is no obvious difference between lines. After low temperature treatment, the leaves of two overexpression lines have larger staining area and darker staining degree than WT, indicating that the overexpression lines accumulate more OFR / H2O2 than WT plants; while the leaves of two knockout lines have smaller staining area and lighter staining degree than WT, indicating that the two knockout lines accumulate less OFR / H2O2 than WT plant leaves.

[0157] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended 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. Application of knocking out NtTCP7 gene in regulating tobacco cold tolerance, characterized in that, The NtTCP7 gene sequence is shown as SEQ ID NO.

1.

2. Use of biological material knocking out NtTCP7 gene in regulating tobacco cold tolerance, characterized in that, The NtTCP7 gene sequence is shown as SEQ ID NO.

1. The biological material is any one of the following: A: an expression cassette capable of silencing the NtTCP7 gene with a nucleotide sequence shown as SEQ ID NO. 1; B: a 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.

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