Application of NtMPK4 gene in tobacco cold tolerance

Abstract

This invention discloses the application of the NtMPK4 gene in tobacco cold tolerance, belonging to the field of molecular biology. The NtMPK4 gene sequence is shown in SEQ ID NO.1. The NtMPK4 gene negatively regulates tobacco cold tolerance. The discovery and functional identification of this gene provide important genetic resources and a theoretical basis for low-temperature tolerance breeding of tobacco.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and more specifically to the application of the NtMPK4 gene in tobacco cold tolerance. Background Technology

[0002] Tobacco (Nicotiana tabacum L.), native to tropical and subtropical regions, is an important leaf crop and model organism. It is susceptible to low-temperature stress, with an optimal growth temperature of 25-28℃. Low temperatures inhibit tobacco growth, leading to reduced quality and yield. Seedling tobacco is particularly vulnerable to low-temperature stress; exposure to 12℃ for two weeks in 6-7 leaf stage seedlings can cause premature flowering, reducing leaf yield and quality, resulting in significant economic losses for agricultural production and tobacco farmers. Therefore, comprehensively utilizing molecular biotechnology to discover low-temperature tolerant genes in tobacco, developing molecular markers, and achieving targeted improvement of tobacco varieties to cultivate new low-temperature tolerant varieties has become an indispensable method for developing superior germplasm resources.

[0003] The mitogen-activated protein kinase (MAPK, MPK) cascade pathway is widely distributed and highly conserved in eukaryotes, playing a crucial role in plant growth, development, and responses to environmental stimuli. During signal transduction, the MPK cascade pathway integrates and amplifies signals through stepwise phosphorylation, transmitting them to downstream substrates such as protein kinases and transcription factors, ultimately activating specific physiological responses in plant cells to external stimuli. The basic MPK cascade pathway generally consists of three parts: MAP kinase kinases (MAPKKK, MAP3K, MEKK), MAP kinase kinases (MKK, MAP2K, MEK), and MAP kinases (MAPK, MPK). Extracellular stimulation activates the plasma membrane receptor, which in turn activates the upstream MAPKKK. Once activated, MAPKKK activates downstream MAPKK by phosphorylating two serine or threonine residues in the S / T-X5-S / T motif of MAPKK. MAPKK is a dual phosphorylation specific kinase that phosphorylates the threonine and tyrosine residues in the TXY motif of downstream MAPK. MAPK is a conserved serine / threonine protein kinase that, upon activation, acts on different substrates through phosphorylation, thereby transferring extracellular signals into the cell and enabling the cell to respond to external stimuli, thus forming a complete MPK cascade pathway.

[0004] The MAPK cascade pathway is widely involved in plant responses to biotic and abiotic stresses. In Arabidopsis, some MAPK cascade pathways have been reported to participate in stress responses to pathogen infection, wounding, cold, drought, hyperosmolarity, high salinity, heavy metals, ozone, and UV radiation. The same MAPK can be activated by different upstream cascade pathways under different backgrounds. The MAPK cascade pathway also plays an important role in plant hormone signal transduction. Mizoguchi et al. found that treatment of auxin-deficient tobacco with the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in rapid and transient activation of a 46-kDa protein kinase, which can squalize myelin basic protein (MBP). MAPKs have also been reported to participate in the synthesis and signal transduction of jasmonic acid (JA) and salicylic acid (SA). Studies have also found that MAPKs are involved in ABA signal transduction; MAPK activity was observed in the protoplasts of ABA-treated guard cells.

[0005] Therefore, providing an application of the NtMPK4 gene in tobacco cold tolerance is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides the application of the NtMPK4 gene in tobacco cold tolerance.

[0007] This invention, based on previous analysis of phosphorylated proteomics in tobacco under low-temperature stress, identified a candidate gene, NtMPK4, which responds to low-temperature stress. Building upon this, this study identified the function of this gene and elucidated its function, providing genetic resources and a theoretical basis for molecular breeding of tobacco for low-temperature tolerance.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Application of the NtMPK4 gene in tobacco cold tolerance, wherein the NtMPK4 gene sequence is shown in SEQ ID NO.1.

[0010] Furthermore, the application of knocking out the NtMPK4 gene in the positive regulation of cold tolerance in tobacco, the NtMPK4 gene sequence of which is shown in SEQ ID NO.1.

[0011] Furthermore, the application of biomaterials with the NtMPK4 gene knocked out in the positive regulation of cold tolerance in tobacco, wherein the NtMPK4 gene sequence is shown in SEQ ID NO.1;

[0012] The biomaterial is any one of the following:

[0013] A: An expression cassette capable of silencing the NtMPK4 gene with a nucleotide sequence as shown in SEQ ID NO.1;

[0014] B: A recombinant vector containing the expression cassette described in A;

[0015] C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

[0016] Furthermore, the application of overexpression of the NtMPK4 gene in the negative regulation of cold tolerance in tobacco, wherein the NtMPK4 gene sequence is shown in SEQ ID NO.1.

[0017] Furthermore, the application of biomaterials overexpressing the NtMPK4 gene in negatively regulating cold tolerance in tobacco, wherein the NtMPK4 gene sequence is shown in SEQ ID NO.1;

[0018] The biomaterial is any one of the following:

[0019] A: An expression cassette capable of overexpressing the NtMPK4 gene with a nucleotide sequence as shown in SEQ ID NO.1;

[0020] B: A recombinant vector containing the expression cassette described in A;

[0021] C: Recombinant microorganisms containing the expression cassette described in A or the recombinant vector described in B.

[0022] Furthermore, the application of the NtMPK4 gene in tobacco breeding, the NtMPK4 gene sequence of which is shown in SEQ ID NO.1.

[0023] Furthermore, the application of the NtMPK4 gene in the breeding of cold-resistant tobacco germplasm, the NtMPK4 gene sequence of which is shown in SEQ ID NO.1.

[0024] As can be seen from the above technical solution, compared with the prior art, this invention discloses the application of the NtMPK4 gene in tobacco cold tolerance. A low-temperature response gene, NtMPK4, was isolated from tobacco. Sequence results show that the CDS sequence of this gene is 1122 bp in length, encoding 373 amino acids. Phylogenetic analysis results show that the protein encoded by this gene has high homology with NtoMMK2 from Tobacco velutipes and NaMMK2 from Tobacco stenoptera. By constructing an overexpression vector and transforming it into Xiangyan 7, two overexpression lines, OE#8 and OE#13, were obtained. Using Xiangyan 7 as a background, two knockout lines of this gene, KO#2 and KO#15, were obtained using CRISPR / Cas9 technology. Phenotypic identification results show that under low-temperature stress, compared with Xiangyan 7 (WT), the two overexpression lines exhibited more severe wilting, while the two knockout lines showed the opposite. Physiological and biochemical results showed that under low-temperature stress, the contents of malondialdehyde (MDA), relative conductivity (EL), hydrogen peroxide (H2O2), and superoxide anion (OFR) in the leaves of the overexpressing lines were significantly higher than those in the WT lines, while the two knockout lines showed the opposite. DAB and NBT staining results indicated that, compared to WT, the two overexpressing lines had larger stained leaf areas, while the two knockout lines showed the opposite. In summary, these results indicate that the NtMPK4 gene negatively regulates cold tolerance in tobacco. The discovery and functional identification of this gene provide important genetic resources and a theoretical basis for low-temperature tolerance breeding in tobacco. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0026] Figure 1Phylogenetic tree of NtMPK4; where (Nt)Nicotianatabacum; (Nto)Nicotianatomentosiformis; (Na)Nicotiana attenuata; (Lb)Lyciumbarbarum; (Ca)Capsicumannuum; (Sp)Solanum pennellii; (Sl)Solanum lycopersicum; (Cs)Camellia sinensis; (Lt)Ipomoeatriloba; (Cp)Caricapapaya; (Ha)Helianthus annuus; (Vv)Vitis vinifera; (St)Solanum tuberosum; (Cm)Camellia amiltiorrhiza;

[0027] Figure 2 Similarity comparison of NtMPK4 homologous proteins;

[0028] Figure 3 Analysis of NtMPK4 overexpression lines;

[0029] Figure 4 Sequence alignment and peak plot analysis were performed near the target site in the knockout strains.

[0030] Figure 5 For sequence alignment of NtMPK4 protein in knockout lines and WT;

[0031] Figure 6 Results of cold tolerance phenotype identification and physiological and biochemical index determination of tobacco knockout lines;

[0032] Among them, A: Phenotype after 4 hours of low-temperature treatment: the left figure is a top view and the right figure is a side view; B: NBT and DAB staining results after 3 days of low-temperature treatment; C: H2O2 change analysis after 3 days of low-temperature treatment; D: OFR change analysis after 3 days of low-temperature treatment; E: MDA change analysis after 3 days of low-temperature treatment; F: EL change analysis after 3 days of low-temperature treatment.

[0033] Figure 7 Results of cold tolerance phenotype identification and physiological and biochemical index determination of tobacco overexpression lines;

[0034] Among them, A: Phenotype after 3h of low temperature treatment: the left figure is a top view and the right figure is a side view; B: NBT and DAB staining results after 3d of low temperature treatment; C: H2O2 change analysis after 3d of low temperature treatment; D: OFR change analysis after 3d of low temperature treatment; E: MDA change analysis after 3d of low temperature treatment; F: EL change analysis after 3d of low temperature treatment. Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] The NtMPK4 CDS sequence is shown in SEQ ID NO.1.

[0037] ATGGAAGCAATTTCAGGTGATCAAGGTGTAC AAAGTAATTTCAAAGGAGTTCCAACACATGGGGGTCGTTATGCGCAGTATAATGTGTATGGTAATCTCTTTGAAGTTTCCAA AAAGTATGTTCCCCTTAGGCCGGTTGGTCGTGGAGCTTATGGCATCGTTTGTGCCGCTATGAACTCTGAAACACGTGAGGAAGTAGCTATTAAGAAGATTGGCAATGCATTTGATAATAGAATTGATGCGAAAAGGACACTACGAGAGATAAAGCTTCTTCGTCACATGGATCATGACAATGTGATTGCCACTAAAGATATTATAAGGCCTCCACAAACTGAGAATTTCAATGATGTCTACATTGTTTATGAATTGATGGACACTGATCTTCATCAGATAATTCGTTCCAACCAACAGTTGACTGATGATCACTGTCGGTATTTCCTATACCAAATATTACGAGGACTTAAGTACATTCACTCTGCCAACGTCCTGCATCGTGATCTAAAACCTAGCAATTTGTTTCTCAATGCGAATTGTGACCTTAAAGTTGGAGATTTTGGGCTTGCAAGGACAACATCCGAGACAGATTTCATGACGGAGTATGTCGTAACGCGGTGGTATCGGGCACCGGAGTTGCTCCTAAATTGTTCAGAATATACAGCAGCAATTGATATCTGGTCAGTAGGTTGCATACTGGGTGAGATGATGACAAGACAACCTCTCTTTCCCGGCAAGGACTATGTTCACCAGTTGAAACTTATCACAGAGCTCATAGGATCACCTGATGATGCCAGTCTTGGATTTCTCCGGAGTGATAATGCTCGGAGATATGTTAGACAGCTCCCCCAGTATCCAAGACAACAATTTGCTGCTAGATTCCCCAATTCATCTCCTGGAGCTGTTGATTTGCTTGAAAAAATGCTTGTCTTTGATCCAAGCAGGCGTGTTACAGTTGATCAAGCGCTCTGCCACCCCTACTTGGCGCCTCTTCATGATATCAATGAGGAGCCCATTTGTCCTAAACCTTTCAGTTTTGACTTTGAGCAGCCATCTTTTACTGAAGAAAATATCAAGGAGCTCATCTGGAGGGAAT CCGTGAAATTTAATCCAGATCCAACTCACTGA ;SEQ IDNO.1。

[0038] The NtMPK4 protein sequence is shown in SEQ ID NO.2.

[0039] MEAISGDQGVQSNFKGVPTHGGRYAQYNVYGNLFEVSKKYVPLRPVGRGAYGIVCAAMNSETREEVAIKKIGNAFDNRIDAKRTLREIKLLRHMDHDNVIATKDIIRPPQTENFNDVYIVYELMDTDLHQIIRSNQQLTDDHCRYFLYQILRGLKYIHSANVLHRDLKPSNLFLNANCDLKVGDFGLA RTTSETDFMTEYVVTRWYRAPELLLNCSEYTAAIDIWSVGCILGEMMTRQPLFPGKDYVHQLKLITELIGSPDDASLGFLRSDNARRYVRQLPQYPRQQFAARFPNSPGAVDLLEKMLVFDPSRRVTVDQALCHPYLAPLHDINEEPICPKPFSFDFEQPSFTEENIKELIWRESVKFNPDPTH; SEQ ID NO.2.

[0040] Example 1: Gene Sequence Feature Analysis

[0041] The NtMPK4 protein sequence used in this study was obtained from the National CeNtoer for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / BLAST / ). Software was used to analyze gene homology and the neighbor-to-neighbor merging (NJ) method was used to construct a phylogenetic tree. Software was also used to perform protein similarity comparisons.

[0042] Phylogenetic analysis revealed that NtMPK4 is most closely related to NtoMMK2 (XP_070038289.1) of Nicotiana pubescens. Figure 1 Homology analysis of the protein sequence revealed that the protein sequence possesses a typical MAPK domain and shows high homology with NtoMMK2 from Nicotiana fluff and NaMMK2 from Nicotiana narrower leaf. Figure 2 ).

[0043] Example 2 Construction of overexpression vector

[0044] 1) cDNA Acquisition

[0045] (1) RNA extraction

[0046] This experiment mainly used the FastPure Universal PlaNto 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.

[0047] (2) Reverse transcription

[0048] This experiment used the HiScript III reverse transcription kit. st Use the Strand cDNA SyNtohesis Kit (+gDNAwiper) to synthesize cDNA (Novozymes, catalog number: R312-02). Refer to the instruction manual for specific steps.

[0049] 2) Synthetic primers

[0050] Synthesize the following target gene amplification primers:

[0051] PrimerF1:

[0052] AACACGGGGGACTTTGCAACatggaagcaatttcaggtgatcaaggtgtac;SEQ ID NO.3;

[0053] Primer R1:

[0054] TGAAGACAGAGCTAGTTACAtcagtgagttggatctggattaaatttcacgg; SEQ ID NO.4.

[0055] 3) Amplify the target fragment

[0056] The amplification reaction was carried out according to the following system and procedure:

[0057] PCR system: Nuclease-free Water 20 μL, BiorunPfu PCR Mix 25 μL, Primer F12 μL, Primer R12 μL, cDNA 1 μL, Total volume 50 μL.

[0058] 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.

[0059] Then, the Gname (1122bp) fragment was extracted by 1% agarose gel electrophoresis at 5V / cm for 20 minutes under UV light and placed in a system for sol-gel recovery. The recovery procedure is as per the instructions of the specific manufacturer's DNA recovery kit (Novozymes, catalog number: DC301-01). The recovered DNA was dissolved and recovered with 40μL of water (the recovered product was labeled as: rDNAG1). After verification, it was recombined with the vector.

[0060] 4) Recombination of target fragment with vector

[0061] (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.

[0062] (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 dish and incubate overnight at 37°C.

[0063] (3) Selecting positive clones

[0064] Select single colonies for colony PCR. Primer sequences are as follows:

[0065] Primer F2: tTCATTTGGAGAGAACACGGGggac; SEQ ID NO.5;

[0066] Primer R2: ctccggtgcccgataccac; SEQ ID NO. 6.

[0067] PCR system: Nuclease-free Water 9.5μL; Biorun Magic PCR Mix 12.5μL; PrimerF2 (100μM) 1μL; Primer R2 (100μM) 1μL; Template 1μL; Total volume 25μL.

[0068] 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.

[0069] Then, positive clones were identified using 1% agarose gel electrophoresis. The target band was a fragment of approximately 1122 bp. 200 μL of bacterial culture corresponding to 1-3 positive bands was sent to a sequencing company for validation.

[0070] 5) Extract plasmid pBWA(V)HS-ccdB(D)-NtMPK4

[0071] 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).

[0072] Example 3: Construction of gene editing vector

[0073] 1) Target design

[0074] 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:

[0075] Target1: TCAAAGGAGTTCCAACACATCGG; SEQ ID NO.7;

[0076] Target2:AAAGTATGTTCCCCTTAGGC CGG ; SEQ ID NO.8.

[0077] Then, primers for the CRSIPR vector were designed, and their sequence information is as follows:

[0078] F1(+):cagtGGTCTCatgcaTCAAAGGAGTTCCAACACAT; SEQ ID NO.9;

[0079] R1(-): cgatGGTCTCaaaacGCCTAAGGGGAACATACTTT; SEQ ID NO. 10.

[0080] 2) PCR amplification

[0081] Prepare a 50 μL system according to Table 1 and perform the amplification reaction according to the procedure in Table 2.

[0082] Table 1 PCR system

[0083]

[0084] Table 2 PCR Procedure

[0085]

[0086] 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.

[0087] 3) Enzyme 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 Enzyme digestion and ligation system

[0090]

[0091] Table 4 Enzyme digestion and ligation reaction conditions

[0092]

[0093] 4) Transformation and identification

[0094] Transform 5-10 μL of the ligation product into competent E. coli cells according to the manufacturer's 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.

[0095] 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. The identification system and reaction conditions are shown in Tables 5 and 6.

[0099] Table 5 Colony PCR Reaction System

[0100]

[0101]

[0102] Table 6 PCR reaction procedure

[0103]

[0104] The target band is a fragment of approximately 800bp.

[0105] 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.

[0106] Example 4 Tobacco Genetic Transformation

[0107] 1) Preparation of Agrobacterium

[0108] 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.

[0109] 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 was reached. 600 The value is approximately 0.6, which is used as a pre-treatment solution.

[0110] 2) Genetic transformation

[0111] Select plump and uniform "Xiangyan No. 7" tobacco seeds, disinfect them with 10% sodium hypochlorite solution for 15 minutes, rinse them 5 times with sterile water, and slightly dry them 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.

[0112] Example 5: Screening and Identification of Positive Seedlings

[0113] 1) Screening and identification of overexpression-positive seedlings

[0114] (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 Hyg B. Finally, the tubes were sealed with sealing film and cultured in a tissue culture room for 7 days.

[0115] (2) The green seedlings that grow from the culture medium are transferred to soil for further cultivation. After the seeds mature, the T1 generation seeds are harvested by division.

[0116] (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.

[0117] (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.

[0118] (5) RNA was extracted from transgenic tobacco to verify the expression of the target gene. The methods for total RNA extraction and cDNA synthesis were the same as above. The expression of the target gene NtMPK4 was detected using real-time quantitative PCR.

[0119] NtMPK4 fluorescence quantitative primers:

[0120] NtMPK4-qF: GGATCATGACAATGTGATTG; SEQ ID NO.13;

[0121] NtMPK4-qR: TGGTTGGAACGAATTATCTG; SEQ ID NO. 14.

[0122] Primers for quantitative real-time analysis of the tobacco internal reference gene ACTIN7:

[0123] NtACT7-qF: CCACACTGGTGTTATGGTTG; SEQ ID NO.15;

[0124] NtACT7-qR: AATACCGTGCTCAATTGGG; SEQ ID NO. 16.

[0125] The amplification system and procedure for real-time quantitative PCR (Thermo Fisher Scientific kit, catalog number: A25742) are shown in Tables 7 and 8.

[0126] Table 7 Real-time quantitative PCR reaction system

[0127]

[0128] Table 8 Real-time quantitative PCR reaction program

[0129]

[0130] (5) Once the seeds of this generation of plants mature, collect seeds from lines with high expression levels for subsequent phenotypic identification.

[0131] Screening results of NtMPK4 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, OE#8 and OE#13, were identified through expression analysis. Further harvesting of T1 generation seeds yielded T2 generation seeds (pure lines), which were then treated with 150 μg / mL hygromycin. Figure 3 ).

[0132] 2) Screening and identification of gene knockout positive seedlings

[0133] Cut leaves from sterile rooted tissue culture seedlings and use... The PlaNto DNA Isolation Mini Kit (Nanjing Novizan, catalog number: DC104-01) was used to extract total DNA from plants. Detailed instructions were provided in the product manual. Knockout materials were amplified by PCR using PrimeSTAR Max DNA Polymerase, a high-fidelity enzyme from Baori Biotechnology (Beijing) Co., Ltd. The templates were DNA from Xiangyan No. 7, KO#2, and KO#15.

[0134] Primer sequence information is as follows:

[0135] MPK4-crispr-F: ATGGGGGGTGGTGGTACATT; SEQ ID NO.17;

[0136] MPK4-crispr-R: CCGATCCACAAATAGCTGGAGC; SEQ ID NO. 18.

[0137] Fragment length: 1161bp.

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

[0139] Table 9 PCR Reaction System

[0140]

[0141] Table 10 PCR Procedure

[0142]

[0143] Screening results of NtMPK4 homozygous knockout lines: Sequencing analysis of 30 positive seedlings revealed two successfully edited lines, KO#2 and KO#15. Furthermore, it was found that mpk4-2 (KO#2) had a base deletion at target site 1, while mpk4-15 (KO#15) had a base insertion at target site 1. The clean single peaks near the target sites indicate that these two lines are homozygous mutants. Figure 4 Protein sequence alignment revealed that the translation of NtMPK4 protein terminated prematurely in the mpk4-2 and mpk4-15 lines. Figure 5 This indicates that the NtMPK4 gene function was disrupted in both strains.

[0144] Example 6: Identification of tobacco cold tolerance phenotype and determination of physiological and biochemical indicators

[0145] Using Xiangyan 7 as a background, two knockout lines, KO#2 and KO#15, for this gene were obtained using CRISPR / Cas9 technology. Overexpression vectors were constructed and transformed into Xiangyan 7 to obtain two overexpression lines, OE#8 and OE#13. Seeds from WT plants, the two knockout lines, and the two overexpression lines were washed, disinfected, and then subjected to a 4°C low-temperature treatment for 48 hours. They were then sown in small square boxes pre-filled with tobacco-specific substrate and fully watered, covered for warmth and humidity control, and cultured in an artificial climate chamber (temperature 25°C, relative humidity 75%, light / dark = 16h / 8h) for 30 days until further treatment. Seedlings with uniform growth were selected, placed at 4°C for low-temperature treatment, and their phenotypes were observed and photographed.

[0146] At 0h and 3d time points, the third leaf (counting from the top down) was taken as a plant sample for physiological and biochemical index detection, with 5 biological replicates for each treatment. After sampling, the samples were quickly wrapped in aluminum foil and placed in liquid nitrogen for storage at -80℃.

[0147] Malondialdehyde (MDA) content was determined using a plant-based MDA test kit (Nanjing Jiancheng, catalog number: A003-3-1); hydrogen peroxide (H2O2) content was determined using a hydrogen peroxide test kit (Nanjing Jiancheng, catalog number: A064-1-1); and 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.

[0148] 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%).

[0149] 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 incubation time can be longer). Finally, transfer the leaves to anhydrous ethanol, photograph and observe them under a microscope or camera, and preserve them.

[0150] 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.

[0151] like Figure 6 As shown in Figure A, under control conditions (temperature 25℃), the phenotypes of the two knockout lines were not significantly different from WT (Xiangyan 7). After 4 hours of low-temperature treatment at 4℃, compared with WT, the leaves of the two knockout lines only showed slight drooping, with no significant change from before the treatment. This result indicates that the cold tolerance of the two knockout lines was significantly enhanced compared with WT, and NtMPK4 negatively regulates the cold tolerance of tobacco. Furthermore, physiological and biochemical results showed that under control conditions, the contents of H2O2, OFR, EL, and MDA in the two knockout lines were not significantly different from those in WT. Three days after low-temperature stress treatment, the contents of H2O2 (OFR, EL, and MDA in the two knockout lines were significantly lower than those in WT.) Figure 6 C), OFR ( Figure 6 D), EL Figure 6 F) and MDA Figure 6 E) lower than WT. ROS tissue staining was performed on knockout lines and wild-type plants treated with 4℃ (3d). NBT staining results showed that under normal conditions, there was no significant difference between the leaves of the knockout lines and WT leaves. After low-temperature stress, the stained area and lightest staining of the leaves of both knockout lines were smaller than those of WT, indicating that WT plants accumulated more OFR than knockout plants. DAB staining results showed that under normal conditions, there was no significant difference between the leaves of the knockout lines and WT leaves. After low-temperature stress, the stained area and lightest staining of the leaves of both knockout lines were smaller than those of WT, indicating that WT plants accumulated more H2O2 ( Figure 6 B).

[0152] like Figure 7 As shown in Figure A, under control conditions, the two overexpression lines showed no significant difference in phenotype compared to WT (Xiangyan 7). After 3 hours of low-temperature treatment at 4℃, compared to WT, the leaves of the two overexpression lines wilted more severely. This result further indicates that the two overexpression lines had significantly weaker cold tolerance, and NtMPK4 negatively regulates the cold tolerance of tobacco. Furthermore, physiological and biochemical results showed that under control conditions, the contents of H2O2, OFR, EL, and MDA in the two overexpression lines were not significantly different from those in WT. However, 3 days after low-temperature stress treatment, the contents of H2O2 (OFR, EL, and MDA) in the two overexpression lines significantly increased. Figure 7 C), OFR ( Figure 7 D), EL Figure 7 F) and MDA Figure 7E) higher than WT. ROS tissue staining was performed on the overexpression lines and wild-type plants treated with 4℃ (3d). NBT staining results showed that under normal conditions, there was no significant difference between the leaves of the overexpression lines and WT leaves. After low-temperature stress, the leaves of both overexpression lines had a larger stained area and a deeper staining degree than WT, indicating that the overexpression plants accumulated more OFR. DAB staining results showed that under normal conditions, there was no significant difference between the leaves of the overexpression lines and WT leaves. After low-temperature stress, the leaves of both overexpression lines had a larger stained area and a deeper staining degree than WT, indicating that the overexpression plants accumulated more H2O2 ( Figure 7 B).

[0153] 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. Knocking out NtMPK4 the application of a gene in the positive regulation of tobacco cold tolerance, characterized in that, The NtMPK4 The gene sequence is shown as SEQ ID NO.

1.

2. Knockout NtMPK4 The use of the biological material of the gene in regulating the cold tolerance of tobacco, characterized in that, The NtMPK4 The gene sequence is shown as SEQ ID NO. 1; The biomaterial is any one of the following: A: capable of causing the nucleotide sequence as shown in SEQ ID NO. 1 NtMPK4 gene silencing expression cassette; 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 NtMPK4 Application of genes in breeding of tobacco for cold tolerance, characterized in that, The NtMPK4 The gene sequence is shown in SEQ ID NO.

1.

4. Knockout NtMPK4 The application of genes in breeding tobacco cold-tolerant germplasm, characterized in that, The NtMPK4 The gene sequence is shown in SEQ ID NO.1.

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