Application of NtMPK4 gene in cold resistance of tobacco

Abstract

The invention discloses application of an NtMPK4 gene in cold resistance of tobacco, and belongs to the technical field of molecular biology. According to the application of the NtMPK4 gene disclosed by the invention in the cold resistance of the tobacco, the sequence of the NtMPK4 gene is as shown in SEQ ID NO. 1. The NtMPK4 gene negatively regulates the cold resistance of the tobacco, and the excavation and functional identification of the gene provide important gene resources and theoretical basis for low-temperature-resistant breeding of the tobacco.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of molecular biology, and more particularly to application of NtMPK4 gene in tobacco cold tolerance. BACKGROUND

[0002] Tobacco (Nicotiana tabacum L.) is an important leaf economic crop and model organism, which is originally from tropical and subtropical regions, and is susceptible to low temperature stress, with the optimum growth temperature being 25-28 DEG C. Under low temperature conditions, the growth of tobacco is hindered, and the quality and yield of tobacco are reduced. The tobacco seedlings of 6-7 leaf age are more susceptible to low temperature stress, and if the tobacco seedlings are subjected to 12 DEG C low temperature stress for two weeks, the tobacco will appear early flowering phenomenon, the yield and quality of tobacco leaves are reduced, and great economic losses are caused to agricultural production and tobacco farmers. Therefore, comprehensive utilization of molecular biological technology to mine tobacco low temperature resistant genes, development of molecular markers, realization of directional improvement of tobacco varieties and cultivation of new tobacco varieties with low temperature resistance have become an indispensable method for cultivating excellent germplasm resources.

[0003] The mitogen-activated protein kinase (MAPK, MPK for short) cascade pathway widely exists in eukaryotes and is highly conserved, and plays a very important role in the growth and development of plants and response to environmental stimuli. In the process of signal transmission, the MPK cascade pathway amplifies and transmits signals to downstream protein kinases, transcription factors and other substrates through step-by-step phosphorylation, and finally activates specific physiological responses of plant cells to external stimuli. The basic MPK cascade pathway is generally composed of three parts: MAP kinase kinase kinase (MAPKKK, MAP3K, MEKK), MAP kinase kinase (MKK, MAP2K, MEK) and MAP kinase (MAPK, MPK). After the extracellular stimulation activates the plasma membrane receptor, the uppermost MAPKKK is activated, the activated MAPKKK activates the downstream MAPKK by phosphorylating two serine or threonine residues in the S / T-X5-S / T motif of MAPKK, and the MAPKK is a double phosphorylation specific kinase, which phosphorylates the threonine and tyrosine residues in the T-X-Y motif of the downstream MAPK. The MAPK is a conserved serine / threonine protein kinase, which is activated by phosphorylation on different substrates, thereby converting extracellular signals to intracellular, so that the cell responds to external stimuli, and constitutes a complete MPK cascade pathway.

[0004] The MAPK cascade pathway is widely involved in the response of plants to biological and non-biological stresses. It has been reported that some MAPK cascade pathways in Arabidopsis are involved in pathogen infection, wound, cold, drought, high osmotic, high salt, heavy metal, ozone and UV radiation stress responses. The same MAPK can be activated by different upstream cascade pathways in different contexts. The MAPK cascade pathway also plays an important role in the signal transduction of plant hormones. Mizoguchi et al. found that when auxin-deficient tobacco was treated with synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), rapid and transient activation of a 46-kDa protein kinase activity was detected, which could phosphorylate myelin basic protein (MBP). MAPK is also reported to be involved in the synthesis and signal transduction of JA (Jasmonic acid) and SA (Salicylic acid). Studies have found that MAPKs are also related to ABA signal transduction. MAPK activity is observed in guard cell protoplasts after ABA treatment.

[0005] Therefore, providing the application of NtMPK4 gene in tobacco cold tolerance is an urgent problem for those skilled in the art. SUMMARY

[0006] Therefore, providing the application of NtMPK4 gene in tobacco cold tolerance is an urgent problem for those skilled in the art.

[0007] Based on the previous phosphoproteomic analysis of tobacco under low temperature stress, a candidate gene NtMPK4 responding to low temperature stress was mined. On this basis, the function of the gene was identified, and the function of the gene was analyzed, which provided gene resources and theoretical basis for tobacco low temperature tolerance molecular breeding.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows:

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

[0010] Further, the application of knocking out NtMPK4 gene in regulating tobacco cold tolerance, wherein the sequence of the NtMPK4 gene is shown as SEQ ID NO. 1.

[0011] Further, the application of biological material for knocking out NtMPK4 gene in regulating tobacco cold tolerance, wherein the sequence of the NtMPK4 gene is shown as SEQ ID NO. 1.

[0012] The biological material is any one of the following:

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

[0014] B: a recombinant vector comprising the expression cassette of A;

[0015] C: a recombinant microorganism comprising the expression cassette of A or the recombinant vector of B.

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

[0017] Further, the application discloses an application of a biomaterial of overexpression of the NtMPK4 gene in negative regulation of cold tolerance of tobacco, wherein the NtMPK4 gene sequence is shown as SEQ ID NO. 1.

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

[0019] A: an expression cassette capable of overexpression of the NtMPK4 gene with the nucleotide sequence shown as SEQ ID NO. 1;

[0020] B: a recombinant vector comprising the expression cassette of A;

[0021] C: a recombinant microorganism comprising the expression cassette of A or the recombinant vector of B.

[0022] Further, the application discloses an application of the NtMPK4 gene in tobacco breeding, wherein the NtMPK4 gene sequence is shown as SEQ ID NO. 1.

[0023] Further, the application discloses an application of the NtMPK4 gene in screening of cold-tolerant germplasm of tobacco, wherein the NtMPK4 gene sequence is shown as SEQ ID NO. 1.

[0024] Compared with the prior art, the application provides application of the NtMPK4 gene in tobacco cold tolerance, a low temperature response gene NtMPK4 is isolated from tobacco, sequence results show that the length of the CDS sequence of the gene is 1122 bp, and 373 amino acids are encoded. The phylogenetic tree analysis result shows that the protein encoded by the gene has higher homology with NtoMMK2 of Nicotiana tomentosiformis and NaMMK2 of Nicotiana attenuata. By constructing an overexpression vector, Xiangyan No. 7 is transformed, and two overexpression lines OE#8 and OE#13 are obtained. Taking Xiangyan No. 7 as the background, two knockout lines KO#2 and KO#15 of the gene are obtained by using the CRISPR / Cas9 technology. The phenotype identification result shows that, under low temperature stress, compared with Xiangyan No. 7 (WT), the wilting degree of the two overexpression lines is more serious, and the two knockout lines are on the contrary. The physiological and biochemical results show 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 overexpression lines are significantly higher than those of WT, and the two knockout lines are on the contrary. The DAB and NBT staining results show that, compared with WT, the staining area of the leaves of the two overexpression lines is larger, and the two knockout lines are on the contrary. The above results show that the NtMPK4 gene negatively regulates the cold tolerance of tobacco, and the mining and functional identification of the gene provide important gene resources and theoretical basis for tobacco low temperature breeding. BRIEF DESCRIPTION OF DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or the prior art description will be briefly introduced. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can be obtained by those skilled in the art without creative labor on the basis of the provided drawings.

[0026] Figure 1Figure 1 is a phylogenetic tree of NtMPK4; wherein, (Nt) Nicotiana tabacum; (Nto) Nicotiana tomentosiformis; (Na) Nicotiana attenuata; (Lb) Lycium barbarum; (Ca) Capsicum annuum; (Sp) Solanum pennellii; (Sl) Solanum lycopersicum; (Cs) Camellia sinensis; (Lt) Ipomoea triloba; (Cp) Carica papaya; (Ha) Helianthus annuus; (Vv) Vitis vinifera; (St) Solanum tuberosum; (Cm) Camellia miltiorrhiza;

[0027] Figure 2 Figure 2 is a similarity alignment of NtMPK4 homologous proteins;

[0028] Figure 3 Figure 3 is an expression analysis of NtMPK4 overexpression lines;

[0029] Figure 4 Figure 4 is a sequence alignment and peak analysis of the target site of knockout lines;

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

[0031] Figure 6 Figure 6 is the results of phenotype identification and physiological and biochemical index determination of tobacco knockout lines;

[0032] A: Phenotype after 4h of low temperature treatment: the left image is a top view and the right image 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;

[0033] Figure 7 Figure 7 is the results of phenotype identification and physiological and biochemical index determination of tobacco overexpression lines;

[0034] A: Phenotype after 3h of low temperature treatment: the left image is a top view and the right image 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 DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described in combination with the accompanying drawings of the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

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

[0037] ATGGAAGCAATTTCAGGTGATCAAGGTGTAC AAAGTAATTTCAAAGGAGTTCCAACACATGGGGGTCGTTATGCGCAGTATAATGTGTATGGTAATCTCTTTGAAGTTTCCAA AAAGTATGTTCCCCTTAGGCCGGTTGGTCGTGGAGCTTATGGCATCGTTTGTGCCGCTATGAACTCTGAAACACGTGAGGAAGTAGCTATTAAGAAGATTGGCAATGCATTTGATAATAGAATTGATGCGAAAAGGACACTACGAGAGATAAAGCTTCTTCGTCACATGGATCATGACAATGTGATTGCCACTAAAGATATTATAAGGCCTCCACAAACTGAGAATTTCAATGATGTCTACATTGTTTATGAATTGATGGACACTGATCTTCATCAGATAATTCGTTCCAACCAACAGTTGACTGATGATCACTGTCGGTATTTCCTATACCAAATATTACGAGGACTTAAGTACATTCACTCTGCCAACGTCCTGCATCGTGATCTAAAACCTAGCAATTTGTTTCTCAATGCGAATTGTGACCTTAAAGTTGGAGATTTTGGGCTTGCAAGGACAACATCCGAGACAGATTTCATGACGGAGTATGTCGTAACGCGGTGGTATCGGGCACCGGAGTTGCTCCTAAATTGTTCAGAATATACAGCAGCAATTGATATCTGGTCAGTAGGTTGCATACTGGGTGAGATGATGACAAGACAACCTCTCTTTCCCGGCAAGGACTATGTTCACCAGTTGAAACTTATCACAGAGCTCATAGGATCACCTGATGATGCCAGTCTTGGATTTCTCCGGAGTGATAATGCTCGGAGATATGTTAGACAGCTCCCCCAGTATCCAAGACAACAATTTGCTGCTAGATTCCCCAATTCATCTCCTGGAGCTGTTGATTTGCTTGAAAAAATGCTTGTCTTTGATCCAAGCAGGCGTGTTACAGTTGATCAAGCGCTCTGCCACCCCTACTTGGCGCCTCTTCATGATATCAATGAGGAGCCCATTTGTCCTAAACCTTTCAGTTTTGACTTTGAGCAGCCATCTTTTACTGAAGAAAATATCAAGGAGCTCATCTGGAGGGAAT CCGTGAAATTTAATCCAGATCCAACTCACTGA ; SEQ ID NO. 1.

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

[0039] MEAISGDQGVQSNFKGVPTHGGRYAQYNVYGNLFEVSKKYVPLRPVGRGAYGIVCAAMNSETREEVAIKKIGNAFDNRIDAKRTLREIKLLRHMDHDNVIATKDIIRPPQTENFNDVYIVYELMDTDLHQIIRSNQQLTDDHCRYFLYQILRGLKYIHSANVLHRDLKPSNLFLNANCDLKVGDFGLARTTSETDFMTEYVVTRWYRAPELLLNCSEYTAAIDIWSVGCILGEMMTRQPLFPGKDYVHQLKLITELIGSPDDASLGFLRSDNARRYVRQLPQYPRQQFAARFPNSSPGAVDLLEKMLVFDPSRRVTVDQALCHPYLAPLHDINEEPICPKPFSFDFEQPSFTEENIKELIWRESVKFNPDPTH; SEQ ID NO. 2.

[0040] Example 1: Analysis of gene sequence characteristics

[0041] The NtMPK4 protein sequence involved in this study is from the National CeNtoer for Biotechnology Information website (http: / / www.ncbi.nlm.nih.gov / BLAST / ), and the software is used to analyze the gene homology and construct the phylogenetic tree using the NJ method. The software is used for protein similarity comparison.

[0042] Phylogenetic tree analysis found that the NtMPK4 has the closest genetic relationship with NtoMMK2 (XP_070038289.1) of Nicotiana tomentosiformis ( Figure 1 ). Protein sequence homology analysis found that the protein sequence has a typical MAPK domain, and has high homology with NtoMMK2 and NaMMK2 of Nicotiana attenuata ( Figure 2 ).

[0043] Example 2: Construction of overexpression vector

[0044] 1) cDNA acquisition

[0045] (1) RNA extraction

[0046] The experiment mainly uses the kit FastPure Universal PlaNto Total RNA Isolation Kit to extract plant total RNA (Novozyme, item number: RC411-01), and the specific steps refer to the instruction manual.

[0047] (2) Reverse transcription

[0048] The experiment uses the reverse transcription kit HiScriptIII 1 st Strand cDNA SyNtohesis Kit (+gDNAwiper) to synthesize cDNA (Novozyme, item number: R312-02), and the specific steps refer to the instruction manual.

[0049] 2) Synthesis of primers

[0050] The following target gene amplification primers are synthesized:

[0051] Primer F1:

[0052] AACACGGGGGACTTTGCAACatggaagcaatttcaggtgatcaaggtgtac; SEQ ID NO. 3;

[0053] Primer R1:

[0054] TGAAGACAGAGCTAGTTACAtcagtgagttggatctggattaaatttcacgg; SEQ ID NO. 4.

[0055] 3) Amplification of target fragments

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

[0057] PCR system: Nuclease-free water 20 μL, Biorun Pfu PCR Mix 25 μL, Primer F 12 μL, Primer R 12 μL, cDNA 1 μL, total volume 50 μL.

[0058] PCR program: 94℃ 5min; 94℃ 30sec, 50℃ 45sec, 72℃ 106sec, 30 cycles; 72℃ 10min; 16℃ 30min.

[0059] Then use 1% agarose gel electrophoresis, 5v / cm voltage, 20 minutes, electrophoresis fragments of Gname(1122bp) are cut out under the ultraviolet lamp, placed in a system for gel recovery, see the manufacturer's DNA recovery kit(Novagen, item number: DC301-01) instructions, dissolve the recovered DNA with a total volume of 40μL of water(recovery product labeled: rDNAG1), and after testing no error, recombine with the vector.

[0060] 4) Recombination of the target fragment and the vector

[0061] (1) The target fragment obtained in step 3) and the BsaI / EcoI enzyme-purified linearized vector pBWA(V)HS-ccdB(D) are added to the recombination system in a molar ratio of 2:1(Novagen, IIOne StepCloning Kit) for reaction, refer to the instructions for specific methods.

[0062] (2) Take 1μL of homologous recombination connection product and add it to 20μL of E. coli chemically competent cells(Fast-T1 chemically competent cells, Novagen, item number: C505-03) for transformation, and spread on 50μg / mL Km-resistant LB solid culture dishes and incubate at 37℃ overnight.

[0063] (3) Select positive clones

[0064] Pick 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; Primer F2(100μM) 1μL; Primer R2(100μM) 1μL; Template 1μL; Total volume 25μL.

[0068] PCR program: 94℃ 5min; 94℃ 30sec, 50℃ 45sec, 72℃ 100sec, 30 cycles; 72℃ 10min; 16℃ 30min.

[0069] Then, the positive clones were identified by 1% agarose gel electrophoresis. The target band was about 1122bp. 1-3 positive bands were taken for sequencing verification.

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

[0071] The correct and complete clones were added to 5-10ml LB liquid medium containing 50μg / mL Km, and cultured overnight at 200rpm and 37°C. 500μl of fresh bacterial solution was added to an equal volume of sterilized 50% glycerol, and the E. coli solution was stored at -80°C. The remaining bacterial solution was extracted using a plasmid extraction kit (FastPure EndoFree Plasmid Mini Kit-Box2, item number DC203-01).

[0072] Example 3 Construction of gene editing vector

[0073] 1) Target design

[0074] 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 after analysis:

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

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

[0077] Further, the CRSIPR vector construction primers were designed, and the sequence information is as follows:

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

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

[0080] 2) PCR amplification

[0081] According to Table 1, 50μL system was prepared and amplification reaction was carried out according to Table 2 procedure.

[0082] Table 1 PCR system

[0083]

[0084] Table 2 PCR procedure

[0085]

[0086] The target band of about 250 bp was recovered from the gel, and the total volume of 30 μL of water was used to dissolve the DNA. After the test was correct, the vector was connected.

[0087] 3) Enzyme cutting and connection

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

[0089] Table 3 Enzyme cutting and connection system

[0090]

[0091] Table 4 Enzyme cutting and connection reaction conditions

[0092]

[0093] 4) Transformation and identification

[0094] 5-10 μL of the connection 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 was cultured at 37°C for 12 hours, and the 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 picked for PCR identification, and the identification system and reaction conditions were referred to Table 5 and Table 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 about 800 bp.

[0105] Select 1-3 positive bands corresponding to the bacterial liquid, take 100 μL for sequencing, the rest 400 μL of bacterial liquid is inoculated into 5-10 ml kanamycin resistant LB, and the test tube is shaken. After the sequencing result is obtained, the corresponding correct sequencing is taken out to extract the plasmid. After extracting the plasmid, the strain and the plasmid are stored.

[0106] Example 4 Tobacco Genetic Transformation

[0107] 1) Agrobacterium preparation

[0108] Take 1 μL plasmid (overexpression vector or gene editing vector) and add it to 50 μL GV3101 Agrobacterium competent cells. Transform and coat with 50 μg / mL kanamycin-resistant LB plate, incubate at 28°C for 48h, and perform colony PCR identification. The amplification primer, reaction system and reaction program are the same as above. The PCR product is detected by gel electrophoresis. The electrophoresis results of positive control (using the above sequencing correct plasmid as template) and sample electrophoresis band 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 tobacco infection.

[0109] Select single colony in liquid LB medium containing 25 μg / mL rifampicin and 100 μg / mL kanamycin (Km) antibiotics, and shake at 28°C for 24h. Centrifuge the shaken bacterial liquid at 4000r / min for 10min, discard the supernatant, resuspend in infiltration buffer (10mM 2-(N-morpholino) ethanesulfonic acid (MES) containing 10mM MgCl2, pH=5.2 and 0.1mM acetyl-syringone), incubate at room temperature for more than 3h, and use OD 600 value about 0.6 as the infection liquid.

[0110] 2) Genetic transformation

[0111] Select full consistent "Xiangyan 7" 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 the rooting medium (MS + 50mg / L kanamycin + 500mg / L carbenicillin + 0.2mg / L IAA), and the roots are formed after about 7d. When the seedlings grow to about 6cm, the bottle opening is opened, and the seedlings are hardened for 2d. Then the seedlings are transplanted into the tobacco special substrate (Hunan Tianliang Agricultural Technology Development Co., Ltd.) sterilized by high temperature, covered with plastic film to keep moist, and cultured under light at 25-27℃. Further harvest seeds.

[0112] Example 5 Positive seedling screening and identification

[0113] 1) Overexpression positive seedling screening and identification

[0114] (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 Hyg B. Finally, seal with sealing film and put into the tissue culture room for 7 days.

[0115] (2) The green seedlings grown from the medium are transferred to the soil for further culture. Wait for the seeds to mature and collect T1 generation seeds.

[0116] (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.

[0117] (4) Ten possible positive seedlings were selected from each dish and transferred to soil for further culture. Meanwhile, the remaining small green seedlings in the dish were collected to extract RNA, reverse transcribed into cDNA, and quantitatively detected the expression level of the target gene.

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

[0119] NtMPK4 fluorescence quantitative primers:

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

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

[0122] Tobacco internal reference gene ACTIN7 fluorescence quantitative primers:

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

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

[0125] The real-time quantitative PCR (kit Thermo Fisher Scientific, catalog number: A25742) amplification system and procedure 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) When the seeds of the current generation of plants mature, collect the seeds of the strains with high expression levels for subsequent phenotypic identification.

[0131] Results of screening of NtMPK4 homozygous overexpression strains: After 15 positive seedlings were harvested, T0 seeds were screened with 150 μg / mL hygromycin and two strains with high expression levels, OE#8 and OE#13, were identified through expression analysis. T1 generation seeds were further harvested and T2 generation seeds (pure lines) were obtained by treating T1 seeds with 150 μg / mL hygromycin (Figure 3 )。

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

[0133] Cut the sterile rooted tissue culture seedlings leaves, use PlaNto DNA Isolation Mini Kit (Nanjing Nuowezan, item number: DC104-01) plant total DNA extraction kit to extract DNA, and the detailed method is referred to the instruction. Use high-fidelity enzyme PrimeSTAR Max DNA Polymerase of Baoray Biotechnology (Beijing) Co., Ltd. to perform PCR amplification on the knockout material, and the template is the DNA of Xiangyan 7, KO#2 and KO#15.

[0134] The 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: 1161 bp.

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

[0139] Table 9 PCR reaction system

[0140]

[0141] Table 10 PCR program

[0142]

[0143] NtMPK4 homozygous knockout strain material screening results: through sequencing analysis of 30 positive seedlings, two successfully edited strains KO#2 and KO#15 were screened from them. It was found that mpk4-2 (KO#2) lacked one base at the target point 1 position, and mpk4-15 (KO#15) had one base insertion at the target point 1 position, and the target point was clean single peak, indicating that the two strains were homozygous mutation. Figure 4 ) Protein sequence alignment found that the NtMPK4 protein translation of mpk4-2 and mpk4-15 strains was terminated early ( Figure 5 ), indicating that the function of NtMPK4 gene in the two strains was destroyed.

[0144] Example 6 Tobacco cold tolerance phenotype identification and physiological and biochemical index determination

[0145] Xiangyan 7 as the background, using CRISPR / Cas9 technology to obtain two knockout lines KO#2 and KO#15 of the gene. By constructing overexpression vectors, Xiangyan 7 was transformed to obtain two overexpression lines OE#8 and OE#13. After seed cleaning and disinfection of WT plants, two knockout lines and two overexpression lines, they were placed at 4℃ for 48h. Then they were sowed in small boxes filled with tobacco special substrate and covered with lids to keep warm and humid. They were cultured in an artificial climate chamber (temperature 25℃, relative humidity 75%, light / dark = 16h / 8h) for 30d for subsequent treatment. Selecting tobacco seedlings with consistent growth, they were placed at 4℃ for phenotype observation and photographing.

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

[0147] Malondialdehyde (MDA) content was determined using Plant Malondialdehyde (MDA) Test Kit (Nanjing Jiancheng, Item No.: A003-3-1); hydrogen peroxide content (H2O2) was determined using Hydrogen Peroxide Test Kit (Nanjing Jiancheng, Item No.: A064-1-1); superoxide anion capacity (OFR) was determined using Oxygen Free Radical (OFR) Kit (Jiangsu Aidsen Biological Technology, Item No.: ADS-W-YH008); specific method: refer to the kit instructions.

[0148] Relative conductivity: Fresh leaf of the 4th true leaf (counted from the top) of seedlings was taken with a circular puncher, 0.2g fresh tissue leaf was placed in a centrifuge tube containing 25ml ddH2O, and treated at 37℃ on a shaker for 24h. The first exosmosis liquid conductivity value L1 was measured, and then the EP tube was placed in an autoclave for 15min at 120℃. After the temperature cooled to room temperature, the second exosmosis liquid conductivity value L2 was measured. The relative conductivity (EL = L1 / L2*100%) was calculated.

[0149] 3,3'-diaminobenzidine (DAB) staining: First prepare 0.1mg / ml DAB dissolved in 50mM Tris-acetic acid buffer (pH5.0). 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 10min (if the chlorophyll is difficult to dehydrate, the time can be longer). Finally, transfer the leaves into anhydrous ethanol, and observe under a microscope or camera, and save.

[0150] Chloride nitro tetrazolium blue (NBT) staining: NBT staining solution 1 mg / mL was prepared in 10 mM PBS (pH 7.8). Leaves were cut and put into NBT staining solution, and then were dyed under light for 1-2 h. When the leaves showed blue color, the staining solution was removed, and anhydrous ethanol was added. The leaves were boiled in boiling water for 10 min, and then were stored in 70% ethanol. The leaves were observed under microscope or camera.

[0151] As shown in Figure 6 A, under control conditions (temperature 25℃), the two knockout lines had no obvious difference in phenotype with WT (Xiangyan 7). After 4℃ low temperature treatment for 4 h, the leaves of the two knockout lines only showed slight drooping, and had no obvious change compared with before treatment. The results showed that the cold tolerance of the two knockout lines was significantly enhanced compared with WT, and NtMPK4 negatively regulated the cold tolerance of tobacco. Further, the physiological and biochemical results showed that under control conditions, the contents of H2O2, OFR, EL and MDA in the two knockout lines had no obvious difference with WT. Three days after low temperature stress treatment, the contents of H2O2 (C), OFR (D), EL (F) and MDA (E) in the two knockout lines were lower than those in WT. Figure 6 Figure 6 Figure 6 Figure 6 Figure 6

[0152] As shown in Figure 7 A, under control conditions, the two overexpression lines had no obvious difference in phenotype with WT (Xiangyan 7). After 4℃ low temperature treatment for 3 h, the leaves of the two overexpression lines wilted more severely compared with WT. The results further showed that the cold tolerance of the two overexpression lines was significantly weaker, and NtMPK4 negatively regulated the cold tolerance of tobacco. Further, the physiological and biochemical results showed that under control conditions, the contents of H2O2, OFR, EL and MDA in the two overexpression lines had no obvious difference with WT. Three days after low temperature stress treatment, the contents of H2O2 (C), OFR (D), EL (F) and MDA (E) in the two overexpression lines were lower than those in WT. Figure 7 Figure 7 Figure 7 Figure 7 ​​​​​​​​E) higher than WT. ROS tissue staining was performed between overexpression lines and wild type after 4°C (3d) low temperature treatment. NBT staining results showed that there was no obvious difference between overexpression lines and wild type under normal conditions. After low temperature stress, the two overexpression lines had larger staining area and darker staining degree than WT, indicating that overexpression plants accumulated more OFR. DAB staining results showed that there was no obvious difference between overexpression lines and wild type under normal conditions. After low temperature stress, the two overexpression lines had larger staining area and darker staining degree than WT, indicating that overexpression plants accumulated more H2O2 Figure 7 Figure 7 B).

[0153] 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 NtMPK4 gene in tobacco cold tolerance, characterized in that, The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

2. Application of knocking out the NtMPK4 gene in positively regulating tobacco cold tolerance, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

3. Application of biomaterials with NtMPK4 gene knockout in positively regulating tobacco cold tolerance, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.1; The biological material is any one of the following: A: an expression cassette capable of silencing the NtMPK4 gene whose nucleotide sequence is shown in SEQ ID NO.1; B: recombinant vector containing the expression cassette described in A; C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

4. Application of overexpression of NtMPK4 gene in negatively regulating tobacco cold tolerance, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

5. Application of biomaterials overexpressing the NtMPK4 gene in negatively regulating tobacco cold tolerance, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.1; The biological material is any one of the following: A: an expression cassette capable of overexpressing the NtMPK4 gene with the nucleotide sequence shown in SEQ ID NO.1; B: recombinant vector containing the expression cassette described in A; C: A recombinant microorganism containing the expression cassette described in A or the recombinant vector described in B.

6. Application of NtMPK4 gene in tobacco breeding, characterized in that, The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

7. Application of NtMPK4 gene in breeding tobacco cold-tolerant germplasm, characterized in that, The NtMPK4 gene sequence is shown in SEQ ID NO.1.

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