Application of NtMPK4 gene in drought tolerance of tobacco

Abstract

The invention discloses application of an NtMPK4 gene in tobacco drought tolerance, and belongs to the technical field of molecular biology. According to the application of the NtMPK4 gene disclosed by the invention in the drought tolerance of the tobacco, the sequence of the NtMPK4 gene is as shown in SEQ ID NO. 1. The NtMPK4 gene positively regulates the drought tolerance of the tobacco, and the excavation and functional identification of the gene provide important gene resources and theoretical basis for drought-tolerant 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 drought resistance. BACKGROUND

[0002] Tobacco is an important economic crop and is widely planted all over the world. Drought stress is one of the main adverse factors limiting its yield and quality. Climate change has led to frequent seasonal droughts, especially in hilly and mountainous tobacco-growing areas, and water resource shortage is a prominent problem. Drought not only inhibits the growth of tobacco but also changes its chemical composition and reduces the industrial value of tobacco leaves. Therefore, it has become an indispensable method to comprehensively utilize molecular biological techniques to mine tobacco drought-resistant genes, develop molecular markers, realize directional improvement of tobacco varieties, and cultivate new drought-resistant tobacco varieties.

[0003] Mitogen-activated protein kinase (MAPK, for short, MPK) is a kind of conservative serine / threonine protein kinase. After activation, it phosphorylates different substrates, thereby transferring extracellular signals to intracellular, so that the cell responds to external stimuli, and constitutes a complete MPK cascade pathway. The MAPK cascade pathway is an important signal transduction pathway and is involved in various life activities of plants. A typical MAPK cascade signal pathway is composed of three components, including MAPKKK (MAP3K), MAPKK (MKK) and MAPK (MPK). The MAPK cascade pathway plays a connecting role in the process of plant signal transduction, amplifies and transmits signals through successive phosphorylation reactions, thereby combining external stimuli with cell response. After the receptor on the plant cell membrane receives the stress signal, it transmits it to the inside of the cell, activates the upstream kinase MAP3Ks through phosphorylation, and transmits the stress signal to the downstream components MKKs and MPKs through successive phosphorylation activation. The activated MPKs can function in the cytoplasm or nucleus, such as phosphorylating downstream protein kinases, cytoskeleton proteins or transcription factors, etc. These downstream substrates often have docking sites and phosphorylation motifs that can bind to MPKs proteins.

[0004] The influence of drought stress on tobacco agronomic characters mainly occurs in the early and middle stages of tobacco plant growth. The most sensitive period of tobacco to drought is the vigorous growth period, and if drought occurs at this time, it will cause irreversible damage to tobacco. Under drought stress, the plant height, stem girth, leaf length and leaf width of tobacco at the rosette stage and vigorous growth stage all show a downward trend. Under drought stress, the nicotine content of tobacco increases, the total sugar content decreases, that is, the sugar-alkali ratio decreases, resulting in reduced tobacco yield and deteriorated quality. Drought stress inhibits the plant's capture of light energy, reduces its electrical energy conversion and transmission efficiency, and ultimately leads to a decrease in plant photosynthesis. Under drought stress, the stomatal conductance of the plant's leaf surface decreases, causing the stomata to close and limiting the entry of CO2 into the leaf, which reduces the conversion of inorganic carbon to organic carbon. At the same time, due to the inability to timely absorb CO2 in the environment, the amount of chemical energy formed by the conversion of light energy is reduced, the leaf is inhibited, the chloroplast structure is destroyed, and ultimately irreversible damage to the chloroplast is caused. Drought stress also causes tobacco to produce a large amount of active oxygen, leading to membrane lipid peroxidation and inhibiting plant photosynthesis and respiration. Studies have shown that under drought stress, a large number of MAPK cascade pathway genes are involved in the ABA signal transmission process, and through ABA signal induction, a series of drought response genes are expressed to regulate plant drought stress response. The MAPK cascade pathway plays an important role in regulating the closure of stomata by integrating hormone signals, thereby enhancing the drought resistance of plants.

[0005] Therefore, providing the application of NtMPK4 gene in tobacco drought resistance is a problem that those skilled in the art urgently need to solve. SUMMARY

[0006] Therefore, the application provides the application of NtMPK4 gene in tobacco drought resistance.

[0007] Based on the previous phosphoproteomic analysis of tobacco under drought stress, a drought stress response candidate gene NtMPK4 was mined. On this basis, the function of the gene was identified, and the gene function was analyzed, providing gene resources and theoretical basis for tobacco drought resistance 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 drought resistance, wherein the sequence of NtMPK4 gene is shown as SEQ ID NO. 1.

[0010] Further, the application of overexpressing NtMPK4 gene in positively regulating tobacco drought resistance, wherein the sequence of NtMPK4 gene is shown as SEQ ID NO. 1.

[0011] Further, the application discloses a biological material overexpressing the NtMPK4 gene, and the NtMPK4 gene sequence is shown as SEQ ID NO. 1.

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

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

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

[0015] C: a recombinant microorganism containing the expression cassette shown in A or the recombinant vector shown in B.

[0016] Further, the application discloses an application of knocking out the NtMPK4 gene in negative regulation of tobacco drought resistance, and the NtMPK4 gene sequence is shown as SEQ ID NO. 1.

[0017] Further, the application discloses an application of knocking out the NtMPK4 gene in negative regulation of tobacco drought resistance, and the NtMPK4 gene sequence is shown as SEQ ID NO. 1.

[0018] The biological material is any of the following:

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

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

[0021] C: a recombinant microorganism containing the expression cassette shown in A or the recombinant vector shown in B.

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

[0023] Further, the application discloses an application of the NtMPK4 gene in breeding of tobacco drought-resistant germplasm, and 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 drought resistance, a drought response gene NtMPK4 is separated 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 and NaMMK2 of Nicotiana glauca and 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 drought stress, compared with Xiangyan No. 7 (WT), the wilting degree of the two knockout lines is more serious, and the two overexpression lines are on the contrary. The physiological and chemical results show that under drought 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 lower 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 smaller, and the two knockout lines are on the contrary. The above results show that the NtMPK4 gene positively regulates the drought resistance of tobacco. The mining and functional identification of the gene provide important gene resources and theoretical basis for tobacco drought resistance 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 drought tolerance phenotype identification and physiological and biochemical index determination of tobacco overexpression lines;

[0032] wherein, A: phenotype after 4h of simulated drought treatment: the left graph is a top view and the right graph is a side view; B: NBT and DAB staining results after 5d of simulated drought treatment; C: H2O2 change analysis after 5d of simulated drought treatment; D: OFR change analysis after 5d of simulated drought treatment; E: MDA change analysis after 5d of simulated drought treatment; F: EL change analysis after 5d of simulated drought treatment;

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

[0034] wherein, A: phenotype after 3h of simulated drought treatment: the left graph is a top view and the right graph is a side view; B: NBT and DAB staining results after 3d of simulated drought treatment; C: H2O2 change analysis after 3d of simulated drought treatment; D: OFR change analysis after 3d of simulated drought treatment; E: MDA change analysis after 3d of simulated drought treatment; F: EL change analysis after 3d of simulated drought treatment. DETAILED DESCRIPTION

[0035] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in 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 of ordinary skill 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 F1 2 μL, Primer R1 2 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 program

[0085]

[0086] The target fragment (about 250bp) was recovered from the gel, and the total volume of 30μL of water was used to dissolve the recovered DNA. After the DNA was confirmed to be correct, it was ligated with a vector.

[0087] 3) Enzymatic digestion and ligation

[0088] The enzymatic 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] 5-10μL of the ligation product was transformed into E. coli competent cells according to the instructions (Fast-T1 chemical competent cells, Novagen, Catalog No.: C505-03). The transformation was coated with 50μg / mL kanamycin-resistant LB solid culture dish, and incubated at 37℃ for 12 hours. 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 are shown in Table 5 and Table 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 800bp.

[0104] Pick 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.

[0105] Example 4 Tobacco Genetic Transformation

[0106] 1) Preparation of Agrobacterium

[0107] Take 1 μL of plasmid (overexpression vector or gene editing vector) and add it to 50 μL of GV3101 Agrobacterium competent cells. Transform the LB plate containing 50 μg / mL kanamycin resistance and incubate at 28°C for 48h. 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.

[0108] Pick single colony in liquid LB medium containing 25 μg / mL of rifampicin and 100 μg / mL of kanamycin (Km) antibiotics, and shake at 28°C for 24h; centrifuge the shaken bacterial liquid at 4000r / min for 10min, discard the supernatant, and 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 an infection liquid for standby.

[0109] 2) Genetic transformation

[0110] Select full and 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 small pieces of about 5mmx5mm, 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.

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

[0114] (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.

[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) 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.

[0117] (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.

[0118] NtMPK4 fluorescence quantitative primers:

[0119] NtMPK4-qF: GGATCATGACAATGTGATTG; SEQ ID NO.13;

[0120] NtMPK4-qR: TGGTTGGAACGAATTATCTG; SEQ ID NO. 14.

[0121] Tobacco internal reference gene ACTIN7 fluorescence quantitative primers:

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

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

[0124] The real-time quantitative PCR (kit Thermo Fisher Scientific, catalog number: A25742) amplification system and procedure 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 program

[0128]

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

[0130] Results of screening of 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 )。

[0131] 2) Gene knockout positive seedling screening and identification

[0132] 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 the 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.

[0133] The primer sequence information is as follows:

[0134] MPK4-crispr-F: ATGGGGGGTGGTGGTACATT; SEQ ID NO. 17;

[0135] MPK4-crispr-R: CCGATCCACAAATAGCTGGAGC; SEQ ID NO. 18.

[0136] Fragment length: 1161 bp.

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

[0138] Table 9 PCR reaction system

[0139]

[0140] Table 10 PCR program

[0141]

[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 1 position, and mpk4-15 (KO#15) had one base insertion at the target 1 position, and the target nearby was a 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 drought tolerance phenotype identification, physiological and biochemical index determination

[0145] Taking Xiangyan 7 as the background, two knockout lines KO#2 and KO#15 of the gene were obtained by using CRISPR / Cas9 technology. By constructing an overexpression vector, 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°C for 48h low temperature treatment. Then they were sowed in small boxes filled with tobacco special substrate and fully watered, and covered with a lid to keep warm and humid, and cultured in an artificial climate room (temperature 25°C, relative humidity 75%, light / dark = 16h / 8h) for 30d for subsequent treatment.

[0146] Mannitol simulated drought experiment: select tobacco seedlings with consistent growth, and pour 200mL of 400mM mannitol solution into each tobacco seedling for penetration, observe the phenotype and take pictures. Pour 200ml of H2O into each tobacco seedling as a control (CK).

[0147] At 0h and 3d time points, the third leaf (counted from the top) samples of Xiangyan 7 and knockout lines were taken for physiological and biochemical index detection; at 0h and 5d time points, the third leaf (counted from the top) samples of Xiangyan 7 and overexpression lines were taken for physiological and biochemical index detection; each treatment had 5 biological replicates. After sampling, quickly wrap with tin paper and put into liquid nitrogen, and store at -80°C.

[0148] Malondialdehyde (MDA) content was determined by plant malondialdehyde (MDA) test kit (Nanjing Jiancheng, item number: A003-3-1); hydrogen peroxide content (H2O2) was determined by hydrogen peroxide test kit (Nanjing Jiancheng, item number: A064-1-1); superoxide anion capacity (OFR) was determined by superoxide anion (Oxygen free radical, OFR) kit (Jiangsu Aidesen Biological Technology, item number: ADS-W-YH008); specific method: refer to the kit instruction.

[0149] Relative conductivity: fresh leaf of the 4th true leaf (counted from the top leaf) of seedling, 0.2g fresh tissue leaf was taken with a circular puncher, and was put into a centrifuge tube containing 25ml ddH2O, and was treated at 37°C for 24h on a shaker, and the first exosmosis liquid conductivity value L1 was measured, and then the EP tube was put into an autoclave for 15min at 120°C, and 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.

[0150] 3,3'-diaminobenzidine (DAB) staining: Prepare 0.1 mg / ml DAB in 50 mM Tris-acetate buffer (pH 5.0). Soak the leaves in the staining solution, and incubate in the dark at room temperature overnight. Remove the staining solution, and add absolute ethanol. Boil in boiling water for 10 min (if the chlorophyll is difficult to dehydrate, the time can be longer). Finally, transfer the leaves into absolute ethanol, and observe under microscope or camera.

[0151] Nitro blue tetrazolium chloride (NBT) staining: Prepare NBT staining solution 1 mg / mL in 10 mM PBS (pH 7.8). Cut the leaves and put them into the NBT staining solution, and incubate under light for 1-2 h. When the color phenotype appears (the leaves are blue), remove the staining solution, and add absolute ethanol. Boil in boiling water for 10 min, and store in 70% ethanol. Observe under microscope or camera.

[0152] As shown in Fig. 1A, under control conditions, the phenotypes of the two overexpression lines were not significantly different from that of WT (Xiangyan No. 7). After 4 h of mannitol treatment, the wilting degree of the wild type leaves was more serious than that of the overexpression lines, and the leaves of the two overexpression lines only showed slight drooping, which was not significantly different from that before treatment. This result indicated that the drought tolerance of the two overexpression lines was significantly enhanced compared with WT, and NtMPK4 positively regulated the drought tolerance of tobacco. Figure 6 As shown in Fig. 1A, under control conditions, the phenotypes of the two overexpression lines were not significantly different from that of WT (Xiangyan No. 7). After 4 h of mannitol treatment, the wilting degree of the wild type leaves was more serious than that of the overexpression lines, and the leaves of the two overexpression lines only showed slight drooping, which was not significantly different from that before treatment. This result indicated that the drought tolerance of the two overexpression lines was significantly enhanced compared with WT, and NtMPK4 positively regulated the drought tolerance of tobacco. Figure 6 Further, the 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. After 5 d of mannitol simulated drought 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 6 Figure 6 Figure 6 Further, the 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. After 5 d of mannitol simulated drought 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 6 Further, the 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. After 5 d of mannitol simulated drought treatment, the contents of H2O2 (C), OFR (D), EL (F) and MDA (E) in the two overexpression lines were lower than those in WT.

[0153] As shown in Fig. 1A, under control conditions, the phenotypes of the two overexpression lines were not significantly different from that of WT (Xiangyan No. 7). After 4 h of mannitol treatment, the wilting degree of the wild type leaves was more serious than that of the overexpression lines, and the leaves of the two overexpression lines only showed slight drooping, which was not significantly different from that before treatment. This result indicated that the drought tolerance of the two overexpression lines was significantly enhanced compared with WT, and NtMPK4 positively regulated the drought tolerance of tobacco. Figure 7 ​​As shown in Fig. 1A, under control condition, the two knockout lines had no obvious difference in phenotype with WT (Xiangyan 7). After 3h mannitol treatment, the wilting degree of the leaves of the two knockout lines was more serious than that of WT. This result further indicated that NtMPK4 positively regulated the drought tolerance of tobacco. Further, the physiological and biochemical results showed that under control condition, the contents of H2O2, OFR, EL and MDA in the two knockout lines had no obvious difference with WT. After 3d mannitol simulated drought treatment, the contents of H2O2( Figure 7 C), OFR( Figure 7 D), EL( Figure 7 F) and MDA( Figure 7 E) in the two knockout lines were higher than those in WT. ROS tissue staining was performed on the knockout lines and wild type after 3d mannitol simulated drought treatment. The NBT staining results showed that under normal conditions, there was no obvious difference between the leaves of the knockout lines and WT. After drought stress, the leaves of the two knockout lines had larger staining area and darker staining degree than WT, indicating that WT accumulated less OFR than the knockout plants. The DAB staining results showed that under normal conditions, there was no obvious difference between the leaves of the knockout lines and WT. After drought stress, the leaves of the two knockout lines had larger staining area and darker staining degree than WT, indicating that WT accumulated less H2O2( Figure 7 Figure 7 B) than the knockout lines.

[0154] The above description of disclosed embodiments enables one skilled in the art to make or use the application. Numerous modifications to these embodiments will be 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. Therefore, 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 drought tolerance, characterized in that, The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

2. Application of overexpression of NtMPK4 gene in positively regulating tobacco drought tolerance, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

3. Application of biomaterials overexpressing the NtMPK4 gene in positively regulating tobacco drought 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.

4. Application of knocking out the NtMPK4 gene in negatively regulating tobacco drought tolerance, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.

1.

5. Application of biomaterials with NtMPK4 gene knockout in negatively regulating tobacco drought 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.

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 drought-tolerant germplasm, characterized in that: The NtMPK4 gene sequence is shown in SEQ ID NO.1.

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