LU600447B1 - APPLICATION OF NtMPK8 GENE IN COLD TOLERANCE OF NICOTIANA TABACUM L. - Google Patents

Abstract

The present disclosure discloses an application of an NtMPK8 gene in cold tolerance of Nicotiana tabacum L., falling within the technical field of plant genetic engineering. The present disclosure discloses the application of an NtMPK8 gene in cold tolerance of Nicotiana tabacum L., and an NtMPK8 gene sequence is as shown in SEQ ID NO. 1. The NtMPK8 gene positively regulates cold tolerance of Nicotiana tabacum L. The excavation and functional identification of the gene provide an important gene resource and a theoretical basis for low temperature tolerance breeding in the Nicotiana tabacum L.

Description

APPLICATION OF NtMPK8 GENE IN COLD TOLERANCE OF NICOTIANA !-U600447

TABACUM L.

TECHNICAL FIELD

[0001] The present disclosure relates to the technical field of plant genetic engineering, in particular to an application of an NIMPKS gene in cold tolerance of Nicotiana tabacum L.

BACKGROUND ART

[0002] Nicotiana tabacum L., native to the tropics and subtropics, is an important economic crop for leaves and a model organism. The Nicotiana tabacum L. is vulnerable to low temperature stress, and an optimal growth temperature is 25-28°C. Under low temperature conditions, the growth of Nicotiana tabacum L. is impeded, and the quality and yield declined. At the seedling stage, Nicotiana tabacum L. is more vulnerable to low temperature stress. Nicotiana tabacum L. seedlings at the age of 6-7 leaves can be subjected to low temperature stress of 12°C for two weeks, which may lead to early flowering of Nicotiana tabacum L., reducing the yield and quality of

Nicotiana tabacum L. leaves and causing huge economic losses to agricultural production and

Nicotiana tabacum L. farmers. Therefore, the comprehensive utilization of molecular biotechnology means to excavate low temperature resistant genes of Nicotiana tabacum L., develop molecular markers, achieve a targeted improvement of Nicotiana tabacum L. varieties, cultivate new varieties of Nicotiana tabacum L. with low temperature tolerance, and has become an indispensable way to cultivate excellent germplasm resources at present.

[0003] A mitogen-activated protein kinase (MPK) cascade pathway widely exists in eukaryotes and is highly conserved, playing a very important role in the growth and development of plants and in response to environmental stimuli. In the signal transmission process, the MPK cascade pathway integrates and amplifies the signal through step-by-step phosphorylation and transmits the signal to downstream protein kinases, transcription factors and other substrates. Finally, plant cells are activated to produce a specific physiological response to external stimuli. A basic MPK cascade pathway generally consists of three parts: MAP kinase kinase kinase (MAPKKK,

MAP3K, MEKK), MAP kinase kinase (MKK, MAP2K, MEK) and MAP kinase (MAPK, MPK).

Extracellular stimulation activates the most upstream MAPKK after activating plasma membrane receptors. The MAPKK is activated to activate the downstream MAPKK by phosphorylating two serine or threonine residues in the S/T-Xs-S/T motif of MAPKK, but the MAPKK is a dual phosphorylation-specific kinase that phosphorylates threonine and tyrosine residues in the T-X-¥U600447 motif of MAPK downstream. MAPK is a conserved serine/threonine protein kinase that acts on different substrates through phosphorylation after activation, thereby transferring extracellular signals to the inside of the cell, making the cell respond to external stimuli and constituting a complete MPK cascade pathway.

[0004] The MAPK cascade pathway is widely involved in the response of plants to biotic and abiotic stresses. Some MAPK cascade pathways have been reported in Arabidopsis thaliana to participate in stress responses such as pathogen infection, trauma, cold, drought, high osmolarity, high salinity, heavy metals, ozone, and UV radiation. 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 Nicotiana tabacum L. was treated with synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D), a rapid and instantaneous activation of 46-kDa protein kinase was detected, which could scale myelin basic protein (MBP). MAPK has also been 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, and MAPK activity is observed in protoplasts of guard cell treated with ABA.

[0005] Therefore, providing an application of an N/MPKS8 gene in cold tolerance of Nicotiana tabacum L. is an urgent problem to be solved by those skilled in the art.

SUMMARY

[0006] In view of this, the present disclosure provides an application of an NMPK8 gene in cold tolerance of Nicotiana tabacum L.

[0007] Based on a previous analysis of phosphorylated proteomic analysis of Nicotiana tabacum L. under low temperature stress, the present disclosure has excavated a candidate gene

NtMPKS responding to low temperature stress. On this basis, the present disclosure carries out functional identification of the gene, analyzes the function of the gene, providing a genetic resource and theoretical basis for Nicotiana tabacum L. low temperature tolerance molecular breeding.

[0008] In order to achieve the foregoing objects, the present disclosure adopts the following technical solutions.

[0009] An application of an NtMPKS gene in cold tolerance of Nicotiana tabacum L., where ahV600447

NIMPKS8 gene sequence is as shown in SEQ ID NO. 1.

[0010] Further, an application of an overexpression NIMPKS gene in a positive regulation of cold tolerance in Nicotiana tabacum L., where the NIMPKS gene sequence is as shown in SEQ

IDNO. 1.

[0011] Further, an application of biomaterials of an overexpression N/MPK8 gene expression in a positive regulation of cold tolerance in Nicotiana tabacum L., where the NiMPK8 gene sequence is as shown in SEQ ID NO. 1;

[0012] the biomaterials are any one of following:

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

[0014] B: a recombinant vector including the expression cassette described in A; and

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

[0016] Further, an application of an NMPKS gene in Nicotiana tabacum L. breeding, where the

NIMPKS8 gene sequence is as shown in SEQ ID NO. 1.

[0017] Further, an application of an N/MPK8 gene in breeding of cold tolerance germplasm in

Nicotiana tabacum L., where the NIMPK8 gene sequence is as shown in SEQ ID NO. 1.

[0018] According to the foregoing technical solution, compared with the prior art, the present disclosure publicly provides the application of the N/MPK8 gene in cold tolerance of Nicotiana tabacum L., and low temperature stress seriously affects the yield and quality of Nicotiana tabacum L. The use of biological technology to excavate low temperature tolerance genes and explore gene functions is of great theoretical and practical significance for breeding and cultivating low temperature tolerance varieties. In the present disclosure, a low temperature response gene MMPK8 was isolated from Nicotiana tabacum L., and the sequence results showed that a gene CDS sequence was 1,773 bp in length and encoded 590 amino acids. An evolutionary tree analysis showed that the protein encoded by the gene had high homology with Nicotiana sylvestris NSMPK9-X1 and stenophylious Nicotiana tabacum L. NaMPK9L. Two overexpression strains, MPKS8-OE#1 and MPK8-OE#3, were obtained by constructing overexpression vectors and transforming Xiangyan NO. 7. Phenotypic identification results showed that a wilting degree of the two overexpression strains was less than that of Xiangyan NO. 7 (WT) under 1owU600447 temperature stress. Physiological results showed that the contents of malondialdehyde (MDA), relative electrical conductivity (EL), hydrogen peroxide (H2O2) and oxygen free radical (OFR) in leaves of overexpression strains were obviously lower than those of WT under low temperature stress. The results of DAB and NBT staining showed that the leaves of two overexpression strains had smaller staining areas compared with that of WT under low temperature stress. The results of photosynthetic identification showed that the transpiration rate (Tr) and stomatal conductance (gsw) of the two overexpression strains were obviously lower than those of WT Xiangyan NO. 7 under low temperature stress. In summary, the results showed that the N/MPK&8 gene was positively regulating the cold tolerance of Nicotiana tabacum L. The excavation and functional identification of this gene provided important gene resources and a theoretical basis for breeding

Nicotiana tabacum L. with low temperature tolerance.

BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to more clearly describe the technical solutions in the examples of the present disclosure or in the prior art, the accompanying drawings required for describing the examples or the prior art are briefly described below. Obviously, the accompanying drawings in the following description show only some examples of the present disclosure, and for those of ordinary skill in the art, other accompanying drawings may also be obtained according to the provided accompanying drawings without creative efforts.

[0020] FIG. 1 is a drawing showing an evolutionary tree analysis of an N/MPK8 system of the present disclosure;

[0021] FIG. 2 is a drawing showing a homology comparison analysis of an N/MPK8 protein sequence of the present disclosure;

[0022] FIG. 3 is a drawing showing an expression level analysis of an NMPK8 overexpression strain of the present disclosure;

[0023] FIG. 4 is a drawing showing a phenotypic analysis after low temperature (4°C) treatment for 5 h of the present disclosure;

[0024] FIG. 5 is a drawing showing an analysis of changes in MDA content (left) and EL (right) after low temperature treatment of the present disclosure;

[0025] FIG. 6 is a drawing showing an analysis of changes in OFR content (left) and H202

(right) after low temperature treatment of the present disclosure; LU600447

[0026] FIG. 7 is a drawing showing results of NBT (left) and DAB (right) staining after low temperature treatment of the present disclosure; and

[0027] FIG. 81sa drawing showing changes of photosynthetic parameters after low temperature 5 treatment of the present disclosure.

[0028] In the figures: A: leaf net photosynthetic rate; B: intercellular CO2 concentration; C: transpiration rate; and D: gsw.

DETAILED DESCRIPTION OF EMBODIMENTS

[0029] The following technical solutions in the examples of the present disclosure are described clearly and completely by reference to the drawings in the examples of the present disclosure. It is obvious that the described examples are only some of the examples of the present disclosure, rather than all examples of the present disclosure. Based on the examples in the present disclosure, all other examples obtained by those of ordinary skill in the art without making creative efforts belong to the scope of protection of the present disclosure.

[0030] An NtMPKS CDS sequence is as shown in SEQ ID NO. 1.

[0031] ATGGGGGGTGGTGGTACATTTGTGGATGGTGTTCTTCGCTGGTTTCAACGTC

GTCATAGTAATGAAGATGCGATCTTGACTGATCTTCAGAAACCCCATAATACCCATTT

ACAAGAAAGAGCAGAAGATAATAATCAAGAGTTTACCATTACTGAGGATTTTGACAT

TACAGGTCTAAAGCTTATCAAAGTACCCAAACGGCTTAGTTTCCCTATTTCCGCCCAT

TCTTCTATGGATCCTCTCAAAAAGAATGCGTTGGAGACGGAATTCTTTACGGAATATG

GAGAGGCAAGTAGATACCAAGTTCAGGAAGTAATTGGAAAAGGCAGCTATGGAGTC

GTGGGATCTGCTGTCGATACTCATACTGGTGAAAGAGTTGCAATCAAGAAAATTAAT

GATGTCTTTGATCATGTTTCTGATGCTACAAGAATCTTGAGAGAAATCAAGCTTCTTC

GGCTACTTAGGCATCCAGATATTGTAGAAATAAAGCACATTATGTTGCCTCCTTCTCG

GAGAGAGTTTAAAGATATTTATGTTGTTTTTGAATTGATGGAATCAGATCTCCATCAG

GTGATTAAGGCCAATAATGATCTTACTGCTGAGCATTATCAGTTTTTCCTATACCAGCT

TATGCGTGGACTAAAATATATTCATACAGCAAATATTTTCCACCGGGATTTAAAGCCTA

AGAATATTCTTGCTAATGCTGACTGTAAGTTGAAGATTTGTGATTTTGGTCTTGCTCGT

GTATCATTCAATGATGTGCCATCAGCTATTTTCTGGACTGATTATGTTGCAACTCGATG

GTATCGTGCCCCTGAGCTATGTGGCTCCTTTTTCTCTAAGTATACTCCTGCTATTGATAT

TTGGAGCATCGGATGCATATTTGCAGAATTGCTTTCTGGAAAACCGTTATTTCCTGGALU600447

AAGAATGTGGTGCATCAATTAGACCTAATCACAGATTTGCTTGGGACACCTCCTCCCG

AAACAGTTGCAAAGATCAGAAATGAAAAGGCAAGAAGATACCTCAGTAGCATGCGG

AAGAAACAACCAGTTCCATTTGAAAAAAAGTTTCCAAATGCAGATCCTTTGGCGTTA

CGGCTACTTGAACGACTGATTGCATTTGACCCTAAAGATCGGCCATCGGCAGAAGAG

GCATTGTCGGATCCATATTTCCGTGGTTTATCAAATGCTGATCATGAACCATCTAGGCC

ACCAATATCAAAGCTTGAGTTTGAATTTGAGAAAAGAAAACTGGCAAAAGAAGATG

TTAGAGAACTCATCTATCGCGAGATTTTAGAATATCATCCTCAGATGCTTCAGGAGTAT

CTTAGTGGTGGAGATCAGACAAGTGGCTTTATGTACCCAAGTGGTGTTGATCGGTTC

AAGCGACAATTTGCACATCTGGAGGAGCACTACGGTAAAGGTGAACGTAGCACCCC

GCTTCAGAGGCAGCATGCTTCCTTGCCTAGGGAGCGAGTTCCTGCACCAAAAAATGA

CACCTCTTCCCAAAATAATGATTGTGAAAAGCGAACTGTTTCAACAGCTCTTCAGAG

CTCACCAGGGCAGTCTGAGGGATCGGAGAACTCAATTGTCGGTACACAAAATGGAA

GTAATCAGGCAAACAACAGTGCTCGTAGCTTGCTGAAAAGTGCTAGCATCAGTGCTT

CTAAGTGTGTGGAAGTTAAAAACAGAAACACAGAGGAAGAGCCAATTGAAGAGGC

AAACGAGGAAGTTGATGATTTGTCTCAAAAAGTTGCAGCTCTTCATACTTAA; SEQ ID

NO. 1.

[0032] An MMPKS8 protein sequence is as shown in SEQ ID NO. 2.

[0033] MGGGGTFVDGVLRWFQRRHSNEDAILTDLQKPHNTHLQERAEDNNQEFTITE

DFDITGLKLIKVPKRLSFPISAHSSMDPLKKNALETEFFTEYGEASRYQVQEVIGKGSYG

VVGSAVDTHTGERVAIKKINDVFDHVSDATRILREIKLLRLLRHPDIVEIKHIMLPPSRREF

KDIYVVFELMESDLHQVIKANNDLTAEHY QFFLYQLMRGLKYIHTANIFHRDLKPKNIL

ANADCKLKICDFGLARVSFNDVPSAIFWTDYVATRWYRAPELCGSFFSK YTPAIDIWSIG

CIFAELLSGKPLFPGKNVVHQLDLITDLLGTPPPETVAKIRNEKARRYLSSMRKKQPVPFE

KKFPNADPLALRLLERLIAFDPKDRPSAEEALSDPYFRGLSNADHEPSRPPISKLEFEFEK

RKLAKEDVRELIYREILEYHPQMLQEYLSGGDQTSGFMYPSGVDRFKRQFAHLEEHYG

KGERSTPLQRQHASLPRERVPAPKNDTSSQNNDCEKRTVSTALQSSPGQSEGSENSIVGT

QNGSNQANNSARSLLKSASISASKCVEVKNRNTEEEPIEEANEEVDDLSQKVAALH,;

SEQ ID NO. 2.

[0034] Example 1: Analysis of Gene Sequence Features

[0035] An NtMPK8 CDS sequence was as shown in SEQ ID NO. 1, and an NiMPK8 proteihU600447 sequence was as shown in SEQ ID NO. 2. The NtMPKS protein sequence was derived from the

National Center for Biotechnology Information website (http://www.ncbi.nlm.nih.gov/BLAST/),

MEGA7.0 software was used to analyze gene homology and a neighbor joining method (NJ) was used to construct an evolutionary tree. DNAMAN software was used for protein similarity comparison.

[0036] A system evolution tree analysis found that NIMPKS and Nicotiana sylvestris NSMPK9-

X1: (XP_009799687.1) and stenophyllous Nicotiana tabacum L. NaMPKOL (XP 0192315251) had the closest genetic relationship (Fig. 1). Analysis of homologous alignment of a protein sequence found that the protein sequence had a typical MAPK domain, and had high homology with Nicotiana sylvestris NSMPK9-X1 and stenophylious Nicotiana tabacum L. NaMPKOL (Fig. 2).

[0037] Example 2: Construction of Overexpression Vector

[0038] 1) cDNA Acquisition

[0039] (1) RNA extraction

[0040] Total RNA was extracted from leaves of Xiangyan NO. 7 using a FastPure Universal

Plant Total RNA Isolation Kit (Vazyme, article number: RC411-01), and specific steps were referred to instructions.

[0041] (2) Reverse transcription

[0042] cDNA (Vazyme, article number: R312-02) was synthesized using a reverse transcription kit HiScript III 1* Strand cDNA Synthesis Kit (+gDNA wiper), and specific steps were referred to instructions.

[0043] 2) Synthetic Primers

[0044] The following target gene amplification primers were synthesized:

[0045] Primer F1:

[0046] AACACGGGGGACTTTGCAACatggggggtggtegtacatttgtg; SEQ ID NO. 3;

[0047] Primer R1:

[0048] TGAAGACAGAGCTAGTTAC Attaagtatgaagagctgcaactttttgagacaaatcatc; SEQ ID NO. 4.

[0049] 3) Amplification of Target Fragments

[0050] An amplification reaction was performed according to following systems anHU600447 procedures:

[0051] APCR amplification reaction system was 20 uL of Nuclease-free Water, 25 uL of Biorun

Pfu PCR Mix, 2 uL of Primer F1 (100 uM), 2 uL of Primer R1 (100 uM), 1 pL of cDNA, and 50 uL of Total volume.

[0052] A PCR reaction procedure was 94°C for 5 min, 94°C for 30 sec, 50°C for 45 sec, 72°C for 106 sec, 30 cycles, 72°C for 10 min, and 16°C for 30 min.

[0053] PCR products were analyzed by 1% agarose gel electrophoresis at a voltage of 5 v/cm for 20 minutes. An electrophoresis fragment of Gname (1,773 bp) was cut out under an ultraviolet lamp and put into a system for sol recovery. A recovery procedure was described in the instructions of a DNA recovery kit (Vazyme, article number: DC301-01), and DNA was recovered by dissolving a total volume of 40 uL of water (the recovered product was labeled as rDNAG1), and then recombined with a vector after detecting for errors.

[0054] 4) Recombination of Target Fragments and Vectors

[0055] (1) The target fragment obtained in step 3) was added to a recombination system (Vazyme ClonExpress® II One Step Cloning Kit) with a linearization vector pPBWA(V)HS after enzyme digestion and purification of Bsal/Ecol according to a molar ratio of 2:1 for the reaction, and specific steps were referred to instructions.

[0056] (2) 1 pL of homologous recombination linked products was taken to add to 20 pL of chemically competent cells of Escherichia coli for transformation and coated with LB medium containing 50 pg/mL Km resistance, and cultured at 37°C overnight.

[0057] (3) Selection positive clones

[0058] A monoclonal was selected for colony PCR, and primer sequences were as follows:

[0059] Primer F2: tTCATTTGGAGAGAACACGGGggac; SEQ ID No. 5; and

[0060] Primer R2: gcataagctggtataggaaaaactg; SEQ ID NO. 6.

[0061] A PCR reaction system was 9.5 uL of Nuclease-free Water, 12.5 pL of Biorun Magic

PCR Mix, 1 pL of Primer F2 (100 uM), 1 pL of Primer R2 (100 uM), 1 pL of Template, and 25 uL of Total volume.

[0062] A PCR reaction program was 94°C for 5 min, 94 °C for 30 sec, 50°C for 45 sec, 72°C for 100 sec, 30 cycles, 72°C for 10 min, and 16°C for 30 min.

[0063] Positive clones were detected and identified by 1% agarose gel electrophoresis, andV600447 target bands were fragments of about 662 bp. Bacterial solutions were taken corresponding to 1- 3 positive bands, and 200 uL was taken to send to a sequencing company for verification.

[0064] 5) Extraction Plasmid pBWA(V)HS-cedB(D)-NtMPK8

[0065] Sequenced correct and complete clones were added to 5-10 ml of LB medium containing 50 ug/mL KM in a triangular flask at 200 rpm, and cultured overnight at 37°C on a shaker, and 500 ul of fresh bacterial solution was taken to add to 50% glycerol with an equal volume of sterilized bacteria. An Escherichia coli bacterial solution was stored at -80°C, and remaining bacterial solutions were extracted using a plasmid extraction kit (FastPure EndoFree Plasmid Mini

Kit-Box2, article number DC203-01) to extract plasmid.

[0066] Example 3

[0067] 1) Genetic Transformation of Nicotiana tabacum L.

[0068] (1) Agrobacterium preparation

[0069] 1 uL of plasmid was taken to add into 50 uL of GV3101 Agrobacterium competent cells, transformed into an LB dish coated with 50 pg/mL kanamycin resistance, cultured at 28°C for 48 h, and colonies were identified by PCR. The amplification primer, reaction system and reaction procedure were the same as above. The PCR products were detected by gel electrophoresis, electrophoresis results of a positive control (using the plasmid with sequenced correct as a template ) and electrophoresis bands of samples were clear and sizes were correct, and a negative control (the template was water) had no bands, indicating that the sample could enter the next step and could be used to infect Nicotiana tabacum L.

[0070] A single colony was selected in a liquid LB medium containing 25 pg/mL rifampicin and 100 pg/mL Km antibiotic, and bacteria were shaken at 28°C for 24 h. The shaken bacterial solution was centrifuged at 4000 r/min for 10 min, after the supernatant was discarded. The shaken bacterial solution was re-suspended in an infiltration buffer (containing 10 mM MgClz, 10 mM 2- (N-morpholine) ethanesulfonic acid (MES) at pH=5.2, and 0.1 mM acetosyringone. After incubation at room temperature for more than 3 h, the OD600 value was about 0.6 as an infiltration solution.

[0071] (2) Genetic transformation

[0072] The plump and uniform "Xiangyan No. 7" Nicotiana tabacum L. seeds were selected to disinfect with a 10% sodium hypochlorite solution for 15 min, rinsed with sterile water five times-V600447 slightly dried with sterile absorbent paper, and sown on MS + 30 g/L sucrose + 8 g/L. agar (pH = 5.8) medium at 4 grains/bottle. A constant temperature incubator was placed at 25°C, a culture condition was a light intensity of 1600 Ix, and a photoperiod was 16 h (light)/8 h (dark) cultured for 45 d. After aseptic seedlings had grown four leaves, the leaves were cut into small pieces of about 5 mm x 5 mm, and leaf veins were removed. After pre-culture in MS medium containing 2 mg/L 6-BA and 0.2 mg/L IAA for 2 d, then infiltrated in Agrobacterium infiltration solution. The infected explants were placed to culture in MS medium containing 2 mg/L. 6-BA and 0.2 mg/L

IAA for 2 d in the dark. After co-culture, 50 mg/L. kanamycin and 500 mg/L carbenicillin were added to the medium for screening. Culture conditions for inducing resistant callus were the same as above, and were subcultured once every 14 d. When resistant buds on the callus grew to 2 cm, the buds were transferred to a rooting medium (MS + 50 mg/L kanamycin + 500 mg/L carbenicillin + 0.2 mg/L IAA), and rooting was performed for about 7 d. Plantlets grew about 6 cm long, and a mouth of a culture flask was opened for hardening for 2 d. Then the seedlings were transplanted into a high-temperature sterilized Nicotiana tabacum L. special substrate (Hunan

Tianliang Agricultural Technology Development Co., Ltd.), covered with a plastic film for moisturization, and cultured under a light condition at 25-27°C for further seed harvesting.

[0073] 2) Positive Seedlings Screening and Identification

[0074] (1) The harvested contemporary transgenic Nicotiana tabacum L. seeds were germinated on the medium containing Hyg B. The whole process needs to be carried out in a sterile environment. Firstly, the seeds were soaked in ddH20 for 12 h. The ddH2O in an EP tube was poured out, and then 1 mL of 75% alcohol was added to vortex for 30 sec. The alcohol was poured out, and 1 mL ddH20 was added to rinse 3 times. 1 mL of a NaClO solution was added and mixed evenly, the process did not exceed 5 min, then rinsed four times with sterilized ddH2O, and the rinsed seeds were spread flat on an MS medium containing 50 mg/L. Hyg B. Finally, a para film was used to seal and placed into an Arabidopsis thaliana chamber for culture for 7 days.

[0075] (2) Green seedlings grown from the medium were transferred into the soil for further cultivation. After the seeds were matured, T1 generation seeds were collected by ramets.

[0076] (3) The collected T1 generation seeds were further screened according to the method of step (1), a Nicotiana tabacum L. tissue culture chamber was placed for 7 d, and a culture dish with all green seedlings in the same dish was regarded as possible positive seedlings. LU600447

[0077] (4) 10 strains of possible positive seeds were selected from each dish and transferred into soil for further cultivation. Simultaneously, remaining small green seedlings in a collection dish were collected to extract RNA and reverse transcription into cDNA, and the expression of a target gene NIMPKS8 was detected by real-time quantitative PCR.

[0078] MMPKS fluorescent quantitative primers:

[0079] N/MPK8-q-F: CGCCCATTCTTCTATGGATCCT, SEQ ID NO. 7; and

[0080] N/MPK8-q-R: AGTATCGACAGCAGATCCCAC; SEQ ID NO. 8.

[0081] A fluorescent quantitative primer for Nicotiana tabacum L. internal reference gene

ACTIN7;

[0082] NtACT7-q-F: TGCTGATCGTATGAGCAAGG; SEQ ID NO. 9; and

[0083] NtACT7-g-R: ATCCTCCGATCCAGACACTG; SEQ ID NO. 10.

[0084] (5) Two strains of MPK8-OE # 1 and MPK8-OE # 3 with high expression levels were screened out (Fig. 3). The seeds of strains with high expression levels (T2) were collected for subsequent phenotypic identification when the seeds of a current generation of strains were matured.

[0085] 3) Identification of Low Temperature Tolerance Phenotype of Nicotiana tabacum L.

[0086] The seeds of transgenic pure-line plants and wild-type plants with plump and uniform sizes were selected, disinfected and sterilized, and treated at 4°C for 48 h. The seeds were then sown in a small square box pre-filled with a specific machine quality for Nicotiana tabacum L. and fully absorbed water, covered with a lid for heat preservation and moisture retention, and incubated and germinated in a greenhouse (temperature of 25°C, humidity of 75%, light for 16 h and darkness for 8 h). After sowing for 30 d (six leaves and one heart stage), Nicotiana tabacum

L. seedlings with consistent growth were selected and placed in an incubator at 4°C for low temperature treatment, and the phenotype was observed and photographed for recording. The phenotypic analysis after 5 h of low temperature treatment (4°C) is shown in Fig. 4.

[0087] Fig. 4 shows that under control conditions (temperature of 25°C, humidity of 75%), the overexpression strains MPK8-OE#1 and MPK8-OE#3 had no obvious differences in phenotypes from Xiangyan No. 7 (WT). After 5 h of low temperature treatment at 4°C, a wilting degree of

WT strains was more obvious, while the leaves of two overexpression strains only showed slight drooping, which had no significant change compared to before treatment. The results showed thdt/600447 compared with WT, the cold tolerance of MPK8-OE#1 and MPKS8-OE#3 strains was obviously enhanced.

[0088] At the time points of O h and 3 d, the third leaf (counting from a top end) was taken as a plant sample to detect physiological and biochemical indexes, and each treatment had 5 biological repetitions. After sampling, the samples were quickly wrapped in tinfoil and placed in liquid nitrogen and stored at -80°C.

[0089] 4) Determination of Physiological and Biochemical Indexes

[0090] The content of MDA was determined by a plant MDA test box (Nanjing Jiancheng, article number: A003-3-1). The content of H202 was determined by a H20: test box (Nanjing

Jiancheng, article number: A064-1-1). OFR was determined by an OFR kit (Jiangsu Edison

Biotechnology Co., Ltd., article number: ADS-W-YH008). Specific methods refer to kit instructions.

[0091] The relative electrical conductivity (EL): fresh leaves from the fourth true leaf of seedlings (from a top leaf to a bottom), 0.2 g of fresh tissue leaves were taken with a circular hole puncher, loaded into a centrifuge tube containing 25 ml ddH2O, and processed in a shaker at 37°C for 24 h. A conductivity value L1 of a first extravasation liquid was measured, and then the EP tube was put into an autoclave for treatment at 120°C for 15 min, and the conductivity value L2 of a second extravasation liquid was measured after the temperature was cooled to room temperature. The EL (EL = L1/L2 * 100%) was calculated.

[0092] Physiological and biochemical results showed that under the control conditions (temperature of 25°C, humidity of 75%, light for 16 h and darkness for 8 h), a MDA content and

EL in overexpressing strains MPK8-OE#1 and MPK8-OE#3 were not obviously different from

WT. After low temperature stress treatment, the MDA content and EL in the two overexpression strains were obviously lower than those in WT (Fig. 5).

[0093] As shown in Fig. 6, the contents of OFR and H2O2in the overexpression strains and WT before low temperature treatment were not obviously different. After low temperature stress treatment, the contents of neutralization OFR and H202 in the two overexpression strains were obviously lower than those in WT.

[0094] 3, 3'-Diaminobenzidine (DAB) staining: firstly, 0.1 mg/ml DAB was prepared and dissolved in 50 mM Tris-acetic acid buffer solution (pH 5.0). Leaves were soaked in a staininkU600447 solution and darkened overnight at room temperature. The staining solution was removed, absolute ethanol was added, and a boiling water bath was used for 10 min (if chlorophyll was difficult to dehydrate, the time could be longer). Finally, the leaves were transferred into anhydrous ethanol, photographed and observed under a microscope or camera, and preserved.

[0095] Nitrotetrazolium blue chloride (NBT) staining: firstly, 1 mg/mL of an NBT staining solution was prepared and dissolved in 10 mM PBS (pH 7.8), and the leaves were cut off and put into the NBT staining solution, stained for 1-2 h under light. Decolorization was performed when there was a chromogenic phenotype (blue leaves). The staining solution was removed, anhydrous ethanol was added, and boiled in boiling water for 10 min, preserved in 70% ethanol, and photographed for observation under a microscope or camera.

[0096] ROS histochemical staining was performed on the overexpression strains and wild types treated at 4°C (5 d). NBT staining results showed (left in Fig. 7) that under normal conditions (temperature of 25°C, humidity of 75%, light for 16 h, darkness for 8 h), although both the leaves of the overexpression strains and the WT leaves had slight blue spots, no obvious difference was found among the strains. After low temperature treatment, the leaves of the two overexpression strains were stained with a smaller staining area and lighter degree than the WT. The results showed that the overexpression strains accumulated less the OFR than a WT plant. The results of

DAB staining (right in Fig. 7) showed that at the normal temperature, the leaves of the overexpression strains were not obviously different from those of WT. After low temperature stress, the leaves of the overexpression strains MPK8-OE#1 and MPK8-OE#3 were stained with the smaller area and lighter degree. The results showed that the overexpression strains accumulated less H202 than the WT plants.

[0097] Determination of photosynthesis parameters: photosynthetic indexes were determined using a LI-6800 portable photosynthesizer (LI-COR Company, USA), a 2 cm x 3 cm red and blue light source leaf chamber was used, and a light source intensity was 300 umol/(m°-s). Five strains were randomly selected for each treatment, and the determined parameters included leaf net photosynthetic rate [umol/(m?-s))], Transpiration rate [mmol/(m*-s)], Stomatal conductance [mol/(m°:s)], and Intercellular CO» concertration [umol/mol].

[0098] Photosynthetic parameters of overexpression and wild-type strains treated with low temperature at 4°C (2.5 h) were measured. The net photosynthetic rate and intercellular CO:V600447 concentration of WT and MPK8-OE#1 were not obviously different under the normal conditions (temperature of 25°C, humidity of 75%, light for 16 h, and darkness for 8 h). Low temperature treatment decreased the net photosynthetic rate and intercellular CO2 concentration of Nicotiana tabacum L., but the net photosynthetic rate and intercellular CO2 concentration of the two overexpression strains were not obviously different from those of WT (Fig. 8 A-B). Differently, under low temperature stress, the transpiration rate and gsw of the two overexpression strains were obviously lower than those of WT (Fig. 8C-D).

[0099] The foregoing description of the disclosed examples enables those skilled in the art to realize or use the present disclosure. A variety of modifications to these examples will be obvious to those of ordinary skill in the art, and the general principles defined herein can be realized in other examples without departing from the spirit or scope of the present disclosure. Therefore, the present disclosure is not to be limited to the examples shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (5)

CLAIMS LU600447

1. An application of an NMPK8 gene in cold tolerance of Nicotiana tabacum L., wherein an NIMPKS8 gene sequence is as shown in SEQ ID NO. 1.

2. An application of an overexpression NtMPKS gene in a positive regulation of cold tolerance in Nicotiana tabacum L., wherein the NIMPK8 gene sequence is as shown in SEQ ID

NO. 1.

3. An application of biomaterials of an overexpression NMPK8 gene expression in a positive regulation of cold tolerance in Nicotiana tabacum L., wherein the NIMPKS gene sequence is as shown in SEQ ID NO. 1; the biomaterials are any one of following: A: an expression cassette capable of overexpressing an NtMPKS gene with a nucleotide sequence as shown in SEQ ID NO. 1; B: a recombinant vector comprising the expression cassette described in A; and C: a recombinant microorganism comprising the expression cassette described in A or a recombinant vector described in B.

4. An application of an MMPK8 gene in Nicotiana tabacum L. breeding, wherein the NIMPKS8 gene sequence is as shown in SEQ ID NO. 1.

5. An application of an MMPK8 gene in breeding of cold tolerance germplasm in Nicotiana tabacum L., wherein the NtMPKS gene sequence is as shown in SEQ ID NO. 1.