Use of hog genes to improve plant resistance to abiotic stress

Abstract

The present application belongs to the technical field of plant molecular biology, and particularly relates to the use of HOG gene in improving the non-biological stress resistance of plants. The nucleotide sequence of the HOG gene is shown as SEQ ID NO. 1, the non-biological stress includes salt stress, drought stress and low temperature stress, and the plants include Arabidopsis thaliana and wheat. In the present application, the HOG gene from Eurotium lavalum in the Dead Sea is introduced into Arabidopsis thaliana Columbia ecotype (Col-0) and wheat fielder variety by means of Agrobacterium-mediated genetic transformation, and the HOG gene driven by CaMV 35S promoter is introduced into the plants. After PCR identification, a plurality of transgenic positive lines are obtained. The non-biological stress resistance of the transgenic Arabidopsis thaliana and wheat is significantly improved, and the growth and some physiological indexes thereof under salt, drought and low temperature stress are superior to those of the wild type lines. The present application provides a new idea for improving the resistance of plants.

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology technology, specifically relating to the use of the HOG gene in improving plant resistance to abiotic stresses. Background Technology

[0002] The Dead Sea has a salt concentration of up to 348 g / L at its bottom, making the living environment extremely harsh. Furthermore, the seawater has a very unique ionic composition, including high levels of magnesium. 2+ and Ca 2+ The concentration is higher than that of Na. + and K + *Eurotium herbariorum*, a fungus isolated from the Dead Sea, exhibits halophilicity and salt tolerance. A MAPK encoding the HOG gene was cloned from *E. herbariorum*. Introducing the HOG gene into wild-type *Saccharomyces cerevisiae* and the hog1 mutant revealed that the HOG gene compensates for the deficiency in hog1, and the transgenic yeast showed superior resistance to lithium salts and peroxides. Therefore, it is believed that the HOG gene of *Eurotium herbariorum* possesses the ability to withstand abiotic stresses. As a unique and extremely harsh environment on Earth, the key genes of species that survive in the Dead Sea will serve as a valuable genetic resource to enhance resilience to abiotic stresses.

[0003] When yeast and animal cells are exposed to environments with continuously increasing solution concentrations (i.e., hyperosmolar stress), a conserved MAPK pathway within the cell is activated. Eukaryotes use different MAPKs to perform different functions. Kinases that specifically transmit environmental stress signals are called SAPKs. In budding yeast (Saccharomyces cerevisiae), this is called the HOG pathway. The HOG pathway is activated when microorganisms are exposed to many abiotic stresses and is crucial for their survival in various extreme environments. HOG1 is a protein kinase and the most important core of the HOG pathway. Phosphorylated HOG1 rapidly translocates from the cytoplasm to the nucleus. Once in the nucleus, HOG1 interacts with nucleoproteins. The duration of HOG1 kinase residence in the nucleus is directly proportional to the degree of cellular stress; that is, the higher the osmotic stress, the longer the residence time. It can rapidly activate more than 300 target genes, including stress-response genes, transcription factors, cell cycle regulatory genes, and genes encoding metabolic enzymes. When Saccharomyces cerevisiae is subjected to osmotic stress, 70% of the stress response genes are activated by Hog1.

[0004] The HOG gene is known as the switch of the HOG pathway. Its homologs have been reported in fungi and mammals. However, the HOG pathway and HOG gene have not been reported in plants. The use of the HOG gene in plants, especially in the context of abiotic stress, has not been reported. Summary of the Invention

[0005] One objective of this invention is to provide the use of the HOG gene in enhancing plant resistance to abiotic stresses, the nucleotide sequence of which is shown in SEQ ID NO.1. The HOG gene was cloned from the Dead Sea fungus *Eurotium herbariorum* and encodes mitogen-activated protein kinase.

[0006] Furthermore, the abiotic stresses include salt stress, drought stress, and low temperature stress.

[0007] Furthermore, the plants include Arabidopsis thaliana and wheat.

[0008] In this invention, the HOG gene, driven by the CaMV 35S promoter, was introduced into Arabidopsis thaliana Columbia ecotype (Col-0) and wheat Fielder variety via Agrobacterium-mediated genetic transformation. After PCR identification, multiple transgenic positive lines were obtained. Three transgenic Arabidopsis lines #20, #41, and #77, and two transgenic wheat lines #11 and #18 were randomly selected for further analysis.

[0009] The salt tolerance of transgenic Arabidopsis and transgenic wheat was significantly improved. Germination and survival rates were significantly higher in transgenic Arabidopsis under 175 mM NaCl and 19 mM LiCl stress than in wild-type Arabidopsis. Physiological indicators showed that the salt-tolerant transgenic Arabidopsis had lower MDA content, indicating that the transgenic plants experienced less photosynthetic inhibition and damage under salt stress, exhibiting stronger salt tolerance. After 3 weeks of LiCl stress, the transgenic wheat showed better growth than wild-type wheat, demonstrating greater salt tolerance.

[0010] The drought resistance of transgenic Arabidopsis thaliana has been significantly improved. In terms of germination rate and survival rate, under 400mM mannitol stress, the germination rate and survival rate of transgenic Arabidopsis thaliana are significantly higher than those of wild-type Arabidopsis thaliana. In the physiological index test, the drought-resistant transgenic Arabidopsis thaliana has a lower MDA content, showing stronger drought resistance.

[0011] The resistance to low-temperature stress of transgenic Arabidopsis and transgenic wheat has been significantly improved. In terms of germination rate and survival rate, after 1 day of stress at 4℃, followed by a gradient cooling to -12℃, a gradient warming to 4℃ for 1 day, and 4 days of recovery to normal conditions, the germination rate and survival rate of transgenic Arabidopsis were significantly higher than those of wild-type Arabidopsis. In the physiological index test, the transgenic Arabidopsis with strong resistance to low temperatures had lower MDA content, showing stronger resistance to low temperatures.

[0012] The second objective of this invention is to provide a plant expression vector containing the HOG gene.

[0013] Furthermore, the plant expression vector contains the CaMV 35S promoter. In a specific embodiment of the present invention, the plant expression vector is pCAMBIA3301.

[0014] A third objective of this invention is to provide an engineered bacterium containing the plant expression vector. In a specific embodiment of this invention, the engineered bacterium is Agrobacterium GV3101.

[0015] The fourth objective of this invention is to provide the use of the plant expression vector or the engineered bacteria in improving the resistance of plants to abiotic stresses, including salt stress, drought stress and low temperature stress, and the plants including Arabidopsis thaliana and wheat.

[0016] The fifth objective of this invention is to provide a method for improving the resistance of plants to abiotic stresses, including the step of overexpressing the HOG gene in plants, resulting in transgenic plants with enhanced resistance to salt, drought, and low temperature stresses; for example: operably linking the HOG gene to an expression regulatory element to obtain a recombinant plant expression vector that expresses the encoding gene in plants; transforming the recombinant plant expression vector into a target plant to overexpress the HOG gene in the plant.

[0017] The target plants include the dicotyledonous Arabidopsis thaliana and the monocotyledonous wheat, but are not limited to other monocotyledonous or dicotyledonous plants. More preferably, the target plants include crops, vegetables or ornamental plants, fruit trees, etc., such as corn, rice, sorghum, soybeans, potatoes, barley, green beans, tomatoes, peanuts, or sugarcane.

[0018] The transformation scheme and the scheme for introducing the gene into the plant can vary depending on the type of plant (monocot or dicot) or plant cell used for transformation. Suitable methods for introducing the gene into plant cells include electroporation, microinjection, Agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment. In certain embodiments, various transient transformation methods can be used to deliver the gene to the plant.

[0019] The nucleotide sequence of the HOG gene described in this invention is shown in SEQ ID NO.1. Furthermore, those skilled in the art can optimize the nucleotides shown in SEQ ID NO.1 to enhance expression efficiency in plants (especially wheat).

[0020] The beneficial effects of this invention are as follows:

[0021] This invention discloses a novel application of the HOG gene in enhancing plant resistance to abiotic stresses. The HOG pathway is a crucial pathway in the response of fungi and animals to salt stress, and the HOG gene is one of the key genes in this pathway. However, plants do not possess the HOG gene or the HOG pathway. Considering the critical role of the HOG pathway in fungi and animals, and the enhanced stress resistance brought about by the HOG gene, we integrated HOG as a foreign gene into the plant genome. Transgenic plants overexpressing HOG exhibited better tolerance to salt, drought, and freezing stresses. This demonstrates that adding a key fungal / animal regulatory gene can not only improve plant resistance to abiotic stresses, but also that the genomes of extremophiles have the potential to become a valuable resource for genetic engineering and biotechnology. Attached Figure Description

[0022] Figure 1 The figure shows PCR amplification of the HOG gene. In the figure, M: 2000bp Marker; 1-2: HOG single clone verification.

[0023] Figure 2 This is a schematic diagram of the construction of the plant expression vector pBI121-HOG.

[0024] Figure 3 PCR verification of transgenic Arabidopsis thaliana, where 1-24 are transgenic Arabidopsis thaliana with the HOG gene.

[0025] Figure 4 Western blot analysis of transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana.

[0026] Figure 5 Germination rates of transgenic Arabidopsis and wild-type Arabidopsis on different stress treatment media.

[0027] Figure 6 The root lengths of transgenic Arabidopsis and wild-type Arabidopsis were measured on media containing different concentrations of NaCl (A), LiCl (B), and Mannitol (C).

[0028] Figure 7 To assess the salt, drought and freezing resistance of transgenic Arabidopsis and wild-type Arabidopsis seedlings.

[0029] Figure 8 To determine the chlorophyll content of wild-type and transgenic lines under different stress treatments.

[0030] Figure 9 To determine the proline content in wild-type and transgenic lines under different stress treatments.

[0031] Figure 10 To determine the MDA content of wild-type and transgenic lines under different stress treatments.

[0032] Figure 11 PCR identification of transgenic wheat, where 1-29 are transgenic HOG wheat.

[0033] Figure 12 For the identification of stress resistance in transgenic wheat, A is the identification of frost resistance in HOG transgenic lines; B is the identification of lithium salt resistance in HOG transgenic lines; C is the identification of heavy metal (Cd) resistance in HOG transgenic lines. 2+ Resistance identification. Detailed Implementation

[0034] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0035] The test materials involved in the following embodiments of the present invention:

[0036] Arabidopsis thaliana L. was of the Columbia ecotype (Col-0). The plant expression vectors were PBI121 and pCAMBIA3301. The Escherichia coli strain was DH5α. E. herbariorum was isolated from Dead Sea water and is published in the following literature:

[0037] Yan J,Song W,Eviatar NA MAPK gene from Dead Sea fungus confersstress tolerance to lithium salt and freezing-thawing:Prospects for salineagriculture.[J].Proceedings of the National Academy of Sciences of the UnitedStates of America,2005,102(52):18992-7.

[0038] Example 1: Obtaining the gene encoding mitogen-activated protein kinase (HOG)

[0039] 1. Extract total RNA

[0040] Activated *Eurotium herbariorum* was isolated from the Dead Sea and cultured at 25°C on GY medium (1% yeast extract + 10% sucrose) for 2 weeks. The culture was then centrifuged at 10,000 rpm for 30 min to collect the cells. The collected cells were then soaked in GY containing 2M NaCl for a certain period of time to induce stress. Samples were then taken, quickly placed in liquid nitrogen, and stored at -80°C. Total RNA extraction from *Cyclocarya cereus*: Take 100 mg of fungal sample, place the sample in liquid nitrogen, and quickly grind it into powder. Immediately add 500 μL of RB, vortex vigorously, and transfer the liquid to a homogenization column. Centrifuge at 13,000 rpm for 5 min, transfer the supernatant, then add 0.5 volumes of anhydrous ethanol, vortex to mix, transfer the mixture to an RNA column, centrifuge at 10,000 rpm for 1 min, discard the filtrate, add 300 μL of RNA Wash Buffer I to the column, centrifuge at 10,000 rpm for 1 min, discard the filtrate, add DNase to the column membrane at room temperature, incubate for 15 minutes, add 500 μL of RNA Wash Buffer I to the column, centrifuge at 10,000 rpm for 1 min, discard the filtrate and collection tube, place the column in a new 2 ml collection tube, add 700 μL of RNA Wash Buffer II, centrifuge at 10,000 rpm for 1 min, discard the filtrate, add 500 μL of RNA Wash Buffer... II. Centrifuge at 10,000 rpm for 1 min, discard the filtrate, and centrifuge at 13,000 rpm for 2 min to completely remove anhydrous ethanol. Attach the column to a new 1.5 ml centrifuge tube, add 50 μL of DEPC Water, incubate at room temperature for 2 min, and centrifuge at 10,000 rpm for 1 min. Store the obtained RNA at -80℃.

[0041] 2. Synthesize cDNA

[0042] In a 200 μL PCR tube, add the following in sequence: 2 μL Reverse Transcription 10× Buffer, 4 μL MgCl2 (25 mM), 2 μL dNTP Mixture (10 mM), 0.5 μL Ribonuclease Inhibitor, 15 U AMV Reverse Transcriptase, 0.5 μg Oligo Primer OR Random Primers, and 1 μg RNA. Mix thoroughly by pipetting, incubate at 42°C for 15 min, then at 95°C for 5 min, incubate on ice for 5 min, centrifuge briefly, analyze, and store at -20°C.

[0043] 3. PCR amplification

[0044] Using cDNA from *Cyclocarya cereus* as a template, and primers F and R at a final concentration of 1 μmol / L, PCR amplification was performed in a 50 μl reaction system. The amplification program was as follows: 94℃ pre-denaturation for 4 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for a total of 30 cycles; 72℃ extension for 10 min.

[0045] The specific primers are designed as follows:

[0046] HOG-F (SEQ ID NO.2): 5′-ATGGCGGAATTCGTGGCGTGCCACGATT-3′

[0047] HOG-R (SEQ ID NO.3): 5′-GGCCGCGAATGCCTGCTGGCCATCCCC-3′

[0048] 4. Recover the fragments obtained from PCR.

[0049] Cut off a piece of gel containing DNA fragments and weigh it. Add sol solution at a 1:1 ratio and incubate at 55°C for 10 minutes until the gel is completely dissolved. Add 500 μL of equilibration buffer to the collection tube and centrifuge at 12,000 rpm for 1 minute. Discard the equilibration buffer. Add gel buffer to the collection tube and let stand for 2 minutes. Centrifuge at 12,000 rpm for 1 minute and discard the liquid. Add 600 μL of wash buffer and let stand for 2 minutes. Centrifuge at 12,000 rpm for 1 minute and centrifuge at 12,000 rpm for 2 minutes to remove residual alcohol. Place the collection column containing DNA fragments into a new centrifuge tube and let stand for 10 minutes to allow the alcohol to evaporate completely. Add 30 μL of ddH2O, let stand for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes. Store the recovered DNA at -20°C.

[0050] 5. Ligation of the target fragment with the T-vector

[0051] The recovered DNA was ligated into the pMD18-T vector (TaKaRa): the reagents were thawed on ice and briefly centrifuged. To a 10 μL reaction mixture, add 5 μL of Ligase Mix, 4.5 μL of the recovered PCR product, and 0.5 μL of the pMD18 vector. Mix gently and ligate at 16°C for 16 h to obtain the recombinant plasmid pMD-HOG.

[0052] 6. Transformation of Escherichia coli DH5α

[0053] Dissolve 100 μL of DH5α competent cells on ice. Add 4 μL of pMD18-DNA ligation solution to the competent cells, mix well, and incubate on ice for 30 min. Heat shock at 42°C for 90 s, immediately incubate on ice for 2 min, add 900 μL of LB liquid medium, and incubate at 37°C with shaking at 175 rpm for 1 h. Spread 200 μL of the bacterial culture onto solid medium (100 μg / ml ampicillin) and incubate overnight at 37°C. Identify the colonies by PCR.

[0054] 7. Plasmid extraction and sequencing

[0055] Pick a single colony with a toothpick and add it to 1 ml of LB liquid medium (100 μg / ml ampicillin). Incubate overnight at 37°C and 200 rpm. Add 500 μL of BL to the adsorption column CP4, centrifuge at 12000 rpm for 1 min, discard the waste liquid, centrifuge the culture at 4°C and 12000 rpm for 1 min, collect the bacterial cells, discard the supernatant, resuspend the bacteria in 500 μL of pre-chilled PI, shake thoroughly to suspend the precipitate, add 500 μL of P2, seal the tube tightly, and gently invert 10 times. Add 700 μL of P3, seal the tube tightly, and gently invert 7 times. Centrifuge the reaction mixture at 12000 rpm for 10 min, add the supernatant to the adsorption column CP4, and centrifuge at 12000 rpm for 1 min. Discard the waste liquid. Add 600 μL of PW to the CP4 adsorption column, centrifuge at 12000 rpm for 1 min, discard the waste liquid, and centrifuge the CP4 column at 12000 rpm for 2 min to completely remove the PW. Open the CP4 column and incubate at room temperature for 10 min. Add 30 μL of ddH2O to the adsorption membrane and place the membrane in a clean centrifuge tube. Incubate at room temperature for 2 min, then centrifuge at 12000 rpm for 2 min. Sequencing the extracted plasmid and using the plasmid as a template and M13 as a primer, PCR amplification was performed. The sequence is as follows:

[0056] M13-F (SEQ ID NO.4): 5′-TGTAAAACGACGGCCAGT-3′

[0057] M13-R (SEQ ID NO.5): 5′-CAGGAAACAGCTATGACC-3′

[0058] 8. Results and Analysis

[0059] Colony PCR identification results as follows Figure 1 As shown, sequencing indicates that the fragment obtained above is the mitogen-activated protein kinase encoding gene (HOG) of the present invention. The nucleotide sequence is shown in SEQ ID NO.1. The full-length sequence is 1101 nucleotides, encoding a protein with a length of 366 amino acids and a molecular weight of approximately 41 kDa, which is the mitogen-activated protein kinase. This gene is named HOG.

[0060] Example 2: Construction and transformation of plant expression vector pBI121-HOG

[0061] 1. Amplification of the target fragment

[0062] Using recombinant plasmid pMD-HOG as a template, the HOG gene with added restriction sites BamHI and SacI was amplified by PCR using specific primers B / HOG-F and S / HOG-R.

[0063] B / HOG-F (SEQ ID NO.6): 5′-CGGGATCCATGGCTGAATTCGTTAGAGCTAC-3′;

[0064] S / HOG-R (SEQ ID NO. 7): 5′-CGAGCTCTTAAGCAGCAAAAGCTTGTTGTCC-3′.

[0065] 2. Construction of expression vector by double enzyme digestion

[0066] The amplification product of HOG was recovered, and the HOG and PBI121 plasmids were digested with BamHI and SacI, respectively. The concentrations were recovered and determined to establish the ligation system. Ligation was performed at 16℃ for 16 hours to obtain the expression vector pBI121-HOG. The construction diagram is shown below. Figure 2 As shown.

[0067] 3. Preparation of Agrobacterium competent cells by CaCl2 method

[0068] Streak the preserved GV3101 on a plate containing 50 mg / ml rifampicin and incubate at 28°C. When colonies appear, take a single colony and add it to 1 ml of liquid LB medium (50 mg / ml rifampicin). Incubate at 28°C with shaking at 200 rpm for 12-16 hours. Then, add 0.5 ml of fresh bacteria to 50 ml of liquid LB medium (50 mg / ml rifampicin) and incubate at 28°C with shaking at 200 rpm for 1-2 days. Measure the OD to ensure the OD600 is approximately 0. Collect the cells by centrifuging at approximately 0.5 μL at 4°C and 5000 rpm for 10 min. Remove the supernatant. Pre-cool the cells with 0.1 mol / L CaCl2, then add 10 ml to the cells. Suspend the cells by aspiration and incubation on ice for 30 min. Centrifuge at 4°C and 5000 rpm for 10 min. Add 1 ml of pre-cooled 0.1 mol / L CaCl2 and 1 ml of 30% glycerol. Gently aspirate the suspended cells. Aliquot the suspension and store at -80°C.

[0069] 4. Transformation of Agrobacterium using the freeze-thaw method

[0070] Take two tubes of 100 μL Agrobacterium competent cells, thaw them on ice, add 5-8 μL of pBI121-HOG or ddH2O to each tube, gently aspirate and mix, then place on ice for 30 min, place in liquid nitrogen for 2 min, then quickly transfer to a constant temperature water bath, heat shock at 37℃ for 5 min, place on ice for 2 min, add 900 μL of LB liquid culture medium, shake at 200 rpm for 4-6 h, centrifuge at 5000 rpm for 2 min, discard the supernatant, suspend the bacterial cells in 100 μL of LB, spread the bacterial solution evenly on LB solid medium containing antibiotics (containing 50 mg / ml kanamycin and 50 mg / ml rifampin), and incubate at 28℃ inverted for 36-48 h. Transformants were identified by PCR. Positive clones that amplified the correct bands were selected and added to LB liquid medium (containing 50 mg / ml kanamycin and 50 mg / ml rifampin). The medium was incubated overnight at 28°C and 200 rpm. Plasmids were extracted and double enzyme digestion was performed for verification. 700 μL of the verified positive bacterial culture pBI121-HOG-GV3101 was taken and 300 μL of 50% glycerol was added. After mixing, the mixture was flash-frozen in liquid nitrogen and stored at -80°C.

[0071] Example 3: Obtaining transgenic Arabidopsis and identifying its resistance to abiotic stress

[0072] 1. Infection of Arabidopsis thaliana by dipping flowers

[0073] After the flower buds of the wild-type plants sprout, cut off the main stem. When the flower buds of the lateral branches begin to sprout, prepare for infection. Take the preserved strain pBI121-HOG-GV3101 and place it on ice to dissolve. Take 1 μL of pBI121-HOG-GV3101 and add it to 1 ml of LB liquid medium (containing 50 mg / ml kanamycin and 50 mg / ml rifampin). Incubate overnight at 28°C and 200 rpm. Add all 1 ml of the culture solution to 50 ml of LB liquid medium (containing 50 mg / ml kanamycin and 50 mg / ml rifampin). Incubate overnight at 28°C and 200 rpm. Water the wild-type plants to be transformed thoroughly. When the concentration of the bacterial solution reaches OD600 = 0.8-0.9, stop shaking. Centrifuge at 5000 rpm for 15 min, discard the supernatant, suspend the bacterial cells in 50 ml of transformation buffer, immerse the flower buds of the wild-type plants to be transformed in the transformation buffer for 1 min, incubate in the dark for 24 h, and then culture normally. Infect once a week, for a total of 2 infections.

[0074] 2. Identification of transformed strains

[0075] T0 generation transgenic seeds were sterilized with 75% anhydrous ethanol, and the process was repeated three times. The sterilized seeds were sown on MS medium (50 mg / ml kanamycin) for screening, with about 500 seeds sown in each dish. After sealing with sealing film, the dishes were vernalized at 4°C for 3 days. The dishes were then grown under normal conditions. After about two weeks, the resistant green seedlings were transplanted into the substrate to continue growing. When the plants grew to 8 leaves, PCR molecular detection was performed. After the T1 generation transgenic plants grew to 8 leaves, the DNA of Arabidopsis thaliana was extracted using the CTAB method modified in our laboratory and identified by PCR: 0.2g of leaves were placed in a mortar, liquid nitrogen was added and the mixture was ground into powder. Before the liquid nitrogen had completely evaporated, the powder was quickly transferred into a 1.5ml centrifuge tube, 500μL of pre-cooled DS buffer was added, and the mixture was inverted to mix. Then 750μL of phenol-chloroform-isoamyl alcohol was added, the mixture was inverted for 20-30 seconds, placed on ice for 20 minutes, and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected, 600μL of chloroform was added, the mixture was gently mixed, and centrifuged at 10,000 rpm for 10 minutes at 4°C. The supernatant was collected, an equal volume of isoamyl alcohol and 1 / 10 volume of NaAc were added, the centrifuge tube was gently inverted several times, and then centrifuged at 10,000 rpm for 10 minutes at 4°C. The liquid phase was discarded, and the DNA was washed twice with 70% ethanol. The mixture was then air-dried at room temperature, and 50μL of ddH2O was added to dissolve the DNA. The primers for PCR detection of Arabidopsis thaliana transgenic with the HOG gene are HOG2-F and HOG2-R, and the primer sequences are as follows:

[0076] HOG2-F (SEQ ID NO.8): 5′-AGAACATTATCTGCCTGAGCG-3′;

[0077] HOG2-R (SEQ ID NO. 9): 5'-TTAGGAAGCGACTTGACGA-3'.

[0078] T1 generation plants selected through antibiotic screening and positive plants (T0 generation) were continuously self-pollinated. After self-pollination of T0 generation seeds, the harvested seeds from individual plants were designated as T1, and each seed from an individual plant constituted a line. When kanamycin was used for screening, non-transgenic plants showed yellowing and died after germination, while transgenic plants could grow and survive normally. Therefore, each generation of seeds was screened using MS with kanamycin. After germination, the ratio of green seedlings to yellow seedlings was calculated to be 1:3. This process was repeated until homozygous transgenic lines that did not segregate in the offspring were selected.

[0079] 3. Identification of abiotic stress resistance in transgenic Arabidopsis thaliana

[0080] Seed germination rate determination under different abiotic stresses: Transgenic Arabidopsis #20, #41, and #77 seeds and WT seeds were disinfected with 75% alcohol for 1 min, rinsed twice with ddH2O, and repeated 3 times. Sodium salt stress treatment: 120 seeds were evenly sown on 0 mM, 50 mM, 100 mM, 150 mM, and 175 mM NaCl MS medium; Lithium salt stress treatment: 120 seeds were evenly sown on 0 mM, 10 mM, 15 mM, 17 mM, and 19 mM LiCl MS medium; Drought stress treatment: 120 seeds were evenly sown on 0 mM, 200 mM, 300 mM, and 400 mM Mannitol MS medium. Freezing stress: 120 seeds were evenly sown on MS medium and incubated at 4℃. 30 seeds were planted per line, and the germination rate was recorded after one week.

[0081] Root growth rate determination under different abiotic stresses: Transgenic Arabidopsis lines #20, #41, and #77, as well as WT seeds, were sterilized and sown on MS medium, with 10 plants from each line. After germination, the seeds were transferred to MS medium containing different stresses: 0 mM, 50 mM, 100 mM, 150 mM, and 175 mM NaCl; 0 mM, 10 mM, 15 mM, 17 mM, and 19 mM LiCl; and 0 mM, 200 mM, 300 mM, and 400 mM Mannitol. The culture dishes were incubated vertically, and root length was measured daily.

[0082] Growth under different abiotic stresses: Three transgenic Arabidopsis lines (#20, #41, #77) and wild-type seeds were sown in a substrate. After two weeks of cultivation under normal conditions, salt stress treatment was performed by increasing the salt concentration by 50 mM every three days, with final NaCl concentrations of 100 mM, 150 mM, 200 mM, 250 mM, and 300 mM. Plants grown under normal conditions for two weeks were treated with drought stress by withholding water for 10 days, followed by rehydration for 4 days. Plants under freezing stress were moved to 4 degrees Celsius and grown for two weeks. Growth and different physiological indicators of transgenic Arabidopsis lines #20, #41, and #77 and wild-type plants were measured after different abiotic stresses. Six plants of each type were placed in each pot.

[0083] Determination of chlorophyll content in Arabidopsis thaliana: Weigh 0.2g of leaf tissue from specimens subjected to salt, drought, low-temperature stress, and normal conditions. Place the tissue in a mortar, add a small amount of quartz sand and 13ml of 95% ethanol, and grind into a homogeneous slurry. Let stand for 5 minutes, filter the slurry, and rinse the mortar and other materials with 95% ethanol, ensuring all chlorophyll is washed into the volumetric flask. Finally, bring the volume to 25ml, shake well, and transfer 3ml of chlorophyll to a cuvette. Measure the absorbance at 665nm and 649nm. Use 95% ethanol as a blank control. Calculate the concentrations of chlorophyll a and chlorophyll b using formulas 1-1 and 1-2, respectively.

[0084] Ca = 13.95 * OD665 - 6.88 * OD649 (Equation 1-1)

[0085] Cb = 24.96 * OD649 - 7.32 * OD665 (Equation 1-2)

[0086] Calculate the chlorophyll content according to Equation 1-3:

[0087] Chlorophyll content (mg / g) = [(Ca+Cb)*V*n] / W (Equation 1-3)

[0088] Where: V: volume of extract; n: dilution factor; W: fresh weight of sample.

[0089] Determination of proline content in Arabidopsis thaliana: Preparation of standard curve: Take 2 ml of proline standard solutions of different concentrations, add 2 ml, 2 ml, and 4 ml of 3% sulfosalicylic acid, glacial acetic acid, and 2.5% ninhydrin respectively, mix well, and develop color in boiling water for 1 hour. After 1 hour, remove and cool, add 4 ml of toluene, shake thoroughly, and let stand at room temperature for layering. The last red toluene layer is used as the test sample, and the control is without proline standard solution. Measure the OD520 value. Weigh 0.5 g of leaf tissue, take 5 ml of 3% sulfosalicylic acid, grind into a homogenate, and boil in water for 10 min. After cooling, centrifuge at 3000 rpm for 10 min, take 2 ml of supernatant, add 2 ml, 2 ml, and 4 ml of distilled water, glacial acetic acid, and 2.5% ninhydrin respectively, boil in water for 60 min, take the supernatant, and measure the OD520 absorbance value. Calculate the proline content according to the standard curve.

[0090] Arabidopsis thaliana MDA determination: Weigh 1g of leaf tissue, add 10ml of 10% trichloroacetic acid, grind, centrifuge the homogenate at 4000rpm for 10min, collect the supernatant, take 2ml of the supernatant, add 2ml of 0.6% thiobarbituric acid. Mix well, boil in water for 15min, cool, and centrifuge at 4000rpm for 10min. Take the supernatant and measure the OD532, OD600, and OD450 values. Calculate the MDA and soluble sugar concentrations according to formulas 1-4.

[0091] MDA = 6.45 * (A532 - A600) - 0.56 * A450 (Equation 1-4)

[0092] The unit of MDA is μmol·L⁻¹. -1 .

[0093] 4. Results and Analysis

[0094] In this embodiment, the HOG gene driven by the CaMV 35S promoter was ligated into the plant binary expression vector PBI121 and introduced into the Arabidopsis thaliana Columbia ecotype (Col-0) variety via Agrobacterium-mediated genetic transformation. Multiple transgenic positive lines were obtained after PCR identification. Figure 3 Three transgenic Arabidopsis lines, #20, #41, and #77, were randomly selected. Figure 4 Further analysis was conducted.

[0095] like Figure 5As shown, the transgenic Arabidopsis lines #20, #41, and #77 exhibited higher seed germination rates than wild-type Arabidopsis under abiotic stress. When NaCl concentrations exceeded 150 mM, LiCl concentrations exceeded 17 mM, and mannitol concentrations exceeded 400 mM, the germination rates of transgenic Arabidopsis lines #20, #41, and #77 reached over 75%, while the transgenic lines showed virtually no germination. After exposure to salt, drought, and low-temperature stress, the transgenic Arabidopsis lines #20, #41, and #77, after two weeks of growth, all showed good growth, while the wild-type Arabidopsis showed significant wilting, yellowing, and even death.

[0096] like Figure 6 As shown, when the NaCl concentration is greater than 100 mM, the LiCl concentration is greater than 15 mM, and the mannitol concentration is greater than 300 mM, the root lengths of transgenic Arabidopsis lines #20, #41, and #77 are significantly longer than those of wild-type Arabidopsis.

[0097] like Figures 7-10 As shown, transgenic Arabidopsis thaliana lines exhibited significantly higher chlorophyll and proline content and significantly lower malondialdehyde content than wild-type Arabidopsis thaliana under salt, drought, and freezing stress, indicating that the transgenic Arabidopsis thaliana lines have improved stress resistance.

[0098] Example 4: Obtaining transgenic wheat and identifying its resistance to abiotic stress

[0099] 1. Constructing genetically modified wheat

[0100] Design primers containing the pCAMBIA3301 homologous arm:

[0101] HOG-F (SEQ ID NO. 10): 5′-ggtacccggggatcctctagaATGGCGGAATTCGT GCGT-3′

[0102] HOG-R (SEQ ID NO. 11): 5′-acgacggccagtgccaagcttTTAGGCCGGAATG CCTG-3′

[0103] Using Dead Sea fungus *Eurotium herbariorum* DNA as a template, the HOG gene fragment was cloned, and the HOG gene was ligated into pCAMBIA3301 via homologous cloning to construct an expression vector. The constructed vector was then transformed into *Agrobacterium* EHA105 to transform the wheat variety Fielder. The specific steps for HOG gene transformation in wheat followed the method of Ishida et al. (Ishida Y, Tsunashima M, Hiei Y, Komari T. Wheat (Triticum aestivum L.) transformation using immature embryos. Methods Mol). Biol.2015;1223:189-98.doi:10.1007 / 978-1-4939-1695-5_15.PMID:25300841.) Transgenic seedlings with well-developed root systems were transplanted into 15cm diameter pots and cultured in a greenhouse under the following conditions: temperature: 22℃, light intensity: 30000lx, 16h light / 8h dark.

[0104] 2. Identification of genetically modified wheat

[0105] Genomic DNA was extracted from transgenic wheat and Fielder wild-type wheat. Specific detection primers were used to verify by PCR whether the HOG gene was integrated into the wheat genome. DNA extraction was performed using the DS method (Weining S, Langridge P. Identification and mapping of polymorphisms in cereals based on the polymerase chain reaction. Theor Appl Genet. 1991 Aug; 82(2):209-16. doi:10.1007 / BF00226215.PMID:24213068.). The specific steps are as follows: Take an appropriate amount of wheat leaves into a 2ml centrifuge tube, add 2 steel beads, freeze quickly in liquid nitrogen, vortex and grind into dry powder, add 800μl of DNA extraction buffer, vortex and mix well, remove the steel beads with a magnet, and add an equal volume of phenol. Chloroform:isoamyl alcohol (25:24:1), vortex vigorously to mix, incubate on ice for 20 min, centrifuge at 10000g for 10 min at 4°C, aspirate the supernatant and transfer to a new 1.5 ml centrifuge tube, add an equal volume of chloroform, vortex vigorously, transfer the supernatant to a new 1.5 ml centrifuge tube, add an equal volume of pre-chilled isopropanol and 1 / 10 volume of 3M NaAc, gently invert to mix, precipitate DNA, slowly discard the supernatant, add 1 ml of 70% ethanol to the tube to wash the DNA, centrifuge at 10000g for 10 min at 4°C, slowly discard the 70% ethanol, and air dry the DNA. Add 50 μl of sterile ddH2O to the dried DNA to dissolve the DNA, mix well, and determine the quality of the extracted DNA using a Nano Drop ND-1000 ultra-micro spectrophotometer and 1% agarose gel electrophoresis. PCR identification (1213 bp) was performed using specific HOG detection primers.

[0106] HOG-F (SEQ ID NO. 12): 5′-CCCCTCCCACAACCTCTTT-3′;

[0107] HOG-R (SEQ ID NO. 13): 5'-CCTCTCCTGCACGTATCCCT-3'.

[0108] Multiple transgenic positive lines were obtained ( Figure 11 Two transgenic wheat lines, #11 and #18, were randomly selected.

[0109] 3. Identification of abiotic stress resistance in transgenic wheat

[0110] Lithium resistance identification: T3 homozygous HOG transgenic wheat and Fielder seedlings cultured normally to the three-leaf stage in soil were treated with 80 mM LiCl for 7 days, and then the LiCl concentration was increased to 100 mM for 14 days.

[0111] Freeze resistance assessment: Transgenic wheat and Fielder seedlings that had been normally cultured in soil to the three-leaf stage were placed in a low-temperature incubator at 4°C for 24 hours. Then the temperature was decreased by 1°C every 45 minutes until it reached -12°C (12 hours). Subsequently, the temperature was increased by 1°C every 45 minutes until it reached 4°C (12 hours). The seedlings after freezing were cultured at 4°C for 24 hours and then cultured under normal conditions for 7 days. The survival rate of transgenic seedlings and Fielder seedlings was observed.

[0112] Salt tolerance (Na+) assessment: Transgenic wheat and Fielder seedlings cultured normally in soil to the three-leaf stage were treated with three concentration gradients of NaCl (100 mM, 200 mM, and 250 mM, with the concentration increased every three days), and the growth and survival rate of the seedlings were observed.

[0113] Heavy metal resistance identification: Transgenic wheat and Fielder seedlings cultured normally to the three-leaf stage in soil were treated with 50 μM cadmium chloride solution for 7 days, and then treated with 80 μM cadmium chloride solution for 14 days.

[0114] Drought resistance assessment: Transgenic wheat and Fielder seedlings cultured normally in the soil to the three-leaf stage were stopped from watering until they wilted, and then re-watered, and the seedling survival rate was observed.

[0115] 4. Results and Analysis

[0116] like Figure 12 As shown, after freezing, wild-type wheat died completely, while transgenic wheat lines #11 and #18 suffered less frost damage and could recover under normal culture conditions later. Under lithium salt stress, wheat growth and development were severely inhibited due to ion toxicity; wild-type wheat almost completely lost its green color and died, while transgenic wheat lines #11 and #18 showed faster growth and development than the wild type, and their leaves remained green. Under heavy metal cadmium stress, the growth of transgenic wheat lines #11 and #18 and the wild type were significantly inhibited, but they could still grow normally, indicating that the transgenic wheat lines have improved stress resistance.

[0117] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the invention.

[0118] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. HOG The use of genes in improving plant resistance to abiotic stresses is characterized by, By overexpressing the above in plants HOG Genes that enhance plant resistance to abiotic stresses, the aforementioned HOG The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the abiotic stress is drought stress and low temperature stress; the plant is Arabidopsis thaliana or wheat.

2. A method for improving plant resistance to abiotic stress, characterized in that, Includes overexpression in plants of the form described in claim 1 HOG The steps of gene generation, wherein the abiotic stress is drought stress and low temperature stress, and the plant is Arabidopsis thaliana or wheat.

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