CN115927453A

CN115927453A - Application of malic acid dehydrogenase gene in improving absorption and metabolism capacity of plant formaldehyde

Info

Publication number: CN115927453A
Application number: CN202310046688.6A
Authority: CN
Inventors: 李昆志; 赵星; 汪子扬; 年洪娟
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2023-01-31
Filing date: 2023-01-31
Publication date: 2023-04-07
Anticipated expiration: 2043-01-31
Also published as: CN115927453B

Abstract

The invention discloses an application of a malate dehydrogenase gene AtMDH1 in improving the absorption and metabolism capacity of plant formaldehyde, wherein the nucleotide sequence of the malate dehydrogenase gene AtMDH1 is shown as SEQ ID NO 1; the experimental result shows that under the same concentration and stress and the same time, the formaldehyde absorption effect of the tobacco and the arabidopsis thaliana overexpressed by the AtMDH1 is better than that of the wild type; tobacco and arabidopsis thaliana leaf oxidation damage index H with AtMDH1 overexpression ₂ 0 ₂ The content is obviously lower than that of a wild type; the transcription level and the protein level of AtMDH1 of wild arabidopsis are obviously increased under the stress of liquid formaldehyde; fruit of Chinese wolfberryExperiments prove that the At14-3-3PSI protein interacts with AtMDH1, the transcriptional level of AtMDH1 is reduced by the over-expression of At14-3-3PSI, and the interaction of the At14-3-3PSI protein and AtMDH1 is reduced by the stress of HCHO.

Description

Application of malic acid dehydrogenase gene in improving absorption and metabolism capacity of plant formaldehyde

Technical Field

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to an application of an arabidopsis thaliana malate dehydrogenase gene AtMDH1 in improving formaldehyde absorption and metabolism capacity of plants.

Background

Formaldehyde (HCHO) is the simplest saturated aldehyde and is one of the most important common carcinogenic environmental pollutants. Formaldehyde is widely present in gasoline exhaust, fumes, coatings and industrial products, resulting in the production of large amounts of HCHO-containing wastewater and HCHO air pollution. Formaldehyde is one of the most typical environmental pollutants and has a pungent odor. Due to its use in industrial and consumer products, formaldehyde is widely distributed in living organisms. In addition, formaldehyde has been classified as the first human carcinogen by the international cancer research institute. HCHO, a ubiquitous environmental pollutant, binds non-specifically to a wide variety of lipids, nucleic acids and proteins. Thus, prolonged exposure to formaldehyde can lead to health problems such as upper respiratory illness, allergies, cancer, leukemia, and possibly death. Formaldehyde may also cause dysfunction in many tissues and organs of animals and humans. The development of formaldehyde pollution remediation technology is receiving wide attention, and in the practically applied remediation technology, phytoremediation is considered as an environment-friendly and sustainable development technology. In a plant purification system, the leaves can absorb HCHO in the contaminated air and convert it to carbohydrates and amino acids through the calvin cycle and C1 metabolism. The existing view considers that the absorption capacity of plant formaldehyde is related to the formaldehyde metabolism capacity of the plant formaldehyde, so that the method for improving the formaldehyde metabolism level of the plant by using the modern genetic engineering technology and promoting the continuous high-level formaldehyde metabolism capacity of the plant is an important way for improving the formaldehyde absorption capacity of the plant.

Malate dehydrogenase is widely present in nature, promotes the reversible conversion of malate into oxaloacetate, is critical to energy balance, plant growth and development, and plays an important role in abiotic stress responses. Malate is an important intermediate that plays a key role in plant nutrition, metabolism and cellular energy supply. ROS are chemically reactive molecules containing oxygen, which are harmful to plant cells due to the generation of oxidative stress. Under environmental stress conditions, the regulation of multiple redox and Reactive Oxygen Species (ROS) signals in plants requires a high degree of coordination and balance between the signals and metabolic pathways in the different cell compartments. MDH responses are involved in central metabolism and redox homeostasis between organelle compartments, and cytoplasmic MDH is thought to be a key role in the transfer of reducing equivalents to other destinations by chloroplasts or mitochondria in plant cells. Research shows that after the MDH is over-expressed, the increase of the GSH content of the non-enzymatic antioxidant is beneficial to directly eliminating ROS and protecting plants from oxidative stress induced by stress. Plants may have increased MDH expression when subjected to some abiotic stress.

At present, no relevant report that the effect of an arabidopsis thaliana malate dehydrogenase gene AtMDH1 in the process of improving the absorption and metabolism of formaldehyde by plants and the effect of an At14-3-3PSI protein in the process of improving the absorption and metabolism of formaldehyde by plants through regulating and controlling the arabidopsis thaliana malate dehydrogenase gene are found.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a new application of an arabidopsis thaliana malate dehydrogenase gene AtMDH1, namely the arabidopsis thaliana malate dehydrogenase gene AtMDH1 is applied to the improvement of the absorption and metabolism capability of plants, and the GenBank accession number of the arabidopsis thaliana malate dehydrogenase gene AtMDH1 is as follows: AT1G04410; the transgenic plant with enhanced formaldehyde absorption capacity is constructed by utilizing the AtMDH1 gene, can be used for further research on the gene, and can also be planted in soil with serious formaldehyde pollution.

In order to achieve the above object of the present invention, the technical solution of the present invention is as follows:

1. selecting a malic dehydrogenase gene AtMDH1 of a model plant Arabidopsis thaliana as an experimental material, and constructing a prokaryotic expression vector pGEX-4T-1-AtMDH1 of the AtMDH1 gene

Cloning a malic dehydrogenase gene AtMDH1 full length from arabidopsis leaves, successfully constructing an AtMDH1 gene prokaryotic expression vector, inducing expression protein in BL21, and purifying to obtain AtMDH1 protein; the enzyme characteristic analysis shows that the optimum temperature of AtMDH1 is 40 ℃, the enzyme can be seen to form white flocculent precipitate due to denaturation at 65 ℃, and the enzyme activity can not be basically detected; the optimum pH value of AtMDH1 is alkaline and is about 8.0; k is ⁺ Has obvious activation effect on enzyme; mg (Mg) ²⁺ And Ca ²⁺ Has no influence on enzyme activityLarge; zn ²⁺ And Cu ²⁺ Inhibiting the enzyme activity to different degrees; cu (copper) ²⁺ The inhibition degree is strongest; and polyclonal rabbit antibody is prepared by using the purified protein, and can be used as primary antibody for subsequent analysis after detection;

2. construction of plant expression vector pSPYCE-AtMDH1 of AtMDH1 Gene

Total RNA from Arabidopsis was extracted using RNAioso Plus (Takara) reagent according to the instructions; and carrying out reverse transcription to obtain cDNA of AtMDH1; searching a CDS sequence (AT 1G 04410) of Arabidopsis thaliana AtMDH1 AT NCBI, and designing double enzyme cutting sites (Sal I and Sma I) and gene specific primers by DNAman software;

upstream primer 5' -GTCGACATGGCGAAAGACAGTTCG (containing SalI cleavage site)

A downstream primer 3' -CCCGGGGAGAGAGGCATAGTACGCC (containing SmaI enzyme cutting site);

PCR amplification is carried out by using the primer to obtain a full-length DNA fragment (999 bp, shown as SEQ ID NO: 1) of the coding region of the AtMDH1; connecting to pMD18-T vector by enzyme digestion, transforming Escherichia coli DH5 alpha by heat shock, and obtaining TA clone containing correct sequence by resistance screening and sequencing; and then enzyme digestion connection is carried out to connect the fragment to a pSPYCE-35S vector, and a plant expression vector pSPYCE-AtMDH1 is obtained through resistance screening and sequencing. Transferring pSPYCE-AtMDH1 into agrobacterium tumefaciens by a freeze-thaw method, transforming wild tobacco into the agrobacterium tumefaciens by a leaf disc transformation method by using agrobacterium tumefaciens with correct screening detection, transforming wild arabidopsis thaliana into the agrobacterium tumefaciens by a floral disc transformation method, obtaining a regeneration plant, and performing the following screening and detection;

3. detecting the expression level of genome and protein of transgenic plants obtained by resistance screening to obtain a plurality of transgenic AtMDH1 gene tobacco and Arabidopsis thaliana, and propagating;

4. selecting robust plants with consistent growth from plants of wild type tobacco, wild type arabidopsis thaliana, atMDH1 gene-transferred tobacco, atMDH1 gene-transferred arabidopsis thaliana, atMDH1 expression inhibiting arabidopsis thaliana mutant Atmdh1, at14-3-3PSI gene-transferred tobacco and At14-3-3PSI expression inhibiting arabidopsis thaliana mutant, taking 1g of leaves, washing with tap water for 3-4 times to remove dirty dust, absorbing residual water on the surface with sterile absorbent paper, transferring to treatment solution containing 2mM HCHOIn a 50mL culture flask, light irradiation (100. Mu. Mol. M) was continued at 25 ℃ ^-2 ·s ^-1 ) Performing shaking culture at 100rpm for 0h, 6h, 12h, 24h, 36h, 48h and 60h, taking a sample without plant leaves and only added with formaldehyde as a control (BC), and comparing formaldehyde absorption efficiency and physiological and biochemical indexes of different types of tobacco and arabidopsis thaliana;

5. the yeast double-hybrid, pull-down, LCA and BiFC experiments prove that At14-3-3PSI protein and AtMDH1 are interacted, HCHO stress weakens the interaction of the At14-3-3PSI protein and AtMDH1, and the At14-3-3PSI is proved to change the transcription level of enzyme through the interaction with the metabolism key enzyme so as to regulate formaldehyde metabolism; the types of metabolites of wild type, transgenic tobacco and transgenic arabidopsis leaves treated by 2mM HCHO liquid for 24h are the same, and the over-expressed metabolites are higher than that of the wild type, and the result shows that the over-expressed transgenic tobacco and arabidopsis have stronger formaldehyde absorption capacity than that of the wild type tobacco and arabidopsis.

The invention has the beneficial effects that:

the gene AtMDH1 is over-expressed in tobacco and arabidopsis thaliana, the resistance of the plant to formaldehyde is improved, the formaldehyde metabolism capability of the plant is also improved, atMDH1 expression is regulated and the formaldehyde tolerance of the plant is enhanced through the interaction of AtMDH 14-3-3PSI and AtMDH1 protein, the formaldehyde absorption and metabolism capability of the plant is improved, and the AtMDH 14-3-3PSI and AtMDH1 gene can be used as a potential important modification target in the improvement of the formaldehyde tolerance of the plant, so that the application of the AtMDH1 gene provides a simple and quick way for molecular breeding for improving the formaldehyde absorption and metabolism capability of the plant, the obtained seeds after cultivation are easy to store and convenient to popularize and plant, the transgenic tobacco and arabidopsis thaliana can be planted in soil with serious formaldehyde pollution, and have important significance for purifying formaldehyde pollution.

Drawings

FIG. 1 is a schematic diagram showing the results of the electrophoretic detection of PCR verification (A) of the pMD-18T-AtMDH1 vector, verification (B) of the pGEX-4T-1-AtMDH1 vector and transformation detection (C) of BL 21;

FIG. 2 is a schematic diagram showing the results of the detection of the induced expression (A) and the purification (B) of the recombinant protein pGEX-4T-1-AtMDH 1;

FIG. 3 is the effect of temperature on the activity of AtMDH1 enzyme;

FIG. 4 shows the results of thermal stability analysis of AtMDH1 enzyme;

FIG. 5 is the effect of pH on the enzymatic activity of AtMDH1;

FIG. 6 shows Western blot (A) and two-way immunodiffusion assay (B) for detecting the titer of AtMDH1 antibody;

FIG. 7 is a schematic diagram showing the results of the electrophoretic detection of PCR verification (A) for the pMD-18T-AtMDH1 vector, verification (B) for the pSPYCE-AtMDH1 plant expression vector, and transformation detection (C) for Agrobacterium GV 3101;

FIG. 8 is a schematic diagram of the tobacco genome PCR (A) and western blot (B) detection of the over-expressed AtMDH1 gene and the results of the Arabidopsis genome PCR (C) and western blot (D) of the AtMDH1 gene; 3-12 in the A picture are tobacco transgenic lines, and 2-11 in the C picture are Arabidopsis transgenic lines;

FIG. 9 shows the results of the absorption of a formaldehyde solution At a concentration of 2mM by leaves of wild type Arabidopsis thaliana (Col-0), at14-3-3 PSI-overexpressing Arabidopsis thaliana (At 14 PSI), atMDH 1-overexpressing Arabidopsis thaliana (AtMDH 1), at14-3-3 PSI-expressing-suppressing Arabidopsis thaliana (At 14 PSI), and AtMDH 1-expressing-suppressing Arabidopsis thaliana (Atmdh 1);

FIG. 10 shows the results of liquid absorption of 2mM formaldehyde concentration into leaves of wild type tobacco (WT) and AtMDH 1-overexpressing tobacco (M8);

FIG. 11 shows H-treated wild type Arabidopsis thaliana (Col-0), at14-3-3PSI overexpressing Arabidopsis thaliana (At 14 PSI), atMDH1 overexpressing Arabidopsis thaliana (AtMDH 1), arabidopsis thaliana (At 14 PSI) inhibiting expression of At14-3-3PSI, and Arabidopsis thaliana (Atmdh 1) blade inhibiting expression of AtMDH1 after HCHO ₂ O ₂ (A scheme) and O ₂ · ^- (panel B) staining results;

FIG. 12 shows H after HCHO treatment of wild type tobacco (WT) and AtMDH1 overexpressing tobacco (M8) ₂ O ₂ (A scheme) and O ₂ · ^- (panel B) staining results;

FIG. 13 shows the relative expression level (graph A) and protein level (graph B) of AtMDH1 in AtMDH1 overexpressing Arabidopsis thaliana after liquid HCHO treatment;

FIG. 14 shows the results of a yeast two-hybrid assay;

FIG. 15 shows the results of Luciferase Complementation Assay (LCA) assay;

FIG. 16 is a metabolic profile of leaves of wild type Arabidopsis thaliana (Col-0), at14-3-3PSI overexpression Arabidopsis thaliana (At 14 PSI), atMDH1 overexpression Arabidopsis thaliana (AtMDH 1), at14-3-3PSI expression inhibition Arabidopsis thaliana (At 14 PSI), atMDH1 expression inhibition Arabidopsis thaliana (Atmdh 1) after 24 hours of formaldehyde treatment;

FIG. 17 is the metabolic profile of wild type tobacco (WT) and AtMDH1 overexpressing tobacco (M8) leaves after 24h formaldehyde treatment;

FIG. 18 shows the relative integration results of the metabolic products of wild type Arabidopsis thaliana (Col-0), at14-3-3PSI overexpression Arabidopsis thaliana (At 14 PSI), atMDH1 overexpression Arabidopsis thaliana (AtMDH 1), at14-3-3PSI expression inhibition Arabidopsis thaliana (At 14 PSI), and AtMDH1 expression inhibition Arabidopsis thaliana (Atmdh 1) leaves treated with formaldehyde; wherein graph A is the relative integral of malic acid, graph B is the relative integral of asparagine, graph C is the relative integral of aspartic acid, and graph D is the relative integral of glutamine;

FIG. 19 is the relative integral of metabolites of wild type tobacco (WT) and AtMDH1 overexpressing tobacco (M8) leaves after formaldehyde treatment, wherein A is the relative integral of malic acid, B is the relative integral of asparagine, C is the relative integral of aspartic acid, and D is the relative integral of glutamine;

FIG. 20 shows the results of the relative expression amounts of AtMDH1 gene in At14-3-3PSI overexpression Arabidopsis thaliana and At14-3-3PSI expression inhibition Arabidopsis thaliana;

FIG. 21 shows the experimental results of the interaction level between At14-3-3PSI and AtMDH1 after HCHO treatment, wherein, graph A is LUC image, and graph B is Westernblot analysis result.

Detailed Description

The invention is explained in more detail below with reference to examples and figures, without limiting the scope of the invention. In the examples, the procedures were carried out in accordance with the usual procedures unless otherwise specified, and all the reagents used were either the conventional commercial reagents or the reagents prepared in accordance with the conventional procedures unless otherwise specified.

Example 1: construction of prokaryotic expression vector of AtIDH3 gene and protein expression purification

Extracting total RNA of arabidopsis thaliana by using an RNAasso Plus kit (Takara) according to the instruction; using the HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wip)er) (Vazyme) reverse transcription kit reverse transcription of RNA into cDNA was performed according to the instructions. The CDS sequence (AT 1G 04410), 999bp, of Arabidopsis thaliana MDH1 was found AT NCBI, and its double cleavage sites (BamH I and Xho I) and gene-specific primers (F-GGATCCATGGCGAAGAACCAGTTCG; R-CTCGAGTTAAGAGAGGGCATAGGAGTACG) were designed by DNAman software. mu.L of cDNA was used as a template, and 50. Mu.L of the AtMDH1 fragment was PCR-amplified using the designed specific primers according to 2 XEs Taq MasterMix (Dye) (Kangshi century) instruction system (annealing temperature 58 ℃). Recovering AtMDH1 gene fragment (999 bp) from gel, performing TA cloning with pMD18-T (Takara) to obtain pMD18-AtMDH1 vector, heat shock transforming DH5 alpha, coating and inoculating in ampicillin (Amp) solid culture medium, and culturing at 37 deg.C in thermostat overnight. And (3) selecting a single bacterial colony in a liquid culture medium for screening culture in the next day, taking 1 mu L of bacterial liquid as a DNA template for PCR detection after the bacterial liquid is turbid (figure 1A), and sending the successfully detected bacterial liquid extracted plasmid to a company for sequencing. The sequencing results were aligned with AtMDH1 using DNAman software, and the correctly aligned pMD18-AtMDH1 plasmid and pGEX-4T-1 vector were double digested using BamH I and Xho I, respectively (25. Mu.L plasmid, 2.5. Mu.L BamH I, 2.5. Mu.L Xho I, 5. Mu.L 10 XBuffer K, 15. Mu.L ddH ₂ O), observing the target fragment by gel electrophoresis, and carrying out gel recovery on the target gene fragment AtMDH1 and the cut pGEX-4T-1 vector fragment. And connecting the recovered fragments in a metal bath at 16 ℃ for 3h, transferring the fragments into DH5 alpha through heat shock, coating the fragments in an Amp solid culture medium, culturing the fragments overnight in a thermostat at 37 ℃, selecting a single colony to be cultured in Amp liquid for mass multiplication culture, taking 1 mu L of culture solution to perform PCR detection (figure 1B), and extracting the plasmid after the detection is successful. The prokaryotic expression vector pGEX-4T-1-AtMDH1 plasmid is transferred into escherichia coli BL21 protein expression bacteria through heat shock transformation, the escherichia coli BL21 protein expression bacteria are coated in an Amp-containing solid culture medium and cultured overnight in a 37 ℃ thermostat, a single colony is selected and cultured in an Amp liquid culture medium, and the culture medium for normal growth of the bacteria is subjected to bacteria liquid PCR detection (figure 1C).

Inoculating the successfully detected bacterial liquid into 25mL LB liquid culture medium, culturing in a shaking table (37 ℃, 200 rpm) until the OD value of the bacterial liquid is 0.5, and then adding IPTG to make the final concentration of the bacterial liquid be 1mmol/L; respectively performing induction expression in shaking tables at 25 ℃ and 37 ℃, centrifuging bacterium solutions after induction for 0, 2, 4, 6 and 8 hours at 12000rpm for 1min, collecting thalli, adding 2mL PBS for resuspension, adding 5 muL of 5 multiplied protein electrophoresis sample buffer solution into 20 muL of resuspended thalli, boiling for 10min in boiling water, cooling, and performing polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis to analyze protein expression (FIG. 2A); experiments show that the AtMDH1 protein induces the maximum expression amount of 8h protein at 25 ℃ by 1mmol/L IPTG.

Inducing a large amount of strains to express proteins under the induction condition of 1mmol/L IPTG at 25 ℃; centrifuging at 4 ℃ and 12000rpm for 15min to collect thallus precipitate, adding PBS for resuspension, placing the resuspended thallus into a 50mL EP tube, and placing the tube in ice to ultrasonically break cells for 15min; after centrifugation of the disrupted cells, the obtained supernatant containing a large amount of the desired expression protein was passed through a GST column to obtain a purified AtMDH1 fusion protein, and 20. Mu.L of the sample was subjected to 12% SDS-PAGE (FIG. 2B).

Example 2: determination of malate dehydrogenase Activity

The protein concentration is measured by using a BCA method protein quantitative kit produced by Beijing kang, a century Biotechnology Co., ltd, taking Bovine Serum Albumin (BSA) as a standard protein, referring to a kit operation manual, performing an experiment, and measuring the enzyme activity by adopting a spectrophotometry method, wherein an enzyme activity measuring system of AtMDH1 contains 0.1mol/L potassium phosphate (pH 7.5), 0.01mol/L LNADH and 0.1mol/L oxaloacetic acid, and detecting the change of the light absorption value at 340nm in the reaction process by using an ultraviolet spectrophotometer at 25 ℃.

1. Optimum temperature and thermal stability of malate dehydrogenase AtMDH1

Measuring the relative enzyme activity of AtMDH1 in the reaction system under the conditions of 25 deg.C, 30 deg.C, 37 deg.C, 40 deg.C, 42 deg.C, 45 deg.C, 55 deg.C, and 65 deg.C in water bath for 10 min; and drawing a curve chart by taking the temperature as an abscissa and the relative enzyme activity as an ordinate, repeating each reaction for 3 times, and calculating an average value to obtain the optimal reaction temperature of the AtMDH1 enzyme. Placing AtMDH1 in water bath at 25 deg.C, 30 deg.C, 37 deg.C, 40 deg.C, 42 deg.C, 45 deg.C, 55 deg.C and 65 deg.C, keeping the temperature for 1h, taking out, immediately placing on ice, determining the residual activity of ATMDH1 enzyme, and drawing a curve graph with the temperature as abscissa and the relative residual enzyme activity of the enzyme as ordinate to determine the thermal stability of the enzyme; setting the enzyme activity of the enzyme solution without heat treatment as 100%, repeating each reaction for 3 times, taking an average value, and observing the thermal stability of the enzyme solution at different temperatures.

The results are shown in fig. 3 and 4, and the results show that the influence of temperature on the reaction speed of enzyme catalysis is actually the comprehensive result of the influence of temperature on the reaction speed and the promotion of enzyme denaturation, and fig. 3 shows that the malic dehydrogenase has the highest enzyme activity at 40 ℃ and can reach the maximum specific enzyme activity of the enzyme. Thus, we can judge that 40 ℃ is the optimum temperature for the enzymatic reaction. In the study of the temperature stability of the enzyme, the temperature which is the same as the specific activity of the enzyme to be measured is selected, the result is shown in figure 4, the enzyme is found to be sensitive to the change of the temperature, the enzyme activity after 42 ℃ is reduced rapidly, the relative enzyme activity after heat preservation at about 45 ℃ is about 50%, and the enzyme activity is reduced rapidly after continuous temperature rise; at 65 ℃, the enzyme is denatured, a white flocculent precipitate is formed, and the enzyme activity is basically not detected.

2. Optimum pH for malate dehydrogenase

Respectively measuring the enzyme activity of the enzyme solution obtained by purification in 20mmol/L Tris-HCl buffer solution with the pH value of 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5 and 10.0 at room temperature, respectively measuring the light absorption value of AtMDH1 at 340nm by an ultraviolet spectrophotometer, determining the optimal pH value of the enzyme reaction by observation, repeating each reaction for 3 times, and averaging to obtain the result shown in figure 5, wherein the result shows that the specific enzyme activity of the malate dehydrogenase is the largest when the pH value is 8.0, and the optimal reaction pH value of the malate dehydrogenase can be determined to be 8.0;

3. effect of Metal ions on the Activity of malate dehydrogenase

Respectively adding MgCl with the final concentration of 5mmol/L into a reaction system under the condition of keeping other components unchanged at 25 DEG C ₂ 、MnCl ₂ 、CoCl ₂ 、CaCl ₂ 、ZnSO ₄ 、CuCl ₂ And NaCl and KCl are used for determining the activity of the ATMDH1 enzyme, each experiment is independently repeated for three times, and the results are counted and the relative enzyme activity of the ATMDH1 is calculated.

The influence of various metal ions on the activity of the malic acid dehydrogenase shows that K ⁺ Has obvious activation effect on enzyme; mg (magnesium) ²⁺ And Ca ²⁺ The influence on the enzyme activity is small; zn ²⁺ And Cu ²⁺ Inhibiting the enzyme activity to different degrees; cu ²⁺ The degree of inhibition is strongest. Different metal ions produce affinity with AtMDH1 in different ways, resulting in a change in enzyme activity. The results show that different metal ions have different effects on the activity of AtMDH1. The experimental indexes were all determined in triplicate, and the results are given in the following table:

example 3: preparation and potency detection of AtMDH1 protein rabbit antibody

1mL of the purified recombinant protein (containing about 5mg of the purified AtMDH1 protein) was emulsified with 1mL of Freund's adjuvant in a ratio of 1. Adding Freund's adjuvant into mortar, and repeatedly emulsifying for several times; until the qualified water-in-oil agent is prepared, rabbit immunization can be carried out. When rabbits are immunized for the first time, after emulsification is carried out by using an equal volume of complete Freund's adjuvant, the rabbits are immunized in a small amount for multiple times, and each point is injected with a small amount of antigen. The antigen is injected to each immunization point uniformly by various modes such as intramuscular injection, subcutaneous injection or intradermal injection. Two weeks after the first immunization, the rabbits were boosted with an equal volume of incomplete Freund's adjuvant to emulsify the AtMDH1 antigen. Weekly boosts were performed for a total of three boosts.

After one week of last immunization, rabbits were anesthetized, their limbs were fixed, rabbit hairs were cut off near the heart, and rabbit skin was disinfected with alcohol-containing cotton balls. Then, the most intense part of the heart beat, which is generally located between the third and fourth ribs, is palpated and a 16 gauge needle is attached with a 50mL syringe, tilted 45 °, and the heart is bled by aiming at the most intense part of the rabbit heart beat. The drawn blood is immediately injected into a sterile container, awaiting the serum to be separated. Placing the obtained rabbit blood in a constant temperature incubator at 37 ℃ for 1h, then placing the rabbit blood in a refrigerator at 4 ℃ for 3-4h until the blood coagulation blood clot shrinks, and then sucking serum; centrifuging at 3000r/min for 15min, collecting supernatant, adding 0.02% sodium azide, preserving, packaging with 2mL sterilized centrifuge tube, and freezing at-20 deg.C.

To verify that the antibody was effective, we performed a specific assay for the AtMDH1 antibody. The purified recombinant proteins of 3. Mu.g, 6. Mu.g and 12. Mu.g were subjected to SDS-PAGE, and western blot analysis was performed using the rabbit antibody as the primary antibody and the commercial goat anti-rabbit as the secondary antibody, the results are shown in FIG. 6A, which indicates that the polyclonal rabbit antibody prepared in this example can specifically react with AtMDH1 antigen and the antibody can be successfully prepared.

In order to further test the effectiveness of the antibody, a two-way immunodiffusion test was performed, and the test result is shown in fig. 6B, a curved white precipitation line can be seen, which indicates that the protein purified by the AtMDH1 has specificity with immune serum of the AtMDH1, and the protein and immune serum freely diffuse in agarose gel, and an antigen-antibody complex is formed after the protein and immune serum meet, and the line bends along the edge of a middle hole, and except for the normal saline, the white line at the position where the protein is diluted by 32 times and the white line at the position where the protein is diluted by 64 times, the white lines are all arranged in other holes diluted by different times, which indicates that the rabbit antibody has higher titer.

Example 4: construction of eukaryotic expression vector pSPYCE-AtMDH1 and identification of over-expressed AtMDH1 gene tobacco and arabidopsis thaliana

1. Construction of eukaryotic expression vector pSPYCE-AtMDH1

The invention constructs a eukaryotic target expression vector pSPYCE-AtMDH1 by using an enzyme digestion ligation technology; the target fragment (999 bp) of the AtMDH1 gene is amplified by PCR. pMD18T and the target fragment are cloned by T/A to obtain pMD18T-AtMDH1 plasmid, PCR is carried out to detect whether the connection is successful (figure 7A), and sequencing is carried out on positive clone to detect whether the gene AtMDH1 is mutated. The successful pMD18T-AtMDH1 plasmid and the pSPYCE plasmid are detected, the target fragment AtMDH1 and the cut pSPYCE are obtained by respectively carrying out double-enzyme gel cutting on Sal I and Sma I, the two fragments are connected for 4 hours at 16 ℃ to form a eukaryotic expression vector pSPYCE-AtMDH1, and PCR is carried out to detect whether the AtMDH1 gene is successfully cloned on the pSPYCE vector (figure 7B).

Transferring the successfully constructed eukaryotic expression vector pSPYCE-AtMDH1 into agrobacterium GV3101 by a freeze-thaw method, obtaining positive clones by screening culture in a kanamycin (Km) solid culture medium, selecting single strains to screen culture in an LB liquid culture medium containing Km (the final concentration is 50 mg/L), detecting the positive clones with exogenous plant expression vector plasmids by bacterial liquid PCR (figure 7C), transforming tobacco by a leaf disc transformation method and transforming wild arabidopsis thaliana by a floral dip method, transfecting the expression vector pSPYCE-AtMDH1 agrobacterium containing target genes into tobacco and arabidopsis thaliana, and screening in a resistance culture medium containing Km (the final concentration is 30 mu g/mL) to obtain transgenic tobacco and arabidopsis thaliana seeds.

2. Identification of over-expressed AtMDH1 gene tobacco and Arabidopsis thaliana

Extracting plant genome from transgenic tobacco and arabidopsis thaliana with long roots in a resistant culture medium by a CTAB method, and detecting the extracted genome DNA by gel electrophoresis. Taking the extracted plant genome DNA as a template, carrying out PCR reaction by using a specific primer of the AtMDH1 gene, and detecting a PCR product on 1% agarose gel electrophoresis; and (3) detecting a plant with the exogenous AtMDH1 gene inserted in a genome by genome PCR, detecting whether the protein is translated, extracting the total plant protein of the transgenic tobacco and the Arabidopsis, and detecting the protein level of the AtMDH1 by using a western blot by using HA-labeled rabbit antibody as a primary antibody.

Transforming tobacco and arabidopsis thaliana with pSPYCE-AtMDH1 to obtain a transgenic strain with kanamycin resistance, and detecting the insertion condition of an exogenous gene by using a specific primer of AtMDH1 gene and using genome DNA as a template; after PCR reaction of the genome, the results are shown in FIG. 8; FIG. 8A shows the AtMDH1 gene insertion in the genome of 5 lines of tobacco (Nos. 4, 8, 10, 11, 12), and FIG. 8C shows the AtMDH1 gene insertion in the genome of 8 lines of Arabidopsis (Nos. 2, 3, 4, 5, 6, 7, 8, 11).

The protein level western blot detection adopts HA-labeled rabbit antibody as a primary antibody, the results are shown in figures 8B and 8D, and the results of western blot analysis show that 3 (M8, M9 and M10) strains of tobacco and 3 (4, 5 and 6) strains of Arabidopsis have normal translation of exogenous AtMDH1 genes in transgenic plants.

The results of DNA and protein level detection and analysis show that the exogenous AtMDH1 gene is accurately inserted into tobacco and Arabidopsis genomes, and the exogenous AtMDH1 gene can be correctly transcribed under a 35S promoter, and a transgenic tobacco strain 8 (M8) and an Arabidopsis strain 6 with higher expression level are selected as transgenic plant test materials in the experiment.

Example 5: formaldehyde stress treatment of transgenic tobacco and Arabidopsis

Selecting plants with the same growth status, collecting 1g of leaves, washing with tap water for 3-4 times to remove dirt, blotting residual water on the surface with sterile absorbent paper, transferring to culture bottles containing 2mM HCHO treatment solution and 50mL each, and continuously irradiating with light (100 μmol. M) at 25 deg.C ^-2 ·s ^-1 ) And culturing for 0h, 6h, 12h, 24h, 36h, 48h and 60h at 100rpm by shaking, and taking a sample which is not added with plant leaves and is only added with formaldehyde as a control (BC).

1. Detection of liquid formaldehyde absorption capacity of transgenic tobacco and arabidopsis

In order to detect the influence of At14-3-3PSI expression change Arabidopsis (At 14-3-3PSI overexpression Arabidopsis At14PSI, and AtMDH 14-3 PSI expression inhibition Arabidopsis At14 PSI), atMDH1 expression change Arabidopsis (AtMDH 1 overexpression Arabidopsis AtMDH1, atmdH1 expression inhibition Arabidopsis AtmdH 1), and AtMDH1 overexpression tobacco absorption of liquid HCHO, 1g of leaf is taken from At14-3-3PSI expression change Arabidopsis, atMDH1 expression change Arabidopsis, wild Arabidopsis (Col-0), atMDH1 overexpression tobacco, and Wild Tobacco (WT), respectively immersed in 50mL of HCHO solution with concentration of 2mM for HCHO absorption detection, the volatilization amount of HCHO is monitored by using the leaf-free Arabidopsis solution as a control, and the average value obtained after measurement is taken to draw a liquid HCHO absorption curve of transgenic tobacco and tobacco. By comparing the HCHO absorbing capacity of the transgenic arabidopsis thaliana and the wild type southwest, the result shows that the HCHO absorbing capacity of the At14-3-3PSI overexpressed arabidopsis thaliana and the AtMDH1 overexpressed arabidopsis thaliana in a 2mM HCHO solution (figure 9) is similar, the overexpressed arabidopsis thaliana is slightly higher than that of the wild type arabidopsis thaliana, the arabidopsis thaliana for inhibiting the expression of At14-3 PSI and the arabidopsis thaliana for inhibiting the expression of AtMDH1 are slightly lower than that of the wild type arabidopsis thaliana, and the HCHO absorbing rate of the At14PSI overexpressed arabidopsis thaliana is fastest.

Comparing the uptake curves of transgenic tobacco and wild type tobacco in 2mM HCHO solution (FIG. 10), it can be seen that the overall tendency of HCHO uptake by tobacco and wild type tobacco is similar to that of AtMDH1 overexpression, which is slightly higher than that of wild type.

2. Transgenic tobacco and transgenic arabidopsis H under formaldehyde stress ₂ O ₂ And O ₂ · ^- Detection of

ROS play a central role in plant stress responses. The accumulation of ROS can be used as an important variable for measuring the degree of adversity stress. We used 3,3' -diaminobenzidine (DAB; H) ₂ O ₂ ) And nitro blue tetrazolium (NBT; o is ₂ · ^- ) Histochemical staining is carried out, and two main active oxygen (ROS) substances, namely hydrogen peroxide (H), before and after HCHO stress of At14PSI overexpression Arabidopsis thaliana, atMDH 14PSI for inhibiting At14-3-3PSI expression, atMDH1 overexpression Arabidopsis thaliana, atMDH1 for inhibiting AtMDH1 expression and AtMDH1 overexpression tobacco are researched ₂ O ₂ ) With hydrogen peroxide anion (O) ₂ · ^- ) In situ accumulation of (a);

O ₂ · ^- content measurement histochemical staining was carried out with nitroblue tetrazolium (NBT), and after the sample was placed in 25mM K-HEPES buffer (pH 7.8) containing 0.1mg/mL NBT and treated in a dark environment at 25 ℃ for 8 hours, it was washed 5 times with 80% ethanol solution at 70 ℃.

H ₂ O ₂ Content measurement histochemical staining was performed with 3,3' -Diaminobenzidine (DAB), and after the sample was placed in 50mM Tris-HCl (pH 3.8) containing 1mg/mL DAB and treated in a dark environment at 25 ℃ for 24 hours, it was washed 5 times with 80% ethanol solution at 70 ℃.

DAB staining of transgenic Arabidopsis lines showed that plants accumulated lower H under normal conditions ₂ O ₂ Wild type, at14-3-3PSI overexpressing Arabidopsis thaliana, atMDH1 overexpressing Arabidopsis thaliana and AtMDH1 overexpressing Arabidopsis thaliana inhibiting expression of At14-3-3PSI and AtMDH1 overexpressing tobacco (FIG. 11) inhibiting expression of AtMDH1 and AtMDH1 overexpressing tobacco (FIG. 12) before HCHO treatment ₂ O ₂ There was no significant difference in the contents. HCHO stress leads to H in Arabidopsis and tobacco lines ₂ O ₂ Levels were elevated, but H in Arabidopsis thaliana inhibiting expression of At14-3-3PSI and Arabidopsis thaliana inhibiting expression of AtMDH1, compared with wild type Arabidopsis thaliana ₂ O ₂ Accelerated accumulation of (a) At14-3-3PSI overexpression Arabidopsis thaliana and overexpression of Arabidopsis thaliana AtMDH1 in the blade H ₂ O ₂ Is the slowest to accumulate.

Blue staining, reflecting NBT staining, deeper in Arabidopsis thaliana inhibiting expression of At14-3-3PSI and Arabidopsis thaliana inhibiting expression of AtMDH1 than in wild type and overexpressed leaves after HCHO treatment, indicating O in the Arabidopsis plants of the suppressor gene ₂ · ^- The accumulation was greater (fig. 11). Therefore, ROS accumulation is promoted when AtMDH 14-3-3PSI and AtMDH1 genes are suppressed, but reduced when AtMDH1, at14-3-3PSI are overexpressed. Similar to AtMDH1 overexpression in arabidopsis, ROS accumulation was reduced in AtMDH overexpression tobacco under formaldehyde stress (fig. 12).

3. Effect of HCHO stress on AtMDH1 expression

In order to detect whether the expression level of AtMDH1 is affected after 2mM formaldehyde stress of AtMDH1 overexpression Arabidopsis thaliana, the transcriptional level and the protein expression level of AtMDH1 are measured by using AtMDH1 overexpression Arabidopsis thaliana which is subjected to formaldehyde treatment for different time periods (0 hour, 12 hour, 24 hour and 36 hour) as a material, and the result is shown in FIG. 13, the transcriptional level of AtMDH1 overexpression Arabidopsis thaliana is increased after formaldehyde treatment, and the protein level of AtMDH1 is increased along with the prolonging of the formaldehyde treatment time.

4. AtMDH1 and At14-3-3PSI interaction analysis

To understand whether At14-3-3PSI interacts with AtMDH1 to affect formaldehyde metabolism, first a yeast two-hybrid analysis was performed (FIG. 14), we first ligated AtMDH1 gene into pGADT7 vector and transformed Y187 yeast strain; the CDS sequence of the At14-3-3PSI gene was cloned into a pGBKT7 vector, and the Y2HGold yeast strain was transformed. We have determined that p35 and large T antigens can interact in a yeast two-hybrid assay, and we set up Y2HGold [ pGBKT7-53] interacting with Y187[ pGADT7-T ] as a positive control, and pGBKT7-Lam and pGADT7-T as negative controls, using the following experimental procedures:

(1) Construction of the vector: the gene At14-3-3PSI is connected into a pGBKT7 vector and the AtMDH1 gene is connected into a pGADT7 vector by enzyme digestion connection, so that the At14-3-3PSI is respectively fused with the DNA binding domain of GAL4 (BD-At 14-3-3 PSI) and the AtMDH1 is fused with the DNA activation domain of GAL4 (AD-AtMDH 1);

and (3) transformation: adding 10 μ L of Y187 or Y2HGold bacterial solution into 2mL0.25 × YPDA, culturing at 28 deg.C and 200rpm overnight for 12 hr;

(2) Taking 250 mu L of bacterial liquid, quickly separating at 12000rpm for 15s, collecting precipitate, adding 1mLddH ₂ O, resuspending, quickly separating and collecting the precipitate;

(3) Slowly adding 240 μ L PEG3350 (50% w/v), 36 μ L LiAc (1M), 20 μ L ss-DNA (2 mg/mL) in that order; 10 μ L of recombinant plasmid, 54 μ L of ddH ₂ O vortex for 1min; centrifuging in 42 deg.C water bath 30min at 3500rpm for 15s, removing supernatant, adding 100 μ L ddH ₂ O, lightly blowing and beating, coating the plasmid containing BD-At14-3-3PSI on a Trp-lacking culture medium, coating the plasmid containing AD-AtMDH1 on a Leu-lacking culture medium, and culturing for 2 to 4 days in a constant temperature box At 28 ℃;

(4) Selecting single colonies, and respectively culturing in Trp-lacking culture solution and Leu-lacking culture solution at 28 deg.C and 200rpm overnight;

(5) And (3) hybridization: the bacterial liquids in the last step are mixed in 2mL of 2 XYPDA culture solution in pairs and cultured overnight at 28 ℃ and 200 rpm;

(6) Centrifuging 1mL of bacterial solution at 3500rpm for 1min, and precipitating with ddH ₂ O washing for 2 times, gently blowing and mixing evenly, sucking 20 mu L of the mixture and coating the mixture on a culture medium lacking Leu and Trp (lacking the culture medium);

(7) Selecting a single bacterial colony to a centrifuge tube, and adding 50-100 mu L ddH ₂ Dissolving O, centrifuging at 3500rpm for 1min, and absorbing ddH ₂ O; reuse of ddH ₂ Washing with O, and adding 30-50 μ L ddH ₂ O is mixed evenly for standby; sucking 1.5-2 μ L of the suspension, dropping on the-Leu and-Trp two-deficient medium plate and the-Ade/-His/-Leu/-Trp four-deficient plate, culturing at 28 deg.C for 2-4 days, and observing;

pGADT7-AtMDH1 and pGBKT7-At14-3-3PSI are taken as experimental groups, and the experimental group can grow colonies on a plate containing four defects as a positive control, so that the At14-3-3PSI and the AtMDH1 have interaction.

Secondly, the luciferase complementation experiment (LCA) is carried out on the recombinant proteins of At14-3-3PSI and AtMDH1, and the At14-3-3PSI gene is connected into a pCAMBIA1300-nLUC vector and the AtMDH1 gene is connected into a pCAMBIA1300-cLUC vector by homologous recombination;

the obtained pCAMBIA1300-nLUC-At14-3-3PSI and pCAMBIA1300-cLUC-AtMDH1 were transformed into Agrobacterium by a freeze-thaw method, and Agrobacterium tumefaciens strain GV3101 cells containing the correctly sequenced fusion vector were cultured in LB liquid medium to OD600=1.0. Two cultures carrying the indicated nLUC and cluuc constructs were mixed in a ratio of 1. Plants were incubated in the dark for 12 hours, then subjected to 16 hours/8 hours light/dark conversion, cycled for 48 hours, imaged for LUC activity, and sprayed with 150. Mu.g/mL potassium D-fluorescein salt on the back of the leaf and kept in the dark for 5 minutes. LUC images were taken using a Tanon 5200 multiple chemiluminescence imaging system (Tanon, china).

The results are shown in FIG. 15, which shows that fluorescence was observed in leaves injected with pCAMBIA1300-nLUC-At14-3-3PSI and pCAMBIA1300-cLUC-AtMDH1, indicating that At14-3-3PSI and AtMDH1 have an interaction.

5. At14-3-3PSI overexpression, at14-3-3PSI expression inhibition, atMDH1 overexpression and AtMDH1 expression inhibition on formaldehyde absorption and metabolism of tobacco and arabidopsis thaliana

By using ¹³ C-NMR technique analyzed H ¹³ CHO metabolism in various transgenic Arabidopsis and tobacco leaves, FIG. 16 shows 2mM liquid H ¹³ CHO treatment for 24h, major metabolic intermediates in wild-type and transgenic Arabidopsis leaves ¹³ The change in the intensity of the C-NMR resonance peak; by comparing wild type and various transgenic Arabidopsis thaliana leaves H ¹³ The change in the amount of each intermediate between the CHO-treated sample and the control sample (FIG. 18) was observed as a result of the reaction in the presence of the intermediate in the CHO-treated sample and the control sample (see the results in the H ¹³ The At14-3-3PSI over-expression Arabidopsis (At 14 PSI) and AtMDH1 over-expression Arabidopsis (AtMDH 1) after CHO treatment for 24h have higher contents than wild type Arabidopsis leaves (Col-0) and wild type leaves (CK) without any treatment, and the intermediate metabolites of the two transgenic Arabidopsis are higher than those without any treatment;

FIG. 17 shows 2mM liquid H ¹³ Main metabolic intermediate product in wild type and transgenic tobacco leaves after 24h CHO treatment ¹³ The change in the intensity of the C-NMR resonance peak; overexpression of tobacco leaf H by comparing wild type with AtMDH1 ¹³ Variation in the amount of each intermediate in the CHO treated and control samples (FIG. 19),as a result, it was found that the compound passes through H ¹³ The intermediate metabolite content of AtMDH1 over-expressed tobacco after CHO treatment for 24h is higher than that of wild type tobacco leaf (WT) and wild type leaf (CK) without any treatment.

The relative content of each main metabolite is analyzed by integrating each peak in the nuclear magnetic spectrum, and the result of FIG. 18 shows that glutamine in the leaves of At14-3-3PSI over-expression Arabidopsis thaliana is 2 times higher than that of wild leaves (Col-0), but malic acid, asparagine and aspartic acid are not obviously changed; the expression of At14-3-3PSI inhibits malic acid in Arabidopsis leaves to be 2.25 times higher than that of wild type leaves (Col-0), and glutamine to be 4.2 times lower than that of wild type leaves (Col-0); malic acid, asparagine, aspartic acid and glutamine in the leaves of AtMDH1 over-expressed Arabidopsis are respectively 6.58 times, 4 times, 2.5 times and 5.65 times higher than those of wild type leaves (Col-0); the expression of AtMDH1 inhibited aspartic acid in Arabidopsis thaliana leaves 19 times higher than that in wild type leaves (Col-0), almost no malic acid and asparagine appeared, and glutamine 4 times lower than that in wild type leaves (Col-0). From these results, we speculate that malic acid, asparagine, aspartic acid, glutamine may be 2mM liquid H in tobacco formaldehyde metabolism ¹³ CHO as the main intermediate.

FIG. 19 shows that malic acid and glutamine in AtMDH1 over-expressed tobacco leaves are 1.83 times and 1.3 times higher than those in wild type leaves (WT), respectively;

in conclusion, the arabidopsis thaliana and tobacco leaf blades have stronger ability to metabolize low-concentration formaldehyde (2 mM) after the overexpression of At14-3-3PSI and the overexpression of AtMDH1 than the wild type.

6. Effect of At14-3-3PSI expression Change on expression amount of AtMDH1

The expression level of AtMDH1 is determined in At14-3-3PSI overexpression Arabidopsis thaliana and At14-3-3PSI expression inhibition Arabidopsis thaliana, the result is shown in figure 20 by taking wild type Arabidopsis thaliana Col-0 as a control, the expression level of AtMDH1 is inhibited by At14-3-3PSI overexpression Arabidopsis thaliana, the expression level of AtMDH1 in At14-3-3PSI expression inhibition Arabidopsis thaliana is higher than that of wild type Arabidopsis thaliana and At14-3-3PSI overexpression Arabidopsis thaliana, and the expression level of AtMDH1 is inhibited by At14-3-3PSI overexpression.

7. Effect of HCHO stress on At14-3-3PSI and AtMDH1 interactions

(1) Tobacco leaves of Bentonium were infected by injecting leaves of Bentonium with Agrobacterium (1. The plants were incubated in the dark for 12 hours, then subjected to 16 hours/8 hours light/dark switching, circulated for 48 hours, after which the leaves were cut into left and right halves, the left leaf was placed in buffer (containing 0.5mM KHCO) ₃ And 1mM MES), the right leaf was placed in 2mM formaldehyde solution, and after 6h, 150 μ g/mL of D-fluorescein potassium salt was sprayed on the back of the leaf, and the leaf was kept in the dark for 5 minutes, and LUC images were taken using a Tanon 5200 multiple chemiluminescence imaging system (Tanon, china), and the results are shown in fig. 21A. The fluorescence signal after formaldehyde treatment (right leaf) was weaker than that of the leaf without formaldehyde treatment (left leaf), indicating that HCHO treatment inhibited AtMDH1 interaction with At14-3-3 PSI.

(2) To further demonstrate the effect of HCHO on their interaction in Arabidopsis thaliana, co-IP analysis was performed using Arabidopsis thaliana leaves, and Co-immunoprecipitation was used to verify the effect of HCHO on the interaction of AtMDH1 with At14-3-3PSI proteins

Treating At14-3-3PSI overexpression Arabidopsis leaves with 2mM HCHO formaldehyde for 0h, 6h, 24h and 36h, respectively extracting the total plant protein, adding 200 μ G of the total plant protein into 2 μ G of Myc specific antibody, adding 20 μ L of agar protein A/G, and shaking with a shaker (40 rpm) At 4 ℃ overnight; the following day, the protein pellet was centrifuged for 5min (4 ℃ C., 3000 rpm), and the protein pellet was collected, washed 3 times with pre-cooled PBS (phosphate buffered saline), and the washed pellet was dissolved in 40. Mu.L of 1 Xloadingbuffer, and after 40. Mu.L of the solution was separated by SDS-PAGE (12%) electrophoresis, westernblot analysis was performed. Transferring the protein onto a PVDF membrane by a semi-dry electrotransfer instrument, incubating an ATMDH1 specific antibody at 4 ℃ overnight, incubating a goat anti-rabbit secondary antibody at normal temperature for 2h, and observing the experimental result by a gel imager. The level of interaction between AtMDH1 and At14-3-3PSI proteins was analyzed based on the gel imaging results, which are shown in FIG. 21B; from the figure it can be seen that AtMDH1 interacts with At14PSI, which is inhibited by HCHO treatment;

the results show that the association of At14-3-3PSI and AtMDH1 can be weakened by HCHO treatment, the interaction between At14-3-3PSI and AtMDH1 is inhibited by HCHO stress, and the inhibition of AtMDH1 transcription by At14-3-3PSI protein is further relieved, so that the metabolism of formaldehyde in plants is enhanced; at14-3-3PSI interacts with AtMDH1 to positively regulate the tolerance of Arabidopsis and tobacco to HCHO stress, and AtMDH1 is also essential for Arabidopsis to metabolize HCHO.

Claims

1. An application of a malate dehydrogenase gene AtMDH1 in improving the formaldehyde absorption and metabolism capability of plants, wherein the nucleotide sequence of the malate dehydrogenase gene AtMDH1 is shown as SEQ ID NO. 1.