CN113502276B - Application of isocitrate dehydrogenase in improving formaldehyde absorption and metabolism capacity of plants - Google Patents

Abstract

The invention discloses an application of an arabidopsis isocitrate dehydrogenase gene AtIDH3 in improving the absorption and metabolism capability of plant formaldehyde, wherein the nucleotide sequence of the arabidopsis isocitrate dehydrogenase gene AtIDH3 is shown as SEQ ID NO 1; recombining AtIDH3 gene into plant expression vector, converting wild tobacco, and screening to obtain AtIDH3 gene-converted tobacco; the experimental result shows that under the same concentration and stress for the same time, the effect of over-expressing tobacco for absorbing formaldehyde is better than that of wild tobacco; the expression level of AtIDH3 transcription levels of wild type tobacco and 14-3-3c over-expression tobacco is obviously increased under the stress of liquid formaldehyde; the interaction between the 14-3-3c protein and AtIDH3 is proved by yeast double-impurity, Pull-down and Co-IP experiments, and the result shows that the over-expressed transgenic tobacco has stronger formaldehyde absorption capacity than wild tobacco.

Description

Application of isocitrate dehydrogenase in improving formaldehyde absorption and metabolism capacity of plants

Technical Field

The invention belongs to the technical field of molecular biology and genetic engineering, and particularly relates to an application of an arabidopsis isocitrate dehydrogenase gene AtIDH3 in improving the formaldehyde absorption and metabolism capability of plants.

Background

Formaldehyde is one of the most frequently contacted pollutants in daily life, so that the treatment of formaldehyde pollution is also widely concerned. Formaldehyde can react non-specifically with various macromolecules in the organism, leading to the inactivation of macromolecular substances, so that formaldehyde has various toxic effects on the organism and is associated with various diseases of human beings. Formaldehyde enters the human body through a respiratory system and reaches the whole body through blood circulation, so that various functional abnormalities of the body are caused. Any animal drinking water polluted by formaldehyde for a long time can cause harm to the body in different degrees, so that the problem of formaldehyde pollution is solved. The method for solving the problem of formaldehyde pollution by using plants is an economic and environment-friendly method, and the existing view points that the absorption capacity of plant formaldehyde is related to the formaldehyde metabolism capacity of the plant, 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.

Isocitrate dehydrogenase catalyzes the oxidative decarboxylation of isocitrate to alpha-ketoglutarate in the TCA cycle. The carbon skeleton can be provided for the ammonia assimilation of the plant, and the generated NADPH can also maintain the redox balance in the cell and help the plant resist various biotic and abiotic stresses. Alpha-ketoglutarate generated by IDH catalysis is an important product for producing carbon dioxide and water by biomacromolecule metabolism such as organism sugars, lipids, proteins and nucleic acids, and therefore isocitrate dehydrogenase has very important significance for organism metabolism. The studies indicate that IDH is a major source of NADPH production and that IDH is closely related to ROS production by organisms. It plays an important role in plants against oxidative stress, and cytoplasmic IDH participates in NADPH required for plants to produce glutathione to resist extreme conditions of stress. IDH activity increases when plants are subjected to some abiotic stresses, such as ionic poisoning.

The 14-3-3c protein is a core protein of protein interaction in plant cells and is involved in various growth and metabolism reactions of plants, including substance transportation, growth and development, nutrition metabolism, cell cycle, stress response, regulation of light signal transmission pathways and the like. In plants, 14-3-3c protein has a wide regulation effect on metabolism, and 14-3-3c protein influences the activity of enzymes such as glutamine synthetase, sucrose phosphate synthase, 6-phosphofructokinase/fructose diphosphate kinase, glucose-1-phosphoadenylyl transferase, glyceraldehyde 3-phosphate dehydrogenase, starch synthase and lipoxygenase through interaction with key enzymes of metabolic pathways, so that the processes of regulating metabolism, influencing starch accumulation, fat oxidation and the like are realized.

At present, the function of the arabidopsis isocitrate dehydrogenase gene AtIDH3 in the process of improving the absorption and metabolism of formaldehyde by plants and the related report that the 14-3-3c protein regulates the arabidopsis isocitrate dehydrogenase gene are not found.

Disclosure of Invention

Aiming at the problems in the prior art, the invention discloses a new application of an arabidopsis thaliana isocitrate dehydrogenase gene AtIDH3, namely, the arabidopsis thaliana isocitrate dehydrogenase gene AtIDH3 is applied to improve the absorption and utilization of formaldehyde by plants, and the GenBank accession number of the arabidopsis thaliana isocitrate dehydrogenase gene AtIDH3 is as follows: at4G 35650; the AtIDH3 gene is used for constructing a transgenic plant with enhanced formaldehyde absorption capacity, 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 purpose of the present invention, the technical scheme of the present invention is as follows:

1. the following experiments were performed by selecting the model plant tobacco, the arabidopsis thaliana isocitrate dehydrogenase gene AtIDH3 as the experimental material:

constructing a prokaryotic expression vector pGEX-4T-1-AtIDH3 of AtIDH3 gene by adopting Gateway technology, cloning the full length of AtIDH3 gene from arabidopsis leaves, successfully constructing AtIDH3 prokaryotic expression vector, inducing expression protein in BL21, and purifying to obtain AtIDH3 protein; the enzyme characteristic analysis shows that the optimum temperature of AtIDH3 is 45 ℃, and the thermal stability is good at 25-35 ℃; the optimum pH value is 8.2; mn (Mn)²⁺And Mg²⁺Has promoting effect on AtIDH3 protease activity and activating effect Mn²⁺>Mg²⁺The other 6 metal ions inhibit the activity of AtIDH3 protein to a certain extent, and the order of inhibition is Ca²⁺>Co²⁺>Zn²⁺>K⁺>Na⁺>Cu²⁺(ii) a 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 pMK-35S-AtIDH3 of AtIDH3 gene by Gateway technology

Extracting arabidopsis thaliana total RNA by using a TRIzol Reagent according to the instruction; and reverse transcription to obtain cDNA of AtIDH 3; searching CDS sequence (At4G35650) of Arabidopsis thaliana IDH3 At NCBI, and designing double enzyme cutting sites (BamH I and Sal I) and gene specific primers by DNAman software;

upstream primer 5' -ATGGCGAGAAGATCCGTTTC (containing BamH I site)

The downstream primer 3' -CTCAAGAGCTGCTATGACAGCA (containing Sal I enzyme cutting site);

carrying out PCR amplification by using the primers to obtain a full-length DNA fragment of the coding region of AtIDH 3; connecting to a pMD18-T vector by enzyme digestion, transforming Escherichia coli DH5 alpha by heat shock, and obtaining TA clone containing a correct sequence by resistance screening and sequencing; then, carrying out enzyme digestion connection to connect the fragment to a pENTR-2B vector, and obtaining an entry vector pENTR-AtIDH3 through resistance screening and sequencing; AtIDH3 was recombined onto the objective vector pMK7WG-cL-2 by LR reaction according to the LR reaction kit LR Clonase TM plus Enzyme Mix (available from Invitrogen, USA) instructions to obtain the plant expression vector pMK-35S-AtIDH 3; transferring pMK-35S-AtIDH3 into Agrobacterium by electric transformation method, transforming wild tobacco by screening and detecting correct Agrobacterium by leaf disc transformation method to obtain regenerated plant, and screening and detecting;

3. detecting the genome, mRNA level, protein expression level and AtIDH3 activity of the transgenic plants obtained by resistance screening to obtain a plurality of transgenic AtIDH3 gene tobaccos, and propagating;

4. healthy plants with consistent growth vigor were selected from wild type tobacco, AtIDH3 transgenic tobacco, 14-3-3c transgenic tobacco, and 14-3-3c expression-inhibited healthy plants (obtained by the method of ZL 201710974873.6 for 14-3-3c transgenic tobacco), 1g of leaf was taken, washed with tap water 3-4 times to remove dust, water remained on the surface was blotted with sterile blotting paper, the blotted leaf was transferred to 50mL culture flasks each containing 2mM, 4mM, and 6mM HCHO treatment solution, and the treatment solution was continuously irradiated with light (100. mu. mol. m.m.^-2·s^-1) And culturing for 0h, 6h, 12h, 24h, 36h, 48h, 60h at 100rpm under shakingTaking a sample which is not added with plant leaves and is only added with formaldehyde as a reference (BC), and comparing the formaldehyde absorption efficiency and physiological and biochemical indexes of different types of tobacco;

5. the interaction of 14-3-3c protein and AtIDH3 is proved through yeast double-hybrid, Pull-down and Co-IP experiments, which shows that 14-3-3c changes the activity of enzyme through the interaction with the metabolism key enzyme to regulate formaldehyde metabolism; the types of metabolites of wild type and transgenic tobacco leaves treated by 2mM HCHO liquid for 24h are the same, and the over-expressed metabolite content is higher than that of the wild type, and the result shows that the over-expressed transgenic tobacco has stronger formaldehyde absorption capacity than that of the wild type tobacco.

The invention has the beneficial effects that: the AtIDH3 gene is transferred into tobacco, so that the formaldehyde absorption efficiency of the tobacco is improved, the AtIDH3 gene transferred tobacco is easy to preserve germplasm resources in an asexual propagation mode, seeds obtained after cultivation are easy to preserve, popularization and planting are facilitated, materials are provided for further research on the gene function, ideas are provided for research on other related genes, and the transgenic tobacco can be planted in soil with serious formaldehyde pollution.

Drawings

FIG. 1 is a schematic diagram showing the results of electrophoresis detection of amplification of the Arabidopsis thaliana AtIDH3 gene (A), vector validation of pMD-18T-AtIDH3 (B), vector validation of pGEX-4T-1-AtIDH3 (C) and transformation detection of BL21 (D);

FIG. 2 is a schematic diagram showing the results of the detection of the recombinant protein pGEX-4T-1-AtIDH3 induced expression (A), soluble expression analysis (B) and purification (C);

FIG. 3 shows the effect of temperature on AtIDH3 enzyme activity;

FIG. 4 shows the results of thermal stability analysis of AtIDH3 enzyme;

FIG. 5 is a graph showing the effect of pH on the enzymatic activity of AtIDH 3;

FIG. 6 shows Western blot (A) and two-way immunodiffusion assay (B) for detecting the antibody titer of AtIDH 3;

FIG. 7 is a diagram showing the results of detection of over-expressed AtIDH3 by tobacco genome PCR (A), qRT-PCR (B) and western blot (C);

FIG. 8 shows the results of liquid absorption of 2mM formaldehyde in wild type tobacco, tobacco overexpressing 14-3-3c (KC), tobacco overexpressing AtIDH3 (IDH3), and transgenic tobacco (RC) expressing 14-3-3 c;

FIG. 9 shows the results of liquid absorption of 4mM formaldehyde concentration in wild type tobacco, tobacco overexpressing 14-3-3c (KC), tobacco overexpressing AtIDH3 (IDH3), and transgenic tobacco (RC) repressing expression of 14-3-3 c;

FIG. 10 shows the results of liquid absorption of 6mM formaldehyde concentration in wild type tobacco, tobacco overexpressing 14-3-3c (KC), tobacco overexpressing AtIDH3 (IDH3), and transgenic tobacco (RC) repressing expression of 14-3-3 c;

FIG. 11 shows the relative expression level of AtIDH3 gene in wild type tobacco;

FIG. 12 shows the results of relative expression levels of AtIDH3 gene in tobacco overexpressing 14-3-3 c;

FIG. 13 is a graph showing the results of suppressing the relative expression amount of AtIDH3 gene in transgenic tobacco expressing 14-3-3 c;

FIG. 14 is a graph of the change in IDH3 enzyme activity in WT tobacco and transgenic tobacco lamina under liquid HCHO;

FIG. 15 shows the results of analysis of 14-3-3c interaction with IDH3 in the Pull-down experiment;

FIG. 16 shows the results of a yeast two-hybrid assay;

FIG. 17 shows the results of Co-immunoprecipitation (Co-IP) experiments;

FIG. 18 is a 2mM formaldehyde treatment 24h metabolic profile of tobacco leaves overexpressing tobacco 14-3-3c, Arabidopsis AtIDH3 overexpressing tobacco, 14-3-3c repressing expression tobacco, and wild type tobacco;

FIG. 19 shows the relative integration results of 2mM formaldehyde-treated metabolites of tobacco leaves overexpressing tobacco 14-3-3c, Arabidopsis AtIDH3 overexpressing tobacco, 14-3-3c repressing tobacco, and wild type tobacco.

Detailed Description

The invention is explained in more detail below by means of examples and figures, without limiting the scope of protection of the invention. The procedures in the examples were carried out in accordance with conventional procedures unless otherwise specified, and the reagents used were all those conventionally purchased or prepared in accordance with conventional procedures unless otherwise specified.

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

Extracting total RNA of the Arabidopsis thaliana leaves by using TRIZOL Reagent (Invitrogen), and performing the operation according to the instruction; reverse transcription of 25. mu.L of RNA into cDNA was carried out using M-MLV Reverse Transcriptase (Fermentas) Reverse transcription kit; the target fragment was amplified by PCR using 1. mu.L of cDNA as a template. The CDS sequence of Arabidopsis thaliana IDH3 was searched At NCBI (At4G35650), and its double cleavage sites (BamHI and SalI) and gene-specific primers (F-ATGGCGAGAAGATCCGTTTC; R-CTCAAGAGCTGCTATGACAGCA) were designed by DNAman software. PCR amplification is carried out by using designed specific primers, AtIDH3 gene segments are recovered by glue (figure 1A), and the AtIDH3 gene segments are stored at-20 ℃ for later use. Recovering AtIDH3 gene from gel, TA cloning with pMD18-T to obtain pMD18-AtIDH3 vector, heat shock transforming DH5 alpha, coating on 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 1B), and sending the successfully detected bacterial liquid extracted plasmid to a company for sequencing. The sequencing results were aligned with AtIDH3 using DNAman, and the correctly aligned pMD18-AtIDH3 plasmid and pGEX-4T-1 vector were double digested using BamH I and Sal I, respectively (25. mu.L of plasmid; 5. mu.L of BamH I; 5. mu.L of Sal I; 7.5. mu.L of 10 XBuffer T; 7.5. mu.L of ddH₂O), observing the target fragment by gel electrophoresis, and carrying out gel recovery on the target gene fragment AtIDH3 and the cut pGEX-4T-1 vector fragment. Recovering the obtained fragments, and connecting the fragments in a metal bath at 16 ℃ overnight to obtain pGEX-4T-AtIDH3 plasmid; pGEX-4T-AtIDH3 plasmid is transferred into DH5 alpha through heat shock, coated in Amp solid culture medium, cultured overnight in 37 ℃ thermostat, single colony is selected to be cultured in Amp liquid for mass multiplication culture, 1 mu L culture solution is taken for PCR detection, and plasmid double enzyme digestion detection is extracted after detection is successful (figure 1C).

The prokaryotic expression vector pGEX-4T-1-AtIDH3 plasmid successfully detected by double enzyme digestion is transferred into escherichia coli BL21 protein expression bacteria through heat shock transformation, the bacteria are coated in an Amp-containing solid culture medium and cultured overnight in a thermostat at 37 ℃, single colonies are selected to be screened and cultured in an Amp liquid culture medium, and the culture medium for normal growth of bacteria is subjected to bacteria liquid PCR detection (figure 1D). Inoculating the successfully detected bacterial liquid into 25mL LB liquid culture medium, culturing in a shaking table (37 ℃, 220rpm) 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 1 mmol/L; centrifuging the induced bacteria solution at 12000rpm for 1min at 2mL respectively after 0, 2, 4, 6 and 8h, collecting thallus, adding 2mL PBS for resuspension, adding 20 μ L of resuspended thallus into 20 μ L of 2 × protein electrophoresis loading buffer solution, boiling in boiling water for 10min, cooling, and performing polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis to analyze protein expression (FIG. 2A); the AtIDH3 protein was found to induce the maximum expression of 6h protein at 1mmol/L IPTG.

The AtIDH3 gene recombinant expression strain is induced and expressed in a shaker at 28 ℃ and 37 ℃ respectively under the condition of 1mmol/L IPTG, the thalli sediment is repeatedly washed three times by 4mL TBS, 2mL PBS is added to resuspend the thalli sediment, ultrasonic crushing is carried out for 15min, the supernatant and the sediment are obtained by centrifugation, and SDS-PAGE analysis is carried out on the supernatant, and the expression amount of the expressed protein is the highest in the supernatant at 28 ℃ under induction (figure 2B). Under the optimal condition, inducing the strain to express protein in a large amount; centrifuging at4 ℃ 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 15 min; after centrifugation of the disrupted cells, the supernatant containing a large amount of the desired expression protein was applied to a GST column to obtain a purified IDH3 fusion protein, and 20. mu.L of the supernatant was subjected to 12% SDS-PAGE (FIG. 2C).

Example 2: determination of isocitrate dehydrogenase Activity

Protein concentration was measured by the Bradford method using a BCA method protein quantification kit produced by shanghai strapdown bioengineering ltd and Bovine Serum Albumin (BSA) as a standard protein, according to the kit manual. The enzyme activity is measured by spectrophotometry, and the enzyme activity measuring system of IDH is 20mmol/L Tris-HCl and 5mmol/L MgCl₂5mmol/L DL-isocitric acid trisodium or 0.5mmol/L DL-isocitric acid trisodium, 2mmol/L NAD⁺Or 0.5mmol/L NADP⁺. The change in the absorbance at 340nm during the reaction was measured using an ultraviolet spectrophotometer at 25 ℃.

(1) Optimum temperature and thermal stability of isocitrate dehydrogenase AtIDH3

The relative enzyme activity of IDH was measured in the above reaction system under the same conditions at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C, 55 deg.C in water bath for 10 min. And drawing a curve graph by taking the temperature as an abscissa and the relative enzyme activity as an ordinate to calculate the optimal reaction temperature of the IDH enzyme. Placing IDH in water bath at 25 deg.C, 30 deg.C, 35 deg.C, 40 deg.C, 45 deg.C, 50 deg.C and 55 deg.C, cooling on ice immediately after 20min, determining the residual activity of IDH enzyme, and drawing a curve graph with temperature as abscissa and relative residual enzyme activity of enzyme as ordinate to determine the thermal stability of enzyme; the enzyme activity without treatment was 100%.

The results of the measurement showed that the effect of temperature on the rate of the enzyme-catalyzed reaction was actually a combination of the effect of temperature on the rate of the reaction and the promotion of enzyme denaturation, and the results are shown in FIGS. 3 and 4, and FIG. 3 shows that the optimal reaction temperature for AtIDH3 enzyme was 45 ℃. IDH can keep more than 80% of activity at 30-55 ℃, which shows that AtIDH3 enzyme activity is less influenced by temperature. FIG. 4 shows that the enzyme activity of AtIDH3 is kept above 60% within the range of 25-40 ℃, and the thermal stability to the enzyme activity is the best at 30-35 ℃, and is all above 80%; after the temperature is over 40 ℃, the enzyme activity stability is gradually reduced, and the thermal stability is obviously reduced, which shows that the stability of AtIDH3 enzyme is relatively greatly influenced by higher temperature.

(2) Optimum pH of isocitrate dehydrogenase

The relative enzyme activity of IDH was measured at 25 ℃ in 20mmol/L Tris-HCl buffer solution with pH 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, respectively, without changing the other conditions in the above reaction system; repeating the pH value for three times, calculating the enzyme activity according to the statistical result, setting the highest point of the enzyme activity as 100%, and drawing by taking the pH value as a horizontal coordinate and taking the relative enzyme activity as a vertical coordinate to obtain the optimal pH value;

the results in FIG. 5 show that the enzyme activity of AtIDH3 reaches the highest at 8.2, which is the optimum pH of AtIDH3 enzyme. When the pH value is within the range of 6-8.2, the activity of the IDH enzyme changes slowly; the pH value is rapidly reduced within the range of 8.2-10, which shows that IDH enzyme can be inactivated by an over-alkali environment.

(3) Effect of Metal ions on Activity of isocitrate dehydrogenase

At 25 deg.C, maintainOn the premise of keeping other components unchanged, MgCl with the final concentration of 5mmol/L is respectively added into the reaction system₂、MnCl₂、CoCl₂、CaCl₂、ZnSO₄、CuCl₂AtIDH3 enzyme activity was determined by NaCl and KCl, each experiment was repeated three times independently, and the results were counted and the relative enzyme activity of IDH was calculated.

The results of the effects of various metal ions on the activity of isocitrate dehydrogenase showed that Mn²⁺And Mg²⁺Has promoting effect on AtIDH3, but has obvious difference on the activation effect of AtIDH3 enzyme, Mn²⁺In the presence of most enzymatically active, Mg^{2 +}Promotion ratio of Mn²⁺To be weak; the rest 6 metal ions inhibit IDH activity to a certain extent, and the specific inhibition sequence is Ca²⁺＞Co²⁺＞Zn²⁺＞K⁺＞Na⁺＞Cu²⁺. Different metal ions produce affinity with AtIDH3 in different ways, resulting in changes in enzyme activity. The results show that different metal ions have different effects on the activity of AtIDH 3. The experimental indexes are determined repeatedly for three times, the experimental data are statistically analyzed by Excel 2010, and the results are shown in the following table:

metal ion	Relative enzyme activity (%) +/-SD
		Control of	100％±0.02
Mg2+	115％±0.03
		Mn2+	172％±0.01
Co 2+	24％±0.04
		Ca 2+	11％±0.02
Zn2+	26％±0.01
		Cu 2+	36％±0.04
Na+	31％±0.03
		K +	30％±0.02

Example 3: preparation and potency detection of AtIDH3 protein rabbit antibody

1mL of the purified recombinant protein (containing about 5mg of the purified AtIDH3 protein) was emulsified with 1mL of Freund's adjuvant in a ratio of 1: 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, rabbits were boosted with an equal volume of incomplete Freund's adjuvant to emulsify AtIDH3 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 at4 ℃ 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 whether the antibodies were effective, we performed a specific assay for the AtIDH3 antibody. The purified recombinant proteins 5 μ L, 10 μ L and 15 μ L (protein concentration is 2.354 μ g, 4.708 μ g and 7.062 μ g respectively) were subjected to SDS-PAGE, and western blot analysis was performed using the rabbit antibody as a primary antibody and a commercial goat anti-rabbit antibody as a secondary antibody, and the results are shown in FIG. 6A, which shows that the polyclonal rabbit antibody prepared in this example can specifically react with AtIDH3 antigen, and the antibody preparation is successful.

To further test the effectiveness of the antibodies, we performed a two-way immunodiffusion test. The results of the experiment are shown in FIG. 6B, and a curved white precipitate line can be seen, indicating that the protein purified with AtIDH3 is specific to the immune serum of AtIDH3, and they diffuse freely in the agarose gel, and after meeting, an antigen-antibody complex is formed. This line is curved along the edge of the middle well and all wells diluted at different times have white lines except for the absence of white lines at the saline, indicating that the rabbit antibody titer is higher.

Example 4: construction of transgenic tobacco strain over-expressing AtIDH3 and identification of tobacco over-expressing AtIDH3 gene

The invention constructs a eukaryotic target expression vector pMK-35S-AtIDH3 by Gateway technology; the target segment (1107bp) of AtIDH3 gene was PCR-amplified. pMD18T and the target fragment are cloned by T/A to obtain pMD18T-AtIDH3, and positive clones are sequenced to detect whether the exogenous gene AtIDH3 is mutated. Detecting the successful non-mutated pMD18T-AtIDH3 plasmid and pENTR plasmid, respectively performing double enzyme gel cutting by BamH I and Sal I to recover a target fragment AtIDH3 and a cut pENTR, connecting the two fragments overnight to form a door-entering cloning vector pENTR-AtIDH3, performing double enzyme digestion, and performing gel electrophoresis to observe whether the AtIDH3 gene is successfully cloned to the pENTR vector. After the detection is successful, the plant binary expression vector pMK7WG-cL-2 and the entry clone vector pENTR-AtIDH3 are subjected to LR reaction under the action of LR Mix Enzyme to form a plant expression vector pMK-35S-AtIDH3 for short.

Transferring a plant expression vector pMK-35S-AtIDH3 into agrobacterium pMP90 by an electrical transformation method, obtaining positive clones by screening and culturing in a spectinomycin (Spe) solid culture medium, selecting single bacteria, screening and culturing in an LB liquid culture medium containing Spe (final concentration is 50mg/L), detecting the positive clones with exogenous plant expression vector plasmids by bacterial liquid PCR, transfecting the expression vector pMK-35S-AtIDH3 agrobacterium containing target genes into tobacco by a leaf disc transformation method, and screening in a Kan resistance culture medium (final concentration is 30 mu g/mL) to obtain transgenic tobacco.

Extracting the genome of the plant from the transgenic tobacco with root growing in the 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 AtIDH3 gene, and detecting a PCR product on 1% agarose gel electrophoresis; detecting a plant with an exogenous AtIDH3 gene inserted in a genome by genome PCR, and analyzing the transcription level of the plant by qRT-PCR; extracting total RNA from the transgenic strain by a TRIzol method, detecting the concentration and the quality of the RNA by using an enzyme-linked immunosorbent assay (ELIAS) reader and performing 1.5% agarose gel electrophoresis, and then performing reverse transcription on 25 mu L of RNA to synthesize first-strand cDNA; taking 1 mu L cDNA as a template, and carrying out qRT-PCR analysis on the transcription level of IDH3 in the transgenic tobacco by taking actin of the tobacco as an internal reference; after IDH3 is identified to be transcribed in tobacco, whether the protein is translated or not is detected, the total plant protein of the transgenic tobacco is extracted, rabbit antibody prepared by chapter II is used as a primary antibody, and the protein level of IDH3 is detected by western blot.

Transforming tobacco with pMK-35S-AtIDH3 to obtain transgenic strain with kanamycin resistance, and detecting the insertion of exogenous gene with genome DNA as template and specific primer of AtIDH3 gene; after PCR reaction of the genome, the following results were obtained (fig. 7A); the results show that the AtIDH3 gene is inserted in the genome of 15 (1, 5, 6, 7, 8, 9, 10, 12, 13, 16, 17, 18, 19, 20 and 21) strains; the results of qRT-PCR analysis (FIG. 7B) show that the exogenous AtIDH3 gene in 6 transgenic plants can be transcribed normally, and the transcription levels of all transgenic tobacco strains are similar. Detection of protein level Using western blot (FIG. 7C) with the antibody obtained in example 3 as the primary antibody, the results of western blot analysis showed that the exogenous AtIDH3 gene was translated normally in 6 (9, 10, 12, 13, 17, 20) lines of transgenic plants, and that the translation levels of each transgenic tobacco line were similar.

The results of detection and analysis of three levels of DNA, RNA and protein show that the exogenous AtIDH3 gene is accurately inserted into the tobacco genome, and under the 35S promoter, the exogenous AtIDH3 gene can be correctly transcribed. In the experiment, a transgenic tobacco strain 10 with a higher transcription level is selected as a transgenic tobacco test material.

Example 5: formaldehyde stress treatment of transgenic plants

Selecting plants with the same growth status, collecting 1g leaf, washing with tap water for 3-4 times to remove dirt, blotting residual water on surface with sterile absorbent paper, transferring to culture flask containing 2mM, 4mM, and 6mM HCHO treatment solution 50mL each, and continuously irradiating at 25 deg.C (100 μmol. m)^-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 absorption capacity of transgenic tobacco to liquid formaldehyde

In order to detect the influence of 14-3-3cc expression-modified tobacco (14-3-3c overexpression tobacco KC for short, and 14-3-3c expression inhibiting tobacco R for short) and overexpression Arabidopsis AtIDH3 on the absorption of liquid HCHO by tobacco, three samples, namely 1g leaf material, are respectively taken from 14-3 c expression-modified tobacco and Arabidopsis AtIDH3 overexpression tobacco, and are respectively immersed in 50mL HCHO solutions with the concentrations of 2mM, 4mM and 6mM for the detection of HCHO absorption, the volatilization amount of HCHO is monitored by taking the HCHO solution without leaf as a control, and the average value obtained after the detection is used for drawing a liquid HCHO absorption curve of transgenic tobacco. By comparing the HCHO absorbing capacity of the transgenic tobacco and the wild type tobacco, the result shows that the HCHO absorbing capacity of the tobacco and the Arabidopsis AtIDH3 overexpression tobacco is changed by expressing 14-3-3c in a 2mM HCHO solution (figure 8), the overexpression tobacco is slightly higher than the wild type tobacco, the inhibition expression is slightly lower than the wild type tobacco, and the HCHO absorbing speed of the wild type tobacco and the HCHO absorbing speed of the transgenic tobacco are obviously different after 24h, wherein the HCHO absorbing speed of the 14-3-3c overexpression tobacco is fastest.

Comparing the uptake curves of transgenic tobacco and wild type tobacco in 4mM HCHO solution (FIG. 9), it can be found that the overall trends of the tobacco with 14-3-3c expression changing tobacco and the Arabidopsis AtIDH3 overexpression and wild type tobacco for absorbing HCHO are similar, the 14-3-3c overexpression is slightly higher than the uptake capacity of AtIDH3, the AtIDH3 overexpression is slightly higher than the uptake capacity of wild type, and the HCHO uptake inhibiting expression by 14-3-3c is weaker than that of wild type.

From the uptake curves of transgenic tobacco and wild type tobacco in 6mM HCHO solution (FIG. 10), we found that the wild type tobacco and the tobacco expressing the gene for inhibiting the uptake of HCHO were significantly weaker than the other two transgenic tobaccos. And the HCHO absorbing capacity of the tobacco over-expressing 14-3-3c is higher than that of the tobacco over-expressing Arabidopsis AtIDH 3. These results indicate that the over-expression of the tobacco 14-3-3c gene and the Arabidopsis AtIDH3 gene in tobacco enhances the absorption capacity of tobacco to liquid HCHO, and the inhibition of the expression of the tobacco 14-3-3c gene reduces the absorption capacity of tobacco to liquid HCHO.

2. Effect of 14-3-3c expression Change on expression amount of AtIDH3

The expression level of AtIDH3 was measured by treating 14-3-3c over-expressed tobacco and 14-3-3c under 2mM liquid formaldehyde for 12h and 24h, and using untreated tobacco as a control, the relative expression level of AtIDH3 was analyzed after 2mM liquid formaldehyde treatment for 0h, 12h and 24h, as shown in FIGS. 11-13. The expression level of AtIDH3 in wild type tobacco increased with increasing treatment time (see FIG. 11); 14-3-3c over-expressed tobacco showed the same tendency as wild type, with longer treatment time and higher AtIDH3 expression (FIG. 12); the expression level of tobacco AtIDH3 in which expression was suppressed by 14-3-3c was also increased, but slightly increased, and there was no high expression level of wild type and overexpression (FIG. 13). After 24h of treatment, the expression level of AtIDH3 of wild type and 14-3-3c over-expressed tobacco is obviously increased, and the expression level of 14-3-3c over-expressed tobacco is higher than that of AtIDH3 of wild type tobacco, but the expression level of AtIDH3 of 14-3-3c over-expressed tobacco is inhibited to be increased, and the difference between the expression level of AtIDH3 of 14-3-3c over-expressed tobacco and the expression level of AtIDH3 of 14-3-3c over-expressed tobacco is not large or even slightly reduced after 12h of treatment; the results prove that the expression quantity of AtIDH3 is increased after formaldehyde treatment after the expression of 14-3-3c is changed, and the increase of 14-3-3c over-expressed tobacco is larger than that of wild tobacco is larger than that of 14-3-3c suppression expression tobacco.

3. Effect of altered 14-3-3c expression on AtIDH3 enzyme Activity

In order to detect whether the enzyme activity of AtIDH3 is affected after formaldehyde stress is carried out on 14-3-3c expression-modified transgenic tobacco, wild type tobacco and 14-3-3c overexpression tobacco which are subjected to formaldehyde treatment for different time and 14-3-3c suppression expression tobacco are used as materials, the relative enzyme activity of AtIDH3 is measured, and the result is shown in figure 14, wherein the enzyme activity of AtIDH3 of 14-3-3c overexpression tobacco subjected to formaldehyde treatment is integrally higher than that of wild type tobacco and 14-3-3c tobacco; except 6h and 60h of formaldehyde treatment, the 14-3-3c inhibits the expression of the tobacco and has higher relative enzyme activity than wild AtIDH 3. After 14-3-3c over-expression tobacco is subjected to formaldehyde treatment, the relative enzyme activity of AtIDH3 is increased at 6h, but is reduced to be slightly higher than that of untreated tobacco at 12h, and then the enzyme activity is increased along with the increase of the formaldehyde treatment time; the 14-3-3c inhibition expression tobacco and the wild type tobacco are just opposite to the 14-3-3c overexpression tobacco before being treated for 24 hours, the enzyme activity is reduced when the tobacco is treated for 6 hours, the enzyme activity is increased when the tobacco is treated for 12 hours, and the enzyme activity is slowly increased after being reduced after 24 hours. The result shows that the enzyme activity of AtIDH3 in the 14-3-3c over-expressed tobacco after formaldehyde treatment is improved, and the wild type and 14-3-3c inhibit the expression of the tobacco to be reduced or slightly improved.

4. In vivo and in vitro AtIDH3 and 14-3-3c interaction analysis

In order to understand whether 14-3-3c interacts with AtIDH3 to affect formaldehyde metabolism, Pull-down analysis was first performed (see FIG. 15), purified 14-3-3c-His fusion proteins were conjugated with GST and GST-AtIDH3 fusion proteins, respectively, in binding buffer, then GST-agarose binding resin was added and centrifuged after incubation, and the precipitated proteins were detected by Western blot analysis, as follows:

(1) construction of expression vector: TA cloning is carried out by using pMD-18T vector, 14-3-3c is inserted into pMD-18T, and the target gene fragment of 14-3-3c is obtained by double digestion with Nco I and BamH I and then gel recovery. Meanwhile, the pET-28a expression vector is subjected to double digestion by Nco I and BamH I, and a linear pET-28a vector is recovered. Connecting for 2h at 16 ℃, transforming DH5 alpha by a heat shock method, screening on an Amp resistance-containing culture medium, and transforming a BL21 expression strain after correct sequencing;

(2) inducing expression of the recombinant protein: transforming BL21 competence with the constructed vector pET-14-3-3c, inducing 14-3-3c recombinant protein expression and detecting the expression condition of the protein;

3. combining: separately mixing purified 14-3-3c-His fusion protein with GST and GST-IDH3 fusion protein 50 μ g, adding binding buffer (50mmol/L Tris-HCl, pH7.5, 100mmol/L NaCl, 0.25%, TritonX-100, 1mmol/L EDTA, 1mmol/L DTT) at room temperature, shaking and binding for 2h, adding 30 μ L GST agarose binding resin, and incubating overnight at4 deg.C in a shaker (40 r/min);

4. washing: centrifuging (3500g) at4 deg.C for 5min to collect precipitate, and washing the collected precipitated protein mixture with binding buffer solution three times;

the settled proteins were boiled in a boiling water bath, and the precipitated proteins were separated on SDS-PAGE gel, transferred to PVDF membrane for Western blot analysis, and then Western blot was performed using Anti-His and Anti-14-3-3 c-specific antibodies (rabbit antibody previously made in the laboratory) as primary antibodies to detect whether 14-3-3c and IDH3 protein interacted in vitro.

As a result, it was found that both His and 14-3-3c were precipitated by GST-AtIDH3 protein, whereas GST control group did not precipitate His and 14-3-3c protein, indicating that interaction between 14-3-3c and AtIDH3 was precipitated.

Next, we performed yeast two-hybrid experiments on the 14-3-3c and AtIDH3 recombinant proteins (see FIG. 16). Firstly, connecting the full length of the 14-3-3c gene into a pGADT7 vector, and transforming a Y187 yeast strain; the CDS sequence of AtIDH3 gene was cloned into pGBKT7 vector, and Y2HGold yeast strain was transformed. Two by two fusions were performed using Leu-Trp-deficient medium. We have determined that p35 and the large T antigen 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, as follows:

(1) construction of the vector: the AtIDH3 gene was ligated into pGBKT7 vector and 14-3-3c gene into pGADT7 vector by enzymatic ligation, such that AtIDH3 was fused to the DNA binding domain of GAL4 (BD-AtIDH3) and 14-3-3c was fused to the DNA activating domain of GAL4 (AD-14-3-3c), respectively.

And (3) transformation: adding 10 μ L of Y187 or Y2H Gold bacteria solution into 2mL of 0.25 XYPDA, culturing at 28 deg.C and 200rpm overnight for 12 h;

(2) taking 250 mu L of bacterial liquid, quickly separating at 12000rpm for 15s, collecting precipitate, adding 1mLddH₂O, resuspending, quickly separating and collecting the precipitate;

(3) 240 μ L of PEG 3350 (50% w/v) was added slowly in sequence; 36 μ L LiAc (1M); 20. mu.L ss-DNA (2 mg/mL); 10. mu.L of the recombinant plasmid; 54 μ L ddH₂O vortex for 1 min; water bath at 42 deg.c for 30 min. Quickly separated for 15s, the suspension removed and 100. mu.L ddH added₂And O, lightly blowing and smearing a piece of culture medium, and culturing for 2 to 4 days in a constant temperature box at the temperature of 28 ℃.

(4) Selecting single colonies, and culturing at 28 deg.C and 200rpm overnight in a single culture medium;

(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) taking 1mL of bacterium liquid for quick separation, ddH₂Washing for 2 times with O, gently blowing, mixing, sucking 20 μ L, and coating on two plates;

(7) picking single colony to centrifuge tube, adding 50-100 μ L ddH2O for dissolving, centrifuging at low speed, and absorbing ddH₂O; reuse of ddH₂And washing once. Adding 30-50 mu L ddH2O and mixing evenly for later use; sucking 1.5-2 μ L, dropping on two-gap plate without AbA, X- α -gal and containing AbA, X- α -gal, culturing at 28 deg.C for 2-4 days, and observing;

as a result of using pGADT7-14-3-3c and pGBKT7-AtIDH3 as experimental groups, blue spots were grown on the two-deficient plate containing AbA and X-alpha-gal in the experimental groups as in the positive control group, and it was confirmed that 14-3-3c and AtIDH3 had an interaction.

Finally, we used co-immunoprecipitation to verify whether AtIDH3 binds to 14-3-3c protein: treating wild tobacco, 14-3-3c over-expression tobacco and leaf blade of 14-3-3c suppression expression tobacco with 2mM formaldehyde for 0h, 6h, 12h, 24h, 36h, 48h and 60h, respectively extracting total plant protein, adding 2 μ G of 14-3-3c protein specific antibody into 200 μ G of total plant protein, adding 20 μ L of agar protein A/G, and shaking at4 deg.C (40rpm) overnight; the next day, the protein pellet was centrifuged for 5min (4 ℃ C., 3000rpm), washed with pre-cooled PBS (phosphate buffered saline) 3 times, and the washed pellet was dissolved in 40. mu.L of 1 × loading buffer, and after 40. mu.L of the pellet was separated by SDS-PAGE (12%) electrophoresis, Western blot analysis was performed. Transferring the protein to a PVDF membrane by a semi-dry electrotransformation instrument, incubating an IDH3 specific antibody at4 ℃ overnight, incubating a goat-anti rabbit-anti antibody at normal temperature for 2h, and observing the experimental result by a gel imaging instrument. The gel imager results were analyzed for AtIDH3 and 14-3-3c protein interaction levels (FIG. 17), and the results indicated that 14-3-3c and IDH3 were able to interact at different formaldehyde treatment times.

5. 14-3-3c overexpression/suppression expression 14-3-3c and AtIDH3 overexpression influence on absorption and metabolism of tobacco formaldehyde

By using¹³C-NMR technique analyzed H¹³CHO metabolism in various transgenic tobacco leaves, FIG. 18 shows 2mM liquid H¹³Main metabolic intermediate products in wild type and transgenic leaves after 24h CHO treatment¹³The change in the intensity of the C-NMR resonance peak; by comparing wild type and various transgenic tobacco leaves H¹³The change in the amount of each intermediate in the CHO treated sample and the control sample was found to have passed through H¹³14-3-3c overexpression (KC), 14-3-3c suppression (RC), AtIDH3 overexpression (IDH3) after CHO treatment for 24h were higher than the wild type leaf (WT) and the wild type leaf (CK) without any treatment, and the intermediary metabolites of the three transgenic tobaccos were higher than those without any treatment.

The relative content of each main metabolite is analyzed by integrating each peak in the nuclear magnetic spectrum, and the results of FIG. 19A, B, C, D show that the wildThe isocitric acid, malic acid, glucose and citric acid in the raw tobacco leaves are respectively 4 times, 1.25 times, 4 times and 2 times higher than that of wild type leaves (CK) which are not treated; the isocitric acid, malic acid, glucose, citric acid in the 14-3-3c over-expressed leaves were 6.5 times, 4.2 times, 22 times and 3.75 times higher than those of the wild type leaves (CK) without any treatment, respectively; 14-3-3c inhibits isocitric acid, malic acid, glucose, citric acid in the expression tobacco leaf blade to be 2 times, 3.25 times, 13 times and 3.75 times higher than that of the wild type tobacco leaf blade (CK) without any treatment, respectively; the AtIDH3 over-expressed leaves showed 6.5-fold, 9.75-fold, 21-fold and 3.75-fold higher isocitrate, malate, glucose and citrate, respectively, than the wild type leaves (CK) without any treatment; from these results, we speculate that isocitric acid, malic acid, glucose, citric acid may be 2mM liquid H in tobacco formaldehyde metabolism¹³CHO as the main intermediate. The tobacco leaves after the overexpression of the 14-3-3c and AtIDH3 genes have stronger capability of metabolizing low-concentration formaldehyde (2mM) than the wild type.

Sequence listing

<110> university of Kunming science

Application of isocitrate dehydrogenase in improving formaldehyde absorption and metabolism of plants

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1107

<212> DNA

<213> Arabidopsis thaliana (Arabidopsis thaliana)

<400> 1

atggcgagaa gatccgtttc gatatttaat cgccttctgg cgaatcctcc ttctccattc 60

acatcactgt cccgatccat cacctacatg cctagacccg gagatggagc tccacgaacc 120

gtaaccctaa ttcccggcga cggaatcgga cctttggtga ccggtgcggt ggaacaggtc 180

atggaagcga tgcacgcgcc agtgcatttc gagagatacg aggttctagg aaacatgaga 240

aaagtccctg aagaagtgat cgagtctgtg aagagaaaca aggtttgtct caaaggtgga 300

ttagcgactc ctgttggtgg gggtgtcagt tctttgaata tgcaattgag gaaagaactc 360

gatatcttcg cttctcttgt caattgcatc aatgtccctg gattagtgac gcgacacgaa 420

aatgttgata tcgttgtgat aagagagaac actgaaggag agtactcagg tctcgagcat 480

gaggttgttc ctggtgttgt cgagagcctt aaggtgataa caaagttttg ttctgagaga 540

atagcgagat atgcatttga gtatgcgtat ctaaacaata ggaagaaagt gactgctgtt 600

cataaagcta acattatgaa gcttgcggat gggctcttcc ttgaatcttg tagagaggtt 660

gctaaacatt attcggggat tacttacaat gaaataatcg tagacaattg ctgtatgcag 720

cttgtcgcca agcctgagca atttgatgtc atggtgacac ctaatttgta tggtaacctt 780

atagcaaaca cggcggctgg aatagccggt ggcactggag tgatgccagg agggaatgtt 840

ggtgcagaac atgcgatatt cgagcaaggt gcatcagcag ggaatgtggg gaatgataag 900

atggtggaac agaagaaagc gaatccggtg gctctacttc tctcgtcggc tatgatgcta 960

agacatctcc ggtttcctac ttttgctgat cggcttgaaa cagcagtgaa acaagtgatt 1020

aaagaaggaa aatatagaac aaaagatctt ggaggagatt gtaccacgca ggaagttgtt 1080

gatgctgtca tagcagctct tgagtga 1107

<210> 2

<211> 20

<212> DNA

<213> Artificial sequence (Artificial)

<400> 2

atggcgagaa gatccgtttc 20

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence (Artificial)

<400> 3

ctcaagagct gctatgacag ca 22

Claims (1)

1. Arabidopsis thaliana isocitrate dehydrogenase geneAtIDH3The application of the plant formaldehyde absorption and metabolism capacity improvement is characterized in that: arabidopsis thaliana isocitrate dehydrogenase geneAtIDH3The nucleotide sequence of (A) is shown as SEQ ID NO. 1.

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