CN114002207A - Application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction - Google Patents
Application of ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction Download PDFInfo
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- CN114002207A CN114002207A CN202111267757.3A CN202111267757A CN114002207A CN 114002207 A CN114002207 A CN 114002207A CN 202111267757 A CN202111267757 A CN 202111267757A CN 114002207 A CN114002207 A CN 114002207A
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- China
- Prior art keywords
- luminol
- active oxygen
- plant
- ascorbic acid
- peroxidase
- Prior art date
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- HWYHZTIRURJOHG-UHFFFAOYSA-N luminol Chemical compound O=C1NNC(=O)C2=C1C(N)=CC=C2 HWYHZTIRURJOHG-UHFFFAOYSA-N 0.000 title claims abstract description 71
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 title claims abstract description 46
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 32
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- 238000000034 method Methods 0.000 claims abstract description 17
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/76—Chemiluminescence; Bioluminescence
Abstract
The invention discloses an application of ascorbic acid peroxidase 1 in catalyzing luminol chemiluminescence reaction, and belongs to the technical field of plant immunity. The invention discloses that the ascorbic acid peroxidase 1 has the activity of catalyzing the oxidation of luminol for the first time, and can be developed into luminol-H2O2A chemiluminescent reaction catalyst. Based on the characteristic of stable expression of the ascorbate peroxidase 1 in plant cells, the dynamics and the content of intracellular active oxygen can be monitored in real time by using a luminol chemiluminescence method, and the dynamic and content can be used as a model for screening induction factors capable of triggering the intracellular active oxygen to burst.
Description
Technical Field
The invention relates to the technical field of plant immunity, in particular to a method for monitoring intracellular active oxygen of a plant based on ascorbic acid peroxidase 1 catalysis luminol chemiluminescence reaction.
Background
Reactive Oxygen Species (ROS) are oxygen-containing radicals generated in the process of aerobic biological evolution, and have high biological activity. The active oxygen mainly comprises singlet oxygen (1O2) Superoxide anion (O)2 -·) Hydrogen peroxide (H)2O2) And hydroxyl radical (OH. cndot.), etc. In plant cells, the major sites for the production of active oxygen are plasma membrane, chloroplast, mitochondria and peroxisomes.
Active oxygen plays a dual role in the life activities of plants: on one hand, the low-concentration active oxygen plays a role in signal regulation on physiological processes of plant such as cell division, programmed death, organ development, biotic and abiotic stress and the like; on the other hand, high concentrations of reactive oxygen species cause oxidative damage to cells, preventing normal metabolism and growth of plants, and even leading to cell death. The plant body has a complex active oxygen generation and elimination mechanism to maintain the delicate balance of the oxidation-reduction state in cells, and can timely and accurately respond to upstream stress and mediate downstream complex physiological processes while avoiding cell damage caused by excessive active oxygen.
The active oxygen burst is one of the most important responses in plant immune response, and not only can be used as a signal molecule to induce other immune responses, but also cell death caused by active oxygen accumulation can directly limit the growth of pathogenic bacteria. Therefore, monitoring the dynamic state and content of active oxygen generation is a very important technical means for active oxygen research.
At present, the method for detecting active oxygen mainly detects the content of the active oxygen indirectly through products generated after different probes react with the active oxygen, and the probes comprise chromogenic dyes, fluorescent probes and chemiluminescent reagents.
Luminol (Luminol) is a commonly used assay H2O2The luminol can emit 425nm blue light after being oxidized, so that the luminous intensity of the luminol can be used for characterizing the H of the reaction2O2Amount of the compound (A). The luminol chemiluminescence method is simple to operate, has high sensitivity, can detect in real time, and is widely applied to detecting the content of active oxygen and generating dynamics in various plant tissues.
However, H is usually produced by a plant2O2The amount is not sufficient to oxidize luminol directly, and some catalyst is required to enhance oxidation of luminol, for example, the commonly used commercial horse radish peroxidase HRP(Horseradish Peroxidase), the progress of which is shown in FIG. 1, and luminol is oxidized in the presence of HRP to generate excited product 3-aminophthalate (3-APA). When the product decays to the ground state, photons are emitted instantaneously. However, since horseradish peroxidase cannot enter cells, the luminol chemiluminescence method can only be used for measuring the accumulation of intercellular active oxygen or the active oxygen released from cells to the outside, so that the application of the method in measuring the content of active oxygen in living cells is limited.
Disclosure of Invention
The invention aims to provide a new luminol-H2O2The catalyst of chemiluminescence reaction, and the application of the catalyst to luminol chemiluminescence method for measuring active oxygen in living cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides application of ascorbic acid peroxidase 1 as a catalyst in luminol-H2O2Application in chemiluminescence reaction.
The research of the invention finds that the ascorbic acid peroxidase 1 has the activity of catalyzing the oxidation of the luminol, so that the ascorbic acid peroxidase can be developed into the luminol-H2O2A catalyst for a chemiluminescent reaction.
Furthermore, the amino acid sequence of the ascorbate peroxidase 1 is shown as SEQ ID NO.1, and the nucleotide sequence of the coding gene thereof is shown as SEQ ID NO. 2. The invention clones ascorbic acid PEROXIDASE 1 gene cAPX1(cytosolic ascorbic acid PEROXIDASE PEROXIDAE 1) from arabidopsis thaliana, and uses an escherichia coli expression system to express and obtain cAPX1 recombinant protein, and the protein has the enzymatic activity of catalyzing luminol oxidation.
Further, luminol-H2O2In a chemiluminescence reaction system, the concentration of ascorbic acid peroxidase 1 is 0.03-0.04 mg/mL, the concentration of luminol is 300-600 mu M, and H is2O2The concentration is 5 to 10 mM.
Further, the application includes: firstly, a 96-pore plate is put into a single photon imaging counter, and the focus is well adjusted in the dark environmentDistance; adding ascorbic acid peroxidase 1, luminol and H into the pore plate2O2And sterile water; and detecting a chemiluminescence signal by using a single photon imaging counter, continuously monitoring for 10min, and recording the total chemiluminescence intensity.
The invention also provides an application of the ascorbic acid peroxidase 1 in detecting the intracellular active oxygen content of the plant, wherein the application is that the ascorbic acid peroxidase 1 in the plant cell is used for catalyzing luminol to perform a chemiluminescence reaction with the intracellular active oxygen, and the intracellular active oxygen content of the plant is obtained through detecting chemiluminescence signal analysis.
Research shows that the ascorbic acid peroxidase 1 existing in the plant cell can be used as a catalyst of luminol chemiluminescence reaction. In the detection process, plant leaf tissues to be detected are added into a luminol solution, the luminol permeates into plant cells, the luminol and intracellular active oxygen generate a chemiluminescence reaction under the catalysis of ascorbic acid peroxidase 1, and the content of the active oxygen is represented by the luminous intensity of the luminol.
Further, the application includes: firstly, placing a plant leaf disc to be detected in sterile water for incubation for 8-12 h, then adding a luminol solution or a mixed solution of luminol and an induction factor into the plant leaf disc, placing the plant leaf disc in a single photon imaging counter to monitor a chemiluminescence signal, and representing the content of active oxygen in a plant cell by using relative light intensity.
The diameter of the leaf disc is 3-5 mm. The plant leaf disk is obtained by a puncher, the plant can be induced to generate active oxygen due to mechanical damage, and in order to relieve the mechanical damage caused by punching, the leaf disk is placed in sterile water for overnight incubation.
The monitoring of the chemiluminescence signal lasts for 1-24 h. Research shows that the burst of active oxygen in the plant body is a continuous process, the ascorbyl peroxidase 1 is continuously expressed in the plant living cell, a catalyst is continuously provided for luminol chemiluminescence reaction, and dynamic monitoring of the active oxygen in the plant cell can be realized.
Further, the concentration of luminol in the solution is 600 μ M. The detection of active oxygen burst can be supported for a long time under the concentration condition.
The invention provides a method for screening an intracellular active oxygen burst induction factor of a plant by taking ascorbyl peroxidase 1 existing in living cells as a catalyst to catalyze luminol to generate a chemiluminescence reaction in the cells as a screening model.
The method comprises the following steps:
(1) placing a plant leaf disc in a pore plate containing sterile water for incubation for 8-12 h;
(2) then adding a mixed solution of luminol and the induction factor to be detected into the plant leaf disc, and taking the luminol solution without the induction factor as a control;
(3) then placing the pore plate in a single photon imaging counter to detect a chemiluminescence signal;
(4) comparing the difference in the chemiluminescent signals with a control to determine whether the induction factor causes intracellular reactive oxygen species to burst;
the statistically significant increase in the chemiluminescence intensity measured in the leaves of the plants treated with the induction factor, as compared to the control, indicates that the induction factor is capable of causing burst of active oxygen in the plants.
Further, the concentration of luminol in the solution is 600 μ M.
The invention has the following beneficial effects:
the invention discloses that the ascorbic acid peroxidase 1 has the activity of catalyzing the oxidation of luminol for the first time, and can be developed into luminol-H2O2A chemiluminescent reaction catalyst. Based on the characteristic of stable expression of the ascorbate peroxidase 1 in plant cells, the dynamics and the content of intracellular active oxygen can be monitored in real time by using a luminol chemiluminescence method, and the dynamic and content can be used as a model for screening induction factors capable of triggering the intracellular active oxygen to burst.
Drawings
FIG. 1 shows HRP-catalyzed luminol-H2O2Reaction scheme.
FIG. 2 shows that the cAPX1 protein of example 2 catalyzes luminol and H in vitro2O2As a result of the reaction of (A) whereinThe reaction results of different groups of reaction liquid are shown in the specification, and HRP is horseradish peroxidase and is used as a positive control; MBP (maltose binding protein) is maltose binding protein and is used as a negative control; MBP-cAPX1 is MBP-labeled ascorbate peroxidase 1; (B) luminol and H catalyzed by cAPX1 for Hydrogen peroxide concentration2O2The effect of the chemiluminescence intensity of the reaction. Data are shown as mean ± sem (n ═ 4, P ≦ 0.05, one-way ANOVA). Different letters indicate significant difference, similar letters indicate insignificant (P.ltoreq.0.05, one-way ANOVA).
FIG. 3 is a schematic representation of the structure of the cAPX1 gene, with black, thin and red boxes representing exons, introns and untranslated regions, respectively. The mutation sites of the single base mutant delt4 and the T-DNA insertion mutant apx1-2 are marked on the figure.
FIG. 4 is a quantitative analysis of cAPX1 gene expression for mutants delt4 and apx1-2 by qRT-PCR. Data are shown as mean ± sem (n ═ 4). The mean values with different letters indicate significant differences (P.ltoreq.0.05, one-way ANOVA).
FIG. 5 shows the amount of cAPX1 protein in mutants delt4 and apx1-2 analyzed by immunoblotting.
FIG. 6 shows the identification of cAPX1 replete delt4 plants. Col-0 is wild type, delt4 is point mutant of cAPX1, pcAPX1, APX1/delt4 is cAPX1 replenisher delt4 plant.
FIG. 7 shows the persistent active oxygen production induced by LPS (lipopolysaccharides) at 50. mu.g/mL, Col-0 as wild type and delt4 as a point mutant of cAPX 1.
FIG. 8 shows the total active oxygen amount 1-21 hr after LPS treatment, Col-0 is wild type, delt4 is point mutant of cAPX1, pcAPX1 shows that cAPX1/delt4 is cAPX1 replenisher delt4 plant.
FIG. 9 shows F resulting from hybridization of delt4 with apx1-21The total active oxygen amount of the generation plant is 1-21 hours after LPS treatment, Col-0 is wild type, delt4 is point mutant of cAPX1, apx1-2 is T-DNA insertion mutant of cAPX1, delt4 x apx 1-2F1F resulting from hybridization of delt4 with apx1-21And (5) plant generation. The values are mean. + -. standard error (n. about.8), different letters indicate significant difference, similar letters indicate insignificant difference(P is less than or equal to 0.05, one-way ANOVA). All experiments were repeated 3 times with similar results.
FIG. 10 shows the total amount of active oxygen in 1 to 21 hours after LPS + HRP treatment of wild type Col-0.
Detailed Description
The present invention is further illustrated by the following specific examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit or essential characteristics thereof.
The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1 obtaining of cAPX1 protein Using prokaryotic expression System
1. Reagent material and instrument device
Carrier: pMAL-c2x-GW (gateway) -for MBP tag protein expression;
coli: BL21(DE 3);
column material: amylose Resin, NEB, Cat#E8021L for MBP-tagged protein purification;
IPTG: solarbio corporation, Cat#18070 preparing 240mg/mL (1M) aqueous solution, filtering, sterilizing, and storing, wherein the concentration is generally 0.2-0.5 mM;
PBS buffer: stock solution 20 ×, pH 7.2-7.6, raw (Sangon Biotech) diluted to 1 ×, with water at the time of use;
protease inhibitors: merck, Cat#4693116001 adding protein extractive solution;
and (3) column emptying of the chromatographic column: specification 20mL, raw works (Sangon Biotech);
protein electrophoresis apparatus, protein tank type transfer apparatus, high pressure crusher, 4 ℃ centrifuge, horizontal shaker (Shanghai Zhichu apparatus Co., Ltd.).
2. Experimental procedure
2.1 construction of recombinant expression engineering bacteria
Cloning an ascorbate peroxidase gene cAPX1(AT1G07890) by taking arabidopsis thaliana leaf cDNA as a template, constructing a prokaryotic expression vector in vitro, transferring into escherichia coli BL21, and screening to obtain the recombinant expression engineering bacteria.
cAPX1F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTACATGACGAAGAACTACCCAAC-3’；
cAPX1R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCAGCATCAGCAAACCCAAGCT-3’。
2.2 Induction of expression
(1) Picking single colony, shaking bacteria, 5mL LB (Ampr), 37 ℃, 200rpm, overnight;
(2) adding 2mL of the bacterial solution into 200mL of LB (Ampr), adding 20% glucose (final concentration of 0.2%) into LB, culturing at 37 deg.C and 220rpm for about 3-4 hr until OD600＝0.6；
(3) Taking out 2mL of bacterial liquid without adding IPTG for induction as a reference, adding 1M IPTG into the remaining 198mL of bacterial liquid to enable the final concentration to be 0.2mM, inducing protein expression at 37 ℃, 220rpm for 4-6 hours;
(4) transferring 198mL of bacterial solution into two 250mL centrifuge tubes in two equal parts for 10min at 5000x g, discarding the supernatant, and adding 30-60mL ddH2O shaking the suspended bacteria (and finally transferring the bacteria liquid into a 250mL centrifuge tube), 5000x g, 10min, removing the supernatant, and storing at-20 ℃ overnight;
(5) adding 80-100mL PBS buffer solution (containing protease inhibitor dissolved) into the frozen thallus in the centrifuge tube the next day, shaking the suspended thallus, placing on ice (the subsequent steps are all carried out on the ice), and crushing for 1.0-1.5min by using a high-pressure crusher (700 plus 800Pa) until the solution becomes clear;
(6) transferring the clear liquid into three 50mL centrifuge tubes in three equal parts, centrifuging at 12000 Xg for 10min at 4 ℃, pouring the supernatant in the centrifuge tubes into 250mL centrifuge tubes, and storing at 4 ℃.
2.3 purification of proteins
(1) Cleaning a chromatographic column empty column: washing 20mL of the hollow column of the chromatographic column by PBS buffer solution for 4 times, 10mL each time, covering the plug at the top after washing, and leaving about 5mL of PBS buffer solution in the chromatographic column;
(2) washing MBP resin for binding to protein of interest: first, 500. mu.L of MBP resin (which should be gently shaken before suction) is pipetted into the PBS buffer solution in the column, and the column base is opened to allow the liquid to flow out normally. When the liquid in the chromatographic column is to flow out and the MBP resin is remained, the MBP resin is washed by PBS buffer solution for 3-4 times, 5-10mL each time, and the humidity of the MBP resin is kept in the washing process. After the washing is finished, covering a plug, adding a proper amount of PBS, transferring the MBP resin into a 250mL centrifuge tube containing the supernatant, and placing the 250mL centrifuge tube into a horizontal shaking table at 4 ℃ for incubation for at least 2 h;
(3) eluting and collecting induced protein: the liquid in the centrifugal tube is shaken gently and mixed evenly, the cover and the base of the chromatographic column are opened, and the liquid is transferred into the chromatographic column in batches; when the liquid in the chromatographic column is completely drained, passing the chromatographic column through PBS buffer solution, washing the chromatographic column containing MBP resin for 4 times, wherein 10mL of the chromatographic column is used each time, and keeping the MBP resin wet in the washing process to ensure the combination of protein; after washing, sealing the bottom, adding 1mL of solution buffer (50mM Tris-HCl pH 8.0, 10mM maltose) into a chromatographic column, incubating for 5-10min (repeatedly eluting for 3-4 times), opening the base, collecting by using a centrifuge tube, and detecting the concentration by A280;
(4) and (3) column cleaning: 5-10mL PBS buffer for 4 washes, followed by 5-10mL ddH2O cleaning for 4 times;
(5) storing the column with 20% ethanol;
(6) and (3) electrophoresis detection: 20. mu.L of the eluate was subjected to electrophoresis.
Example 2cAPX1 protein catalyzes luminol and H ex vivo2O2Reaction of (2)
The luminescence intensity after catalysis of luminol was determined using the purified cAPX1 protein of example 1.
1. Reagent material and instrument device
96-well plates (white) (horizontal macrobiology, Cat)#WHB-96)；
Purifying maltose binding protein MBP from Escherichia coli;
purifying MBP-cAPX1 recombinant protein marked by MBP from escherichia coli;
10mg/mL HRP (Peroxidase from horse radish dish, horseradish Peroxidase, Sigma-Aldrich): 20mg of HRP powder was weighed and dissolved in 2mL of ddH2In OThen subpackaging into 1.5mL light-proof centrifuge tubes (50 mu L per tube) and storing at-20 ℃ as a storage solution;
30％H2O2(Shanghai test, 30% converted to 10M): it is diluted to a final concentration of 100mM when used;
100mM luminol (luminol, Sigma-Aldrich, S-A4685-25G): 177mg of luminol powder (MW 177.16) was weighed out and dissolved in 10mL KOH, and then dispensed into 1.5mL light-shielding centrifuge tubes (100 μ L per tube) and stored at-20 ℃ as stock solution. When in use, the extract is diluted to a final concentration of 600 mu M;
single photon imaging counter HRPCS5 (available from Photok, UK);
final concentration of each component of the detection system: the concentration of the protein is unified to be 0.04 mg/mL; 10mMH2O2(ii) a 600 μ M luminol.
2. Experimental procedure
(1) Opening a single photon imaging counter, and debugging the focal length in a dark environment;
(2) different groups of reaction liquid are prepared according to the needs
Class i (50 μ L, n ═ 4): 600 mu M luminol; ② 600 mu M luminol +0.04mg/mL HRP; ③ 600. mu.M luminol +0.04mg/mL MBP; 600 mu M luminol +0.04mg/mL MBP-cAPX 1; (class I is directly added into a 96-well plate to be detected);
class II the class I system was reduced to 45. mu.L, four groups of reaction solutions (n-4) were added to 96-well plates, and 5. mu.L of 100mM H was added to each class II well reaction solution before assay2O2；
(3) Rapidly placing a 96-well plate in a photo k camera HRPCS5 to detect a chemiluminescence signal, and continuously monitoring for 10 min;
(4) the instrument was stopped and the file saved. Experimental data were derived using Image32 software analysis. Relative Light units RLU (relative Light units) according to the Photok camera display represent ROS content.
3. Results of the experiment
As shown in FIG. 2, it contained 600. mu.M luminol and 10mM H2O2The reaction solution of (2) generates an intense chemiluminescent signal in the presence of cAPX1 or HRP (0.04 mg/mL). Indicating that cAPX1 can catalyze LuAnd (4) oxidation luminescence of the minox.
Luminol and H catalyzed by concentration of hydrogen peroxide on cAPX12O2The effect of the chemiluminescence intensity of the reaction. Conditions are as follows: 0.04mg/mL MBP-cAPX 1; 600 μ M luminol.
Example 3 application of cAPX1 to luminol monitoring of the dynamic reactive oxygen species production in plant cells
To determine whether cAPX1 could catalyze luminol and H intracellularly2O2In response, we used LPS to induce intracellular H in plants2O2And (4) accumulating.
Lipopolysaccharide is the main component of gram-negative bacteria cell outer membrane, and after the plant leaves are treated, the lipopolysaccharide induces the intracellular active oxygen accumulation and is mainly positioned at the periphery of chloroplast in cytoplasm by observing under a microscope by a fluorescent staining method.
The lipopolysaccharide can cause two-phase active oxygen burst by monitoring with luminol chemiluminescence method, and the first active oxygen burst is rapidly increased within 30min and has short duration; the second burst of active oxygen (late burst of active oxygen) started 3 hours after the lipopolysaccharide treatment, reached a maximum value for about 8 hours, and then continued to decrease for about 20 hours, which is a continuous dynamic burst of active oxygen.
When we mutated or knocked out cAPX1, the second burst of active oxygen could not be detected using luminol chemiluminescence, suggesting that cAPX1 can be used to monitor the dynamic of active oxygen production in plant cells.
The specific method comprises the following steps:
1. reagent material and instrument device
96-well plates (white) (crouch macrobiosis, Cat # WHB-96);
10MG/mL LPS (L9143-10MG, Sigma, USA): to the brown reagent bottle containing 10mg LPS powder was added 1mL ddH2O, vortexed briefly to dissolve rapidly, and then dispensed into 1.5mL centrifuge tubes (50. mu.L per tube) for storage at-80 ℃ as stock solutions. When in use, the extract is diluted to a final concentration of 50 mug/mL;
100mM luminol: 177mg of luminol (MW 177.16, Sigma, S-A4685-25G) was dissolved in 10mL of KOH and then dispensed into 1.5mL light-shielded centrifuge tubes (100. mu.L per tube) for storage at-20 ℃ as stock solutions. When in use, the extract is diluted to a final concentration of 300 mu M;
10MG/mL HRP (P6782-10MG, Sigma, USA): to a reagent bottle containing 10mg HRP (horseradish peroxidase) powder was added 1mL ddH2O, vortexed briefly to dissolve rapidly, and then dispensed into 1.5mL light-shielded centrifuge tubes (50. mu.L per tube) for storage at-20 ℃ as stock solutions. When in use, the extract is diluted to a final concentration of 0.02 mg/mL;
single photon imaging counter HRPCS5 (available from Photok, UK);
2. experimental procedure
2.1 transforming the Arabidopsis cAPX1 gene by using a biological technology to obtain a mutant plant.
Arabidopsis thaliana wild type Col-0;
delt 4: point mutants of cAPX1 obtained by EMS mutagenesis screening;
apx 1-2T-DNA insertion mutant of cAPX 1;
as shown in fig. 3, which is a schematic representation of the campx 1 gene structure, black boxes, thin lines, and red boxes represent exons, introns, and untranslated regions, respectively.
The mutation sites of the cAPX1 mutant-point mutant delt4 and T-DNA insertion mutant apx1-2 are marked on the figure.
Quantitative analysis of cAPX1 gene expression of point mutant delt4 and T-DNA insertion mutant apx1-2 by qRT-PCR. The results are shown in fig. 4, where the transcription level of the campx 1 gene in the point mutant delt4 and the T-DNA insertion mutant apx1-2 was significantly reduced compared to the wild type.
The amount of cAPX1 protein in the point mutant delt4 and the T-DNA insertion mutant apx1-2 was analyzed by immunoblotting. The results are shown in fig. 5, and compared with wild type, the expression level of the cAPX1 protein in the point mutant delt4 and the T-DNA insertion mutant apx1-2 is significantly reduced.
pcAPX1, cAPX1/delt4 shows that the gene of cAPX1 driven by the self promoter is transferred into the delt4 mutant, thereby obtaining a transgenic plant complemented with delt 4.
The anaplerotic plants were identified by immunoblot analysis using ponceau S staining of the α -cAPX antibody and membrane as a loading control and the results are shown in FIG. 6, identifying that the cAPX1 protein was detected in four independent pcAPX1: cAPX1/delt4 transgenic lines # 2, #3, #4, # 11.
2.2 luminol monitoring the dynamic Generation of reactive oxygen species in plant cells
1) Selecting leaf of Arabidopsis thaliana with growth period of about 3 weeks, and obtaining leaf with size of 0.25cm by puncher2Leaf disks of (5) were placed in a container containing 50. mu.L of ddH2O in 96-well plates overnight.
2) Preparing a reaction solution: that is, 6. mu.L of luminol (100mM), and 10. mu.L of LPS (10mg/mL) were added per mL of the reaction solution, finally with ddH2And (4) metering the volume of O to 1mL, and expanding the total volume of the reaction system according to the number of samples by corresponding times.
3) And opening the single photon imaging counter, and debugging the focal length in a dark environment.
4) 50 μ L of the reaction solution was added to each well of the 96-well plate, and then the 96-well plate was quickly placed in a Photek camera HRPCS5 to detect fluorescence signals, and the monitoring was continued for about 20 hours.
5) The instrument was stopped and the file saved. Experimental data were derived using Image32 software analysis. Relative light units RLU according to the photo camera display represent ROS content.
2.3 analysis of the Effect of the absence or addition of Horseradish peroxidase on the monitoring of reactive oxygen species in plant cells
The experimental procedure described in 2.2 was repeated, but with the exception of the reaction system type H2O/LPS-luminol, and also set up experiment group H2O/LPS-luminol-HRP, i.e. 4. mu.L of 10mg/mL horseradish peroxidase HRP was additionally added to the control reaction system.
3. Results of the experiment
As shown in FIG. 7, Col-0 is wild type, persistent active oxygen production begins to occur after 3h of LPS (50. mu.g/mL) induction, delt4 is cAPX1 point mutant, and luminol luminescence signal is not monitored after LPS induction.
As shown in fig. 8, campx 1 can complement the luminol-emitting signal in delt 4. The bar graph represents the total amount of active oxygen within 1-21 hours after LPS treatment.
As shown in FIG. 9, by performing allele testing by crossing delt4 with apx1-2, F1 plants crossed with delt4 with apx1-2 exhibited significantly less total luminol luminescence than wild type within 1-21 hours after LPS treatment, indicating that point mutant delt4 and T-DNA insertion mutant apx1-2 are allelic mutations, which are allelic to each other.
As shown in fig. 10, the additional addition of HRP did not enhance the intensity of reactive oxygen species induced by LPS as monitored by luminol, indicating that HRP was unable to enter cells to exert catalytic action.
Sequence listing
<110> Zhejiang university
Application of <120> ascorbic acid peroxidase 1 in catalysis of luminol chemiluminescence reaction
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 250
<212> PRT
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 1
Met Thr Lys Asn Tyr Pro Thr Val Ser Glu Asp Tyr Lys Lys Ala Val
1 5 10 15
Glu Lys Cys Arg Arg Lys Leu Arg Gly Leu Ile Ala Glu Lys Asn Cys
20 25 30
Ala Pro Ile Met Val Arg Leu Ala Trp His Ser Ala Gly Thr Phe Asp
35 40 45
Cys Gln Ser Arg Thr Gly Gly Pro Phe Gly Thr Met Arg Phe Asp Ala
50 55 60
Glu Gln Ala His Gly Ala Asn Ser Gly Ile His Ile Ala Leu Arg Leu
65 70 75 80
Leu Asp Pro Ile Arg Glu Gln Phe Pro Thr Ile Ser Phe Ala Asp Phe
85 90 95
His Gln Leu Ala Gly Val Val Ala Val Glu Val Thr Gly Gly Pro Asp
100 105 110
Ile Pro Phe His Pro Gly Arg Glu Asp Lys Pro Gln Pro Pro Pro Glu
115 120 125
Gly Arg Leu Pro Asp Ala Thr Lys Gly Cys Asp His Leu Arg Asp Val
130 135 140
Phe Ala Lys Gln Met Gly Leu Ser Asp Lys Asp Ile Val Ala Leu Ser
145 150 155 160
Gly Ala His Thr Leu Gly Arg Cys His Lys Asp Arg Ser Gly Phe Glu
165 170 175
Gly Ala Trp Thr Ser Asn Pro Leu Ile Phe Asp Asn Ser Tyr Phe Lys
180 185 190
Glu Leu Leu Ser Gly Glu Lys Glu Gly Leu Leu Gln Leu Val Ser Asp
195 200 205
Lys Ala Leu Leu Asp Asp Pro Val Phe Arg Pro Leu Val Glu Lys Tyr
210 215 220
Ala Ala Asp Glu Asp Ala Phe Phe Ala Asp Tyr Ala Glu Ala His Met
225 230 235 240
Lys Leu Ser Glu Leu Gly Phe Ala Asp Ala
245 250
<210> 2
<211> 753
<212> DNA
<213> Arabidopsis thaliana (Arabidopsis thaliana)
<400> 2
atgacgaaga actacccaac cgtgagcgaa gattacaaga aggctgttga gaagtgcagg 60
aggaagctca gaggtttgat cgctgagaag aactgtgcac ccatcatggt ccgactcgca 120
tggcactctg ctggaacttt cgattgtcaa tcaaggactg gaggtccatt cggaacaatg 180
aggtttgacg ctgagcaagc tcatggagcc aacagtggta tccacattgc tcttaggttg 240
ttggacccca tcagggagca attccctacc atctcttttg ctgatttcca tcagcttgct 300
ggtgttgtgg ccgttgaagt tactggtggc cctgacattc ctttccaccc tggaagagag 360
gacaagcccc aaccacctcc agagggtcgt cttcctgatg ctaccaaggg ttgtgaccat 420
ttgagagatg tctttgctaa gcagatgggc ttatctgaca aagacattgt cgctttatct 480
ggtgcccaca ctctgggacg atgccacaag gataggtctg gcttcgaagg tgcatggaca 540
tcaaaccctc taatcttcga caactcttac ttcaaggaac tcttgagcgg agagaaggaa 600
ggccttcttc agcttgtctc tgacaaagca ctattggacg accctgtttt ccgtcctttg 660
gtcgagaaat acgctgctga tgaagatgcc tttttcgctg attacgctga ggcccacatg 720
aagctttctg agcttgggtt tgctgatgct taa 753
<210> 3
<211> 51
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
ggggacaagt ttgtacaaaa aagcaggcta catgacgaag aactacccaa c 51
<210> 4
<211> 50
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
ggggaccact ttgtacaaga aagctgggtc agcatcagca aacccaagct 50
Claims (9)
1. Ascorbic acid peroxidase 1 as luminol-H2O2The application of the chemiluminescent reaction catalyst.
2. The use of claim 1, wherein the amino acid sequence of ascorbate peroxidase 1 is as set forth in SEQ ID No. 1.
3. The use according to claim 2, wherein the nucleotide sequence of the gene encoding ascorbate peroxidase 1 is as shown in SEQ ID No. 2.
4. The use of claim 1, wherein luminol-H is2O2The concentration of the ascorbic acid peroxidase 1 in a chemiluminescence reaction system is 0.03-0.04 mg/mL, the concentration of luminol is 300-600 mu M, and H is2O2The concentration is 5 to 10 mM.
5. The application of claim 1, wherein the application comprises: firstly, putting a 96-well plate into a single photon imaging counter, and debugging the focal length in a dark environment; adding ascorbic acid peroxidase 1, luminol and H into the pore plate2O2And sterile water; and detecting a chemiluminescence signal by using a single photon imaging counter, continuously monitoring for 10min, and recording the total chemiluminescence intensity.
6. The application of the ascorbic acid peroxidase 1 in detecting the intracellular active oxygen content of the plant is characterized in that the ascorbic acid peroxidase 1 in the plant cell is used for catalyzing luminol to perform a chemiluminescence reaction with the intracellular active oxygen, and the intracellular active oxygen content of the plant is obtained through detecting the chemiluminescence signal intensity and analyzing.
7. The application of claim 6, wherein the application comprises: firstly, placing a plant leaf disc to be detected in sterile water for incubation for 8-12 h, then adding a luminol solution or a mixed solution of luminol and an induction factor into the plant leaf disc, placing the plant leaf disc in a single photon imaging counter to monitor a chemiluminescence signal, and representing the content of active oxygen in a plant cell by using relative light intensity.
8. The use of claim 7, wherein said luminol solution has a concentration of 600 μ M.
9. A method for screening an intracellular active oxygen burst-inducing factor of a plant, comprising the steps of:
(1) placing a plant leaf disc in a pore plate containing sterile water for incubation for 8-12 h;
(2) then adding a mixed solution of luminol and the induction factor to be detected into the plant leaf disc, and taking the luminol solution without the induction factor as a control;
(3) then placing the pore plate in a single photon imaging counter to detect a chemiluminescence signal;
(4) comparing the difference in the chemiluminescent signals with a control to determine whether the induction factor causes intracellular reactive oxygen species to burst;
the statistically significant increase in the chemiluminescence intensity measured in the leaves of the plants treated with the induction factor, as compared to the control, indicates that the induction factor is capable of causing burst of active oxygen in the plants.
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