A redox sensor for monitoring the change of h2o2 at the subcellular level of plants and its preparation method and application

Abstract

The application discloses an oxidation-reduction sensor for monitoring H2O2 change at a plant subcell level and a preparation method and application thereof. The sensor is a fusion protein roGFP2-PRXIIB formed by fusing oxidation-reduction sensitive green fluorescent protein roGFP2 and plant endogenous peroxidase PRXIIB. The application positions the fusion protein in different cell compartments of plants and finds that the fusion protein can be used for monitoring H2O2 change between different cell compartments. Specifically, the roGFP2-PRXIIB can monitor H2O2 change in cytoplasm, nucleus, mitochondria and chloroplast of plants triggered by an inducing agent and pathogenic bacteria effect protein; the roGFP2-PRXIIB can also monitor H2O2 change in cytoplasm of plants induced by abiotic stress ABA and NaCl. In addition, the roGFP2-PRXIIB can also detect H2O2 change in pollen tubes in the process of plant growth.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and particularly relates to an oxidation-reduction sensor for monitoring H2O2 changes at the subcellular level of plants and a preparation method and application thereof. BACKGROUND

[0002] As an important signal molecule, active oxygen plays an important role in the growth, development, disease resistance and stress resistance of plants, for example, regulating hypocotyl elongation, root hair development, cell death, stomatal closure, systemic acquired resistance and plant-pathogen interaction. Active oxygen is composed of a series of molecular oxygen derivatives, including H2O2, O2 - 、 1 O2, ·OH, etc., among which H2O2 is relatively stable and has a long half-life, and is often used as an intracellular and intercellular signal molecule to regulate downstream biological activities. Therefore, to study the biological function of H2O2, it is necessary to analyze the generation time, location, distribution and function of H2O2.

[0003] H2O2 is ubiquitous in plants, and the main production sites of H2O2 are apoplast, mitochondria, chloroplasts and peroxisomes. Therefore, it is very important to develop a tool to measure the dynamics of intracellular H2O2 and accurately identify H2O2 at the subcellular level. The method for studying active oxygen is usually to use some synthetic dyes such as luminol, nitro blue tetrazolium chloride (NBT), 2,7-dichlorofluorescein diacetate (DCFHDA), diaminobenzidine (DAB), etc., to qualitatively analyze active oxygen by the degree of change of the reactants or products. However, due to the unevenness of dye uptake, the inaccuracy of subcellular localization, and the non-specificity and irreversibility of the reaction with H2O2, it is difficult to accurately locate the organelles and monitor the dynamic changes of H2O2.

[0004] With the development of fluorescent protein sensors, great progress has been made in monitoring H2O2 at the subcellular level. For example, the first sensor HyPer developed in 2006 is composed of a circularly arranged yellow fluorescent protein (cpYFP) inserted into the regulatory region of prokaryotic H2O2 sensitive protein OxyR. The inter-cysteine disulfide bond formation induced by H2O2 causes the ratio change of fluorescence excitation spectrum, thereby monitoring the level of H2O2. However, cpYFP is highly sensitive to pH, and the change of pH value seriously destroys its hypersensitive response to H2O2. Currently, pH-insensitive Hyper7 has also been developed and applied to plant research. In addition, another type of fluorescent protein sensor, redox-sensitive green fluorescent protein (roGFP), also monitors H2O2 changes in a similar principle. Since the two cysteines of roGFP are in a low affinity state when reacting with H2O2, roGFP needs to be coupled with a high affinity H2O2 reaction enzyme to form an oxidation-reduction relay system, such as roGFP2-Orp1 and roGFP2-Tsa2ΔC R , roGFP2 is coupled with the peroxidase Orp1 or Tsa2ΔC R of yeast, the exchange of peroxidase and roGFP2 disulfide bond mediated by the reaction of peroxidase and H2O2, and the fluorescence ratio change of roGFP2 responds to the change of H2O2 in the cell. However, there is no plant endogenous peroxidase applied to the sensor at present, and the sensitivity of the sensor is improved, and efforts are made to create a better sensor to monitor the dynamic level of H2O2 in plant cells. SUMMARY

[0005] The purpose of the present application is to apply the plant endogenous peroxidase PRXIIB to the fluorescent protein sensor to achieve more sensitive monitoring of the dynamic change level of H2O2 in plant cells.

[0006] In order to achieve the above purpose, the present application first provides a fusion protein.

[0007] The fusion protein provided by the present application comprises peroxidase PRXIIB and fluorescent protein roGFP2;

[0008] The amino acid sequence of the peroxidase PRXIIB is any one of the following A1)-A3):

[0009] A1) the amino acid sequence is a protein represented by SEQ ID NO: 2 274-434;

[0010] A2) a protein with the same function obtained by substitution and / or deletion and / or addition of one or more amino acid residues of the amino acid sequence represented by SEQ ID NO: 2 274-434;

[0011] A3) a protein having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, positions 274-434, and having the same function.

[0012] In the fusion protein, the amino acid sequence of the fluorescent protein roGFP2 is shown in SEQ ID NO: 2, positions 1-239.

[0013] The fusion protein further comprises a linker connecting the peroxidase PRXIIB and the fluorescent protein roGFP2. In some embodiments, the amino acid sequence of the linker is shown in SEQ ID NO: 2, positions 240-273.

[0014] The fusion protein can be any one of the following B1) to B5):

[0015] B1) a protein having the amino acid sequence shown in SEQ ID NO: 2;

[0016] B2) a fusion protein having the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2;

[0017] B3) a fusion protein having the same function obtained by connecting a signal peptide or a transit peptide or a leader peptide to the N-terminus and / or C-terminus of the amino acid sequence shown in SEQ ID NO: 2;

[0018] B4) a protein having the same function obtained by substitution and / or deletion and / or addition of one or several amino acid residues to the amino acid sequence shown in SEQ ID NO: 2;

[0019] B5) a protein having 80% or more identity to the amino acid sequence shown in SEQ ID NO: 2, and having the same function.

[0020] In the protein of B2), the tag refers to a polypeptide or a protein fused and expressed with the fusion protein of interest by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the fusion protein of interest. The tag includes but is not limited to: a GST (glutathione S-transferase) tag protein, a His6 tag protein (His-tag), an MBP (maltose binding protein) tag protein, a Flag tag protein, a SUMO tag protein, an HA tag protein, a Myc tag protein, a GFP (green fluorescent protein), a CFP (cyan fluorescent protein), a YFP (yellow-green fluorescent protein), a mCherry (monomeric red fluorescent protein) or an AviTag tag protein.

[0021] In the protein of A2) or B4), the substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 or 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1 amino acid residues.

[0022] In the protein described in A3) or B5) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page of the NCBI home page website. For example, the identity (%) of a pair of amino acid sequences can be calculated by searching using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, in Advanced BLAST 2.1, and then the value of the identity (%) is obtained. The identity includes an amino acid sequence having 80% or more, or having 85% or more, or having 90% or more, or 91% or more, or 92% or more, or 93% or more, or 94% or more, or 95% or more, or 96% or more, or 97% or more, or 98% or more, or 99% or more identity to the amino acid sequence shown in SEQ ID NO: 2 or SEQ ID NO: 2.

[0023] In the protein described in B3) above, the signal peptide or the transit peptide or the leader peptide can be any one of the following polypeptides: SV40 signal peptide, β-ATPase signal peptide, RBCS1A transit peptide.

[0024] The amino acid sequence of the SV40 signal peptide is shown in SEQ ID NO: 9.

[0025] The amino acid sequence of the β-ATPase signal peptide is shown in SEQ ID NO: 10.

[0026] The amino acid sequence of the RBCS1A transit peptide is shown in SEQ ID NO: 11.

[0027] In some embodiments, the fusion protein is fusion protein roGFP2-PRXIIB, and the amino acid sequence of the fusion protein roGFP2-PRXIIB is shown in SEQ ID NO: 2.

[0028] In some embodiments, the fusion protein is fusion protein Nuc-roGFP2-PRXIIB, and the fusion protein Nuc-roGFP2-PRXIIB is fusion protein roGFP2-PRXIIB with an SV40 signal peptide at the N terminus.

[0029] In some embodiments, the fusion protein is fusion protein Mit-roGFP2-PRXIIB, which is a fusion protein roGFP2-PRXIIB with a β-ATPase signal peptide at the N-terminus.

[0030] In some embodiments, the fusion protein is fusion protein Chloro-roGFP2-PRXIIB, which is a fusion protein roGFP2-PRXIIB with an RBCS1A transit peptide at the N-terminus.

[0031] To achieve the above object, the present application further provides a nucleic acid molecule encoding the above fusion protein.

[0032] The nucleic acid molecule encoding the above fusion protein provided by the present application is any one of the following 1) or 2):

[0033] 1) a DNA molecule as shown in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8;

[0034] 2) a DNA molecule having 75% or more identity to the DNA molecule defined in 1) and encoding the above fusion protein.

[0035] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; or the nucleic acid molecule can be RNA, such as mRNA or hnRNA, etc.

[0036] Those skilled in the art can easily mutate the nucleotide sequence encoding the above fusion protein of the present application by using known methods, such as methods of directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence encoding the above fusion protein, as long as they encode the above fusion protein and have the same function, are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application.

[0037] The term "identity" used herein refers to sequence similarity to the natural nucleic acid sequence. The 75% or more identity can be 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or more.

[0038] To achieve the above object, the present application further provides a recombinant expression vector.

[0039] The recombinant expression vector provided by the present application can express the above fusion protein.

[0040] Further, the recombinant expression vector is a vector obtained by ligating the above nucleic acid molecule into a plant expression vector. The recombinant vector containing the expression cassette of the nucleic acid molecule can be constructed using an existing plant expression vector. The plant expression vector includes Agrobacterium binary vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector can also contain the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase gene Nos), the 3' end of the plant gene (such as the soybean storage protein gene). Transcription of the untranslated region has similar functions. When using the gene of the present application to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent regions of start codons, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence.

[0041] Further, the recombinant expression vector is a vector obtained by ligating the above nucleic acid molecule into a plant expression vector. The recombinant vector containing the expression cassette of the nucleic acid molecule can be constructed using an existing plant expression vector. The plant expression vector includes Agrobacterium binary vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector can also contain the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium crown gall tumor-inducing (Ti) plasmid gene (such as the nopaline synthase gene Nos), the 3' end of the plant gene (such as the soybean storage protein gene). Transcription of the untranslated region has similar functions. When using the gene of the present application to construct a plant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent regions of start codons, but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence.

[0042] The recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB is a vector obtained by replacing the DNA fragment between the KpnI and Bstb1 enzyme cutting sites of the pCAMBIA1300-35S vector with the DNA molecule shown in SEQ ID NO: 1, and keeping other sequences of the pCAMBIA1300-35S vector unchanged.

[0043] The recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB is a vector obtained by replacing the DNA fragment between the Pst1 and Kpn1 enzyme cutting sites of the pCAMBIA1300-Lat52 vector with the DNA molecule shown in SEQ ID NO: 1, and keeping other sequences of the pCAMBIA1300-Lat52 vector unchanged.

[0044] The recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB is a vector obtained by replacing the DNA fragment between the Kpnl and Bstbl restriction sites of the pCAMBIA1300-35S vector with the DNA molecule shown in SEQ ID NO: 6, and keeping other sequences of the pCAMBIA1300-35S vector unchanged.

[0045] The recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB is a vector obtained by replacing the DNA fragment between the Kpnl and Bstbl restriction sites of the pCAMBIA1300-35S vector with the DNA molecule shown in SEQ ID NO: 7, and keeping other sequences of the pCAMBIA1300-35S vector unchanged.

[0046] The recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB is a vector obtained by replacing the DNA fragment between the Kpnl and Bstbl restriction sites of the pCAMBIA1300-35S vector with the DNA molecule shown in SEQ ID NO: 8, and keeping other sequences of the pCAMBIA1300-35S vector unchanged.

[0047] To achieve the above-mentioned purpose, the present application further provides a recombinant bacterium.

[0048] The recombinant bacterium provided by the present application contains the recombinant expression vector mentioned above.

[0049] Further, the recombinant bacterium is a bacterium obtained by introducing the recombinant expression vector mentioned above into a recipient bacterium. The recipient bacterium can be a yeast, a bacterium, an alga or a fungus. The bacterium can be specifically Agrobacterium, such as Agrobacterium GV3101.

[0050] To achieve the above-mentioned purpose, the present application further provides a new use of the fusion protein mentioned above, the nucleic acid molecule mentioned above, the recombinant expression vector mentioned above or the recombinant bacterium mentioned above.

[0051] The present application provides the use of the fusion protein mentioned above, the nucleic acid molecule mentioned above, the recombinant expression vector mentioned above or the recombinant bacterium mentioned above in any one of K1) to K4) below:

[0052] K1) monitoring the change of H2O2 at the subcellular level of plants;

[0053] K2) preparing a product for monitoring the change of H2O2 at the subcellular level of plants;

[0054] K3) monitoring the change of H2O2 at the organ or tissue or cell level of plants;

[0055] K4) a product for monitoring H2O2 change in plant organ or tissue or cell level.

[0056] To achieve the above object, the present application finally provides a method for monitoring H2O2 change in plant subcellular level or monitoring H2O2 change in plant organ or tissue or cell level.

[0057] The method for monitoring H2O2 change in plant subcellular level or monitoring H2O2 change in plant organ or tissue or cell level provided by the present application comprises the step of introducing the above-mentioned recombinant expression vector into a recipient plant as a redox sensor.

[0058] In any of the above-mentioned applications or methods, the monitoring of H2O2 change in plant subcellular level is embodied as any one of M1) to M3) below:

[0059] M1) monitoring H2O2 change in plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplast triggered by an inducer (elicitor);

[0060] M2) monitoring H2O2 change in plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplast triggered by an effector protein of pathogenic bacteria;

[0061] M3) monitoring H2O2 change in plant cytoplasm induced by abiotic stress;

[0062] Further, in the M1), the inducer (elicitor) can be at least one of the following polypeptides: flg22 polypeptide, chitin, nlp20 polypeptide, Pep2 polypeptide.

[0063] In the M2), the effector protein can be at least one of the following proteins: AvrRpt2 protein, HopZ1a protein, and AvrB protein.

[0064] In the M3), the abiotic stress can be at least one of the following stresses: NaCl stress, ABA stress.

[0065] In any of the above-mentioned applications or methods, the monitoring of H2O2 change in plant organ or tissue or cell level is embodied as monitoring H2O2 change in plant pollen tube induced by RALF4 polypeptide.

[0066] In any of the above-mentioned applications or methods, the H2O2 change includes whether H2O2 is produced and the dynamic change level of H2O2.

[0067] In any of the above-mentioned applications or methods, the plant is a dicotyledonous plant or a monocotyledonous plant.

[0068] In some embodiments, the dicotyledonous plant is Arabidopsis thaliana.

[0069] In some embodiments, the Arabidopsis is wild-type Arabidopsis Col-0.

[0070] The present application provides a redox sensor for monitoring the change of H2O2 at subcellular level of plants, and its preparation method and application. The sensor is a fusion protein roGFP2-PRXIIB formed by fusing the redox-sensitive green fluorescent protein roGFP2 and the plant endogenous peroxidase PRXIIB. Experiments prove that roGFP2-PRXIIB is more sensitive and more suitable for application in plants than the reported H2O2 detection fluorescent probes roGFP2-Orp1 and roGFP2-Tsa2ΔC R and the probe roGFP2-GPX2 composed of the plant glutathione peroxidase GPX2. The present application further discovers that roGFP2-PRXIIB can be used for monitoring the change of H2O2 between different cell compartments by locating it in different cell compartments of plants, which specifically embodies that roGFP2-PRXIIB can monitor the change of H2O2 in cytoplasm, nucleus, mitochondria and chloroplast of plants triggered by inducers and pathogenic protein effectors; roGFP2-PRXIIB can also monitor the change of H2O2 in cytoplasm of plants induced by abiotic stress ABA and NaCl. In addition, roGFP2-PRXIIB can also detect the change of H2O2 in pollen tube during the growth of plants. BRIEF DESCRIPTION OF DRAWINGS

[0071] Figure 1 For screening of transgenic plants and detection of GFP expression. a is for screening of transgenic plants with relatively high fluorescence value (roGFP2-PRXIIB, roGFP2-Orp1, roGFP2-GPX2 and roGFP2-Tsa2ΔC R ) by using a microplate reader. b is for detection of GFP expression of transgenic plants in a by using Western blotting. c is for screening of transgenic plants with relatively high fluorescence value (Nuc-roGFP2-PRXIIB, Mit-roGFP2-PRXIIB and Chloro-roGFP2-PRXIIB) by using a microplate reader. d is for detection of GFP expression of transgenic plants in c by using Western blotting.

[0072] Figure 2 For comparison of sensitivity of different H2O2 detection fluorescent probes in detecting H2O2. a is that roGFP2-PRXIIB responds to the change of oxidation level induced by elicitors more greatly than roGFP2-Orp1 and roGFP2-GPX2. b is that roGFP2-PRXIIB responds to the change of oxidation level induced by elicitors more greatly than roGFP2-Tsa2ΔC RThe changes in oxidation levels induced by elicitors were more dramatic. Leaves from 4-week-old transgenic plants expressing roGFP2-PRXIIB, roGFP2-Orpl, roGFP2-GPX2 and roGFP2-Tsa2AC, respectively, were treated with 2 μΜ flg22, 200 μg / mL chitin, 2 μΜ Pep2 and 2 μΜ nlp20, respectively, and the changes in cytosolic roGFP2 oxidation levels were monitored. R The changes in oxidation levels induced by elicitors were more dramatic. Leaves from 4-week-old transgenic plants expressing roGFP2-PRXIIB, roGFP2-Orpl, roGFP2-GPX2 and roGFP2-Tsa2AC, respectively, were treated with 2 μΜ flg22, 200 μg / mL chitin, 2 μΜ Pep2 and 2 μΜ nlp20, respectively, and the changes in cytosolic roGFP2 oxidation levels were monitored.

[0073] Figure 3 Confocal microscopy of roGFP2-PRXIIB subcellular localization. Representative confocal images of roGFP2-PRXIIB localization in cytosol, nucleus, mitochondria and chloroplast, and corresponding subcellular markers. The images of cytosol, mitochondria and chloroplast were obtained from leaf epidermal cells of 7-day-old seedlings, and the image of nucleus was taken from protoplasts of 4-week-old seedlings. The fluorescence intensity along the yellow line in the merged images was plotted on the right side, respectively. The white line is a scale bar with a size of 20 μΜ.

[0074] Figure 4 Dynamic changes of H2O2 in different subcellular compartments induced by elicitors detected by roGFP2-PRXIIB. Leaves from 4-week-old transgenic plants expressing roGFP2-PRXIIB in different subcellular compartments were treated with 2 μΜ flg22, 200 μg / mL chitin, 2 μΜ Pep2 and 2 μΜ nlp20, respectively, and the changes in roGFP2-PRXIIB oxidation levels in cytosol (a), chloroplast (b), nucleus (c) and mitochondria (d) were monitored.

[0075] Figure 5 Dynamic changes of H2O2 in different subcellular compartments induced by effectors detected by roGFP2-PRXIIB. Leaves from 4-week-old transgenic plants expressing roGFP2-PRXIIB in different subcellular compartments were injected with ddH2O, D36E, D36E avrRpt2, D36E hopZla and D36E avrB bacterial solutions at a concentration of 2.5 x 10 8 CFU / mL, respectively. The changes in roGFP2-PRXIIB oxidation levels in cytosol (a), chloroplast (b), nucleus (c) and mitochondria (d) were monitored every hour after inoculation.

[0076] Figure 6Dynamic changes of cytosolic H2O2 induced by NaCl and ABA were monitored by roGFP2-PRXIIB. a Dynamic changes of cytosolic H2O2 induced by NaCl were monitored by roGFP2-PRXIIB and roGFP2-Orp1. The leaves of roGFP2-PRXIIB and roGFP2-Orp1 transgenic plants grown for four weeks were treated with 200 mM NaCl, and the changes of oxidation level of roGFP2-PRXIIB were monitored. b Dynamic changes of cytosolic H2O2 induced by ABA were monitored by roGFP2-PRXIIB and roGFP2-Orp1. The leaves of roGFP2-PRXIIB and roGFP2-Orp1 transgenic plants grown for four weeks were treated with water or 100 μM ABA, respectively, and the pictures were taken by microscope after 90 min. The pictures were representative results. c The significant analysis of changes of oxidation level of roGFP2-PRXIIB and roGFP2-Orp1 in guard cells.

[0077] Figure 7 Dynamic changes of cytosolic H2O2 in pollen tubes were monitored by roGFP2-PRXIIB. a The pictures were taken by high-resolution spinning-disk confocal microscope after ddH2O or 2 μM RALF4 was applied to the pollen tubes of roGFP2-PRXIIB transgenic plants. The images were representative of the oxidation of pollen tubes, and the scale bar was 5 μM. b The significant analysis of oxidation level of roGFP2-PRXIIB in pollen tubes under different treatment conditions. DETAILED DESCRIPTION

[0078] The application will be further described in conjunction with the preferred embodiments thereof, and the examples given are only intended to illustrate the application, but not to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0079] In the following examples, the experimental methods are conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents, etc. used in the following examples can be obtained commercially, unless otherwise specified.

[0080] In the following examples, the experiments were set up in triplicate, unless otherwise specified.

[0081] In the following examples, the data were processed using GraphPad Prism statistical software, and the experimental results were expressed as mean ± standard deviation, t-test was used, and P<0.05 indicated a significant difference.

[0082] The vector pCAMBIA1300-35S and Agrobacterium GV3101 in the following examples are described in the literature “Bi, G., Hu, M., Fu, L., Zhang, X., Zuo, J., Li, J., Yang, J.*, and Zhou, J. M.*. The cytosolic thiol peroxidase PRXIIB is an intracellular sensor for H2O2 that regulates plant immunity through a redox relay. Nat. Plants 8, 1160-1175. (2022)”, which is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the biological material is only used for repeating the experiments related to the present application and cannot be used for other purposes.

[0083] The vector pCAMBIA1300-Lat52 in the following examples is described in the literature “Meng, J., Liang, L., Jia, P., Wang, Y., Li, H.*, and Yang, W. C.*. Integration of ovular signals and exocytosis of a Ca 2+ channel by MLOs in pollen tube guidance. Nat. Plants 6, 143-153. (2020)”, which is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the biological material is only used for repeating the experiments related to the present application and cannot be used for other purposes. The strain D36E in the following examples is described in the literature “Wei, H. L., Chakravarthy, S., Mathieu, J., Helmann, T. C., Stodghill, P., Swingle, B., Martin, G. B.*, and Collmer, A.*. Pseudomonas syringae pv. tomato DC3000 Type III secretion effector polymutants reveal an interplay between HopAD1 and AvrPtoB. Cell Host Microbe 17, 752-762. (2015)”, which is publicly available from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and the biological material is only used for repeating the experiments related to the present application and cannot be used for other purposes.

[0084] The strains D36E hopZ1a, D36E avrRpt2 and D36E avrB in the following examples are obtained by introducing the recombinant plasmids pUCP20tk-HopZ1a, pUCP20tk-AvrRpt2 and pUCP20tk-AvrB into the strain D36E by electroporation, respectively. The recombinant plasmids pUCP20tk-HopZ1a, pUCP20tk-AvrRpt2 and pUCP20tk-AvrB are obtained by ligating the DNA molecules shown in SEQ ID NO: 12 (the first to 500th nucleotides are the HopZ1a self promoter, and the 501st to 1604th nucleotides are the HopZ1a gene sequence), SEQ ID NO: 13 (the first to 171st nucleotides are the AvrRpt2 self promoter, and the 172nd to 939th nucleotides are the AvrRpt2 nucleic acid sequence) or SEQ ID NO: 14 (the first to 249th nucleotides are the AvrB self promoter, and the 250th to 1215th nucleotides are the AvrB gene sequence) into the vector pUCP20tk, and expressing the effector proteins HopZ1a, AvrRpt2 and AvrB, respectively. The vector pUCP20tk is described in the document “West, S. E.*, Schweizer, H. P., Dall, C., Sample, A. K., and Runyen-Janecky, L. J. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18 / 19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148, 81-86. (1994)”, and the public can obtain the material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The obtained biological material is only used for repeating the experiments of the present application, and cannot be used for other purposes.

[0085] The flg22 polypeptide (the amino acid sequence of the flg22 polypeptide is TRLSSGLKINSAKDDAAGLQIA), the nlp20 polypeptide (the amino acid sequence of the nlp20 polypeptide is AIMYSWYFPKDSPVTGLGHR), the Pep2 polypeptide (the amino acid sequence of the Pep2 polypeptide is DNKAKSKKRDKEKPSSGRPGQTNSVPNAAIQVYKED), and the RALF4 polypeptide (the amino acid sequence of the RALF4 polypeptide is RRYIGYDALKKNNVPCSRRGRSYYDCKKRRRNNPYRRGCSAITHCYRYAR) in the following examples are synthesized by Shenguo Bioengineering (Shanghai) Co., Ltd.

[0086] The chitin in the following examples is a product of Sigma, with the item number C9752-5G.

[0087] Example 1, construction of a recombinant vector

[0088] I. Construction of recombinant vectors pCAMBIA1300-35S-roGFP2-PRXIIB and pCAMBIA1300-Lat52-roGFP2-PRXIIB

[0089] 1. Construction of recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB

[0090] The DNA fragment between the Kpnl and Bstbl enzyme cutting sites of the pCAMBIA1300-35S vector is replaced with the DNA molecule (roGFP2-PRXIIB) shown in SEQ ID NO: 1, and the other sequences of the pCAMBIA1300-35S vector remain unchanged, to obtain the recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB. The recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB expresses the fusion protein roGFP2-PRXIIB, and the amino acid sequence of the fusion protein roGFP2-PRXIIB is shown in SEQ ID NO: 2, wherein the 1st-239th amino acid sequences are the amino acid sequence of the roGFP2 protein, the 240th-273rd amino acid sequences are the amino acid sequence of a linker, and the 274th-434th amino acid sequences are the amino acid sequence of the PRXIIB protein.

[0091] 2. Construction of recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB

[0092] The DNA fragment between the Pstl and Kpnl enzyme cutting sites of the pCAMBIA1300-Lat52 vector was replaced with the DNA molecule shown in sequence 1 (roGFP2-PRXIIB), and other sequences of the pCAMBIA1300-Lat52 vector were kept unchanged, to obtain the recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB. The recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB expresses the fusion protein roGFP2-PRXIIB, and the amino acid sequence of the fusion protein roGFP2-PRXIIB is shown in sequence 2.

[0093] 3. Construction of the recombinant vector pCAMBIA1300-35S-roGFP2-Orpl

[0094] The DNA fragment between the Kpnl and Bstbl enzyme cutting sites of the pCAMBIA1300-35S vector was replaced with the DNA molecule shown in sequence 3 (roGFP2-Orpl), and other sequences of the pCAMBIA1300-35S vector were kept unchanged, to obtain the recombinant vector pCAMBIA1300-35S-roGFP2-Orpl. The recombinant vector pCAMBIA1300-35S-roGFP2-Orpl expresses the fusion protein roGFP2-Orpl.

[0095] 4. Construction of the recombinant vector pCAMBIA1300-35S-roGFP2-GPX2

[0096] The DNA fragment between the Kpnl and Bstbl enzyme cutting sites of the pCAMBIA1300-35S vector was replaced with the DNA molecule shown in sequence 4 (roGFP2-GPX2), and other sequences of the pCAMBIA1300-35S vector were kept unchanged, to obtain the recombinant vector pCAMBIA1300-35S-roGFP2-GPX2. The recombinant vector pCAMBIA1300-35S-roGFP2-GPX2 expresses the fusion protein roGFP2-GPX2.

[0097] 5. Construction of the recombinant vector pCAMBIA1300-35S-roGFP2-Tsa2AC R

[0098] The DNA fragment between the Kpnl and Bstbl enzyme cutting sites of the pCAMBIA1300-35S vector was replaced with the DNA molecule shown in sequence 5 (roGFP2-Tsa2AC R ​The DNA fragment between the Kpnl and Bstbl sites of the pCAMBIA1300-35S vector is replaced by the DNA molecule shown in SEQ ID NO. 6 (Nuc-roGFP2-PRXIIB), and other sequences of the pCAMBIA1300-35S vector remain unchanged, to obtain the recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB. The recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB expresses the fusion protein Nuc-roGFP2-PRXIIB, which is the fusion protein roGFP2-PRXIIB with an SV40 signal peptide (the amino acid sequence of the SV40 signal peptide is shown in SEQ ID NO. 9) at the N-terminus. R The recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB R Expression of the fusion protein Nuc-roGFP2-PRXIIB R .

[0099] II. Construction of the recombinant vectors pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB, pCAMBIA1300-35S-Mit-roGFP2-PRXIIB and pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB

[0100] 1. Construction of the recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB

[0101] The DNA fragment between the Kpnl and Bstbl sites of the pCAMBIA1300-35S vector is replaced by the DNA molecule shown in SEQ ID NO. 6 (Nuc-roGFP2-PRXIIB), and other sequences of the pCAMBIA1300-35S vector remain unchanged, to obtain the recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB. The recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB expresses the fusion protein Nuc-roGFP2-PRXIIB, which is the fusion protein roGFP2-PRXIIB with an SV40 signal peptide (the amino acid sequence of the SV40 signal peptide is shown in SEQ ID NO. 9) at the N-terminus.

[0102] 2. Construction of the recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB

[0103] The DNA fragment between the Kpnl and Bstbl sites of the pCAMBIA1300-35S vector was replaced with the DNA molecule shown in SEQ ID NO: 7 (Mit-roGFP2-PRXIIB), while keeping other sequences of the pCAMBIA1300-35S vector unchanged, to obtain the recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB. The recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB expresses the fusion protein Mit-roGFP2-PRXIIB, which is the fusion protein roGFP2-PRXIIB with the mitochondrial β-ATPase signal peptide (the amino acid sequence of the mitochondrial β-ATPase signal peptide is shown in SEQ ID NO: 10) at the N terminus.

[0104] 3. Construction of the recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB

[0105] The DNA fragment between the Kpnl and Bstbl sites of the pCAMBIA1300-35S vector was replaced with the DNA molecule shown in SEQ ID NO: 8 (Chloro-roGFP2-PRXIIB), while keeping other sequences of the pCAMBIA1300-35S vector unchanged, to obtain the recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB. The recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB expresses the fusion protein Chloro-roGFP2-PRXIIB, which is the fusion protein roGFP2-PRXIIB with the chloroplast RBCS1A transit peptide (the amino acid sequence of the chloroplast RBCS1A transit peptide is shown in SEQ ID NO: 11) at the N terminus.

[0106] Example 2. Obtaining of Arabidopsis thaliana transgenic materials with different subcellular localization of roGFP2-PRXIIB

[0107] I. Construction of the recombinant Agrobacterium

[0108] The recombinant vectors pCAMBIA1300-35S-roGFP2-PRXIIB, pCAMBIA1300-Lat52-roGFP2-PRXIIB, pCAMBIA1300-35S-roGFP2-Orp1, pCAMBIA1300-35S-roGFP2-GPX2, pCAMBIA1300-35S-roGFP2-Tsa2ΔC RpCAMBIA1300-35S-Nuc-roGFP2-PRXIIB, pCAMBIA1300-35S-Mit-roGFP2-PRXIIB, and pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB were introduced into Agrobacterium GV3101. The transformed strains were then spread onto LB solid culture dishes containing kanamycin (50 mg / L) and gentamicin (50 mg / L) and cultured at 28°C for 36 hours. Positive monoclonal strains were screened to obtain Agrobacterium carrying the corresponding recombinant vectors, and then stored at -80°C.

[0109] II. Culture and Transformation of Recombinant Agrobacterium

[0110] 1. Culture of recombinant Agrobacterium

[0111] The recombinant Agrobacterium carrying the recombinant vector was removed from the -80℃ freezer and streaked onto LB solid medium containing kanamycin and gentamicin, and incubated at 28℃ for 12 hours. A small amount of activated cells was inoculated into 2 mL of LB liquid medium containing the corresponding antibiotic and incubated on a shaker at 28℃ for 12 hours. Then, all 2 mL of LB liquid medium was transferred to 300 mL of LB liquid medium containing the corresponding antibiotic and incubated on a shaker at 28℃ for about 10 hours. After centrifugation at 4000 rpm for 10 min, the bacterial suspension was collected, resuspended in 5% sucrose solution, and diluted to OD. 600 Approximately equal to 1, and add surfactant Silwet L-77 at a ratio of 0.00017:1, shake well for conversion.

[0112] 2. Transformation

[0113] Select wild-type Arabidopsis thaliana Col-0 at the appropriate flowering period, remove the open flowers, and immerse the Arabidopsis thaliana with only flower buds upside down in the bacterial solution for 5 minutes. Place the infected plants horizontally in a tray and keep them in the dark for 24 hours before they grow normally upright.

[0114] III. Screening of resistant plants

[0115] First, the harvested transgenic plant seeds were disinfected with 70% alcohol, and then evenly spread on 1 / 2 MS medium containing 25 mg / L hygromycin and 50 mg / L carbenicillin for positive seedling screening. The seedlings were then cultured in an incubator at 23°C for about 10 days. The seedlings with normal growth were then selected and transferred to a greenhouse for further cultivation.

[0116] IV. Obtaining Transgenic Plants

[0117] The four-week-old transgenic Arabidopsis leaves were punched with a puncher with a diameter of 4.5 mm, and the small discs were loaded into a 96-well black plate. The fluorescence value of roGFP2 was detected by a Perkin Elmer microplate reader (excitation wavelength: 488 nm; emission wavelength: 510 nm). The plants with relatively high fluorescence values were selected. Then the selected plants were punched with a puncher with a diameter of 7 mm, and the small discs were loaded into a centrifuge tube containing 80 μL cell lysate. The small discs were ground thoroughly with a sample grinder to extract the proteins of the small discs. 20 μL 4x Protein Loading Buffer was added to each tube. After mixing, the sample was boiled at 100 ℃ for 5 min. Then the expression amount of GFP in the sample was detected by Western blotting (GFP antibody is a product of Abmart Company, product number P30010M). The results of the screening of transgenic plants and the detection of GFP expression amount are shown in Figure 1 .

[0118] The GFP-expressing Arabidopsis plants were individually collected seeds. The T2 generation seeds were placed on 1 / 2MS medium containing antibiotics, and the separation ratio was detected to be close to 3:1. The positive seedlings were transplanted and individually collected seeds (single copy insertion of the target gene), and the expression separation ratio of the next generation was recorded. The homozygous plants with stable expression (stable homozygous line T3) were selected.

[0119] V. roGFP2-PRXIIB is more sensitive to H2O2

[0120] Transgenic plant lines stably expressing roGFP2-PRXIIB, roGFP2-Orp1, roGFP2-GPX2 and roGFP2-Tsa2ΔC R , respectively, were selected, and were planted in a short-day plant room. The four-week-old leaves were punched with a puncher with a diameter of 4.5 mm, and were placed on a 96-well black plate pre-added with 100 μL ddH2O with the front side up for overnight incubation in weak light. The next day, different excitors (2 μM flg22 polypeptide, 200 μg / mL chitin, 2 μM Pep2 polypeptide or 2 μM nlp20 polypeptide) were used for treatment, and the specific treatment method included the following steps: the plate was first scanned in a Perkin Elmer microplate reader for 15 minutes on the next day, and then the polypeptide solution was added. The leaves floated on the polypeptide solution in each well of the plate, and then the plate was immediately placed in the microplate reader for continuous scanning for 6 hours. The fluorescence value change of roGFP2 was detected by the microplate reader (excitation wavelength: 405 nm and 488 nm; emission wavelength: 510 nm). The fluorescence ratio I 405 / I 488 , which can reflect the relative amount change of H2O2 in the cytoplasm.

[0121] The results are shown inFigure 2 As shown in Figure 6, the results showed that roGFP2-PRXIIB responded to the changes in the level of oxidation induced by various elicitors more greatly than roGFP2-Orpl and roGFP2-GPX2 Figure 2 a). While roGFP2-Tsa2AC R had too high background oxidation level and too much noise (b). In summary, roGFP2-PRXIIB was more suitable for monitoring the dynamic changes in intracellular H202 induced by elicitors. Figure 2 b). In summary, roGFP2-PRXIIB was more suitable for monitoring the dynamic changes in intracellular H202 induced by elicitors.

[0122] VI. Analysis of different subcellular localization of roGFP2-PRXIIB

[0123] Transgenic plant lines stably expressing fusion proteins roGFP2-PRXIIB, Nuc-roGFP2-PRXIIB, Mit-roGFP2-PRXIIB and Chloro-roGFP2-PRXIIB were selected respectively, and confocal microscopy was used to observe the different subcellular localization of fusion proteins roGFP2-PRXIIB, Nuc-roGFP2-PRXIIB, Mit-roGFP2-PRXIIB and Chloro-roGFP2-PRXIIB.

[0124] The results of confocal microscopy observation are shown in Figure 7. Figure 3 As shown in Figure 7, the results showed that fusion protein roGFP2-PRXIIB was localized in cytoplasm and nucleus, and did not co-localize with chloroplast; fusion protein Nuc-roGFP2-PRXIIB was localized in nucleus, and co-localized with nuclear protein AHL22-mcherry; fusion protein Mit-roGFP2-PRXIIB was localized in mitochondria, and co-localized with mitochondrial dye MitoTracker; fusion protein Chloro-roGFP2-PRXIIB was localized in chloroplast, and co-localized with chloroplast autofluorescence. The above results demonstrated the correctness of the expression of roGFP2-PRXIIB with different subcellular localization.

[0125] The transgenic homozygous line stably expressing obtained by introducing recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB was a roGFP2-PRXIIB transgenic Arabidopsis thaliana line targeting cytoplasm.

[0126] The transgenic homozygous line stably expressing obtained by introducing recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB was a roGFP2-PRXIIB transgenic Arabidopsis thaliana line targeting pollen tube.

[0127] The stable transgenic homozygous line obtained by introducing the recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB is a Nuc-roGFP2-PRXIIB transgenic Arabidopsis thaliana line targeting nucleus.

[0128] The stable transgenic homozygous line obtained by introducing the recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB is a Mit-roGFP2-PRXIIB transgenic Arabidopsis thaliana line targeting mitochondria.

[0129] The stable transgenic homozygous line obtained by introducing the recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB is a Chloro-roGFP2-PRXIIB transgenic Arabidopsis thaliana line targeting chloroplast.

[0130] Example 3, roGFP2-PRXIIB monitors the change of H2O2 at the subcellular level of plants

[0131] I. roGFP2-PRXIIB monitors the production of H2O2 induced by elicitors

[0132] The stable transgenic plant lines targeting cytoplasm, chloroplast, nucleus and mitochondria respectively are selected, planted in a short-day plant house, and the leaves grown for four weeks are punched with a puncher with a diameter of 4.5 mm, placed in a 96-well black plate with 100 μL ddH2O added in advance, and incubated in weak light overnight. The next day, different elicitors (flg22 polypeptide, chitin, nlp20 polypeptide, Pep2 polypeptide) are used for treatment, and the treatment method is the same as step five of Example 2.

[0133] The results are shown in Figure 4 The results show that in cytoplasm, chloroplast and nucleus, the production of H2O2 induced by different elicitors can be detected, and the time and regularity of the production of H2O2 induced by different compartments and different elicitors are different; and the curve change of mitochondria is not obvious due to its high degree of oxidation, and no obvious production of H2O2 is detected.

[0134] II. roGFP2-PRXIIB monitors the production of H2O2 induced by effector proteins

[0135] The transgenic Arabidopsis thaliana lines targeting cytoplasm, chloroplast, nucleus and mitochondria are selected, planted in a short-day plant house, and the leaves grown for four weeks are injected with D36E, D36E hopZ1a, D36E avrRpt2 and D36E avrB bacterial solution respectively (the concentration of the bacterial solution is 2.5×10 8The concentration of roGFP2 was measured (CFU / mL), with ddH2O injection serving as a control. One hour after injection, wells were punched using a 0.45 cm punch and placed face up in a 96-well black plate containing 200 μL of ddH2O. Two hours after injection, the fluorescence change of roGFP2 was detected using a microplate reader for 10 minutes each time, and once every hour.

[0136] The results are as follows Figure 5 As shown, the results indicated that the effector proteins AvrRpt2, HopZ1a, and AvrB all induced H2O2 accumulation in the cytoplasm, chloroplasts, nucleus, and mitochondria. Notably, the oxidative changes induced by effector proteins were significantly higher than those induced by elicitors. Among these organelles, effector proteins induced rapid H2O2 accumulation in chloroplasts. For example, AvrB induced H2O2 accumulation in chloroplasts approximately 2–3 hours post-transfection, while AvrRpt2 and HopZ1a induced it approximately 5–6 hours post-transfection. Furthermore, H2O2 induced by AvrB also accumulated in the nucleus approximately 4–5 hours post-transfection, while AvrRpt2 and HopZ1a induced it approximately 6–7 hours post-transfection. Additionally, all effectors induced H2O2 accumulation in mitochondria approximately 7–8 hours post-transfection. In contrast, the degree of roGFP2-PRXIIB oxidation induced by D36E was similar to that ddH2O treatment. These results indicate that large amounts of H2O2 accumulated in the cytoplasm, chloroplasts, nucleus, and mitochondria during ETI.

[0137] III. Monitoring H2O2 Production Induced by Abiotic Stress ABA and NaCl Using roGFP2-PRXIIB

[0138] 1. roGFP2-PRXIIB monitoring of H2O2 production induced by abiotic stress NaCl

[0139] Transgenic Arabidopsis lines stably expressing roGFP2-PRXIIB and roGFP2-Orp1 targeting the cytoplasm were selected and planted in a short-day plant room. Leaves from the fourth week of growth were taken, and holes were punched using a 0.45 cm diameter punch. The leaves were placed face up in 96-well black plates pre-filled with 100 μL ddH2O and incubated overnight under low light. The next day, the plates were treated with 100 mM NaCl. The specific treatment method included the following steps: On the second day, the plates were first scanned in a microplate reader for 15 minutes, then removed and NaCl solution was added. The leaves in each well floated on the NaCl solution. The plates were then immediately placed back into the microplate reader for further scanning to detect changes in roGFP2 fluorescence for 6 hours.

[0140] The results are as follows Figure 6As shown in Figure a, the results indicate that within one hour of NaCl treatment, the oxidation levels of both roGFP2-PRXIIB and roGFP2-Orp1 increased rapidly and remained at a high level for several hours. Furthermore, the oxidation level change of roGFP2-PRXIIB was significantly higher than that of roGFP2-Orp1. This suggests that roGFP2-PRXIIB can effectively monitor the dynamic changes of H2O2 induced by NaCl and is more sensitive than roGFP2-Orp1.

[0141] 2. roGFP2-PRXIIB monitoring of ABA-induced H2O2 production under abiotic stress

[0142] Abscisic acid (ABA) is a plant hormone crucial for plant adaptation to various abiotic stresses. Within minutes, ABA induces guard cells to produce large amounts of reactive oxygen species (ROS) to promote stomatal closure, thereby reducing water loss due to transpiration and conserving water. To detect ABA-induced H2O2 production in guard cells, transgenic Arabidopsis lines stably expressing roGFP2-PRXIIB and roGFP2-Orp1 targeting the cytoplasm were selected and planted in a short-day plant house. Leaves from four weeks of growth were harvested, cut from the petiole, and immersed in a solution containing 100 μM ABA. After 90 minutes, the fluorescence intensity changes of roGFP2-PRXIIB in guard cells were observed using a confocal microscope (excitation wavelengths: 405 nm and 488 nm; emission wavelengths: 493-533 nm).

[0143] The results are as follows Figure 6 As shown in b, the results indicate that after ABA treatment, roGFP2-PRXIIB in guard cells underwent strong oxidation, significantly higher than roGFP2-Orp1. This suggests that roGFP2-PRXIIB can effectively monitor ABA-induced stomatal H2O2 production and is more sensitive than roGFP2-Orp1.

[0144] IV. Monitoring H2O2 production in RALF4-induced pollen tubes using roGFP2-PRXIIB

[0145] ROS in pollen tube has been studied in depth, which plays a key role in the process of pollen water absorption, germination and tube growth. In order to observe the production of H2O2 in growing pollen tube, the roGFP2-PRXIIB transgenic Arabidopsis line targeting pollen tube was selected, and the pollen was cultured on the germination substrate (solvent is water, solute and its concentration are 1 mM CaCl2, 1 mM Ca(NO3)2, 1 mM MgSO4, 0.01% H3BO4, 18% sucrose and 1% agarose) at 28°C in the dark for 4 hours, and then treated with 2 μM RALF4 polypeptide. The specific treatment method includes the following steps: drop 2 μM RALF4 polypeptide solution on the germination substrate, and observe the fluorescence intensity change of roGFP2-PRXIIB in pollen tube under microscope after 2 min (excitation wavelength: 405 nm and 488 nm; emission wavelength: 493-533 nm).

[0146] The results are shown in Figure 7 The results show that RALF4 polypeptide treatment significantly improves the oxidation level of roGFP2-PRXIIB. It is proved that roGFP2-PRXIIB is suitable for observing the dynamic change of H2O2 in pollen tube.

[0147] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wider range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that the present application can be further improved. In general, according to the principle of the present application, the present application intends to include any change, use or improvement of the present application, including the change made by the conventional technology known in the art, which is out of the range disclosed in the present application. Some basic features can be applied according to the scope of the following attached claims.

Claims

1. A fusion protein consisting of a peroxidase PRXIIB and a fluorescent protein roGFP2; the amino acid sequence of the peroxidase PRXIIB is a protein shown in SEQ ID NO: 2 from 274th to 434th; the fluorescent protein roGFP2 is located at the N-terminal; and the peroxidase PRXIIB is located at the C-terminal.

2. A fusion protein being any one of the following B1) - B3): B1) a protein with an amino acid sequence shown in SEQ ID NO: 2; B2) a fusion protein with the same function obtained by connecting a tag at the N-terminal and / or C-terminal of the amino acid sequence shown in SEQ ID NO: 2; B3) a fusion protein with the same function obtained by connecting a signal peptide or a transit peptide or a leader peptide at the N-terminal of the amino acid sequence shown in SEQ ID NO:

2.

3. A nucleic acid molecule encoding the fusion protein of claim 1 or 2.

4. The nucleic acid molecule of claim 3, wherein: the nucleic acid molecule being any one of the following 1) or 2): 1) a DNA molecule shown in SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 7 or SEQ ID NO: 8; 2) a DNA molecule having more than 75% identity with the DNA molecule defined in 1) and encoding the fusion protein of claim 1 or 2.

5. A recombinant expression vector capable of expressing the fusion protein of claim 1 or 2.

6. The recombinant expression vector of claim 5, wherein: the recombinant expression vector being a vector obtained by linking the nucleic acid molecule of claim 3 or 4 into a plant expression vector.

7. A recombinant bacterium containing the recombinant expression vector of claim 5 or 6.

8. Use of the fusion protein of claim 1 or 2 or the nucleic acid molecule of claim 3 or 4 or the recombinant expression vector of claim 5 or 6 or the recombinant bacterium of claim 7 in any one of the following K1) - K4): K1) monitoring H2O2 change in plant subcellular level; K2) preparing a product for monitoring H2O2 change in plant subcellular level; K3) monitoring H2O2 change in plant organ or tissue or cell level; K4) preparing a product for monitoring H2O2 change in plant organ or tissue or cell level.

9. Use according to claim 8, characterized in that: the monitoring H2O2 change in plant subcellular level being any one of the following M1) - M3): M1) monitoring H2O2 change in plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplast triggered by an inducer; M2) monitoring H2O2 change in plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplast triggered by an effector protein of a pathogenic bacterium; M3) monitoring H2O2 change in plant cytoplasm induced by abiotic stress.

10. Use according to claim 8, characterized in that: the monitoring H2O2 change in plant organ or tissue or cell level being monitoring H2O2 change in plant pollen tube induced by RALF4 polypeptide.

11. A method for monitoring H2O2 change in plant subcellular level or monitoring H2O2 change in plant organ or tissue or cell level, the method comprising the step of introducing the recombinant expression vector of claim 5 or 6 into a recipient plant.

12. The method of claim 11, wherein: the monitoring H2O2 change in plant subcellular level being any one of the following M1) - M3): M1 ) monitoring H2O2 changes in the plant cytosol and / or nucleus and / or mitochondria and / or chloroplast triggered by an elicitor; M2) monitoring H2O2 changes in the plant cytosol and / or nucleus and / or mitochondria and / or chloroplast triggered by an effector protein of a pathogenic bacterium; M3) monitoring H2O2 changes in the plant cytosol induced by an abiotic stress.

13. The method of claim 11, wherein: The monitoring of H2O2 changes in the plant organ or tissue or cell level is embodied in the monitoring of H2O2 changes in the plant pollen tube induced by a RALF4 polypeptide.

Images