Redox sensor for monitoring change of plant subcellular level H2O2 as well as preparation method and application of redox sensor

Abstract

The invention discloses a redox sensor for monitoring the change of plant subcellular level H2O2 as well as a preparation method and application of the redox sensor. The sensor is a fusion protein roGFP2-PRXIIB formed by fusing a redox sensitive green fluorescent protein roGFP2 and a plant endogenous peroxidase PRXIIB, the sensor is positioned in different cell compartments of a plant, and it is found that the sensor can be used for monitoring H2O2 changes among the different cell compartments; specifically, the roGFP2-PRXIIB can monitor H2O2 change in plant cytoplasm, cell nucleus, mitochondria and chloroplast triggered by the inducer and pathogenic bacteria effect protein; the roGFP2-PRXIIB can also be used for monitoring the change of H2O2 in plant cytoplasm induced by the ABA and the NaCl under abiotic stress. In addition, the roGFP2-PRXIIB can also be used for detecting the change of H2O2 in a pollen tube in the plant growth process.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to a redox sensor for monitoring H 2 O 2 changes at the subcellular level of plants, and a preparation method and application thereof. Background Art

[0002] As an important signaling molecule, reactive oxygen species play an important role in the processes of plant growth, development, disease resistance and stress resistance. For example, they regulate hypocotyl elongation, root hair development, cell death, stomatal closure, systemic acquired resistance, and plant-pathogen interaction. Reactive oxygen species are composed of a series of derivatives of molecular oxygen, including H 2 O 2 、O 2 - 、 1 O 2 、·OH, etc. Among them, H 2 O 2 is relatively stable and has a longer half-life, and is often used as a signaling molecule intracellularly and intercellularly to regulate downstream biological activities. Therefore, to study the biological function of H 2 O 2 , it is first necessary to analyze its production time, production location, distribution, and where it functions.

[0003] H 2 O 2 is ubiquitous in plants, and the main production sites of H 2 O 2 are apoplast, mitochondria, chloroplasts and peroxisomes. Therefore, it is very important to develop tools for measuring the dynamics of intracellular H 2 O 2 and accurately identify H 2 O 2 at the subcellular level. Usually, the methods for studying reactive oxygen species are to use some synthetic dyes, such as luminol, nitroblue tetrazolium chloride (NBT), 2,7-dichlorofluorescein diacetate (DCFHDA), diaminobenzidine (DAB), etc., to indirectly qualitatively analyze reactive oxygen species through the degree of change of reactants or products. However, due to the uneven uptake of dyes, inaccurate subcellular localization, and non-specific and irreversible reactions with H 2 O 2 , it is difficult to accurately localize to organelles and monitor the dynamic changes of H 2 O 2 .

[0004] With the development of fluorescent protein sensors, monitoring H 2 O 2Great progress has been made. For example, the first sensor, HyPer, developed in 2006, consists of a circularly arranged yellow fluorescent protein (cpYFP) inserted into the regulatory region of the prokaryotic H 2 O 2 sensitive protein OxyR. It monitors H 2 O 2 levels by forming disulfide bonds between cysteines in the H 2 O 2 -induced domain and causing a ratio change in the fluorescence excitation spectrum. However, cpYFP is highly sensitive to pH, and pH changes severely disrupt its hypersensitive response to H 2 O 2 . Currently, the pH-insensitive Hyper7 has also been developed and applied to plant research. In addition, another type of fluorescent protein sensor, the redox-sensitive green fluorescent protein (roGFP), also monitors H 2 O 2 changes based on a similar principle. Since the two cysteines of roGFP are in a low-affinity state when reacting with H 2 O 2 , roGFP needs to be coupled with a high-affinity reaction enzyme for H 2 O 2 to form a redox relay system, such as roGFP2-Orp1 and roGFP2-Tsa2ΔC R . roGFP2 is coupled with the yeast peroxidase Orp1 or Tsa2ΔC R . The reaction between the peroxidase and H 2 O 2 mediates the exchange of disulfide bonds between the peroxidase and roGFP2, and the fluorescence ratio change of roGFP2 reflects the H 2 O 2 changes in the cell. However, currently, there is no plant endogenous peroxidase applied to the sensor to improve its sensitivity. Efforts are being made to create better sensors to monitor the H 2 O 2 dynamic levels in plant cells. Summary of the Invention

[0005] The object of the present invention is to apply the plant endogenous peroxidase PRXIIB to a fluorescent protein sensor to achieve more sensitive monitoring of the H 2 O 2 dynamic change levels in plant cells.

[0006] To achieve the above object, the present invention first provides a fusion protein.

[0007] The fusion protein provided by the present invention 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) A protein whose amino acid sequence is the one shown at positions 274-434 of Sequence 2;

[0010] A2) A protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown at positions 274-434 of Sequence 2;

[0011] A3) A protein that has 80% or more identity with the amino acid sequence shown at positions 274-434 of Sequence 2 and has the same function.

[0012] In the above-mentioned fusion protein, the amino acid sequence of the fluorescent protein roGFP2 is as shown at positions 1-239 of Sequence 2.

[0013] The above-mentioned fusion protein further includes a linker that connects the peroxidase PRXIIB and the fluorescent protein roGFP2. In some embodiments, the amino acid sequence of the linker is as shown at positions 240-273 of Sequence 2.

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

[0015] B1) A protein whose amino acid sequence is the one shown in Sequence 2;

[0016] B2) A fusion protein with the same function obtained by connecting a tag to the N-terminus and / or C-terminus of the amino acid sequence shown in Sequence 2;

[0017] B3) A fusion protein with 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 Sequence 2;

[0018] B4) A protein with the same function obtained by substituting and / or deleting and / or adding one or several amino acid residues to the amino acid sequence shown in Sequence 2;

[0019] B5) A protein that has 80% or more identity with the amino acid sequence shown in Sequence 2 and has the same function.

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

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

[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 homology search sites on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained. The identity includes an amino acid sequence having 80% or higher, or 85% or higher, or 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher identity with amino acid positions 274-434 of Sequence 2 of the present invention or the amino acid sequence shown in Sequence 2.

[0023] In the protein described in B3) above, the signal peptide or transit peptide or 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 as shown in Sequence 9.

[0025] The amino acid sequence of the β-ATPase signal peptide is as shown in Sequence 10.

[0026] The amino acid sequence of the RBCS1A transit peptide is as shown in Sequence 11.

[0027] In some embodiments, the fusion protein is the fusion protein roGFP2-PRXIIB, and the amino acid sequence of the fusion protein roGFP2-PRXIIB is as shown in Sequence 2.

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

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

[0030] In some embodiments, the fusion protein is the fusion protein Chloro-roGFP2-PRXIIB, and the fusion protein Chloro-roGFP2-PRXIIB is the fusion protein roGFP2-PRXIIB with an RBCS1A transit peptide linked to its N-terminus.

[0031] To achieve the above object, the present invention 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 invention is any one of the following 1) or 2):

[0033] 1) The DNA molecule shown in Sequence 1, Sequence 6, Sequence 7 or Sequence 8;

[0034] 2) A DNA molecule having more than 75% identity with the DNA molecule defined in 1) and encoding the above fusion protein.

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

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

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

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

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

[0040] Furthermore, the recombinant expression vector is a vector obtained by ligating the above nucleic acid molecule into a plant expression vector. Existing plant expression vectors can be used to construct recombinant vectors containing the nucleic acid molecule expression cassette. The plant expression vectors include binary Agrobacterium vectors and vectors suitable for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb, etc. The plant expression vector may further comprise a 3'-untranslated region of a foreign gene, that is, it contains a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylate to the 3'-end of the mRNA precursor. For example, the non-translated regions transcribed at the 3'-ends of Agrobacterium tumefaciens Ti plasmid genes (such as the nopaline synthase gene Nos) and plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translational enhancers or transcriptional enhancers. These enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence.

[0041] Furthermore, the recombinant expression vector is recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB or recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB or recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB or recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB or recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB.

[0042] The recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB is a vector obtained by replacing the DNA fragment between the KpnI and Bstb1 restriction enzyme sites of the pCAMBIA1300-35S vector with the DNA molecule shown in Sequence 1, while keeping the 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 restriction enzyme sites of the pCAMBIA1300-Lat52 vector with the DNA molecule shown in Sequence 1, while keeping the 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 KpnI and Bstb1 restriction enzyme sites of the pCAMBIA1300-35S vector with the DNA molecule shown in Sequence 6, while keeping the 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 KpnI and Bstb1 restriction enzyme sites of the pCAMBIA1300-35S vector with the DNA molecule shown in Sequence 7, while keeping the 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 KpnI and Bstb1 restriction enzyme sites of the pCAMBIA1300-35S vector with the DNA molecule shown in Sequence 8, while keeping the other sequences of the pCAMBIA1300-35S vector unchanged.

[0047] To achieve the above object, the present invention also provides a recombinant bacterium.

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

[0049] Furthermore, the recombinant bacterium is a bacterium obtained by introducing the above recombinant expression vector into a recipient bacterium. The recipient bacterium can be yeast, bacteria, algae or fungi. The bacteria can specifically be Agrobacterium, such as Agrobacterium tumefaciens GV3101.

[0050] To achieve the above object, the present invention also provides a new use of the above fusion protein or the above nucleic acid molecule or the above recombinant expression vector or the above recombinant bacterium.

[0051] The present invention provides an application of the above fusion protein or the above nucleic acid molecule or the above recombinant expression vector or the above recombinant bacterium in any one of the following K1)-K4):

[0052] K1) Monitoring the change of H 2 O 2 in plant subcellular level;

[0053] K2) Preparing a product for monitoring the change of H 2 O 2 in plant subcellular level;

[0054] K3) Monitoring the change of H 2 O 2 in plant organ or tissue or cell level;

[0055] K4) Preparing a product for monitoring the change of H 2 O 2 in plant organ or tissue or cell level.

[0056] To achieve the above object, the present invention finally provides a method for monitoring the change of H 2 O 2 in plant subcellular level or monitoring the change of H 2 O 2 in plant organ or tissue or cell level.

[0057] The method for monitoring the change of H 2 O 2 in plant subcellular level or monitoring the change of H 2 O 2 in plant organ or tissue or cell level provided by the present invention includes the step of introducing the above recombinant expression vector as a redox sensor into a recipient plant.

[0058] In any of the above applications or methods, the monitoring of the change of H 2 O 2 in plant subcellular level is embodied as any one of the following M1)-M3):

[0059] M1) Monitoring the change of H 2 O 2 in plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplasts triggered by an inducer (elicitor);

[0060] M2) Monitoring the change of H 2O 2 Change;

[0061] M3) Monitoring of H in the cytoplasm of plants induced by abiotic stress 2 O 2 Change;

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

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

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

[0065] In any of the above applications or methods, the monitoring of H at the level of plant organs or tissues or cells 2 O 2 The change is reflected in the monitoring of H in the pollen tubes of plants induced by the RALF4 polypeptide 2 O 2 Change.

[0066] In any of the above applications or methods, the H 2 O 2 The change includes whether H 2 O 2 is generated and the dynamic change level of H 2 O 2 Dynamic change level.

[0067] In any of the above 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 specific embodiments, the Arabidopsis thaliana is wild-type Arabidopsis thaliana Col-0.

[0070] The present invention provides a redox sensor for monitoring the change of H at the subcellular level of plants 2 O 2 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. It is proved by experiments that roGFP2-PRXIIB is more sensitive to H than the reported ones 2 O 2Detection of fluorescent probes roGFP2-Orp1 and roGFP2-Tsa2ΔC R and the probe roGFP2-GPX2 composed of glutathione peroxidase GPX2 in plants, which is more sensitive and more suitable for application in plants. The present invention further locates it in different plant cell compartments and finds that it can be used to monitor the H 2 O 2 changes between different cell compartments, specifically manifested as: roGFP2-PRXIIB can monitor the H 2 O 2 changes in the cytoplasm, nucleus, mitochondria and chloroplasts of plants triggered by inducers and pathogen effector proteins; roGFP2-PRXIIB can also monitor the H 2 O 2 changes in the cytoplasm of plants induced by abiotic stresses ABA and NaCl. In addition, roGFP2-PRXIIB can also detect the H 2 O 2 changes in pollen tubes during plant growth. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 For the screening of transgenic plants and the detection of GFP expression levels. a shows the screening of transgenic plants (roGFP2-PRXIIB, roGFP2-Orp1, roGFP2-GPX2 and roGFP2-Tsa2ΔC R ) with relatively high fluorescence values using a microplate reader. b shows the detection of the GFP expression levels of the transgenic plants in Figure a using immunoblotting. c shows the screening of transgenic plants (Nuc-roGFP2-PRXIIB, Mit-roGFP2-PRXIIB and Chloro-roGFP2-PRXIIB) with relatively high fluorescence values using a microplate reader. d shows the detection of the GFP expression levels of the transgenic plants in Figure c using immunoblotting.

[0072] Figure 2 For the sensitivity comparison of different H 2 O 2 detection fluorescent probes for detecting H 2 O 2 . a shows that roGFP2-PRXIIB shows a greater response to the change in the oxidation level induced by elicitors than roGFP2-Orp1 and roGFP2-GPX2. b shows that roGFP2-PRXIIB shows a greater response to the change in the oxidation level induced by elicitors than roGFP2-Tsa2ΔC R . Four-week-old plants expressing roGFP2-PRXIIB, roGFP2-Orp1, roGFP2-GPX2 and roGFP2-Tsa2ΔC RLeaves of transgenic plants were treated with 2 μM flg22, 200 μg / mL chitin, 2 μM Pep2, and 2 μM nlp20 respectively, and the changes in the oxidized level of cytosolic roGFP2 were monitored.

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

[0074] Figure 4 For roGFP2-PRXIIB to detect the changes in H 2 O 2 dynamics in different subcellular compartments induced by elicitors. Leaves of transgenic plants expressing roGFP2-PRXIIB in different subcellular organelles at 4 weeks of growth were treated with 2 μM flg22, 200 μg / mL chitin, 2 μM Pep2, and 2 μM nlp20 respectively, and the changes in the oxidized level of roGFP2-PRXIIB in the cytoplasm (a), chloroplast (b), nucleus (c), and mitochondria (d) were monitored.

[0075] Figure 5 For roGFP2-PRXIIB to detect the changes in H 2 O 2 dynamics in different subcellular compartments induced by effector proteins. Leaves of transgenic plants expressing roGFP2-PRXIIB in different subcellular organelles at 4 weeks of growth were inoculated by injection with ddH 2 O, bacterial suspensions of D36E, D36E avrRpt2, D36E hopZ1a, and D36E avrB at a concentration of 2.5×10 8 CFU / mL. The changes in the oxidized level of roGFP2-PRXIIB in the cytoplasm (a), chloroplast (b), nucleus (c), and mitochondria (d) were detected every hour after inoculation.

[0076] Figure 6 For roGFP2-PRXIIB to detect the changes in H 2 O 2 dynamics in the cytoplasm induced by NaCl and ABA. a shows the real-time monitoring of H 2 O 2Dynamic changes. Leaves of transgenic plants expressing roGFP2-PRXIIB and roGFP2-Orp1 that had grown for four weeks were selected and treated with 200 mM NaCl to monitor changes in the oxidation level of roGFP2-PRXIIB. b shows the monitoring of H in the cytoplasm of guard cells induced by ABA in roGFP2-PRXIIB and roGFP2-Orp1 2 O 2 Dynamic changes. Leaves of transgenic plants expressing roGFP2-PRXIIB and roGFP2-Orp1 that had grown for four weeks were treated with water or 100 μM ABA respectively, and photographed under a microscope after 90 minutes. The pictures are representative results. c is a significant analysis chart of the changes in the oxidation levels of roGFP2-PRXIIB and roGFP2-Orp1 in guard cells.

[0077] Figure 7 For the monitoring of H in the cytoplasm of pollen tubes by roGFP2-PRXIIB 2 O 2 Dynamic changes. a shows the recording by high-resolution spinning disk confocal microscopy after ddH 2 O or 2 μM RALF4 was applied to the pollen tubes of transgenic plants expressing roGFP2-PRXIIB. The images represent the oxidation of pollen tubes, and the scale bar is 5 μM. b is a significant analysis chart of the oxidation levels of roGFP2-PRXIIB in pollen tubes under different treatment conditions. Specific embodiments

[0078] The present invention will be further described in detail below in combination with specific embodiments. The given embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0079] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial sources unless otherwise specified.

[0080] The tests in the following embodiments are all set with three replicates unless otherwise specified.

[0081] The following embodiments use GraphPad Prism statistical software to process the data. The experimental results are expressed as mean ± standard deviation, and the t-test is used. P < 0.05 indicates significant differences.

[0082] The vector pCAMBIA1300-35S and Agrobacterium tumefaciens GV3101 in the following examples are both described in the literature "Bi, G., Hu, M., Fu, L., Zhang, X., Zuo, J., Li, J., Yang, J.*, and Zhou, J.M.*. The cytosolic thiolperoxidase PRXIIB is an intracellular sensor for H 2 O 2 that regulates plant immunity through a redox relay. Nat. Plants 8, 1160-1175. (2022)". The public can obtain them from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used for repeating the relevant experiments of the present invention 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)". The public can obtain it from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. This biological material is only used for repeating the relevant experiments of the present invention 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)". The public can obtain this material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above-mentioned biological materials are only used for repeating the experiments of the present invention and cannot be used for other purposes.

[0084] The strains D36E hopZ1a, D36E avrRpt2, and D36E avrB in the following examples are bacteria obtained by respectively introducing the recombinant plasmids pUCP20tk-HopZ1a, pUCP20tk-AvrRpt2, and pUCP20tk-AvrB into the strain D36E by electroporation. The recombinant plasmids pUCP20tk-HopZ1a, pUCP20tk-AvrRpt2, and pUCP20tk-AvrB are plasmids obtained by ligating the DNA molecule shown in Sequence 12 (the 1st to 500th positions of Sequence 12 are the self-promoter of HopZ1a, and the 501st to 1604th positions are the gene sequence of HopZ1a), the DNA molecule shown in Sequence 13 (the 1st to 171st positions of Sequence 13 are the self-promoter of AvrRpt2, and the 172nd to 939th positions are the nuclear gene sequence of AvrRpt2), or the DNA molecule shown in Sequence 14 (the 1st to 249th positions of Sequence 14 are the self-promoter of AvrB, and the 250th to 1215th positions are the gene sequence of AvrB) into the vector pUCP20tk, and respectively express the effector proteins HopZ1a, AvrRpt2, and AvrB. Among them, the vector pUCP20tk is described in the literature "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)". The public can obtain this material from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. The above-mentioned biological materials obtained are only used for repeating the experiments of the present invention and cannot be used for other purposes.

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

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

[0087] Example 1. Construction of Recombinant Vectors

[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] Replace the DNA fragment between the KpnI and Bstb1 restriction enzyme sites of the pCAMBIA1300-35S vector with the DNA molecule (roGFP2-PRXIIB) shown in Sequence 1, and keep the other sequences of the pCAMBIA1300-35S vector 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 as shown in Sequence 2. Among them, positions 1-239 of Sequence 2 are the amino acid sequence of the roGFP2 protein, positions 240-273 are the amino acid sequence of the linker, and positions 274-434 are the amino acid sequence of the PRXIIB protein.

[0091] 2. Construction of Recombinant Vector pCAMBIA1300-Lat52-roGFP2-PRXIIB

[0092] Replace the DNA fragment between the PstI and KpnI restriction sites of the pCAMBIA1300-Lat52 vector with the DNA molecule (roGFP2-PRXIIB) shown in Sequence 1, and keep the other sequences of the pCAMBIA1300-Lat52 vector 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-Orp1

[0094] Replace the DNA fragment between the KpnI and BstbI restriction sites of the pCAMBIA1300-35S vector with the DNA molecule (roGFP2-Orp1) shown in Sequence 3, and keep the other sequences of the pCAMBIA1300-35S vector unchanged to obtain the recombinant vector pCAMBIA1300-35S-roGFP2-Orp1. The recombinant vector pCAMBIA1300-35S-roGFP2-Orp1 expresses the fusion protein roGFP2-Orp1.

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

[0096] Replace the DNA fragment between the KpnI and BstbI restriction sites of the pCAMBIA1300-35S vector with the DNA molecule (roGFP2-GPX2) shown in Sequence 4, and keep the other sequences of the pCAMBIA1300-35S vector 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-Tsa2ΔC R

[0098] Replace the DNA fragment between the KpnI and BstbI restriction sites of the pCAMBIA1300-35S vector with the DNA molecule (roGFP2-Tsa2ΔC R), and keeping other sequences of the pCAMBIA1300-35S vector unchanged, the recombinant vector pCAMBIA1300-35S-roGFP2-Tsa2ΔC was obtained R . The recombinant vector pCAMBIA1300-35S-roGFP2-Tsa2ΔC R expresses the fusion protein roGFP2-Tsa2ΔC R .

[0099] II. Construction of Recombinant Vectors pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB, pCAMBIA1300-35S-Mit-roGFP2-PRXIIB and pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB

[0100] 1. Construction of Recombinant Vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB

[0101] The DNA fragment between the KpnI and Bstb1 restriction enzyme sites of the pCAMBIA1300-35S vector was replaced with the DNA molecule (Nuc-roGFP2-PRXIIB) shown in Sequence 6, and keeping other sequences of the pCAMBIA1300-35S vector unchanged, the recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB was obtained. The recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB expresses the fusion protein Nuc-roGFP2-PRXIIB, and the fusion protein Nuc-roGFP2-PRXIIB is a fusion protein roGFP2-PRXIIB with an SV40 signal peptide (the amino acid sequence of the SV40 signal peptide is shown in Sequence 9) linked to the N-terminus.

[0102] 2. Construction of Recombinant Vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB

[0103] Replace the DNA fragment between the KpnI and BstbI restriction sites of the pCAMBIA1300-35S vector with the DNA molecule (Mit-roGFP2-PRXIIB) shown in Sequence 7, and keep the 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, and the fusion protein Mit-roGFP2-PRXIIB is a fusion protein roGFP2-PRXIIB with a mitochondrial β-ATPase signal peptide (the amino acid sequence of the mitochondrial β-ATPase signal peptide is as shown in Sequence 10) linked to the N-terminus.

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

[0105] Replace the DNA fragment between the KpnI and BstbI restriction sites of the pCAMBIA1300-35S vector with the DNA molecule (Chloro-roGFP2-PRXIIB) shown in Sequence 8, and keep the 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, and the fusion protein Chloro-roGFP2-PRXIIB is a fusion protein roGFP2-PRXIIB with a chloroplast RBCS1A transit peptide (the amino acid sequence of the chloroplast RBCS1A transit peptide is as shown in Sequence 11) linked to the N-terminus.

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

[0107] I. Construction of recombinant Agrobacterium

[0108] Using the method of electroporation, respectively, the recombinant vectors pCAMBIA1300-35S-roGFP2-PRXIIB, pCAMBIA1300-Lat52-roGFP2-PRXIIB, pCAMBIA1300-35S-roGFP2-Orp1, pCAMBIA1300-35S-roGFP2-GPX2, pCAMBIA1300-35S-roGFP2-Tsa2ΔC constructed in Example 1 R, pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB, pCAMBIA1300-35S-Mit-roGFP2-PRXIIB, and pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB were introduced into Agrobacterium tumefaciens GV3101. The transformed strains were spread on LB solid plates 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, which were then stored in an -80 °C refrigerator.

[0109] II. Cultivation and Transformation of Recombinant Agrobacterium

[0110] 1. Cultivation of Recombinant Agrobacterium

[0111] The recombinant Agrobacterium carrying the recombinant vector was taken out from the -80 °C refrigerator, streaked on LB solid medium containing kanamycin and gentamicin, and cultured in an incubator at 28 °C for 12 hours. A small amount of activated bacteria was picked and inoculated into 2 mL of LB liquid medium containing the corresponding antibiotics, and cultured on a shaker at 28 °C for 12 hours. Then, the entire 2 mL of LB liquid medium was transferred into 300 mL of LB liquid medium containing the corresponding antibiotics, and cultured on a shaker at 28 °C for about 10 hours. After that, it was centrifuged at 4000 rpm for 10 min to collect the bacterial liquid, and the bacterial liquid was resuspended with 5% sucrose solution and diluted to an OD 600 approximately equal to 1, and surfactant Silwet L-77 was added at a ratio of 0.00017:1, and shaken well for use in transformation.

[0112] 2. Transformation

[0113] Wild-type Arabidopsis thaliana Col-0 at an appropriate flowering stage was selected, and the already opened flowers were removed. The Arabidopsis thaliana with only flower buds was inverted in the bacterial liquid for 5 minutes, and the infected plants were placed horizontally in a tray. After 24 hours of dark treatment, they were allowed to grow vertically normally.

[0114] III. Screening of Resistant Plants

[0115] First, the seeds of the transgenic plants harvested were disinfected with 70% ethanol, and then evenly spread on 1 / 2 MS medium containing 25 mg / L hygromycin and 50 mg / L carbenicillin for screening of positive seedlings. They were cultured in an incubator at 23 °C for about 10 days, and the normally growing seedlings were picked and transferred to the greenhouse for continued cultivation.

[0116] IV. Obtaining of Transgenic Plants

[0117] Select the leaves of transgenic Arabidopsis thaliana that have grown for about four weeks, punch holes with a puncher with a diameter of 4.5 mm, put the small discs into a 96-well black plate, and detect the fluorescence value of roGFP2 with a Perkin Elmer microplate reader (excitation wavelength: 488 nm; emission wavelength: 510 nm), and select the plants with relatively high fluorescence values. Then punch holes in the selected plants with a puncher with a diameter of 7 mm, put the small discs into a centrifuge tube containing 80 μL of cell lysate, and grind the small discs thoroughly with a grinder to extract the protein of the small discs. Add 20 μL of 4×Protein Loading Buffer to each tube. After mixing, boil the samples at 100 °C for 5 min. Then use immunoblotting experiments (the GFP antibody is a product of Abmart, catalog number P30010M) to detect the expression level of GFP in the samples. The screening of transgenic plants and the detection results of GFP expression level are as Figure 1 shown.

[0118] Harvest the seeds of Arabidopsis thaliana plants expressing GFP individually. Spread the T 2 -generation seeds on 1 / 2 MS medium containing antibiotics, detect that the segregation ratio is close to 3:1, transplant the positive seedlings and harvest the seeds individually (the target gene is inserted in a single copy), and record the expression segregation ratio of the next generation, and select the plants with homozygous expression (the homozygous line T 3 ) that stably expresses.

[0119] V. roGFP2-PRXIIB is more sensitive to H 2 O 2 Select transgenic plant lines stably expressing roGFP2-PRXIIB, roGFP2-Orp1, roGFP2-GPX2, and roGFP2-Tsa2ΔC

[0120] respectively, plant them in a short-day plant room, take the leaves that have grown for four weeks, punch holes with a puncher with a diameter of 4.5 mm, and place them face up in a 96-well black plate pre-added with 100 μL of ddH R O, and incubate overnight under weak light. The next day, treat with different elicitors (2 μM flg22 polypeptide, 200 μg / mL chitin, 2 μM Pep2 polypeptide, or 2 μM nlp20 polypeptide). The specific treatment method includes the following steps: Place the plate in a Perkin Elmer microplate reader and scan for 15 minutes the next day, then take it out and add the polypeptide solution. The leaves in each small hole of the plate float on the polypeptide solution, and then immediately put it into the microplate reader and continue to scan for 6 hours. The microplate reader detects the change in the fluorescence value of roGFP2 (excitation wavelength: 405 nm and 488 nm; emission wavelength: 510 nm). The fluorescence ratio I 2 / I 405 / I 488As a relative quantity index of roGFP2 for oxidation / reduction, it can reflect the change in the relative amount of H 2 O 2 in the cytoplasm.

[0121] The results are as Figure 2 shown. The results show that compared with roGFP2-Orp1 and roGFP2-GPX2, roGFP2-PRXIIB shows a greater change in the oxidation level induced by various elicitors ( Figure 2 a). While the basal oxidation level of roGFP2-Tsa2ΔC R is too high and the noise is large ( Figure 2 b). In summary, roGFP2-PRXIIB is more suitable for monitoring the dynamic changes of H 2 O 2 in the cytoplasm induced by elicitors.

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

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

[0124] The results observed by the confocal microscope are as Figure 3 shown. The results show that the fusion protein roGFP2-PRXIIB is localized in the cytoplasm and nucleus and does not co-localize with chloroplasts; the fusion protein Nuc-roGFP2-PRXIIB is localized in the nucleus and co-localizes with the nuclear protein AHL22-mcherry; the fusion protein Mit-roGFP2-PRXIIB is localized in the mitochondria and co-localizes with the mitochondrial dye MitoTracker; the fusion protein Chloro-roGFP2-PRXIIB is localized in the chloroplasts and co-localizes with the chloroplast autofluorescence. The above results illustrate the correctness of the expression of roGFP2-PRXIIB with different subcellular localizations.

[0125] The stably expressed transgenic homozygous line obtained by introducing the recombinant vector pCAMBIA1300-35S-roGFP2-PRXIIB is the transgenic Arabidopsis thaliana line of roGFP2-PRXIIB targeting the cytoplasm.

[0126] The stably expressed transgenic homozygous lines obtained by introducing the recombinant vector pCAMBIA1300-Lat52-roGFP2-PRXIIB are the roGFP2-PRXIIB transgenic Arabidopsis thaliana lines targeting pollen tubes.

[0127] The stably expressed transgenic homozygous lines obtained by introducing the recombinant vector pCAMBIA1300-35S-Nuc-roGFP2-PRXIIB are the Nuc-roGFP2-PRXIIB transgenic Arabidopsis thaliana lines targeting the nucleus.

[0128] The stably expressed transgenic homozygous lines obtained by introducing the recombinant vector pCAMBIA1300-35S-Mit-roGFP2-PRXIIB are the Mit-roGFP2-PRXIIB transgenic Arabidopsis thaliana lines targeting mitochondria.

[0129] The stably expressed transgenic homozygous lines obtained by introducing the recombinant vector pCAMBIA1300-35S-Chloro-roGFP2-PRXIIB are the Chloro-roGFP2-PRXIIB transgenic Arabidopsis thaliana lines targeting chloroplasts.

[0130] Example 3. Monitoring of H in the subcellular level of plants by roGFP2-PRXIIB 2 O 2 Change

[0131] I. Monitoring of the production of H induced by elicitors by roGFP2-PRXIIB 2 O 2 Production

[0132] Select the stably expressed transgenic plant lines targeting the cytoplasm, chloroplasts, nucleus, and mitochondria respectively, plant them in a short-day plant house, take the leaves that have grown for four weeks, punch holes with a puncher with a diameter of 4.5 mm, and place them face up in a 96-well black plate pre-added with 100 μL ddH 2 O, and incubate overnight under weak light. The next day, treat with different elicitors (flg22 polypeptide, chitin, nlp20 polypeptide, Pep2 polypeptide), and the treatment method is the same as step five of Example 2.

[0133] The results are as Figure 4 shown. The results show that in the cytoplasm, chloroplasts, and nucleus, the production of H induced by different elicitors can be detected, and the time and pattern of H 2 O 2 production induced in different compartments and by different elicitors are different; while for mitochondria, due to its high oxidation degree itself, the curve change is not obvious, and no obvious H 2 O 2 production is detected. And due to its high oxidation degree itself, the curve change of mitochondria is not obvious, and no obvious H 2O 2 Generated.

[0134] II. Detection of H 2 O 2 Generated by roGFP2-PRXIIB

[0135] Select transgenic Arabidopsis thaliana lines targeting the cytoplasm, chloroplasts, nucleus, and mitochondria, plant them in a short-day plant house, take the leaves that have grown for four weeks, and inject them with D36E, D36E hopZ1a, D36E avrRpt2, and D36E avrB bacterial suspensions (the bacterial suspension concentration is 2.5×10 8 CFU / mL), and at the same time use ddH 2 O injection as a control. One hour after injection, punch holes using a 0.45 cm puncher, place them face up in a 96-well black plate pre-added with 200 μL of ddH 2 O, and start detecting the fluorescence change of roGFP2 under an enzyme-linked immunosorbent assay (ELISA) reader two hours after injection, detect for 10 minutes each time, and detect once per hour.

[0136] The results are as Figure 5 shown. The results show that the effector proteins AvrRpt2, HopZ1a, and AvrB all induce H 2 O 2 accumulation in the cytoplasm, chloroplasts, nucleus, and mitochondria. It is worth noting that the oxidative changes induced by effector proteins are significantly higher than those of elicitors. Among these organelles, effector proteins induce rapid H 2 O 2 accumulation in chloroplasts. For example, AvrB induces H 2 O 2 accumulation in chloroplasts approximately 2 to 3 hours after transfection, and AvrRpt2 and HopZ1a induce H 2 O 2 accumulation in chloroplasts approximately 5 to 6 hours after transfection. In addition, H 2 O 2 induced by AvrB also accumulates in the nucleus approximately 4 to 5 hours after transfection, and AvrRpt2 and HopZ1a induce H 2 O 2 accumulation in the nucleus approximately 6 to 7 hours after transfection. In addition, all effectors induce H 2 O 2 accumulation in mitochondria approximately 7 to 8 hours after transfection. In contrast, the degree of roGFP2-PRXIIB oxidation caused by D36E is similar to that of ddH 2 O treatment. These results indicate that a large amount of H 2 O 2 accumulates in the cytoplasm, chloroplasts, nucleus, and mitochondria during ETI.

[0137] III. Detection of H 2 O 2 production induced by abiotic stresses ABA and NaCl with roGFP2-PRXIIB

[0138] 1. Detection of H 2 O 2 production induced by abiotic stress NaCl with roGFP2-PRXIIB

[0139] Select transgenic Arabidopsis thaliana lines with stable expression of roGFP2-PRXIIB and roGFP2-Orp1 targeting the cytoplasm, plant them in a short-day plant house, take the leaves that have grown for four weeks, punch holes with a puncher with a diameter of 0.45 cm, and place them face up in a 96-well black plate pre-added with 100 μL ddH 2 O and incubate overnight under weak light. Treat with 100 mM NaCl the next day. The specific treatment method includes the following steps: Scan the plate in a microplate reader for 15 minutes the next day, then take it out and add NaCl solution. The leaves in each small hole of the plate float on the NaCl solution, and then immediately put it into the microplate reader to continue scanning and detecting the fluorescence change of roGFP2 for 6 hours.

[0140] The results are as Figure 6 shown in 2 O 2 a. The results show that within 1 hour after NaCl treatment, a rapid increase in the oxidation levels of roGFP2-PRXIIB and roGFP2-Orp1 was observed and remained at a high level for several hours. And the change in the oxidation level of roGFP2-PRXIIB was significantly higher than that of roGFP2-Orp1. This indicates that roGFP2-PRXIIB can effectively monitor the dynamic changes of H

[0141] 2. Detection of H 2 O 2 production induced by abiotic stress ABA with roGFP2-PRXIIB

[0142] Abscisic acid (ABA) is a plant hormone crucial for plants to adapt to various abiotic stresses. Within a few minutes, ABA can prompt guard cells to generate a large amount of ROS to promote stomatal closure, thereby reducing water loss caused by transpiration and thus conserving water. To detect H 2 O 2For the generation, transgenic Arabidopsis thaliana lines with stable expression of cytoplasm-targeted roGFP2-PRXIIB and roGFP2-Orp1 were selected and planted in a short-day plant house. Leaves grown for four weeks were taken, cut from the petioles, and immersed in a solution containing 100 μM ABA. After 90 minutes, the change in fluorescence intensity of roGFP2-PRXIIB in guard cells was observed by confocal microscopy (excitation wavelengths: 405 nm and 488 nm; emission wavelength: 493 - 533 nm).

[0143] The results are as Figure 6 shown in b. The results showed that after ABA treatment, roGFP2-PRXIIB in guard cells was strongly oxidized, significantly higher than roGFP2-Orp1. This indicates that roGFP2-PRXIIB can effectively monitor ABA-induced stomatal H 2 O 2 production and is more sensitive than roGFP2-Orp1.

[0144] IV. Monitoring of H 2 O 2 production in pollen tubes induced by RALF4 by roGFP2-PRXIIB

[0145] The research on ROS in pollen tubes has been very in-depth, and it plays a key role in the processes of water absorption, germination, and tubular growth of pollen. To observe the production of H 2 O 2 in growing pollen tubes, a transgenic Arabidopsis thaliana line with roGFP2-PRXIIB targeted to pollen tubes was selected. Its pollen was placed on a germination substrate (the solvent is water, and the solutes and their concentrations are 1 mM CaCl 2 , 1 mM Ca(NO 3 ) 2 , 1 mM MgSO 4 , 0.01% H 3 BO 4 , 18% sucrose, and 1% agarose), and cultured in the dark at 28 °C for 4 hours and then treated with 2 μM RALF4 polypeptide. The specific treatment method includes the following steps: Drop the RALF4 polypeptide solution with a concentration of 2 μM on the germination substrate, and observe the change in fluorescence intensity of roGFP2-PRXIIB in pollen tubes under a microscope after 2 min (excitation wavelengths: 405 nm and 488 nm; emission wavelength: 493 - 533 nm).

[0146] The results are as Figure 7 shown. The results showed that treatment with RALF4 polypeptide significantly increased the oxidation level of roGFP2-PRXIIB. This indicates that roGFP2-PRXIIB is suitable for observing H 2 O2 Dynamic changes

[0147] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any variations, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made by conventional techniques known in the art. The application of some basic features can be made within the scope of the following appended claims.

Claims

1. A fusion protein comprising peroxidase PRXIIB and fluorescent protein roGFP2; The amino acid sequence of the peroxidase PRXIIB is any one of the following A1)-A3): A1) The amino acid sequence is the protein shown in positions 274-434 of SEQ ID NO: 2; A2) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in positions 274-434 of SEQ ID NO: 2; A3) A protein having 80% or more identity with the amino acid sequence shown at positions 274-434 of SEQ ID NO: 2 and having the same function.

2. The fusion protein according to claim 1, characterized in that: The fusion protein is any one of the following B1)-B5): B1) The amino acid sequence is the protein shown in SEQ ID NO: 2; 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; B3) A fusion protein having the same function obtained by connecting a signal peptide, a transit peptide or a leader peptide to the N-terminus and / or the C-terminus of the amino acid sequence shown in SEQ ID NO: 2; B4) a protein having the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues of the amino acid sequence shown in SEQ ID NO: 2; B5) A protein that has 80% or more identity with the amino acid sequence shown in SEQ ID NO: 2 and has the same function.

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

4. The nucleic acid molecule according to claim 3, characterized in that: The nucleic acid molecule is any one of the following 1) or 2): 1) The 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 that has 75% or more identity with the DNA molecule defined in 1) and encodes the fusion protein of claim 1 or 2.

5. A recombinant expression vector, which can express the fusion protein according to claim 1 or 2.

6. The recombinant expression vector according to claim 5, characterized in that: The recombinant expression vector is a vector obtained by connecting the nucleic acid molecule according to claim 3 or 4 to a plant expression vector.

7. A recombinant bacterium comprising the recombinant expression vector according to claim 5 or 6.

8. Use of the fusion protein according to claim 1 or 2, the nucleic acid molecule according to claim 3 or 4, the recombinant expression vector according to claim 5 or 6, or the recombinant bacterium according to claim 7 in any of the following K1)-K4): K1) Monitoring H2O2 changes at the subcellular level in plants; K2) preparing products for monitoring H2O2 changes at the subcellular level in plants; K3) Monitoring H2O2 changes in plant organs, tissues or cells; K4) Preparation of products for monitoring H2O2 changes in plant organs, tissues or cells.

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

10. The use according to claim 8 or the method according to claim 9, characterized in that: The monitoring of H2O2 changes in the subcellular level of plants is embodied in any one of the following M1)-M3): M1) monitoring the changes of H2O2 in the plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplasts triggered by the inducer; M2) monitoring the changes of H2O2 in the plant cytoplasm and / or nucleus and / or mitochondria and / or chloroplasts triggered by pathogen effector proteins; M3) monitoring the changes of H2O2 in plant cytoplasm induced by abiotic stress; Alternatively, the monitoring of H2O2 changes at the plant organ, tissue or cell level is embodied by monitoring H2O2 changes in plant pollen tubes induced by RALF4 polypeptide.

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