Application of rice peroxidase gene OsPRX31 or its encoded protein in regulating drought resistance and / or fertility of rice

Abstract

The application relates to a rice peroxidase gene OsPRX31 or application of a coded protein thereof in regulating drought resistance of rice, and belongs to the technical field of genetic engineering. OsPRX31 The rice peroxidase gene OsLEA3 participates in drought stress response through an ABA signal pathway, can reduce oxidative stress damage, including reducing active oxygen and malondialdehyde accumulation, improving proline content, and up-regulating drought resistance gene expression, and enhances drought resistance. OsPRX31 Experimental verification shows that rice overexpressing the gene has a higher survival rate after drought, improved physiological indexes, reduced accumulation of oxidized substances, and up-regulated drought resistance gene expression; and knocking out the gene leads to grain abortion, and overexpressing the gene leads to normal seed setting. OsPRX31 OsPRX31 The application can effectively improve the drought resistance of rice and maintain fertility, and provides gene resources and theoretical basis for drought resistance molecular breeding.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to rice peroxidase genes. OsPRX31 Or the application of its encoded proteins in regulating drought resistance in rice. Background Technology

[0002] Rice ( Oryza sativa Rice (L.) is an important food crop, playing a crucial role in global food security. Drought stress is one of the major abiotic stressors limiting rice yield and planting area. With the intensification of global climate change, the frequency and intensity of droughts are constantly increasing, posing an increasingly serious threat to rice production. Currently, the main methods to improve the drought resistance of rice include traditional hybridization breeding and treatment with exogenous plant growth regulators. However, traditional hybridization breeding has a long cycle, low efficiency, and is limited by germplasm resources, making it difficult to quickly meet production needs; exogenous chemical regulation has potential problems such as high cost, short duration of effect, and environmental pollution. Therefore, how to effectively improve the drought resistance of rice without incurring cost and environmental problems is particularly important for current rice cultivation.

[0003] Genetic improvement using the functional genes of rice itself is a new approach to rice breeding. However, the specific functions of most genes have not yet been revealed, and the key genes available for breeding are very limited. OsPRX31 It is a member of the rice class III peroxidase gene family and has typical peroxidase structural characteristics. There are currently no reports on its drought resistance function. Summary of the Invention

[0004] The purpose of this invention is to address the current lack of effective methods for rice cultivation and breeding that balance cost and environmental concerns when facing drought stress. This invention provides a genetic engineering scheme utilizing endogenous genes in rice for genetic improvement. Specifically, this invention provides a rice peroxidase gene. OsPRX31 Or the application of its encoded proteins in regulating rice drought resistance and / or regulating rice fertility.

[0005] This invention provides a rice peroxidase gene. OsPRX31 The rice peroxidase gene or the application of its encoded protein in regulating rice drought resistance and / or rice fertility. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0006] Preferably, rice peroxidase gene overexpression is preferred. OsPRX31 Applications to improve drought resistance in rice, or knocking out the rice peroxidase gene. OsPRX31 Application in the construction of rice sterile materials.

[0007] This invention also provides overexpression of the rice peroxidase gene. OsPRX31 The application of biomaterials to improve drought resistance in rice, including the rice peroxidase gene. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; The biomaterial includes rice peroxidase genes. OsPRX31 Recombinant expression vectors and / or containing rice peroxidase genes OsPRX31 Recombinant microorganisms.

[0008] Preferably, the improvement of rice drought resistance includes any one or more of the following: ① to ⑥ ① Improve plant survival rate under drought stress; ② Reduce malondialdehyde (MDA) content in leaves under drought stress; ③ Reduce the accumulation of reactive oxygen species in rice plants under drought stress; ④ Increase the proline content in rice plants under drought stress; ⑤ Upregulate the expression levels of drought stress response genes; ⑥ Increase the lignin content in rice plants.

[0009] The drought stress response genes include OsLEA3 , OsERD1 and OsDREB2A At least one of them.

[0010] The present invention also provides a method for improving the drought resistance of rice, comprising the following steps: Overexpression of rice peroxidase gene in rice OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0011] This invention also provides a method for cultivating highly drought-resistant rice, comprising the following steps: Transgenic rice obtained through the method described in the above technical solution is used as a parent for breeding.

[0012] The present invention also provides a drought-resistant rice material in which the rice peroxidase gene is overexpressed. OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0013] This invention also provides a method for knocking out the rice peroxidase gene. OsPRX31 The application of biomaterials in constructing rice sterile materials, wherein the rice peroxidase gene OsPRX31The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; The biomaterials include those used for knockout rice. OsPRX31 Recombinant expression vectors for genes and / or for knockout rice OsPRX31 Microorganisms that regenerate genes.

[0014] This invention also provides a method for constructing rice sterile materials, comprising the following steps: Knockout of peroxidase gene in rice materials OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0015] The present invention also provides a rice sterile material, wherein the rice peroxidase gene is knocked out in the rice sterile material. OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0016] Beneficial effects: This invention provides a rice peroxidase gene. OsPRX31 The application of its encoded protein in regulating rice drought resistance and / or rice fertility. The rice peroxidase gene described in this invention. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2. The rice peroxidase gene of this invention... OsPRX31 Its encoded proteins can participate in the rice response to drought stress through the ABA (abscisic acid) signaling pathway, significantly reducing the damage to cells caused by oxidative stress induced by drought stress. This not only effectively reduces the accumulation of reactive oxygen species in rice plants, lowers malondialdehyde (MDA) content under drought stress, and increases proline content, but also upregulates... OsLEA3 , OsERD1 and OsDREB2A The expression levels of drought-resistant genes are adjusted to enable rice to maintain growth and survival under drought conditions.

[0017] Validated by examples, under rehydration conditions following drought stress: overexpression OsPRX31 Rice plants with the gene showed a higher survival rate compared to wild-type plants; overexpression OsPRX31 Rice plants expressing this gene showed significantly reduced malondialdehyde (MDA) and significantly increased proline content under drought stress; OsPRX31 In rice plants with this gene, the accumulation of superoxide anions and hydrogen peroxide is reduced in anther and leaf tissues; overexpression... OsPRX31 The genes significantly upregulated drought-resistant genes ( OsLEA3 , OsERD1 and OsDREB2A The level of expression of ). At the same time, knockout OsPRX31 Rice mutants of the gene exhibit pod abortion and inability to set grains normally, while lines overexpressing this gene can set grains normally. These results confirm that the rice peroxidase gene provided by this invention... OsPRX31 It can effectively improve the drought resistance of rice and maintain normal fertility, providing important genetic resources and theoretical basis for molecular breeding related to rice drought resistance and fertility. Attached Figure Description To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 for OsPRX31 Basic structural diagram; Figure 2 for OsPRX31 Figure showing the spatiotemporal expression detection results in rice; Figure 3 for OsPRX31 The following are GUS staining results: A shows the GUS staining results of seeds 1 day after germination; B shows the GUS staining results of mature leaves; C shows the GUS staining results of seeds 3 days after germination; D shows the GUS staining results of glumes; E shows the GUS staining results of a single stamen; F shows the GUS staining results of stamens and pistils; and G shows the GUS staining results of a single pistil. For figures A and C, Bar = 5 mm; for figures B, D, E, F, and G, Bar = 1 mm. Figure 4 To knock out mutant lines OsPRX31 A schematic diagram of mutation sites; Figure 5 for OsPRX31 The sterility phenotype of the knockout mutant line; where A represents... OSPRX31 Image of late-stage abortion in mutant caryopsis (Bar=5mm); B represents OSPRX31 Development diagram of the mutant spike (Bar=10cm); Figure 6 for OsPRX31 The results of POD and SOD activity detection in the caryopsis of knockout mutant lines are shown in the figure; This means P < 0.05. This means P < 0.01. This represents P < 0.001; Figure 7 for OsPRX31The image shows the results of reactive oxygen species gene expression detection in the caryae of knockout mutant lines; among them... This means P < 0.05. This means P < 0.01. This represents P < 0.001; Figure 8 for OsPRX31 Images showing the NBT and DAB staining results of anthers and leaves of the knockout mutant line; where the bar of the anther is 1 mm and the bar of the leaf is 1 cm. Figure 9 for OsPRX31 The image shows the results of POD and SOD enzyme activity detection in the leaves of knockout mutant lines; among them... This represents P < 0.001; Figure 10 for OsPRX31 The image shows the results of detecting the expression of starch synthesis genes in the endosperm of knockout mutant lines; among them... This represents P < 0.001; Figure 11 for OsPRX31 Figure showing the lignin content analysis results of the knockout mutant; among which, This represents P < 0.01; Figure 12 for OsPRX31 Schematic diagram of the overexpression material carrier; Figure 13 for OsPRX31 Image showing the identification results of overexpression material strains; among them, This means P < 0.01. This represents P < 0.0001; Figure 14 for OsPRX31 Images showing the NBT and DAB staining results of anthers and leaves of the overexpression material; where the bar value of anthers is 1 mm and the bar value of leaves is 1 cm. Figure 15 for OsPRX31 Figure showing the detection results of POD and SOD enzyme activities in the leaves of the overexpression material; Figure 16 for OsPRX31 The results of lignin content analysis of the overexpression materials are shown in the figure; among them, This means P < 0.05. This means P < 0.01. This represents P < 0.0001; Figure 17 for OsPRX31 The image shows the results of gene expression detection for lignin synthesis-related genes in the overexpression material; where ns represents no significant difference. This means P < 0.05. This means P < 0.01. This represents P < 0.001. This represents P < 0.0001; Figure 18 ABA-induced OsPRX31 The results of the expression detection are shown in the figure; among them, This means P < 0.001. This represents P < 0.0001; Figure 19 for OsPRX31 Drought-sensitive phenotypes of overexpression materials; among them, This means P < 0.01. This represents P < 0.0001; Figure 20 for OsPRX31 Figure showing the results of physiological index analysis of overexpression materials under drought stress; where ns represents no significant difference. This means P < 0.01. This means P < 0.001. This represents P < 0.0001; Figure 21 for OsPRX31 Figure showing the results of drought stress response gene expression analysis in overexpression materials; where ns represents no significant difference; This means P < 0.001. This indicates a significant difference (P < 0.0001). Detailed Implementation

[0019] This invention provides a rice peroxidase gene. OsPRX31 The rice peroxidase gene or the application of its encoded protein in regulating rice drought resistance and / or rice fertility. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0020] The rice peroxidase gene described in this invention OsPRX31 Its encoded proteins can participate in the rice response to drought stress through the ABA signaling pathway, significantly reducing the damage to cells caused by oxidative stress induced by drought stress. This not only effectively reduces the accumulation of reactive oxygen species in rice plants, lowers malondialdehyde (MDA) content under drought stress, and increases proline content, but also upregulates... OsLEA3 , OsERD1 and OsDREB2A The expression levels of drought-resistant genes were measured, enabling rice to maintain growth and survival under drought conditions. This provides important gene resources and theoretical basis for molecular breeding related to drought resistance and fertility in rice. Specifically, SEQ ID NO.2 of this invention is: MRPSFLLLLVVFAGVLAGGARAGPPLAGENLPGQLLPGEKPPGQPLPGQPLPGQPLPGQSLPGQPLPGQPLPGQSLPGQPLVGEKPPGQPLPGQPLPGQSLPGQPLPGQPLVGEKPPGQPLVPGQPLVGEKPPGQPLVPGQPLVGEKPPGQPLGGDKLSPDYYAQTCPRAERIVAEVVQSKQMANPTTAAGVLRLFFHDCFVSGCDASVLVAATAFEKSEQSAEINHSLPGDAFDAV VRAKLALELECPEVVSCADILALAARVLITMTGGPRYPISFGRKDSLTSSPTAPDKEMPQSNFTMDQVIKLFQDKGFTVQEMVALSGGHTLGFSHCKEFAQRIYDYQGKPGNVDP TMNPVLSKGLQTACKEYLKDPTIAAFNDVMTPGKFDNMYFVNLERGLGLLATDEEMWSDKRTQPFVKLYASNPTAFFDDFSRAIDKLSLFGVKTGAAGEIRRRCDTYNHGPMPK.

[0021] As one embodiment, the rice peroxidase gene of the present invention OsPRX31 OsPRX31 It also includes mutants, alleles, and derivatives generated by adding, substituting, inserting, and deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.1.

[0022] As one embodiment, the overexpression of the rice peroxidase gene described in this invention... OsPRX31 Applications to improve drought resistance in rice, or knocking out the rice peroxidase gene. OsPRX31 Application in the construction of rice sterile materials. As one implementation method, under drought stress followed by rehydration conditions, the present invention overexpresses... OsPRX31 Rice plants with the gene showed a higher survival rate compared to wild-type plants; overexpression OsPRX31 Rice plants expressing this gene showed significantly reduced malondialdehyde (MDA) and significantly increased proline content under drought stress; OsPRX31 In rice plants with this gene, the accumulation of superoxide anions and hydrogen peroxide is reduced in anther and leaf tissues; overexpression... OsPRX31 The genes significantly upregulated drought-resistant genes ( OsLEA3 , OsERD1 and OsDREB2A The level of expression of ). At the same time, knockout OsPRX31 Rice mutants of the gene exhibit pod abortion and inability to set grains normally, while lines overexpressing the gene can set grains normally. As one implementation method, the present invention... OsPRX31 Gene expression was upregulated by abscisic acid (ABA). Overexpression was treated with 50 μM ABA. OsPRX31 After transgenic seedlings were introduced, the staining color of GUS (glucuronidase) in the roots, stems, and leaves was significantly darker than that in the untreated control; qRT-PCR detection showed that after 6 hours of ABA treatment... OsPRX31 Gene expression levels increased significantly. These results indicate... OsPRX31 It participates in the rice response to drought stress through the ABA signaling pathway.

[0023] This invention also provides overexpression of the rice peroxidase gene. OsPRX31 The application of biomaterials to improve drought resistance in rice, including the rice peroxidase gene. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; The biomaterial includes rice peroxidase genes. OsPRX31 Recombinant expression vectors and / or containing rice peroxidase genes OsPRX31 Recombinant microorganisms.

[0024] As one embodiment, the base vector of the recombinant expression vector of the present invention is the plant expression vector pCAMBIA1300. As one embodiment, the recombinant expression vector of the present invention uses Kpn I and Xbal I as double restriction enzyme sites, and the inserted fragment is... OsPRX31 The open reading frame of the gene is used to construct a recombinant overexpression vector. As one embodiment, the recombinant microorganism of this invention includes a rice peroxidase gene. OsPRX31 Engineered bacteria with a recombinant expression vector. As one embodiment, the recombinant microorganism of this invention contains the rice peroxidase gene. OsPRX31 Recombinant Agrobacterium with a recombinant expression vector.

[0025] As one implementation method, the improvement of rice drought resistance according to the present invention includes any one or more of the following: ① to ⑥ ① Improve plant survival rate under drought stress; ② Reduce malondialdehyde (MDA) content in leaves under drought stress; ③ Reduce the accumulation of reactive oxygen species in rice plants under drought stress; ④ Increase the proline content in rice plants under drought stress; ⑤ Upregulate the expression levels of drought stress response genes ⑥ Increase the lignin content in rice plants.

[0026] The drought stress response genes include OsLEA3 , OsERD1 and OsDREB2A At least one of them.

[0027] As one implementation method, under drought stress conditions, the overexpression material described in this invention... OsPRX31 -OE exhibited stronger tolerance than the wild type: after drought treatment and rehydration, the survival rate of overexpressing plants was significantly higher than that of wild-type plants; under drought stress, the malondialdehyde (MDA) content in the leaves of overexpressing plants was lower than that of wild-type plants, indicating that their membrane lipid peroxidation damage was less severe; under drought stress, the proline (Pro) content in the leaves of overexpressing plants was higher than that of wild-type plants, indicating that their osmotic regulation and antioxidant capacity were enhanced; at the same time, drought stress response genes ( OsLEA3 , OsERD1 and OsDREB2A The expression level of ) was significantly upregulated. As one implementation, the reactive oxygen species includes superoxide anion (O2). - At least one of ( ) and hydrogen peroxide (H2O2). As one embodiment, the overexpression described in this invention... OsPRX31Under normal growth conditions, the leaf peroxidase (POD) and superoxide dismutase (SOD) activities of the rice lines containing the gene showed no significant changes compared to the wild type. As one implementation method, increasing the lignin content in rice plants includes upregulating the expression levels of genes related to lignin synthesis. As one implementation method, the lignin synthesis-related genes include... OsPAL1 , OsC3H1 , Os4CL3 , OsCAD1 , OsCESA4 and OsCESA7 At least one of them. As one implementation, under drought stress, the overexpression described in this invention... OsPRX31 In rice plants with the gene, OsRbcS1 and OsDip1 Gene expression levels showed a downward trend.

[0028] The present invention also provides a method for improving the drought resistance of rice, comprising the following steps: Overexpression of rice peroxidase gene in rice OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0029] As one embodiment, the present invention describes the overexpression of the rice peroxidase gene in rice. OsPRX31 By introducing and inserting the aforementioned into rice OsPRX31 Overexpression vectors of open reading frames of genes are used. As one implementation method, this invention achieves the expression of the rice peroxidase gene using Agrobacterium-mediated genetic transformation. OsPRX31 Overexpression. As one embodiment, the base vector of the overexpression vector of the present invention is the plant expression vector pCAMBIA1300. As one embodiment, the overexpression vector of the present invention uses Kpn I and Xbal I as double restriction sites, and the inserted fragment is... OsPRX31 Open reading frames of genes were used to construct recombinant overexpression vectors.

[0030] This invention also provides a method for cultivating highly drought-resistant rice, comprising the following steps: Transgenic rice obtained through the method described in the above technical solution is used as a parent for breeding.

[0031] The present invention also provides a drought-resistant rice material in which the rice peroxidase gene is overexpressed. OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0032] As one embodiment, the rice material of the present invention includes rice plants, seeds, tissues, and callus tissue.

[0033] This invention also provides a method for knocking out the rice peroxidase gene. OsPRX31 The application of biomaterials in constructing rice sterile materials, wherein the rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; The biomaterials include those used for knockout rice. OsPRX31 Recombinant expression vectors for genes and / or for knockout rice OsPRX31 Microorganisms that regenerate genes.

[0034] In one embodiment, the recombinant expression vector of the present invention is a CRISPR-Cas9 gene editing vector. In another embodiment, the recombinant expression vector of the present invention contains a targeted... OsPRX31 The sgRNA coding sequence of the gene, the target sequence is shown in SEQ ID NO.3, the PAM sequence is GGG, and the target sequence is located at... OsPRX31 The gene positions are 782 to 804. Specifically, SEQ ID NO. 3 of this invention is: TCCACGACTGCTTCGTCAGCGGG. As one embodiment, the recombinant microorganism of this invention includes engineered bacteria containing the above-mentioned CRISPR-Cas9 editing vector. As one embodiment, the recombinant microorganism of this invention is a recombinant Agrobacterium containing the above-mentioned CRISPR-Cas9 editing vector.

[0035] As one implementation method, the knockout method described in this invention OsPRX31 Rice sterile materials with genes possess at least one of the following characteristics: significantly reduced POD and SOD enzyme activities in caryopsis, increased accumulation of reactive oxygen species (superoxide anion and hydrogen peroxide) in leaves, and starch synthesis-related genes in endosperm ( OsLESV , OsESV1 and OsFLO2 The expression of lignin was significantly downregulated, and the lignin content in the leaves was significantly reduced.

[0036] This invention also provides a method for constructing rice sterile materials, comprising the following steps: Knockout of peroxidase gene in rice materials OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0037] The present invention also provides a rice sterile material, wherein the rice peroxidase gene is knocked out in the rice sterile material. OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0038] As one embodiment, the rice material of the present invention includes rice plants, tissues, and callus tissue.

[0039] To further illustrate the present invention, the rice peroxidase gene provided by the present invention will be described below with reference to the accompanying drawings and embodiments. OsPRX31 The invention describes in detail the application of its encoded proteins in regulating drought resistance and / or fertility in rice, but these descriptions should not be construed as limiting the scope of protection of this invention.

[0040] The following are some of the general experimental methods used in the embodiments of the present invention. Unless otherwise specified, they are all conventional methods.

[0041] 1. Plant materials and growing conditions This study selected wild-type rice Nipponbare, OsPRX31 Gene knockout mutants and OsPRX31 ( LOC_ Os02g50770 The gene overexpression line was used as experimental material and planted in Kunming, Yunnan Province, China, underwent regular irrigation and fertilization management. On the day of flowering, the spikelets were marked, and then the glumes were removed to collect caryopsis fruits at different developmental stages for subsequent experimental analysis. Samples with abnormal appearance were discarded during this process. Endosperm tissue was collected 10 days after flowering for further experimental analysis.

[0042] 2. Genetic transformation methods Generation of CRISPR-Cas9 mutant materials: This study used CRISPR-Cas9 gene editing technology to construct... OsPRX31 Gene knockout mutants. First, CRISPR Primer Designer software is used to design targeted... OsPRX31 Gene editing primers were prepared, and the editing vector was then introduced into Nipponbare rice using Agrobacterium-mediated transformation. In the T0 generation, a 200–300 bp fragment containing the target site was amplified in each transformant, and individuals with successful mutations were screened using Sanger sequencing.

[0043] Production of overexpression mutant materials: for the production OsPRX31 Gene overexpression lines were first amplified from the Nipponbare rice variety using RT-PCR technology. OsPRX31 The open reading frame (ORF) was then cloned into pCAMBIA1300 using Kpn I and Xbal I restriction enzyme sites to construct an overexpression vector. The vector was then transformed into Nipponbare rice using Agrobacterium infection to obtain... OsPRX31 Overexpression of transgenic lines.

[0044] Generation of GUS reporter gene material: This was achieved by using Sal I and Kpn I sites... OsPRX31 A 2000bp promoter fragment upstream of the start codon was cloned into the pCAMBIA1300 vector to construct... OsPRX31pro: GUS The report vector was used to transform rice through Agrobacterium infection.

[0045] 3. DNA extraction methods (1) Take 0.5g of rice leaf tissue, grind it into fine powder with liquid nitrogen, add 600μl of TPS lysis buffer (1M Tris-HCl 10ml, 0.5M EDTA 2ml, 5M KCl 50ml, ultrapure water 38ml), and mix thoroughly.

[0046] (2) Bathe in a 70℃ water bath for 30 minutes, then invert and mix 4 times.

[0047] (3) Centrifuge at 12000×g for 10 minutes and transfer the supernatant to a new centrifuge tube.

[0048] (4) Add 600 μl of isopropanol, invert to mix, and place at -20°C for 10 minutes. Centrifuge at 12000×g for 10 minutes and discard the supernatant.

[0049] (5) Wash the precipitate with 500 μl of 70% ethanol. Centrifuge at 12000×g for 5 minutes, discard the supernatant, and add 30 μl of ultrapure water to dissolve the DNA after precipitation.

[0050] 4. RNA extraction methods Total RNA was extracted from different tissues of rice using the TaKaRa RNAiso Easy Kit (Code No. TCH022).

[0051] 5. Reverse transcription method (1) 2 μg of RNA is needed for one reverse transcription. Calculate the amount of extracted RNA to be added to the EP tube based on the concentration.

[0052] (2) Add 1 μl of Oligo(dT) and add enzyme-free water to a final volume of 16.5 μl.

[0053] (3) Place it at 70℃ for 5 minutes, then transfer it to ice and place it for 10 minutes.

[0054] (4) Next, add 5 μl M-MLV buffer; 2 μl dNTP; 0.5 μl Inhibitor; 1 μl reverse transcriptase.

[0055] (5) Place the EP tube in the PCR instrument and program it at 42℃ for 1 hour and 70℃ for 15 minutes.

[0056] (6) After the procedure is completed, the solution in the EP tube is the reverse transcription product.

[0057] 6. Real-time quantitative PCR method qRT-PCR was performed using the QIAGEN QuantiNova™ SYBR® Green PCR Kit on the QuantStudio 6Flex real-time PCR platform. Rice actin gene assays were used. Actin ( LOC_Os03g50885 (As an endogenous control) The primer sequences used in the embodiments of this invention are shown in Tables 2 to 6.

[0058] 7. Method for Assay of Peroxidase (POD) Activity (1) Enzyme extraction: Take 1g of rice leaves or roots, cut them into small pieces and place them in a pre-cooled mortar. Add 5mL of 0.1mol / L Tris-HCl buffer (pH=8.5) and grind into a homogenate. Centrifuge at 4000×g for 5 minutes and collect the supernatant.

[0059] (2) Preparation of reaction mixture: 2 mol / L phosphate buffer (pH=6.0): Mix 87.7 mL NaH2PO4 (0.2 mol / L) and 12.3 mL Na2HPO4 (0.2 mol / L), and dilute to 200 mL. Reaction solution: 50 mL phosphate buffer + 0.028 mL H2O2 + 0.019 mL guaiacol.

[0060] (3) Enzyme activity assay: Add 3 mL of reaction solution and 1 mL of enzyme solution (boiled enzyme solution for control tube) to a cuvette. Measure the absorbance at 470 nm, record it every minute, for a total of 3 minutes.

[0061] (4) Calculation of results: Enzyme activity (U) = ΔA 470 × Dilution factor / (0.01 × sample fresh weight × reaction time).

[0062] 8. Method for Assay of Superoxide Dismutase (SOD) Activity (1) Enzyme extraction: Take 0.5g of rice leaves, add 5mL of pre-cooled 50mM phosphate buffer (pH=7.8), and grind in an ice bath. Centrifuge at 10000×g for 20 minutes at 4℃ and collect the supernatant.

[0063] (2) Color reaction: Reagent preparation: 130mM methionine (Met), 750μM nitroblue tetrazolium (NBT), 100μM EDTA-Na2, 20μM riboflavin (Riboflavin).

[0064] Reaction system: 3.5 mL buffer + 0.5 mL Met + 0.5 mL NBT + 0.5 mL EDTA-Na2 + 0.5 mL riboflavin + 0.1 mL enzyme solution. After mixing, react under 4000 Lx light for 20 minutes. The control group was treated in the dark.

[0065] (3) Enzyme activity assay: Using a light-proof tube as a blank, the absorbance (A) at 560 nm was measured in each tube.

[0066] Enzyme activity (U / g) = (A control - A sample) × total enzyme volume / (A control × 0.5 × sample fresh weight × enzyme volume).

[0067] 9. Method for determining malondialdehyde content Wash and dry the rice tissues. Weigh 0.05 g (W) of each tissue fraction and add 10 ml of pre-cooled TCA buffer (5% trichloroacetic acid). Grind into a homogenate under ice bath conditions. Centrifuge at 12000×g for 10 minutes, then take 2 mL of the supernatant and add 2 ml of 0.67% thiobarbituric acid (TBA) solution (prepared with 10% TCA). Mix well and heat in a boiling water bath for 20 minutes. After the reaction, quickly cool the reaction tube in an ice bath. Measure the absorbance at 600 nm, 532 nm, and 450 nm using a spectrophotometer. The formula for calculating malondialdehyde content is shown in Formula I. , Formula I.

[0068] Among them, A 532 A 600 A 450 This refers to the absorbance values ​​measured at 532 nm, 600 nm, and 450 nm on a spectrophotometer; W is the weight of the sample.

[0069] 10. Method for determining proline content (1) Sample preparation: Weigh 0.05 g (W) of rice tissue, add 2 ml of 3% sulfosalicylic acid, grind thoroughly and transfer to a centrifuge tube. Heat in a boiling water bath for 15 minutes. After the sample cools to room temperature, centrifuge at 12000×g for 15 minutes and take the supernatant for later use.

[0070] (2) Preparation and reaction of standard solutions: A series of proline standard solutions with different concentrations (0, 20, 40, 60, 80, 100 μg / mL) were prepared. 2 mL of the standard solution was placed in a test tube, 2 mL of acidic ninhydrin solution was added, and the mixture was heated in a boiling water bath for 30 minutes, then immediately cooled. 4 mL of toluene was added, and the mixture was thoroughly shaken for extraction. After separation, the upper toluene phase was collected, and a blank tube without proline was used as a control. The absorbance was measured at a wavelength of 520 nm. A standard curve was plotted with proline concentration on the x-axis and absorbance on the y-axis.

[0071] (3) Sample determination: Take 2 mL of the extracted sample supernatant and proceed according to the reaction steps for preparing the standard curve, i.e., add 2 mL of acidic ninhydrin reagent, heat in a boiling water bath, cool, and extract with toluene, etc. Finally, measure the absorbance at a wavelength of 520 nm and calculate the concentration C of proline in the sample according to the standard curve. The formula for calculating the proline content is shown in Formula II: Formula II.

[0072] Where C refers to the concentration obtained from the standard curve, in μg / mL; W refers to the sample weight, in g; and V refers to the total volume of the extract, in mL.

[0073] 11. Methods for Lignin Determination The determination was performed using the Biosharp brand lignin content assay kit (acetylation method) spectrophotometric method.

[0074] 12. Histochemical staining methods for reactive oxygen species (ROS) NBT staining (for superoxide anion detection): (1) Preparation of NBT staining solution: Dissolve 50 mg NBT powder in 100 mL Tris-HCl buffer (pH 7.4) or phosphate buffer (pH 7.8) to prepare a working solution with a concentration of 0.5 mg / mL. Store at 2~8℃ protected from light.

[0075] (2) Staining: Collect fresh leaves, immerse them completely in NBT staining solution, apply vacuum (negative pressure -0.1MPa) for 20~30 minutes, and then let them stand at room temperature for about 60 minutes for staining.

[0076] (3) Decolorization: Discard the dye solution, add 95% ethanol, and treat at 40°C for 3 to 16 hours, replacing with fresh ethanol 1 to 2 times during the period.

[0077] (4) Observation: After the decolorization is complete, take it out for observation and take pictures. The NBT positive area is blue.

[0078] DAB staining (for detecting hydrogen peroxide): (1) DAB staining solution preparation: Dissolve 100mg DAB powder in 100mL phosphate buffer (pH 5.0, 5.5 or 7.8), store at 4℃ protected from light, and use immediately.

[0079] (2) Staining: Collect fresh leaves, immerse them completely in DAB staining solution, apply vacuum (negative pressure -0.1MPa) for 20~30 minutes, and then let them stand at room temperature in the dark for about 60 minutes for staining.

[0080] (3) Decolorization: Discard the dye solution, add 95% ethanol, and treat at 40°C for 3 to 16 hours, replacing with fresh ethanol 1 to 2 times during the period.

[0081] (4) Observation: After the decolorization is complete, take it out for observation and take pictures. The DAB positive area is brownish-red or dark brown.

[0082] Example 1 OsPRX31 Genes and their encoded proteins The sequence number LOC_Os02g50770 (named) was downloaded from the National Rice Genome Database (website: https: / / www.ricedata.cn / gene / ). OsPRX31 The gene and protein sequences of ).

[0083] OsPRX31, Located on rice chromosome 2, 30999987~31001339, with a full nucleotide length of 1353 bp and a predicted protein length of 450 aa, the structure is as follows: Figure 1 As shown in the figure, this protein has a molecular weight of 47.889 kDa and an isoelectric point of 5.84. Its instability coefficient is 45.98, indicating it is an unstable protein. It contains 78.04% aliphatic amino acids and has a hydrophilicity of -0.238, making it a hydrophilic protein. Analysis of the cis-regulatory elements in the upstream 2000 bp sequence (Table 1) revealed that the most frequent element is the TATA-box, a core initiation element for transcription, with 31 elements. The second most frequent element is the CAAT-box, a common cis-regulatory element in promoter and enhancer regions, with 24 elements. The remaining elements are related to abscisic acid response, light response, and hormone response.

[0084] Table 1 OsPRX31 Starting element

[0085] Example 2 OsPRX31 Expression pattern analysis In order to understand OsPRX31To investigate the expression patterns, this invention extracted RNA from five different developmental stages of wild-type Nipponbare (Nip) roots, stems, leaves, and microspores, and detected RNA in different tissues using qRT-PCR. OsPRX31 The expression level. The primers used are as follows: internal reference gene. Actin upstream primer of (LOC_Os03g50885) Actin-F The downstream primer is TCAGCAACTGGGATGATATGGAG (SEQ ID NO. 6). Actin-R The target gene is GCCGTGTGGTGAATGAGTAAC (SEQ ID NO.7). OsPRX31 upstream primer of (LOC_Os02g50770) OsPRX31-F The downstream primer is TCCAGGACAAGGGTTTCACG (SEQ ID NO. 8). OsPRX31-R is TTGAGGTACTCCTTGCACGC (SEQ ID NO. 9).

[0086] get OsPRX31 The spatiotemporal expression detection results in rice are as follows: Figure 2 As shown, it can be seen OsPRX31 Expression was highest during microspore development and maturation, followed by the binucleate pollen grain stage. Expression was lower in early microspore development and roots, and lowest during meiosis. Next, this invention constructed a vector driving the expression of the glucuronidase (GUS) reporter gene and transformed it into Nipponbare. The specific steps for GUS staining are as follows: By using Sal I and Kpn I sites OsPRX31 A 2000bp promoter fragment upstream of the start codon was cloned into the pCAMBIA1300 vector to construct... OsPRX31pro: GUS The reporter vector was used to transform rice through Agrobacterium infection. Fresh rice tissues carrying the GUS reporter gene were excised using tweezers and a blade, ensuring tissue integrity and avoiding damage as much as possible. The excised tissues were completely immersed in a culture dish containing GUS staining buffer (50 mM sodium phosphate buffer pH 7.0, 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferricyanide, 10 mM EDTA, 0.1% Triton X-100, and 1 mM X-Glucose, 5-bromo-4-chloro-3-indole-D-glucuronide). The culture dish was incubated at a constant temperature of 37°C; a distinct blue staining was observed after 3 hours. After staining, the tissues were gently rinsed with sodium phosphate buffer to remove excess staining substrate. Finally, the tissues were placed on a glass slide for observation and photography under a microscope.

[0087] get OsPRX31 The results of GUS staining are as follows Figure 3As shown, GUS staining results revealed that it was detected in roots, stems, leaves, and coleoptiles. OsPRX31 The GUS activity was measured, and the results were consistent with previous qRT-PCR analysis.

[0088] Example 3 OsPRX31 Construction and phenotypic identification of knockout mutants 1. Construction and identification of knockout mutants In order to study OsPRX31 The biological functions of this invention are described in the construction of OsPRX31 The CRISPR-Cas9 knockout mutant material was obtained. For specific construction methods, please refer to the previous section "Generation of CRISPR-Cas9 Mutant Material". The target sequence is TCCACGACTGCTTCGTCAGCGGG (SEQ ID NO: 3), located at positions 782 to 804 of the gene, and the PAM sequence is GGG. The obtained transgenic seedlings were subjected to Sanger sequencing. Through sequence alignment, this invention obtained two homozygous knockout mutant lines, named... OsPRX31-1 and OsPRX31-2 For use in subsequent research. OsPRX31-1 Compared to the wild-type Nipponbare, the strain inserted an A base in the target sequence region, while OsPRX31-2 Compared to the wild type, the strain lacks the three bases GTC (e.g., Figure 4 (As shown). To verify the two knockout lines... OsPRX31 To determine whether the protein sequence had changed, this invention compared the protein sequences. The results showed that the amino acid sequences of both strains had changed compared to the wild type. The control group was the wild-type Nipponbare, named Nip.

[0089] 2. Phenotypic observation of knockout mutants Throughout the vegetative growth stage, OsPRX31 No significant phenotypic differences were observed between the mutant and the wild type. However, during the later stages of caryopsis development, OsPRX31 The mutant exhibits obvious sterility. Compared to the wild type, the mutant's caryopsis shows significant morphological changes, specifically shrinkage, darkening in color, and poor development of the endosperm. Figure 5 ).

[0090] 3. Detection of physiological and biochemical indicators of knockout mutants Using the method described in the general experimental method, for OsPRX31 Peroxidase (POD) and superoxide dismutase (SOD) activities were measured in caryopsis fruits of the mutant ten days after pollination. The results showed that, compared to the wild type, OsPRX31 The activities of these two enzymes were significantly reduced in the mutant. Figure 6 ).

[0091] RNA was further extracted from leaves, and ROS-related typical genes were detected using reverse transcription and qRT-PCR methods as described in the general experimental procedures. RBOHd , OsMT-1 and OsGPX4 The relative expression levels (primer sequences are shown in Table 2) were found to be significantly downregulated compared to the wild type. Figure 7 ).

[0092] Table 2 Primers used in this study

[0093] The activities of POD and SOD enzymes in leaves were detected using the methods described in the general experimental procedure. The results showed that, consistent with the trend observed in caryopsis, the activities of these two enzymes were also significantly reduced in the mutant leaves. Figure 9 ).

[0094] Leaves of mutant and wild-type organisms were stained with NBT and DAB staining solutions to detect ROS content. Results showed that... OsPRX31 In the absence of gene mutations, OsPRX31 The mutant showed a greater amount and darker color of formazan precipitate in its anthers and leaves, indicating a higher superoxide anion content. Similarly, a darker brown color indicated a higher hydrogen peroxide content. Figure 8 , Figure 9 The above results indicate that the deletion of the OsPRX31 gene leads to increased ROS accumulation in anthers and leaves.

[0095] In addition, the qRT-PCR method described in the general experimental methods was used to investigate starch synthesis-related genes. OsLESV , OsESV1 and OsFLO2 The expression levels of [the gene] were analyzed (primer sequences are shown in Table 3). The results showed that, compared to the wild type, [the expression levels were lower]. OsPRX31 The expression levels of these genes were significantly downregulated in the mutants. Figure 10 Using the method described in the general experimental approach, wild-type and... OsPRX31 The lignin content of the mutant leaves was determined. The results showed that, compared to the wild type, OsPRX31 The lignin content in the mutant leaves was significantly reduced. Figure 11 ).

[0096] Table 3 Primers used in this study

[0097] Example 4 OsPRX31 Construction and functional analysis of overexpression lines Given OsPRX31 The infertility exhibited by gene knockout mutant materials prevents them from producing offspring. This invention further constructs... OsPRX31 Overexpression materials. For specific methods on constructing overexpression vectors, please refer to the previous section "Generation of Overexpression Mutant Materials." Successful construction of the overexpression vector ( Figure 12 The *Acetobacterium* was introduced into Nipponbare rice via Agrobacterium-mediated transformation to obtain T0 generation transgenic plants. Hygromycin markers were screened from the T0 generation plants, and qRT-PCR expression levels were detected, revealing several transgenic plants. OsPRX31 Positive lines with significantly elevated expression levels were identified. T1 generation seeds were harvested by self-pollination of individual plants from these positive lines. After sowing, the expression levels of each individual plant were further assessed using qRT-PCR. The lines with the highest expression levels were selected and self-pollinated again to obtain T2 generation seeds. Identification revealed that the T2 generation plants showed significantly elevated expression levels... OsPRX31 The relative expression level remained high, and the genetic stability was good. In this study, two homozygous overexpression lines, No. 2 and No. 9, with significantly different expression levels were selected and named... OsPRX31- OE-1 and OsPRX31-OE-2 For further research ( Figure 13 The control group consisted of wild-type Nipponbare Nip.

[0098] 1. Analysis of ROS levels and lignin content in overexpression lines for OsPRX31 For the overexpression lines, this invention also conducted ROS level analysis. Using the NBT and DAB histochemical staining methods described in the general experimental methods, the ROS content of anthers and leaves of the overexpression lines and wild-type was detected. The results showed that, regardless of whether NBT or DAB staining was performed, the staining of the overexpression lines was lighter than that of the wild-type (O2). - Both the accumulation of ) and hydrogen peroxide (H2O2) were less than in the wild type ( Figure 14 ).

[0099] The activities of POD and SOD enzymes in the leaves of the overexpression lines were measured using the methods described in the general experimental procedure. The results showed that the activities of POD and SOD enzymes in the leaves of the overexpression lines were not significantly different compared to the wild type. Figure 15 ).

[0100] Using the method described in the general experimental method, for OsPRX31 Lignin content was determined in the roots, stems, and leaves of the overexpression lines. The results showed that, compared with the wild type, the lignin content in the roots, stems, and leaves of the overexpression lines was significantly increased. Figure 16 ).

[0101] Further investigation of lignin synthesis-related genes (such as hydroxylation modification genes) C3H methylation-modified genes COMT Participating in reduction reaction CAD Quantitative analysis was performed on the lignin synthesis-related genes (etc.). RNA was extracted from mature roots, stems, and leaves of overexpressing plants and wild-type plants. The transcriptional levels of these genes in roots, stems, and leaves were determined using reverse transcription and qRT-PCR methods as described in the general experimental procedures. The primer sequences used are shown in Table 4. The results showed: Overexpression of genes related to lignin synthesis in roots, stems, and leaves of plants showed a significant increase compared to wild-type plants. In roots, in addition to... OsCOMT1 The expression level of was significantly reduced. OsPAL1 , OsC3H1 , Os4CL3 , OsCAD1 , OsCESA4 , OsCESA7 The expression levels of all of them increased significantly; although in the stem OsPAL1 , OsC3H1 The expression level of this gene was not significantly different from that of the wild type, but the expression levels of all other genes were significantly upregulated; in the leaves OsPAL1 , OsCESA4 and OsCESA7 The expression level of this molecule was significantly higher than that of the wild type. OsC3H1 , OsCOMT1 Significantly lower than wild type ( Figure 17 ).

[0102] Table 4 Primers used in this study

[0103] 2. ABA induction OsPRX31 Expression Analysis Rice seedlings were treated with 50 μM ABA, and GUS histochemical staining was performed following the same procedure as in Example 2. Histochemical staining showed that the GUS staining in the roots, stems, and leaves of ABA-treated seedlings was deeper than that in untreated seedlings, indicating that… OsPRX31 Gene expression increases after ABA induction.

[0104] Simultaneously, leaf RNA was extracted from wild-type seedlings treated with 50 μM ABA at different time points (0 min, 5 min, 10 min, 30 min, 1 h, 6 h, 9 h, 12 h, 15 h, 18 h, 24 h, and 30 h), and detected using reverse transcription and qRT-PCR methods as described in the general experimental procedures. OsPRX31 The expression levels and primer sequences are shown in Table 5. Results showed that within 0 to 6 hours after treatment... OsPRX31 There was no significant difference in gene expression, but its expression increased significantly after 6 hours. Figure 18 ).

[0105] Table 5 Primers used in this study

[0106] 3. Analysis of drought resistance function in overexpression lines In the complex physiological processes of plant response to drought stress, ABA, as a key signaling molecule, plays multiple regulatory roles and is closely related to the drought resistance of rice. Based on the above theoretical foundation and previous findings, this invention uses three-week-old rice plants planted in nutrient soil, fully watered immediately after transplanting, and then not watered again, to conduct a rice drought resistance experiment, with a focus on detailed monitoring and analysis of the rehydration process. After experiencing 21 days of drought stress (without watering), both the overexpression lines and wild-type materials were simultaneously rehydrated. The experimental results show that the overexpression lines exhibited a higher survival rate than the wild-type materials. Figure 19 ).

[0107] The malondialdehyde (MDA) content in rice leaves under drought conditions was determined using the method described in the general experimental procedure. MDA content can indirectly reflect the oxidative stress level of rice under drought stress. The results showed that, compared to the wild type, OsPRX31 The MDA content in the overexpression lines was significantly reduced. Figure 20 The proline (Pro) content in rice leaves under drought conditions was determined using the methods described in the general experimental procedures. Pro has antioxidant capabilities, which can alleviate the damage to cells caused by oxidative stress, maintain the fluidity and stability of biological membranes, and reduce the occurrence of membrane lipid peroxidation. The results showed that the Pro content of overexpressing plants was significantly higher than that of wild-type plants. Figure 20 ).

[0108] Further investigation was conducted to examine changes in the expression of drought stress response genes. RNA was extracted from leaves of drought-treated overexpressing plants and wild-type plants, and reverse transcription and qRT-PCR methods as described in the general experimental procedures were used to detect changes. OsDREB2A OsRbcS1, OsDip1, OsLEA3 and OsERD1 The expression levels of [the gene] were shown in Table 6 (primer sequences are listed in Table 6). The results indicated that, compared to the wild type, under drought stress, the overexpressing lines [expressed significantly lower levels of the gene]. OsLEA3 , OsERD1 and OsDREB2A The expression levels of these genes generally showed an upward trend, while OsRbcS1 , OsDip1 It shows a downward trend ( Figure 21 The above results indicate that... OsPRX31 Overexpression can activate the expression of drought stress response genes, regulate the plant's water balance, synthesis of osmotic regulators, and antioxidant defense system, thereby enhancing the drought resistance of rice.

[0109] Table 6 Primers used in this study

[0110] As can be seen from the above, the results of this study not only provide insights into the third type of peroxidase genes in rice, but also contribute to a deeper understanding of these genes. OsPRX31 The gene function provides detailed experimental evidence and paves the way for further research. OsPRX31 This study provides new ideas and clues regarding plant growth, development, reproductive processes, and the mechanism of action in rice drought resistance.

[0111] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Rice peroxidase gene OsPRX31 The rice peroxidase gene or the application of its encoded protein in regulating rice drought resistance and / or rice fertility. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, Overexpression of rice peroxidase gene OsPRX31 Applications to improve drought resistance in rice, or knocking out the rice peroxidase gene. OsPRX31 Application in the construction of rice sterile materials.

3. Overexpression of rice peroxidase gene OsPRX31 The application of biomaterials to improve drought resistance in rice, including the rice peroxidase gene. OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; The biomaterial includes rice peroxidase genes. OsPRX31 Recombinant expression vectors and / or containing rice peroxidase genes OsPRX31 Recombinant microorganisms.

4. The application according to claim 2 or 3, characterized in that, The improvement of rice drought resistance includes any one or more of the following: ① to ⑥ ① Improve plant survival rate under drought stress; ② Reduce malondialdehyde (MDA) content in leaves under drought stress; ③ Reduce the accumulation of reactive oxygen species in rice plants under drought stress; ④ Increase the proline content in rice plants under drought stress; ⑤ Upregulate the expression levels of drought stress response genes; ⑥ Increase the lignin content in rice plants. The drought stress response genes include OsLEA3 , OsERD1 and OsDREB2A At least one of them.

5. A method for improving the drought resistance of rice, characterized in that, Includes the following steps: Overexpression of rice peroxidase gene in rice OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

6. A method for cultivating highly drought-resistant rice, characterized in that, Includes the following steps: The transgenic rice obtained by the method described in claim 5 is used as a parent for breeding.

7. A drought-resistant rice material, characterized in that, The rice material overexpresses the rice peroxidase gene. OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

8. Knockout of rice peroxidase gene OsPRX31 The application of biomaterials in constructing rice sterile materials, wherein the rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2; The biomaterials include those used for knockout rice. OsPRX31 Recombinant expression vectors for genes and / or for knockout rice OsPRX31 Microorganisms that regenerate genes.

9. A method for constructing rice sterile materials, characterized in that, Includes the following steps: Knockout of peroxidase gene in rice materials OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.

2.

10. A rice sterile material, characterized in that, The rice peroxidase gene was knocked out in the rice sterile material. OsPRX31 ; The rice peroxidase gene OsPRX31 The amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

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