Application of rice peroxidase OsPrx114 gene promoter

Abstract

The invention discloses application of a rice peroxidase OsPrx114 gene promoter, belongs to the field of rice breeding, and finds that rice can adapt to flood stress by changing the expression quantity of the OsPrx114 gene promoter for the first time, so that a new thought and a new method are provided for later cultivation of flood-resistant rice.

Description

Technical Field

[0001] The present invention relates to the field of rice breeding. Specifically, it relates to the application of a rice peroxidase OsPrx114 gene promoter, and more specifically, to the application of a rice peroxidase OsPrx114 gene promoter in response to waterlogging stress in rice. Background Art

[0002] Rice (Oryza sativa L.) is an annual grass in the Gramineae family and the Oryza genus. It is the main food crop for more than three billion people in the world and also a model plant for studying monocotyledonous plants. As a major food crop, rice not only has edible value but also certain economic and medicinal values. To understand the resistance mechanism of rice when facing environmental stress, we studied peroxidase in downstream genes. PRX peroxidase belongs to the antioxidant protein superfamily and is widely present in prokaryotes and eukaryotes, playing an important role in the growth and development of plants and in the face of various environmental stresses. For example, overexpression of the HvPrx3 gene in wheat makes wheat more resistant to wheat powdery mildew; the OsPrx30 gene in rice, under the regulation of the transcription factor OsATH30, maintains a high level of POD content in the body and reduces the hydrogen peroxide content to enhance the plant's resistance to Xanthomonas oryzae pv. oryzae; in rubber trees, the expression level of the HbPRX53 gene is significantly upregulated after infection with powdery mildew, and it is also upregulated under the induction of abscisic acid, jasmonic acid, and hydrogen peroxide. Previous studies have found that peroxidase also has a certain effect on abiotic stress. For example, ATPRX3 in Arabidopsis thaliana plants has a positive regulatory effect under drought and high-salt stresses; in sugarcane, salt stress can specifically induce the expression of some PRX genes in sugarcane. Existing studies have shown that the OsPrx114 gene can reduce the resistance of rice to Xanthomonas oryzae pv. oryzae, but its role in the response to abiotic stress is still unclear. Therefore, it is of great significance to explore the molecular mechanism of the OsPrx114 promoter in response to waterlogging stress and cultivate new varieties. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of a rice OsPrx114 promoter in response to waterlogging stress in rice.

[0004] Based on this, the first aspect of the present invention provides the application of a rice peroxidase OsPrx114 gene promoter, and the application includes: making rice adapt to waterlogging stress by changing the expression level of the OsPrx114 gene promoter.

[0005] Preferably, the application further includes: cultivating rice varieties resistant to waterlogging stress by increasing the expression level of the OsPrx114 gene promoter.

[0006] Preferably, the nucleotide sequence of the promoter of the rice peroxidase OsPrx114 gene is as shown in SEQ ID No.1.

[0007] Furthermore, in the second aspect of the present invention, there is provided an application of a biological material containing the promoter of the rice peroxidase OsPrx114 gene in improving the flood resistance of rice, and the biological material is selected from any one or more of a to f:

[0008] a: An expression cassette of a nucleic acid molecule containing the nucleotide sequence shown in SEQ ID No.1;

[0009] b: An expression vector containing the nucleotide sequence shown in SEQ ID No.1;

[0010] c: Containing the expression cassette a described in a;

[0011] d: A microorganism containing the expression vector described in b;

[0012] e: Containing the expression cassette described in a;

[0013] f: A cell containing the expression vector described in b.

[0014] In the third aspect of the present invention, there is provided a method for cultivating a rice variety tolerant to flood stress, that is, by transgenic technology, the expression level of the promoter of the rice OsPrx114 gene is increased.

[0015] The beneficial effects of the present invention: By analyzing the promoter sequence of the OsPrx114 gene, it is found that there are multiple stress response elements, and it is predicted that this promoter may participate in flood stress through the response elements. After stress treatment, under the condition of complete submersion to simulate flood stress treatment, the expression activity of the reporter gene GUS in the GUS material is significantly reduced compared with the control group, indicating that OsPrx114-pro is a promoter induced by flood stress. Therefore, the OsPrx114 promoter of the present invention has good application prospects in responding to rice flood stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a diagram of the analysis result of the OsPrx114 promoter sequence in Example 1 of the specific embodiment of the present invention;

[0017] Figure 2 It is an electrophoresis diagram of the cloning of the OsPrx114 promoter in Example 2 of the specific embodiment of the present invention;

[0018] Figure 3 It is a GUS staining analysis diagram of transgenic rice under the condition of submersion treatment in Example 3 of the specific embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0019] To make the above objects, features and advantages of the present invention more obvious and understandable, the following detailed description of the specific embodiments of the present invention is provided. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter range described in the present invention. Reasonable changes derived therefrom are still within the protection scope of the claims of the present invention.

[0020] It should be noted that the endpoints and any values within the ranges disclosed herein are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0021] The specific implementation manner of the present invention provides an application of the OsPrx114 promoter, which specifically includes the following embodiments:

[0022] Example 1

[0023] OsPrx114 Promoter Sequence Analysis

[0024] The OsPrx114 gene is a resistance regulator of rice to Xanthomonas oryzae pv. oryzae. Figure 1 Figure for the analysis result of the OsPrx114 promoter. In order to explore the influence of abiotic stress on the OsPrx114 promoter of rice, the inventor analyzed the promoter of rice OsPrx114 through PlantCARE, and then found many cis-elements related to stress response, such as GC-motif (related to hypoxia stress), ARE element (the key to anaerobic stress), and ABRE element (involved in abscisic acid response).

[0025] Example 2

[0026] Detection of GUS Transgenic Material Plants

[0027] In order to clarify the function of the OsPrx114 promoter in the flood stress response, the inventor constructed a promoter expression line of the OsPrx114 gene. In order to obtain transgenic materials, the inventor amplified the promoter sequence of the OsPrx114 gene from Nip seedlings and cloned it into a vector. Figure 2This is the electrophoresis diagram for the cloning of the OsPrx114 promoter. Among them: Lane M is Trans2KPlusⅡ DNA Marker, and lane 1 is the OsPrx114 promoter. The successfully constructed expression vector was transferred into embryogenic calli with the Nipponbare rice variety as the background through Agrobacterium-mediated genetic transformation to obtain OsPrx114-GUS materials. Transgenic positive plants were identified by GUS staining, and two lines, 21 and 35, were selected from the positive overexpression lines for subsequent experiments.

[0028] Vector construction and Agrobacterium transformation include the following steps:

[0029] (1) PCR amplification: Download the OsPrx114 promoter sequence from NCBI (https: / / www.ncbi.nlm.nih.gov / ), use the genomic DNA of Nipponbare as the template to amplify the OsPrx114 promoter fragment. The PCR reaction system is: Kod buffer (10 μL), dNTP (2 μL), primer - F / R (0.4 μL), Kod FX (0.2 μL), template (1 μL), supplemented with ddH2O to 20 μL. After mixing the system, place it in a PCR instrument. The reaction program is: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 1 min, 35 cycles, extension at 72°C for 7 min, and preservation at 10°C.

[0030] (2) Gel recovery: Recover the fragment through a DNA fragment recovery kit.

[0031] (3) Homologous recombination, transformation, and colony identification: Use homologous recombination to ligate the recovered OsPrx114 promoter fragment into the vector. The system is: infusion (2 μL), vector (1 μL), fragment (3 μL), supplemented with ddH2O to 10 μL. After mixing, react in a 37°C water bath for 15 min. Add the reaction solution to Escherichia coli competent cells. After ice-bathing for 30 min, heat-shock in a 42°C water bath for 90 s. After ice-bathing for 2 min, add 400 μL of SOC and mix well. Incubate in a 37°C / 230 rcf shaker for 1 h and then spread and culture on an LB solid medium. After colonies grow, select monoclonal colonies for colony PCR identification. Sequence the bacterial solution with correct bands, and select positive clones for bacteria preservation and plasmid extraction.

[0032] (4) The successfully constructed expression vector was transferred into calli with the Nipponbare rice variety as the background through Agrobacterium-mediated transformation to obtain GUS transgenic materials of OsPrx114.

[0033] (5) Positive identification by GUS tissue staining method: Take fresh samples of each tissue and soak them in GUS staining solution for more than 4 hours. After the staining is completed, elute with 75% ethanol for 48 hours and then observe the results.

[0034] Example 3

[0035] GUS staining analysis of transgenic rice under submergence treatment conditions

[0036] In this invention, two positive GUS lines (#21, #35) were selected. Submergence experiments were carried out on the GUS lines with a 14-day-old seedling age growing normally and the wild type. After the whole plant was completely submerged 10 cm underwater for 12 hours, the second leaf was subjected to GUS staining. After 7 hours of treatment, decolorization treatment was carried out with 75% ethanol, and the difference in the expression of the GUS reporter gene activity between the GUS plants and the wild type was observed to determine their submergence phenotypes. Figure 3 It is a GUS staining analysis diagram of transgenic rice under submergence treatment conditions. It can be seen that the OsPrx114 promoter can respond to flood stress, which provides new ideas for the subsequent cultivation of new flood-resistant rice varieties.

[0037] Unless otherwise defined, all terms, symbols, and other scientific terms used herein are intended to have the same meaning as commonly understood by those skilled in the art to which this invention belongs. In some cases, for the purpose of clarification or convenient reference, terms with conventional understood meanings are defined herein, and such definitions herein should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or cited herein are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise stated, the use of commercially available kits, reagents, and instruments follows the protocols and parameters given by the manufacturers.

[0038] Although the present disclosure is disclosed as above, the protection scope of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the protection scope of the present invention.

Claims

1. Application of a promoter of rice peroxidase OsPrx114 gene, characterized in that, The application includes: making rice adapt to flooding stress by changing the expression level of the OsPrx114 gene promoter.

2. Use of the promoter of rice peroxidase OsPrx114 gene as described in claim 1, characterized in that, The application further includes: cultivating rice varieties resistant to flooding stress by increasing the expression level of the OsPrx114 gene promoter.

3. The application of the promoter of rice peroxidase OsPrx114 gene according to claim 1, characterized in that, The nucleotide sequence of the promoter of the rice peroxidase OsPrx114 gene is as shown in SEQ ID No.

1.

4. Use of a biological material containing the promoter of rice peroxidase OsPrx114 gene in improving rice resistance to flooding stress, characterized in that, The biological material is selected from any one or more of a to f: a: an expression cassette containing a nucleic acid molecule with the nucleotide sequence shown in SEQ ID No. 1; b: an expression vector containing the nucleotide sequence shown in SEQ ID No. 1; c: containing the expression cassette a described in a; d: a microorganism containing the expression vector b described in b; e: containing the expression cassette described in a; f: a cell containing the expression vector b described in b.

5. A method for cultivating a rice variety tolerant to flooding stress, characterized in that, By transgenic technology, increase the expression level of the OsPrx114 gene promoter in rice.

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