Corn ZmPRX72 gene and application of encoded protein thereof in regulation and control of tolerance to low phosphorus stress

Abstract

The invention discloses an application of a corn ZmPRX72 gene and an encoded protein thereof in regulation and control of tolerance to low phosphorus stress. The polynucleotide sequence of the corn ZmPRX72 gene is as shown in SEQ ID No: 1, and the protein sequence of the corn ZmPRX72 gene is as shown in SEQ ID No: 2. The invention discovers that the relative fresh weights of the overground part and the root system of a ZmPRX72 overexpression plant are obviously increased, which indicates that the ZmPRX72 overexpression material has stronger tolerance to low phosphorus stress. The relative fresh weights of the overground part and the root system of a ZmPRX72 overexpressed plant are obviously higher than those of WT, which indicates that the tolerance of the plant to low phosphorus stress can be enhanced by the overexpression of ZmPRX72.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to maize. ZmPRX72 Application of genes and their encoded proteins in regulating tolerance to low phosphorus stress. Background Technology

[0002] As the core organ for plant anchoring growth and absorbing water and mineral nutrients, the root system's development directly determines a plant's stress resistance, nutrient utilization efficiency, and ultimate productivity, serving as a crucial guarantee for adapting to complex environments and completing its life cycle. Root development is a dynamic process regulated by genetics and involving the synergistic effects of hormonal and environmental signals. It encompasses a series of physiological events, including cell division and differentiation, apical growth, lateral root development, and root hair formation. Precise regulation of redox homeostasis plays an indispensable "molecular switch" role in this process. Reactive oxygen species (ROS), as a core component of the redox system, are not merely metabolic byproducts. Their spatiotemporal accumulation in specific regions of the root system can act as signaling molecules, regulating key aspects such as cell cycle progression, cell wall remodeling, and signal transduction. However, excessive ROS-induced oxidative stress can damage the structure of biological macromolecules, inhibit root growth, and even lead to apoptosis. Therefore, the production and clearance of ROS must maintain a strict dynamic balance.

[0003] Peroxidases (PRXs) are a superfamily of multifunctional redox enzymes in plants. Using heme as a cofactor, they catalyze the decomposition of peroxides such as H2O2, participate in the regulation of ROS homeostasis, and play a central role in physiological processes such as lignin synthesis, phenolic metabolism, and cell wall cross-linking. Based on their structural characteristics and catalytic properties, plant PRXs can be classified into several types, including extracellular secretory, cytoplasmic, and organelle-localized types. Among them, PRXs located in the cell wall or apoplast directly participate in the redox regulation and structural modification of the extracellular environment, becoming a key node connecting ROS signaling and root development. Existing studies have confirmed that PRX-mediated H2O2 scavenging can effectively alleviate root oxidative damage under abiotic stress, while the local ROS signaling catalyzed by PRX can regulate cell differentiation fate during the initiation of lateral root primordia. This "bidirectional regulation" characteristic suggests that PRXs may have a complex mechanism of action in the redox regulatory network of root development.

[0004] Phosphorus, an essential macronutrient for plant growth and development, is easily fixed in soil, resulting in low availability. Phosphorus stress has become one of the major abiotic stress factors limiting agricultural production. To cope with phosphorus-deficient environments, plants initiate root morphology remodeling strategies, including regulating the formation of lateral root primordia, the growth of taproots and lateral roots, changes in the angle of lateral root growth, and increasing root hair density. Previous studies have shown that ROS content in plant roots changes significantly under phosphorus stress, and ROS acts as a key signaling molecule involved in regulating root cell division and differentiation. PRX, as a core regulator of root ROS homeostasis, exhibits significant spatiotemporal specificity in its expression pattern and activity during phosphorus stress responses. Whether the PRX gene in maize regulates root development and participates in the response to low phosphorus stress remains to be further elucidated. Summary of the Invention

[0005] The purpose of this invention is to provide corn ZmPRX72 Application of genes and their encoded proteins in regulating tolerance to low phosphorus stress.

[0006] corn ZmPRX72 Genes, the corn ZmPRX72 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1 of the sequence listing; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating tolerance to low phosphorus stress; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating tolerance to low phosphorus stress.

[0007] The corn ZmPRX72 protein, wherein the amino acid sequence of the corn ZmPRX72 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating tolerance to low phosphorus stress.

[0008] Containing the corn ZmPRX72 The carrier of genes.

[0009] Containing the corn ZmPRX72 Engineered bacteria with gene vectors.

[0010] Amplify the corn ZmPRX72 Primers for any segment of a gene.

[0011] The corn ZmPRX72 Application of genes in regulating maize tolerance to low phosphorus stress.

[0012] A method to enhance maize's tolerance to low phosphorus stress by increasing the expression level of ZmPRX72 protein in maize.

[0013] Beneficial effects of the present invention: The present invention discovers ZmPRX72 The relative fresh weight of the aboveground parts and roots of the overexpressing plants was significantly increased, indicating that... ZmPRX72 Overexpression materials are more tolerant to low phosphorus stress. ZmPRX72 The relative fresh weights of the aboveground parts and roots of the overexpressing plants were significantly higher than the total weight (WT), indicating that... ZmPRX72 Overexpression can enhance the plant's tolerance to low phosphorus stress. Attached Figure Description

[0014] Picture 1 This is a schematic diagram of the CUB-ZmPRX72 carrier.

[0015] Picture 2 for ZmPRX72 Overexpression maize plants ZmPRX72 Gene expression level detection.

[0016] Picture 3 for ZmPRX72 The relative fresh weight of the aboveground parts and roots of maize under different phosphorus treatments was measured by overexpression. Detailed Implementation

[0017] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0018] The maize inbred lines used in the following examples were: B73 and KN5585; strains: Escherichia coli strain DH5α and Agrobacterium strain GV3101; and the overexpression vector was CUB.

[0019] Example 1: Construction of the recombinant vector CUB-ZmPRX72 Using B73 cDNA as a template, PCR amplification was performed using the upstream primer 5'-CGCGGGCCCGGGATCGATGGCAACATCCAGCACGGGC-3' and the downstream primer 5'-GGCAGCAGCCGGATCGTCAGTGGCCGCTGTTGAGCCT-3'. A DNA fragment of approximately 1300 bp was purified and recovered. ZmPRX72 Full-length sequence. The CUB vector was digested with the restriction endonuclease BamHI, and the DNA fragment was inserted into the linearized CUB vector via In-Fusion (Clontech, catalog number 639648) to obtain the recombinant vector CUB-ZmPRX72. A schematic diagram of the vector backbone is shown below. Picture 1 As shown.

[0020] Example 2 ZmPRX72 Obtaining maize materials through overexpression The embryos of maize inbred line KN5585 were transformed using Agrobacterium-mediated transformation. Following infection, co-culture, callus induction, screening, differentiation, and seedling formation, T0 generation seeds were harvested in a greenhouse. After further propagation in Hainan and Beijing, two homozygous transgenic maize lines were obtained. OE #1 and OE #2 Total RNA was extracted from the aboveground parts of OE #1, OE #2, and wild-type KN5585 seedlings, respectively. cDNA was obtained by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). This cDNA was then used as a template for quantitative real-time PCR detection. For quantitative real-time PCR, the primers for ZmPRX72 detection were 5'-AGGCTGTACAACCAGACAGGC-3' and 5'-GAAGTTGTCGAACTTGGCTGG-3'; ZmTub was used as an internal control for the maize gene, with primer sequences of 5'-ACACCACCATTGGGAGTCTA-3' and 5'-TTGTGGGGACCACTACTTTC-3'. Real-time quantitative PCR was performed using the Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific), with each experiment set to three replicates. -ΔΔCT The relative expression level was calculated using this method. The results are as follows: Picture 2 As shown, OE#1 and OE#2 Transgenic pure lines ZmPRX72 Gene expression levels were significantly increased compared to the wild type (KN5585).

[0021] Example 3 ZmPRX72Analysis of the response of maize overexpression materials to low phosphorus stress The experimental plants were: OE#1 , OE#2 T2 generation plants of the strain and wild-type KN5585 material.

[0022] Hydroponic conditions for corn: Select plump corn seeds of uniform size, soak them in 3% NaClO for 20 minutes, and then rinse them three times with distilled water. Soak the seeds in distilled water at room temperature for 6 hours, then place them on moist filter paper; incubate them in the dark at 28℃ for 2 days. When the radicle grows to 1 cm, roll the seedlings in filter paper and place them in distilled water for further cultivation, and cover them with a black plastic bag to block out light. When the corn seedlings have one leaf and one bud, select seedlings of uniform growth, remove the endosperm, and transfer them to a 3 L hydroponic container. First, treat them with a semi-nutrient solution for 2 days, then treat them with a full nutrient solution (250 μM PO4). 3- ) and low phosphorus nutrient solution (5 μM PO4) 3- Treatment: Change the nutrient solution every 2 days during hydroponics. The plant culture conditions are 14 h light / 10 h darkness, 28℃ / 22℃.

[0023] right ZmPRX72 Overexpression lines OE#1 , OE#2 Wild-type (WT) materials were subjected to normal and low-phosphorus hydroponic treatments for 7 days. The fresh weight of the aboveground parts and roots was measured, and the biomass ratio under low-phosphorus and normal-phosphorus conditions was calculated. The relevant results are as follows: Picture 3 As shown, ZmPRX72 The relative fresh weight of the aboveground parts and roots of the overexpressing plants was significantly increased, indicating that... ZmPRX72 Overexpression materials are more tolerant to low phosphorus stress.

[0024] ZmPRX72 overexpression lines (OE#1, OE#2) and wild-type (WT) materials were subjected to normal phosphorus and low phosphorus hydroponic treatments for 7 days. The fresh weight of the aboveground parts and roots was measured, and the biomass ratio under low phosphorus / normal phosphorus conditions was calculated. Results are as follows: Picture 3 show, ZmPRX72 The relative fresh weights of the aboveground parts and roots of the overexpressing plants were significantly higher than the total weight (WT), indicating that... ZmPRX72 Overexpression can enhance the plant's tolerance to low phosphorus stress.

[0025] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Corn ZmPRX72 Genes, characterized by, The corn ZmPRX72 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1 of the sequence listing; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under stringent hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating tolerance to low phosphorus stress; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating tolerance to low phosphorus stress.

2. Corn ZmPRX72 protein, characterized in that, The amino acid sequence of the corn ZmPRX72 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating tolerance to low phosphorus stress.

3. Containing the corn as described in claim 1 ZmPRX72 The carrier of genes.

4. Containing the corn as described in claim 3 ZmPRX72 Engineered bacteria with gene vectors.

5. The maize according to claim 1 ZmPRX72 Primers for any segment of a gene.

6. The corn according to claim 1 ZmPRX72 Application of genes in regulating maize tolerance to low phosphorus stress.

7. A method for enhancing maize's tolerance to low phosphorus stress, characterized in that, Increase the expression level of ZmPRX72 protein in maize.

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