Method for improving heat tolerance of maize anthers

Abstract

The application discloses a method for improving the heat tolerance of corn anthers, and the heat tolerance of the corn anthers can be improved by inhibiting the expression of a gene PRP2 (gene number Zm00001eb080380) in the corn, which indicates that the PRP2 gene is involved in the regulation of the heat stress response process of the corn anthers. The application provides an effective gene target for cultivating high-temperature-resistant corn varieties.

Description

Technical Field

[0001] This invention belongs to the field of agricultural biotechnology and relates to a method for improving the heat resistance of maize anthers, specifically a method for improving the heat resistance of maize anthers by inactivating the maize gene PRP2 (gene number Zm00001eb080380). Background Technology

[0002] Due to the influence of global climate change, the frequency of high-temperature heat damage is increasing. High temperatures have a significant impact on maize yield; for every 1°C increase in global temperature, maize yield will decrease by 7.4% (Zhao C, et al., Temperature increases reduce global yields of major crops in four independent estimates. Proceedings of the National Academy of Sciences of the United States of America. 2017(114):9326-9331.). my country's main maize producing areas include the northern region (approximately 40%), the Huang-Huai-Hai region (approximately 35%), and the southwestern mountainous region (approximately 18%). High-temperature heat damage in China is concentrated from May to September, with frequent occurrences in July and August. The growth and development period of summer maize in the Huang-Huai-Hai region, especially the reproductive growth period (June to September), highly overlaps with the timing of high-temperature heat damage. Compared to the vegetative growth period, the reproductive growth period of maize is more sensitive to high-temperature stress. Brief heat treatment during meiosis in maize anthers can severely impact pollen fertility and germination, leading to a significant decrease in maize seed setting rate (Begcy K, et al., Male Sterility in Maize after Transient Heat Stress during the Tetrad Stage of Pollen Development. Plant Physiol. 2019, 181(2):683-700.). Therefore, identifying and utilizing heat-resistant genes in maize anthers or pollen is of great significance for improving pollen fertility under heat stress and reducing maize yield loss under high temperatures.

[0003] Maize is a warm-season crop, with an optimal growth temperature of 22-30℃. While it can tolerate a certain degree of high temperatures, the reproductive and developmental processes of its anthers are quite sensitive and easily affected by various environmental factors. Therefore, breeding heat-resistant maize varieties using traditional methods is a lengthy and difficult process, requiring significant time and effort, and demanding high physical fitness from researchers. Currently, research on the mechanisms of heat stress response in maize anthers is limited, and the number of genes that can be used to improve heat stress resistance in maize anthers is even smaller. In recent years, researchers have begun to explore using genetic engineering techniques to improve heat stress resistance in maize seedlings. Identifying heat stress response genes using RNA-seq is an important method for discovering heat-resistant genes; however, traditional RNA-seq often uses tissues or organs as experimental materials, masking cellular heterogeneity and preventing the accurate identification of cell-type-specific heat stress response genes. Summary of the Invention

[0004] In our research on heat tolerance genes in maize, we identified a gene, PRP2 (gene number Zm00001eb080380), from maize anthers through single-cell transcriptome sequencing. This gene is involved in regulating the heat stress response of maize anthers. By prematurely terminating the protein coding of PRP2 by mutating a base in its CDS region using EMS, the heat tolerance of maize anthers was significantly improved. Pollen fertility almost returned to normal after heat treatment, without affecting other traits, demonstrating excellent potential for maize germplasm improvement. Based on these findings, this invention includes the following technical solution.

[0005] The first aspect of the present invention provides a method for improving the heat resistance of maize anthers by inhibiting the expression of the gene PRP2 (gene number Zm00001eb080380) in maize to enhance the heat stress resistance of maize anthers.

[0006] The coding region of the PRP2 gene, derived from maize B73, i.e., the CDS nucleotide sequence, is shown in SEQ ID NO:1:

[0007] ATGGCGTCCAAGGTTGAGCTGGTGGTGGAGGTCAAGTCCCCGGCTGACAAGCTGTGGGCGGCGCTGCGTGACTCGACGGAGCTGTTCCCCAAGATCTTCCCCGAGCAGTAC AAGAGCATCGAGACCGTCGAGGGCGACGGCAAGTCGGCCGGCACCGTCCGCCTCCTCAAGTACACCGAGGCGGTGCCGATGCTGACGTTCGCCAAGGAGAAGCTTGAGACGGCGGACGACGAACAAGGTGGTGTCGTACAGCGTGGTGGACGGCGAGCTGGCGGACTTCTACAAGAA CTTCAAGATCACGCTGAAGGTGACTCCGGCCAAGGCGGAGGGCGAGGGCGGCGCCGTCGTCAGCTGGGCCATGGAGTTCGACAAGGCCAACGACCAGGTGCCTGACCCGGACGTCATCAAGGAGACCGCCACCAAGACGTTTCCACGACCTCGACGACTACCTCCTCAAGAACTAG(SEQ ID NO:1).

[0008] The amino acid sequence of the protein PRP2 encoded by the gene PRP2 is shown in SEQ ID NO:2:

[0009] MASKVELVVEVKSPADKLWAALRDSTELFPKIFPEQY K SIETVEGDGKSAGTVRLLKYTEAVPMLTFAKEKLETADDENKVVSYSVVDGELADFYKNFKITLKVTPAKAEGEGGAVVSWAMEFDKANDQVPDPDVIKETATKTFHDLDDYLLKN (SEQ ID NO: 2).

[0010] Specifically, the inhibition of the PRP2 gene refers to the downregulation, inactivation, weakening or knockout of the PRP2 gene expression.

[0011] In one application implementation, the above method uses the gene PRP2 (gene number Zm00001eb080380) as a genetic resource for breeding heat-resistant maize varieties.

[0012] The aforementioned corn varieties include, but are not limited to, corn B73, KN5585, Zheng 958, Chang 72, Mo17, etc.

[0013] The above methods can be used to suppress PRP2 gene activity in the following ways:

[0014] (1) Knock out the PRP2 gene in the maize chromosome;

[0015] (2) Downregulate the expression level of gene PRP2 in maize chromosomes;

[0016] (3) Replace the PRP2 gene in the maize chromosome with a PRP2 mutant that has lost or downregulated coding function; and / or

[0017] (4) Block or interfere with the expression of the PRP2 gene in maize chromosomes.

[0018] Optionally, the above method (2) is selected from the following group:

[0019] (2-1) Mutations in the promoter region and / or coding region of the PRP2 gene lead to downregulation of the expression level of the PRP2 gene;

[0020] (2-2) Mutations in the upstream regulators of the PRP2 gene lead to a downregulation of PRP2 expression levels; or

[0021] (2-3) Introduce interacting proteins of protein PRP2 into maize to alter the function of the PRP2 gene.

[0022] The above method (3) can be selected from the following group:

[0023] (3-1) Base mutations occur in the coding region, resulting in the loss of function of the encoded protein PRP2;

[0024] (3-2) A frameshift mutation occurs in the coding region, resulting in the loss of function of the encoded protein PRP2.

[0025] For example, the above method (3-1) inactivates the PRP2 gene by mutating the base A to T at position 112 of the CDs nucleotide sequence SEQ ID NO:1 in the PRP2 coding region of the maize gene, that is, by mutating the codon AAG to TAG at position 38 of the protein PRP2 (lysine, K), which causes translation to terminate prematurely.

[0026] In the above methods, the inhibition of the gene PRP2 can be implemented through EMS mutation, gene editing technology, antisense nucleic acid, and transcriptional regulation.

[0027] The gene editing technologies mentioned above can be selected from the following group: homologous double crossover, TALEN system, CRISPR-Cas9 system, CRISPR-Cpf1 system, CRISPR-Cas12 system, CRISPR-BEST system, and MuGENT.

[0028] A second aspect of the present invention provides a method for identifying maize varieties with anther heat resistance, comprising the following steps:

[0029] Extract maize genomic DNA, sequence the maize gene PRP2, and / or

[0030] The determination was made to determine whether the protein expressed by the maize cells contained a polypeptide with the amino acid sequence shown in SEQ ID NO:2.

[0031] When the test results show that the coding region of the corresponding gene PRP2 in the maize genome, i.e., the CDS nucleotide sequence, is not SEQ ID NO:1, and / or the protein expressed by maize cells does not contain the protein PRP2 with the amino acid sequence shown in SEQ ID NO:2, it suggests that maize has a tendency to have high anther heat resistance, and this maize variety is regarded as a candidate variety with anther heat resistance.

[0032] In the above identification methods, when sequencing the PRP2 gene in maize, the following primer pairs are used for PCR amplification.

[0033] Forward primer ZmPRP2-EMSF: AGCCAGTCAGCCACATCAC (SEQ ID NO:4),

[0034] Reverse primer ZmPRP2-EMSR: CGCTACAGCAAACATCCGA (SEQ ID NO:5).

[0035] The nucleotide sequence of the PCR fragment obtained by PCR amplification of ZmPRP2-EMSF / ZmPRP2-EMSR using this primer is shown in SEQ ID NO:3:

[0036] AGCCAGTCAGCCACATCACTAGCTATTCTGTGCAGAGGCAATGGCGTCCAAGGTTGAGCTGGTGGTGGAGGTCAAGTCCCCGGCTGACAAGCTGTGGGCGGCGCTGCGTGACTCGACGGAGCTGTTCCCCAAGATCTTCCCCGAGCAGTAC A AGAGCATCGAGACCGTCGAGGGCGACGGCAAGTCGGCCGGCACCGTCCGCCTCCTCAAGTACACCGAGGGTACGGTACTACGTAACCATTCTTGGCTTGGCTAGCGCCTAGCGCCTCTTGGACTGGGACCTGAGTATCTCTTGCATATACATCGGATGTTTGCTGTAGCG (SEQ ID NO: 3).

[0037] In the above identification methods, the PCR amplification can be the PCR MIX method.

[0038] This invention is the first to discover the negative heat stress agronomic trait of the PRP2 gene. Experiments have shown that inactivation of the PRP2 gene in maize can improve the heat stress resistance of maize anthers. Therefore, this property of the PRP2 gene can provide a genetic strategy for improving the heat resistance of maize and breeding high-temperature tolerant maize varieties. Attached Figure Description

[0039] Figure 1 This image shows the identification of the PRP2 gene in maize anther epidermal cells in a specific negative response to heat stress. A: PRP2 expression was downregulated in the anthers after heat treatment; B: Single-cell UMAP plot of PRP2 expression levels; C: Image of epidermal-specific PRP2 expression in a negative response to heat stress.

[0040] Figure 2 The results showed that the pollen heat tolerance of the prp2 mutant was improved compared to that of maize B73. Detailed Implementation

[0041] Anther heat tolerance is an important indicator and agronomic trait of maize's resistance to high-temperature stress. High anther heat tolerance helps ensure stable or increased maize yield. In the investigation of heat tolerance genes / proteins in maize anthers, we subjected maize anthers in the meiotic stage to a 45℃ heat treatment for 24 h, using maize cultured at normal temperature (28℃) as a control. Single-cell transcriptome sequencing was performed on anthers, with two replicates for each treatment. First, marker genes for different cell types in anthers were screened using in situ hybridization. Five main cell types of anthers (epidermis, anther wall, mesonephros, tapetum, and pollen mother cells) were identified. Subsequently, differential gene analysis was performed on different cell types, revealing the PRP2 gene (gene ID: Zm00001eb080380) which was specifically downregulated in the epidermis under heat stress. qPCR and in situ hybridization experiments confirmed that PRP2 does indeed negatively respond to heat stress in maize anther epidermal cells. Figure 1 Therefore, PRP2 may be a negative regulator of the heat stress response of maize anthers, and we speculate that mutating it to inactivate it may improve the heat resistance of maize anthers.

[0042] Therefore, we obtained the PRP2 EMS mutant from the maize EMS mutant library and subjected the homozygous mutant plants to 45℃ heat treatment for 24 hours during the anther meiosis stage (anther length approximately 1.5–2.0 mm), using wild-type B73 as a control. After heat treatment, the plants were returned to normal temperature and cultured until pollen development matured, and pollen fertility was measured. The results showed that the pollen fertility of wild-type B73 decreased by approximately 40–50% after heat stress during anther meiosis, while the pollen fertility of the PRP2 mutant, which was inactivated under normal culture conditions, was almost unaffected. Furthermore, the pollen fertility of the PRP2 mutant was also almost unaffected after heat stress during anther meiosis. Figure 2 This indicates that mutating PRP2 improves the heat resistance of maize anthers.

[0043] The negative regulation of heat stress response by gene PRP2 suggests that gene PRP2 can be used as a genetic resource to create transgenic maize varieties with heat stress tolerance in anthers, i.e., heat-resistant transgenic maize varieties.

[0044] In this article, the term "wild type" refers to native plants such as maize B73 and KN5585 that have a normal growth phenotype and express the normal protein PRP2 gene (Zm00001eb080380).

[0045] Correspondingly, the terms "heat-resistant transgenic maize with anthers", "transgenic plant / maize", and "maize mutant" in this article have the same meaning, all referring to maize with improved anther heat resistance obtained by genetically modifying wild-type maize.

[0046] In some implementations, the terms “(heat resistance) improvement,” “enhancement,” or “increase” can mean an improvement of at least 10% compared to a reference level (such as normal plants), for example, an improvement of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including 100%, or any improvement between 10% and 100%, or an improvement of at least about 2 times, or at least about 3 times, or at least about 4 times, or at least about 5 times, or at least about 10 times compared to a reference level.

[0047] In this document, for the sake of simplicity, the name of a protein, such as protein PRP2, is sometimes used interchangeably with the name of its encoding gene (DNA). Those skilled in the art should understand that they refer to different substances in different descriptive contexts. Their meanings are readily understood by those skilled in the art based on the context. For example, when describing the function or class of the PRP2 protein, it refers to the protein itself; when describing it as a gene, it refers to the gene encoding the enzyme.

[0048] There are various techniques for inactivating, attenuating, and / or preventing the expression of the plant protein PRP2. These techniques can be used individually or in combination.

[0049] In the description of the technical solutions of this invention, the term "and / or" used in terms such as "A and / or B" or "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" used in phrases such as "A, B, and / or C" is intended to cover each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); C (alone).

[0050] On the other hand, identifying the protein PRP2 with a normal sequence or its encoding gene PRP2 has a positive effect on predicting the heat stress tolerance of maize anthers in advance.

[0051] The advantage of the aforementioned PRP2 gene identification scheme lies in the fact that it is possible to pre-assess the potential for anther heat stress resistance in candidate crops, such as maize varieties, solely in the laboratory. Since the entire life cycle of crops like maize is typically 4-5 months, examining their biological traits and phenotypes through field planting would normally require a significant amount of time and resources, including substantial land and labor costs. In contrast, the gene identification scheme can be completed in the laboratory, allowing for gene sequencing on seedlings as young as a few weeks old, or even directly on seeds. This significantly improves efficiency and substantially reduces time, space, and labor costs, resulting in substantial economic benefits.

[0052] The positive effects of this invention are mainly reflected in the following aspects:

[0053] (1) The heat tolerance of the anthers of the PRP2 mutant, which knocks out the PRP2 gene, is significantly improved. After inactivating the PRP2 gene, high-temperature treatment at 45℃ on maize anthers during meiosis does not affect the later pollen development, but the fertility of wild-type pollen decreases by 40-60%. The PRP2 mutant can be crossed with other high-yielding and heat-sensitive varieties to improve the heat tolerance of other heat-sensitive maize varieties.

[0054] (2) Using PRP2 for heat-resistant maize anther improvement is simple and suitable for large-scale operations. Maize hybridization and self-pollination techniques are relatively simple and can be mastered by ordinary workers. DNA is extracted from maize leaves using conventional DNA extraction methods, and then, using our designed PRP2 mutant detection primers, genotype identification can be easily achieved through ordinary PCR reactions and sequencing. The operation is simple, and the experimental methods and equipment are common and readily available.

[0055] (3) Using the mutant pRP2 to improve the heat resistance of maize anthers can greatly reduce costs. Compared with traditional maize genetic improvement, using pRP2 to improve the heat resistance of maize anthers is more effective, stable, time-saving, and labor-saving, thus greatly saving costs.

[0056] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the invention.

[0057] Example

[0058] The examples involve the addition amount, content and concentration of various substances, and unless otherwise specified, the percentage content refers to the mass percentage content.

[0059] In the embodiments, if the operating temperature is not specifically specified, it generally refers to room temperature (15-35°C).

[0060] Materials and methods

[0061] The primer synthesis and gene sequencing in the examples were all completed by Beijing Qingke Biotechnology Co., Ltd.

[0062] The self-pollination, hybridization, transgenic operations, and field breeding of maize are carried out in accordance with conventional breeding methods.

[0063] In this embodiment, the extraction of anther protoplasts and single-cell transcriptome sequencing were performed by Shanghai Meiji Biotechnology Co., Ltd. and Paisennuo Biotechnology Co., Ltd.

[0064] The molecular biology experiments in this article, including plasmid construction, enzyme digestion, competent cell preparation, and transformation, were mainly conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd Edition), edited by J. Sambrook and DW. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002. For example, the methods for competent cell transformation and competent cell preparation were based on Chapter 1, page 96 of *Molecular Cloning: A Laboratory Manual* (3rd Edition). Specific experimental conditions could be determined through simple experiments when necessary.

[0065] The molecular biology experiments in the examples, including PCR amplification, gel electrophoresis, plasmid construction, enzyme digestion, ligation, competent cell transformation, and culture medium preparation, were all performed according to the reaction conditions provided by the reagent supplier or the kit instructions. Adjustments can be made through simple experiments if necessary.

[0066] It should be noted that, for the sake of convenience, in the embodiments, the plant number and the gene number can share the same number, which is easily understood by those skilled in the art, that is, the same number can refer to different biological forms in different environments.

[0067] Example 1: Single-cell transcriptome sequencing and data analysis before and after heat treatment during maize anther meiosis.

[0068] Maize B73 anthers at the meiotic stage (approximately 1.5 mm) were cultured for 24 hours in a heat-treated culture chamber (45℃ daytime / 35℃ nighttime, 12000 lux, 14h light / 10h darkness, 60% humidity). Anthers cultured under normal conditions at the meiotic stage served as a control. After treatment, anthers were harvested for single-cell sequencing, with two biological replicates for each treatment. Fresh maize anthers were placed in 0.4M mannitol solution to extract anther protoplasts. After cell quality control, single-cell transcriptome libraries were constructed using the 10X Genomics platform (this part was completed by Shanghai Meiji Biotechnology Co., Ltd.). Using single-cell analysis R packages such as cellranger, seurat, and hdWGCNA, cell filtering, standardization, normalization, dimensionality reduction, and clustering analyses were performed on the obtained single-cell data to determine cell groups. Based on existing cell type-specific marker genes and the functions of cell groups, the main cell types of maize anthers during meiosis were identified. Differential gene analysis (FindAllmarker() and Findmarker()) functions were used to screen for differentially expressed genes in response to heat stress in different cell types.

[0069] Example 2: In situ hybridization verification of marker genes and PRP2 heat stress response in different cell types of maize anthers. In situ hybridization technology was used to verify the expression specificity of the selected candidate marker genes or PRP2.

[0070] The in situ hybridization procedure was slightly modified from that of Jackson et al. (1994, 2002). The procedure can be summarized as follows: Fresh anthers (approximately 1.5 mm) at the meiotic stage were fixed in 3.7% FAA (Formalin-Aceto-Alcohol) fixative for both the control and heat-treated groups. After dehydration, clearing, and paraffin impregnation, the anthers were fixed in solid paraffin for sectioning. Probes for candidate genes were synthesized in vitro using a DIG RNA Labeling Kit (SP6 / T7) (Roche, Basel, Switzerland), and then purified for later use. Sections were prepared, dried, and spread. The synthesized probes were mixed with hybridization solution and hybridized with the tissue sections at 50°C for 16–20 h. After hybridization, the hybridization solution was washed off, and the sections were blocked and stained for 1–3 days. After staining, the sections were rinsed and photographed under a microscope for recording.

[0071] Example 3: qPCR verification of downregulation of PRP2 gene expression under anther heat stress

[0072] Maize anthers were collected before and after heat treatment, with three biological replicates in each control and treatment group. Total RNA was extracted from the anthers and then reverse transcribed into cDNA. Using maize UBI as an internal reference gene, the expression levels of PRP2 before and after anther heat stress were verified by qPCR. Total RNA extraction and reverse transcription were performed according to the Direct-zol RNA Kits|Microprep (Zymo Research, USA) and [other kits / methods]. The One-Step gDNA Removal and cDNA Synthesis SuperMix (Trugen) procedure was followed. The qPCR reaction was first prepared according to the ChamQ Blue Universal SYBR qPCR Master Mix kit (Novizan), and then the reaction was performed using a real-time PCR instrument to detect the Ct value. The maize UBI gene was used as an internal control gene. The qPCR primer designs for UBI and PRP2 are shown in Table 1.

[0073] Table 1. List of PCR primers

[0074]

[0075] In Table 1, "-F" in the name represents positive; "-R" represents negative.

[0076] Example 4: Obtaining and Genotyping PRP2 Mutants

[0077] The ppr2EMS mutant with inactivated PRP2 gene was purchased from the maize EMS mutant library (http: / / maizeems.qlnu.edu.cn / ). The purchased mutants included three genotypes: AA, Aa, and aa. To identify homozygous mutants, genomic DNA was first extracted from leaves of individual plants using the CTAB method. Primers were then designed to clone a 323 bp sequence upstream and downstream of the mutation site. Finally, the sequences were sent to the company for sequencing to determine the genotype of the ppr2 mutant.

[0078] The PRP2 gene in the prp2 mutant is inactivated by a mutation of A to T at position 112 of the coding region, i.e., the CDS nucleotide sequence SEQ ID NO:1. This mutation results in the inactivation of the PRP2 gene, i.e., the codon AAG to TAG at the 38th amino acid Lys (K) encoding the protein PRP2, leading to premature termination of translation.

[0079] Example 5: Observation of the heat tolerance phenotype of maize pprp2 mutant

[0080] To investigate the phenotype of the prp2 mutant in response to heat stress, we subjected the prp2 mutant and B73 maize in the anthers during meiosis to heat treatment (45℃ / 35℃, 14h / 10h, 12000lux / 0lux) for 24h. After treatment, the plants were returned to the normal environment for cultivation, and then mature pollen was collected for fertility determination.

[0081] The simplified procedure for pollen fertility testing is as follows: Place mature but not yet released anthers in Carnoy's fixative (ethanol:acetic acid = 3:1). Take nine superior flowers and place them in 500 μL of sterile water. Add glass beads and grind the flowers at low speed (20 Hz, 20–40 s). Centrifuge at low speed or allow the pollen to settle naturally. Remove 400 μL of supernatant and add 10–20 μL of potassium iodide, mixing thoroughly. Transfer 40 μL of the pollen mixture to a glass slide and take images from five fields of view using a microscope. Calculate the number of fertile and infertile pollen cells in each image using ImageJ. See also... Figure 1 and Figure 2 .

[0082] Example 6: Extraction of RNA from Maize Anthers

[0083] Total RNA extraction from maize anthers was performed according to the procedures outlined in the Direct-zol RNA Kits|Microprep (Zymoresearch, USA). A brief summary of the procedure is as follows: Anther tissue was flash-frozen in liquid nitrogen and then ground using a sample grinder; 500 μL of TRI Reagent solution was added, mixed, and centrifuged at 12000 rpm for 1 min. The supernatant was transferred to a clean centrifuge tube, and an equal volume of 100% ethanol was added and mixed. The entire mixture was then transferred to a Zymo-Spin containing a collection tube. TM Centrifuge at 12000 rpm for 1 min in an ICColumn centrifuge column, discard the filtrate; add 400 μL RNA Wash Buffer to the centrifuge column, centrifuge at 12000 rpm for 1 min, discard the filtrate; add DNase I to digest genomic DNA; add 400 μL Direct-zol TM Add RNAPreWash to a centrifuge column, centrifuge at 12000 rpm for 1 min, and discard the filtrate; add 700 μl RNA Wash Buffer, centrifuge at 12000 rpm for 1 min, and discard the filtrate; add 15 μl DNase / RNase-Free Water to a centrifuge column, and centrifuge at 12000 rpm for 1 min to elute RNA.

[0084] Example 7: Extraction of maize genomic DNA

[0085] Genomic DNA was extracted from maize leaves using the CTAB method. The specific operating steps are as follows: Take a small amount of tender leaves into a clean centrifuge tube, add steel balls, and quickly freeze in liquid nitrogen. Grind the sample into powder using a grinder. Add 300 μL of CTAB lysis buffer containing 2% β-mercaptoethanol, mix well, and incubate at 65°C for 1 hour, mixing every 15 minutes. Cool the mixture at room temperature, add 400-500 μL of a chloroform:isoamyl alcohol (volume ratio 24:1) mixture, and mix by inversion. Centrifuge at 10,000 rpm for 10 minutes. Transfer the supernatant to a new clean centrifuge tube, add an equal volume of isopropanol, and incubate at -20°C for 30 minutes to precipitate DNA. Centrifuge at 10,000 rpm for 10 minutes. Remove the supernatant, add 70% ethanol to wash the precipitate, and centrifuge at 10,000 rpm for 5 minutes. Repeat this step once. After removing 70% of the ethanol, open the centrifuge tube and place it in a 50°C oven to thoroughly dry the remaining ethanol. Add 1xTE or sterile water to dissolve the precipitate.

[0086] Results and Discussion

[0087] qPCR and in situ hybridization results showed that PRP2 was specifically highly expressed in maize anther epidermal cells under normal temperatures, while its expression was significantly downregulated under high temperatures. Figure 1This indicates that PRP2, specifically expressed in the epidermis, negatively regulates the anther heat stress response. To further verify the function of PRP2 in the anther heat stress response, we performed genetic functional verification using the EMS mutant prp2. Under normal temperatures, the pollen fertility of the prp2 mutant was unaffected, but under high-temperature conditions, the pollen fertility of prp2 was higher than that of the wild type. Figure 2 The results indicate that PRP2 negatively regulates pollen heat tolerance. Therefore, knocking out PRP2 in maize can enhance pollen heat tolerance.

[0088] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention.

Claims

1. A method for improving the heat resistance of maize anthers, characterized in that, Heat tolerance of maize anthers was enhanced by inhibiting the expression of the gene PRP2 (gene number Zm00001eb080380) in maize.

2. The method as described in claim 1, characterized in that, PRP2 gene inhibition refers to the downregulation, inactivation, weakening or knockout of PRP2 gene expression.

3. The method as described in claim 1, characterized in that, The gene PRP2 (gene number Zm00001eb080380) was used as a genetic resource to breed heat-resistant maize varieties.

4. The method as described in claim 1, characterized in that, The corn varieties mentioned include, but are not limited to, corn B73, KN5585, Zheng 958, Chang 72, and Mo17.

5. The method as described in claim 1, characterized in that, PRP2 gene inhibition is implemented in the following manner: (1) Knock out the PRP2 gene in the maize chromosome; (2) Downregulate the expression level of gene PRP2 in maize chromosomes; (3) Replace the PRP2 gene in the maize chromosome with a PRP2 mutant that has lost or downregulated coding function; and / or (4) Block or interfere with the expression of the PRP2 gene in maize chromosomes.

6. The method as described in claim 5, characterized in that, Method (2) is selected from the following group: (2-1) Mutations in the promoter region and / or coding region of the PRP2 gene lead to downregulation of the expression level of the PRP2 gene; (2-2) Mutations in the upstream regulators of the PRP2 gene lead to a downregulation of PRP2 expression levels; or (2-3) Introduce interacting proteins of protein PRP2 into maize to alter the function of the PRP2 gene.

7. The method as described in claim 5, characterized in that, Method (3) is selected from the following group: (3-1) Base mutations occur in the coding region, resulting in the loss of function of the encoded protein PRP2; (3-2) A frameshift mutation occurs in the coding region, resulting in the loss of function of the encoded protein PRP2.

8. The method as described in claim 7, characterized in that, The method (3-1) involves mutating the base A to T at position 112 of the CDS nucleotide sequence SEQ ID NO:1 in the PRP2 coding region of the maize gene, thereby inactivating the PRP2 gene.

9. The method as described in claim 1, characterized in that, The inhibition of the PRP2 gene is achieved through EMS mutation, gene editing technology, antisense nucleic acid, or transcriptional regulation.

10. A method for identifying maize varieties with anther heat tolerance, characterized in that, Includes the following steps: Extract maize genomic DNA and sequence the maize gene PRP2; and / or The determination was made to determine whether the protein expressed by the maize cells contained a polypeptide with the amino acid sequence shown in SEQ ID NO:

2. When the test results show that the coding region of the corresponding gene PRP2 in the maize genome, i.e., the CDS nucleotide sequence, is not SEQ ID NO:1, and / or the protein expressed by maize cells does not contain the protein PRP2 with the amino acid sequence shown in SEQ ID NO:2, it suggests that maize has a tendency to have high anther heat resistance, and this maize variety is regarded as a candidate variety with anther heat resistance.

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