OsERF69 gene for regulating heat tolerance of rice and application of protein thereof

Abstract

The application discloses an OsERF69 gene for regulating heat tolerance of rice and application of a protein thereof, and belongs to the technical field of molecular biology. The application first finds that the OsERF69 gene has a function of negatively regulating heat tolerance of rice; by reducing the expression amount of the OsERF69 gene or using a gene editing technology to knock out the gene, the heat tolerance of rice can be significantly improved. High-temperature stress experiments show that the expression of the OsERF69 gene is significantly inhibited by high temperature, and two independent os erf69 gene knockout mutants both show a survival rate significantly higher than that of a wild type after high-temperature stress in a seedling stage. The application provides a new gene target and a breeding strategy for genetic improvement of heat tolerance of rice, can be applied to improving yield and stability of rice under high-temperature stress, and has important application value in agricultural production.

Description

Technical Field

[0001] This application belongs to the field of molecular biology technology, and relates to the application of the OsERF69 gene and its protein in regulating rice heat resistance. Background Technology

[0002] Rice (Oryza sativa L.) is one of the world's most important food crops, feeding more than half of the world's population. With the intensification of global warming and the increasing frequency of extreme heat events, heat stress has become one of the major abiotic stress factors restricting rice yield and quality. During the vegetative growth stage, high temperatures cause leaf wilting, decreased photosynthetic efficiency, and stunted growth; while during the reproductive growth stage, especially during heading and flowering (daily average temperature exceeding 35℃ or daily maximum temperature exceeding 38℃), high temperatures lead to decreased pollen viability, floret sterility, and a significant reduction in seed setting rate, ultimately resulting in substantial yield reductions. It is estimated that for every 1℃ increase in global average temperature, rice yield will decrease by approximately 3%-10%. Therefore, elucidating the molecular regulatory mechanisms of rice heat tolerance, identifying key heat-tolerant genes, and cultivating heat-tolerant rice varieties through molecular breeding techniques are of significant strategic importance for ensuring national food security.

[0003] APETALA2 / ethylene response factors (AP2 / ERF) are a large superfamily of transcription factors widely distributed in plants. Each member contains one or two highly conserved AP2 / ERF domains, which can specifically bind to GCC-boxes or DRE / CRT cis-acting elements in the promoter regions of downstream target genes, thereby regulating the transcriptional expression of these genes. Based on the number and sequence characteristics of AP2 / ERF domains, this superfamily can be further divided into five subfamilies: AP2, ERF, DREB, RAV, and Soloist. Among them, the ERF subfamily has the largest number of members and the most diverse functional differentiation, widely involved in the regulation of plant growth and development, hormone signal transduction, and responses to biotic and abiotic stresses. In rice, more than 130 ERF family members have been identified, and some members have been reported to participate in responses to abiotic stresses such as drought, salinity, low temperature, and high temperature. However, current research on the regulation of rice heat tolerance by ERF transcription factors is still very limited. The functions of the vast majority of ERF members in rice heat tolerance have not yet been revealed, and further in-depth exploration and systematic identification are urgently needed to enrich the gene resources available for rice heat tolerance breeding.

[0004] OsERF69 (Oryza sativa Ethylene Response Factor 69) is a member of the rice ERF subfamily. Currently, there are no reports on the function of the OsERF69 gene in rice heat tolerance. Summary of the Invention

[0005] This application provides an application of the OsERF69 gene and its protein for regulating heat resistance in rice, aiming to solve the problem of the lack of key heat resistance genes in existing hot-resistant rice breeding processes.

[0006] This application is the first to discover and demonstrate that the OsERF69 gene is a negative regulator of heat tolerance in rice. Specifically, the expression of the OsERF69 gene is significantly inhibited by high temperature stress; two independent loss-of-function mutants (oserf69-1 and oserf69-2) constructed using CRISPR / Cas9 technology both showed significantly higher survival rates than the wild type under high temperature stress during the seedling stage.

[0007] Based on the above findings, this application provides the following technical solution:

[0008] This application first provides an application of the OsERF69 gene, which regulates heat tolerance in rice, in regulating plant heat tolerance. The OsERF69 gene is the rice ERF69 gene.

[0009] In some embodiments, the nucleotide sequence of the OsERF69 gene is shown in SEQ ID NO.1.

[0010] In some embodiments, the amino acid sequence of the protein encoded by the OsERF69 gene is shown in SEQ ID NO.2.

[0011] In some embodiments, the plant is a grass crop, and more specifically, the plant is one of rice, wheat, corn, barley, sorghum, or millet; preferably, the plant is rice.

[0012] In some embodiments, the OsERF69 gene negatively regulates plant heat tolerance, i.e., plant heat tolerance can be improved by reducing the expression level of the OsERF69 gene or knocking out the OsERF69 gene.

[0013] In some embodiments, this application provides a method for improving plant heat resistance, specifically by: reducing the expression level of the OsERF69 gene in the plant or selectively knocking out the OsERF69 gene in the plant, thereby silencing or downregulating the expression level of the OsERF69 gene; or causing all or part of the biological function of the protein encoded by the OsERF69 gene to be lost. The plant is a gramineous crop, and more specifically, the plant is one of rice, wheat, corn, barley, sorghum, or millet; preferably, the plant is rice.

[0014] More preferably, the targeted knockout is performed using gene editing modification.

[0015] In some embodiments, this application provides the application of the above-described method in increasing rice yield under high-temperature stress.

[0016] In some embodiments, this application provides the application of the OsERF69 gene in crop heat tolerance genetic breeding. The resulting heat-tolerant crop varieties can effectively increase crop yield under high-temperature stress.

[0017] The crop having an organism is a grassy crop, which is one of rice, wheat, corn, barley, sorghum or millet; preferably, the crop is rice.

[0018] In some embodiments, the nucleotide sequence of the OsERF69 gene is shown in SEQ ID NO.1.

[0019] In some embodiments, the amino acid sequence of the protein encoded by the OsERF69 gene is shown in SEQ ID NO.2.

[0020] This application reveals for the first time the function of the OsERF69 gene in negatively regulating heat tolerance in rice. Targeted knockout of the OsERF69 gene using gene editing technology can effectively enhance rice heat tolerance, providing important genetic resources and application strategies for heat tolerance genetic breeding in crops, and possessing significant agricultural application value.

[0021] Compared with the prior art, this application has the following beneficial effects:

[0022] This application is the first to discover and demonstrate that the OsERF69 gene is a negative regulator of heat tolerance in rice. On the one hand, the expression of the OsERF69 gene is significantly inhibited by high-temperature stress, indicating that plants activate their own heat tolerance response by downregulating this negative regulator under high-temperature stress. On the other hand, two independent loss-of-function mutants (oserf69-1 and oserf69-2) constructed using CRISPR / Cas9 technology both exhibited significantly higher survival rates than the wild type under high-temperature stress during the seedling stage, and the phenotypes of the two mutants were highly consistent, ruling out off-target effects or site effects. This strongly demonstrates that the loss of function of the OsERF69 gene is the direct cause of enhanced heat tolerance. These results indicate that targeted knockout of the OsERF69 gene using gene editing technology can effectively improve the heat tolerance of rice, and has important application value in crop heat tolerance genetic breeding and agricultural production practices. Attached Figure Description

[0023] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0024] Figure 1 Figure 1 shows the expression patterns of the OsERF69 gene in different rice tissues. The figure shows the relative expression levels of the OsERF69 gene in rice roots, stems, leaves, leaf sheaths, and young panicles under normal growth conditions.

[0025] Figure 2 Figure 1 shows the expression pattern of the OsERF69 gene under high temperature stress. The figure shows the relative expression level of the OsERF69 gene in rice seedling leaves at different time points (0 h, 1 h, 6 h) under 45℃ high temperature stress treatment, indicating that the expression of the OsERF69 gene is continuously downregulated with the extension of high temperature stress time.

[0026] Figure 3 Figure 1 shows the construction and identification results of the OsERF69 gene-editing mutants. It includes a schematic diagram of the CRISPR / Cas9 target design, illustrating the OsERF69 gene structure and the gRNA target sites of two independent mutants (oserf69-1 and oserf69-2); and Sanger sequencing results of the target sites for the two mutants, showing the base deletion mutations occurring at the target sites in oserf69-1 and oserf69-2 and their alignment with the wild-type sequence.

[0027] Figure 4 Figure 1 shows the phenotypic identification results of the oserf69-1 mutant under high temperature stress. The figure shows the phenotypic photos of wild type (WT) and oserf69-1 mutant before and after treatment with 45℃ high temperature stress and after 7 days of recovery.

[0028] Figure 5 Figure 1: Statistical results of high temperature stress survival rate of oserf69-1 mutant. The figure shows the comparison of survival rates of wild type (WT) and oserf69-1 mutant after treatment with 45℃ high temperature stress and subsequent recovery of growth.

[0029] Figure 6 Figure 1 shows the phenotypic identification results of the oserf69-2 mutant under high temperature stress. The figure shows the phenotypic photos of wild type (WT) and oserf69-2 mutant before and after treatment with 45℃ high temperature stress and after 7 days of recovery.

[0030] Figure 7Figure 1: Statistical results of high temperature stress survival rate of oserf69-2 mutant. The figure compares the survival rates of wild-type (WT) and oserf69-2 mutant after treatment with 45℃ high temperature stress and subsequent recovery of growth.

[0031] Figure 8 Figure: Results of detection of relative RNA expression level of ERF69. Detailed Implementation

[0032] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of this application, and not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of this application.

[0033] Furthermore, the technical solutions of the various embodiments can be combined with each other, but only if they are based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed in this application.

[0034] Current research on plant heat tolerance mainly focuses on the plant's survival ability, growth recovery ability, and yield maintenance ability after being subjected to high-temperature stress. Plant heat tolerance is a complex quantitative trait involving multiple levels of molecular and physiological-biochemical mechanisms, including the induction of heat shock protein expression, activation of reactive oxygen species (ROS) scavenging systems, regulation of hormone signaling, and maintenance of cell membrane stability. Under high-temperature stress, proteins in plant cells denature or misfold. Heat shock transcription factors (HSFs) are activated after sensing stress signals, thereby inducing the large-scale expression of downstream heat shock proteins (HSPs). As molecular chaperones, heat shock proteins can help denatured proteins refold correctly or degrade irreparable proteins, thus maintaining intracellular protein homeostasis, which is one of the core mechanisms of plant heat tolerance. In addition, reactive oxygen species (ROS) accumulate in large quantities under high-temperature stress. If they cannot be cleared in time, they will cause severe oxidative damage to cell membrane systems, proteins, and nucleic acids. Therefore, the ability to scavenge ROS is also an important factor determining the strength of plant heat tolerance. Transcription factors play a crucial role in the plant heat tolerance regulatory network. They regulate the expression of a series of downstream heat tolerance-related functional genes, integrate multiple signaling pathways, and coordinate the plant's systemic response to high temperature stress.

[0035] Rice is one of the most important food crops globally and a crucial model plant for functional genomics research in monocotyledonous plants. This application uses rice as the research material for functional verification. In the absence of contrary experimental evidence, the molecular mechanism by which the OsERF69 gene negatively regulates plant heat tolerance, as revealed in this application, is expected to be applicable to various plant species, especially gramineous food crops, including but not limited to wheat, maize, barley, sorghum, and millet.

[0036] The core experimental results of this application are summarized as follows: The rice OsERF69 gene exhibits a specific high expression pattern in leaves and leaf sheaths, showing significant tissue expression specificity; the phenotypic identification results of high temperature stress during the seedling stage show that, compared with wild-type indica rice variety Nanjing 11, the survival rate of the two knockout mutants, erf69-1 and erf69-2, was significantly increased after high temperature stress during the seedling stage.

[0037] This application further illustrates the technical solution through the following embodiments, but no embodiment or combination thereof should be construed as limiting the scope of protection or implementation of this application. In the following embodiments, unless otherwise specified, the experimental methods used are conventional experimental methods in the art; the experimental materials and reagents used, unless otherwise specified, can be obtained through commercial channels.

[0038] Example 1: Obtaining Experimental Materials

[0039] (1) The gene editing vector PMC-OsR41-ccdB can be obtained by the public from the Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences.

[0040] (2) Agrobacterium EHA105 can be obtained by the public from the Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences.

[0041] (3) Rice varieties: Nanjing 11, an indica rice variety, can be obtained by the public from the Shenzhen Institute of Agricultural Genomics, Chinese Academy of Agricultural Sciences.

[0042] (4) Two specific gRNAs were designed using CRISPR / Cas9 gene editing technology and cloned into the editing vector. The vectors were then sent to the company for transformation. T0 generation plants were obtained for identification and finally verified by qRT-PCR to be ERF69 deletion mutants.

[0043] OsERF69 gene sequence information: Total RNA was extracted from leaves of rice variety Nanjing 11, reverse transcribed, and after sequence analysis and functional verification, the full-length coding sequence of the OsERF69 gene was obtained (SEQ ID NO.1). The amino acid sequence of the protein it encodes is shown in SEQ ID NO.2.

[0044] Nucleotide sequence of the OsERF69 gene (full-length nucleotide sequence encoded by CDS, SEQ ID NO.1):

[0045] ATGTGTGGCGGCGCGATTCTGGCTAACATCATACCGGCCACGCCGCCGCGGCCCGCCACGGCGGCGCATGTGTGGCCCGGCGGCGACGGGGAGAAGCGGCGGAAGGTTGGTGGAGGCGGGTGTGATGACGACTTCGAGGCGGCGTTTGAGAGATTCGGACGTGAGGACTCTGAGATGGAGGAGGAGGAGGTGGAGGAGGTGGTGGTTGGGAAGAAGGCGGCGGTGAGGCGGCGGAGGGCGACGCCCGCCGCCGGGCGCCGCGCGAGGCCGAGCAAGTACTGGGGCGTGCGGCGCCGGCCGTGGGGGAAGTGGGCGGCGGAGATCCGCGACCCCGTCGAGGGCGTCCGCGTCTGGCTCGGCACGTTCGCCACCGCCGAGGCCGCCGCCCACGCCTACGACGCCGCCGCCCGCGACCTCCGCGGCGCGACCGCCAAGCTCAACTTCCCCTCCTCCTCCTCCTCCACCGCCGCCACCCCACGCCCCCGCAAGTGCCGCCCCACCACCGCCACCGCCACCCCCAAGGCGACGACACCGAACGTCGTCGTCGTCGTCAACCTCGTCGACAAAGAGGCCGAGGTCAGCGAGAGCTCCGGTGCCAGCAGCAGCGCGCTGCCGGACTTCTCGTGGCAGGGCATGTCGGCGTCGTCCGACGACGACGCCGCGGCGCAGCAGGCACTCCTCGACGCCGCCGGCGGCGCCAAGAAGCGTCCCCGGAGCGAGCCCCACGTCACCTCCGACGACGAAGTGCTCCCGGCGTCATTCGACAGTGACAACAACACCGCCGCCGCCGGCCTGCTCCCGCTCGACGATCCTTTCTTGTTCGGCGACCAGTTCGGCGACCTCAACGGCGGCGCGTTCGCCTCGCTCATGGACGGGCTGTTCGCCGCCGGTGAAGCGAACGTCGCCGGCGAGAGCGTGGGGCTCTGGAGCTTCGGCGACGACTTTCTCAACGCGTCGTACTATTAG。

[0046] The amino acid sequence of the protein encoded by the OsERF69 gene (SEQ ID NO.2):

[0047] MCGGAILANIIPATPPRPATAAHVWPGGDGEKRRKVGGGGCDDFEAAFERFGREDSEMEEEEVEEVVVGKKAAVRRRRATPAAGRRARPSKYWGVRRRPWGKWAAEIRDPVEGVRVWLGTFATAEAAAHAYDAAARDLRGATAKLNFPSSSSSTAATPRP RKCRPTTATATPKATTPNVVVVVNLVDKEAEVSESSGASSSALPDFSWQGMSASSDDDAAAQQALLDAAGGAKKRPRSEPHVTSDDEVLPASFDSDNNTAAAGLLPLDDPFLFGDQFGDLNGGAFASLMDGLFAAGEANVAGESVGLWSFGDDFLNASYY.

[0048] Example 2 Construction of OsERF69 gene knockout material

[0049] In the experiment, two independent gene knockout materials, erf69-1 and erf69-2, were constructed using CRISPR / Cas9 gene editing technology. The mutation forms of the genes were confirmed by Sanger sequencing (e.g., Figure 3 ).

[0050] Example 3 RNA extraction and reverse transcription

[0051] Plant samples were aliquoted into grinding tubes and rapidly ground into powder in liquid nitrogen. RNA was extracted using the Novizan Polysaccharide-Polyphenol Plant RNA Extraction Kit (see instructions). After extraction, RNA concentration and OD260 / 280 ratio were measured using Nanodrop. Reverse transcription was performed using a Novizan Biotech reverse transcription kit. The steps are as follows:

[0052] (1) Genomic DNA removal

[0053] Based on the measured RNA concentration, the reaction system was prepared in RNase-free centrifuge tubes (Table 1). After gently mixing with a pipette, the mixture was incubated at 42°C for 2 min.

[0054] Table 1. Preparation of the reverse transcription system

[0055] 5 × gDNA wiper Mix 2 μL Total RNA 1 ug <![CDATA[RNase-free ddH2O]]> To 10 μL

[0056] (2) Preparation of reverse transcription reaction system

[0057] Add the prepared reaction solution to the mixture after the above reaction, and gently mix by blowing. The formulation of the reaction system is shown in Table 2 below.

[0058] Table 2 Preparation of reverse transcription reaction system

[0059] 10 × RT Mix 2 μL Hiscript Ⅲ Enzyme Mix 2 μL <![CDATA[Olig(dT) 20 VN]]> 1 μL Random hexamers 1 μL <![CDATA[RNase-free ddH2O]]> 4 μL

[0060] (3) Perform reverse transcription reaction

[0061] Place the mixture from step (2) (total 20 μL) into a PCR instrument, react at 37°C for 15 min, react at 85°C for 5 s, and store at 4°C. The product from the reaction should be used immediately for qPCR.

[0062] Example 4: OsERF69 gene expression level detection (qRT-PCR)

[0063] The expression level of the OsERF69 gene in tissues was detected by qRT-PCR using reverse-transcribed cDNA as a template. The ChamQ Universal SYBR qPCR Master Mix kit from Novizan was used. The method is as follows:

[0064] (1) Take 5 μL of the product after the reaction into an octet, add 45 μL of ddH2O, dilute 10 times and place on ice;

[0065] (2) Prepare the reaction system according to Table 3, add it to the Hard-Shell PCR plate, place a layer of aluminum foil under the plate, and add the sample as accurately as possible;

[0066] (3) After adding the samples, cover the PCR plate with a layer of Micro-seal® 'B'seal membrane. Perform the qPCR reaction on the Bio-Rad qRT-PCR instrument according to the reaction procedure in Table 4;

[0067] (4) The CT values ​​obtained in the experiment are expressed in terms of 2 -ΔΔCT The algorithm performs conversions to obtain the relative expression of the gene being measured, with ACTIN as the internal reference gene.

[0068] Table 3 qRT-PCR reaction system

[0069] cDNA 1 μL Forward primer (10 μM) 0.2 μL Reverse primer (10 μM) 0.2 μL 2 × ChamQ Universal SYBR qPCR Master Mix 5 μL <![CDATA[ddH2O]]> 3.6 μL

[0070] Table 4 qRT-PCR reaction procedure

[0071]

[0072] Experimental results:

[0073] ① The expression levels of the OsERF69 gene in different tissues were detected as follows: Figure 1 As shown, the expression level of the OsERF69 gene is relatively high in rice leaves and leaf sheaths, and relatively low in roots, stems and young panicles, indicating that the expression of the OsERF69 gene is tissue-specific.

[0074] ② The expression levels of the OsERF69 gene in rice seedling leaves at different time points under high-temperature stress treatment were detected as follows: Figure 2 As shown, the expression level of the OsERF69 gene was significantly downregulated after 1 h of high temperature stress treatment at 45℃, and the downregulation rate increased further after 6 h of treatment, indicating that OsERF69 is a negative response gene to high temperature stress, and its expression is significantly inhibited by high temperature stress.

[0075] The above results indicate that OsERF69 acts as a negative regulator in the rice's response to high-temperature stress: under normal conditions, OsERF69 maintains a certain level of expression to inhibit heat tolerance; when high-temperature stress occurs, plants relieve this inhibition and initiate a heat tolerance defense response by rapidly downregulating the expression of OsERF69.

[0076] Example 5: High-temperature stress treatment during rice seedling stage and identification of heat tolerance phenotypes

[0077] Seeds of wild-type (Nanjing 11), oserf69-1, and oserf69-2 mutants were placed in germination boxes with 45 mL of purified water and soaked in a 28℃ incubator in the dark for 2 days to promote germination. Seeds with uniform white sprouts were sown in hydroponic boxes and cultured in a light incubator (28℃, 16 h light / 8 h dark) for 2 weeks. After the seedlings reached the two-leaf-one-heart stage, they were subjected to high-temperature stress treatment.

[0078] High-temperature stress treatment method: The hydroponic boxes were transferred to a 45℃ light incubator and subjected to continuous high-temperature treatment for 2 days (16 h light / 8 h darkness). After the treatment, the hydroponic boxes were returned to normal culture conditions at 28℃ to recover growth for 7-10 days. The survival rate of each line was counted (the survival standard was the ability to grow new green leaves after recovery). Each line was set up with 3 biological replicates, and each replicate contained more than 30 seedlings.

[0079] Experimental results: ① Phenotypic identification results of the oserf69-1 mutant are as follows Figure 4 and Figure 5 As shown: After treatment at 45℃ for 2 days and subsequent recovery, the survival rate of the oserf69-1 mutant was significantly better than that of the wild type. Figure 4 The leaves wilted less, and new green leaves appeared earlier and in greater quantities; the survival rate statistics ( Figure 5 The results showed that the survival rate of the oserf69-1 mutant was significantly higher than that of the wild type, exhibiting a significantly enhanced heat resistance phenotype.

[0080] ② Phenotypic identification results of the oserf69-2 mutant are as follows: Figure 6 and Figure 7 As shown: the phenotype of the oserf69-2 mutant under the same high-temperature stress conditions is highly consistent with that of the oserf69-1, and the plant survival rate is significantly better than that of the wild type. Figure 6 Survival rate statistics ( Figure 7 Similarly, the survival rate of the oserf69-2 mutant was significantly higher than that of the wild type.

[0081] ③ For example Figure 8 As shown, oserf69-1 mutants and oserf69-2 The relative RNA expression level of ERF69 in the mutant was significantly lower than that in the WT. The two independent mutants (oserf69-1 and oserf69-2) exhibited consistent enhanced heat tolerance under high-temperature stress, mutually reinforcing each other and ruling out interfering factors such as off-target effects or site effects that might exist in a single mutant. This strongly demonstrates that the loss of function of the OsERF69 gene is the fundamental cause of the enhanced heat tolerance.

[0082] The phenotypic identification results consistently demonstrate that the OsERF69 gene negatively regulates rice heat tolerance. Under normal conditions, OsERF69 gene expression inhibits rice heat tolerance; under high-temperature stress, plants partially relieve this inhibition by downregulating OsERF69 expression to cope with the stress; however, by selectively knocking out the OsERF69 gene through gene editing technology, the inhibition of heat tolerance by this gene can be completely relieved, significantly improving the heat tolerance of rice from the root and reducing the losses to rice growth and development caused by high-temperature stress. Therefore, OsERF69 can serve as an effective target for molecular breeding of rice heat tolerance, possessing significant agricultural application value and broad application prospects.

[0083] This application has made every effort to describe the inventive concept and evidence of its effects. The scope of this application is defined by the appended claims, and those skilled in the art will clearly understand the scope defined by the claims in conjunction with this specification and common knowledge in the field. The above description is merely a preferred embodiment of this application and is not intended to limit this application. Without departing from the spirit and scope of this application, those skilled in the art can make any modifications or changes to the technical solution of this application, and such modifications and changes are also included within the scope of this application.

Claims

1. Regulating the heat resistance of rice OsERF69 Application of genes in regulating plant heat tolerance; The OsERF69 Genes of rice ERF69 Gene; The OsERF69 The nucleotide sequence of the gene is shown in SEQ ID NO.1; The plant in question is rice; The application reduces OsERF69 Gene expression level or knockout OsERF69 Genes enhance the heat resistance of rice.

2. A method for improving the heat resistance of plants, characterized in that: Reduce in plants OsERF69 Gene expression levels or targeted knockout in plants OsERF69 Genes, making OsERF69 Gene expression silencing, downregulation; or causing OsERF69 The protein encoded by the gene has lost all or part of its biological function; The OsERF69 Genes of rice ERF69 Genes; the stated OsERF69 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the plant is rice.

3. The method for improving plant heat resistance according to claim 2, characterized in that: The targeted knockout is performed using gene editing modification.

4. OsERF69 The application of genes in crop heat tolerance genetic breeding, the aforementioned OsERF69 Genes of rice ERF69 Genes; the stated OsERF69 The nucleotide sequence of the gene is shown in SEQ ID NO.1; the crop is rice; the application reduces... OsERF69 Gene expression level or knockout OsERF69 Genes enhance the heat resistance of rice.

Images