Application of rice OsERF141 protein and coding gene thereof in improving low-temperature tolerance of plants

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of a rice OsERF141 protein and a coding gene thereof in improving low-temperature tolerance of plants. Experiments show that the low-temperature resistance of an OsERF141 function deletion mutant strain is remarkably reduced, and the low-temperature resistance of an overexpressed OsERF141 transgenic strain is remarkably improved. The invention provides a new gene resource for the cold resistance problem of rice, and has important significance for the molecular mechanism research of low-temperature stress and the cultivation of new low-temperature-resistant varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and in particular to an application of a rice OsERF141 protein and a gene encoding the same in improving plant low temperature tolerance. Background Art

[0002] Cold stress is a widespread environmental stress that severely impacts the growth, production, and geographic distribution of cash crops (Ding and Yang, 2022; Lesk et al., 2016; Zhu, 2016). As a staple crop native to tropical regions, rice (Oryza sativa L.) is extremely sensitive to cold stress throughout its growth cycle. As global climate change continues to deteriorate and extreme weather conditions such as low temperatures become more frequent, rice growth, development, and yield are under significant threat. Therefore, elucidating the complex regulatory mechanisms of rice cold resistance and identifying novel cold-tolerant genes can provide a theoretical basis for breeding and improving new cold-tolerant rice varieties and have important practical implications for achieving sustainable agricultural development.

[0003] Rice crops encounter a variety of biotic and abiotic stresses during production, among which low temperature stress is one of the most significant abiotic stresses affecting yield. Due to its immobility, rice has evolved a sophisticated regulatory mechanism to respond to low temperature stress and adapt to its environment. Transcription factors, as key regulatory components, precisely control the timely and appropriate expression of downstream target genes to perform specific functions, thus playing a central role in the entire regulatory network. Rice contains a large family of transcription factors, including bZIP, bHLH, NAC, AP2 / ERF, MYB, and WRKY transcription factors known to participate in low temperature stress responses (Ding and Yang, 2022; Hu et al., 2024; Li et al., 2024; Li et al., 2022; Tang et al., 2022; Xu et al., 2024; Zhang et al., 2022; Zhang et al., 2017; Zheng et al., 2023). Numerous studies have demonstrated that CBF-dependent transcriptional regulatory pathways play a crucial role in plant responses to cold stress (Ding et al., 2015, Ding et al., 2020, Jia et al., 2022, Jiang et al., 2022, Thomashow, 1999, Wang et al., 2021). Under cold stress, the expression of CBFs is rapidly induced, and CBF proteins can directly target CRT / DRE cis-elements in the promoters of cold stress-responsive genes (CORs), thereby initiating the cold stress response (Ding et al., 2015, Li et al., 2017, Zhang et al., 2017). Furthermore, CBF gene expression is regulated by multiple transcription factors, while the stability of CBFs and CBF-dependent proteins is modulated by post-translational modifications (Ding et al., 2019). In Arabidopsis, members of the CAMTA (CALMODULIN-BINDING TRANSCRIPTION ACTIVATOR) protein family possess transcriptional activator activity, positively regulating plant cold tolerance by activating CBF expression. Studies have shown that under sudden temperature drops, CAMTA3 and CAMTA5 regulate the expression of CBF1 and CBF2 in response to cold stress, but this pathway becomes inoperative under slow cooling conditions (Kidokoro et al., 2017). ICE1 and its homolog ICE2 enhance plant cold tolerance by positively regulating the expression of CBF genes (Chinnusamy et al., 2003, Fursova et al., 2009).In maize, studies have found that ZmICE1 is a key transcription factor that responds to cold stress. Under low temperature conditions, it can directly inhibit the expression of ZmASs, the genes that synthesize key amino acids (such as glutamate and asparagine), thereby preventing the accumulation of these amino acids and reducing the production of mitochondrial reactive oxygen species induced by these amino acids. This helps maintain the expression of the cold-responsive gene ZmCBF, thereby enhancing maize's cold tolerance (Jiang et al., 2022). Similarly, ZmRR1 can simultaneously regulate ZmCBF and ZmASs to enhance maize's tolerance to cold stress (Zeng et al., 2021). At the same time, researchers have also found that ZmbZIP68 responds to cold stress by negatively regulating the expression of ZmCBF. Therefore, when ZmbZIP68 function is lost, the plant's cold tolerance is significantly enhanced (Li et al., 2022). In rice, Li Jiayang's team discovered that IPA1, a key factor in regulating rice plant architecture, is also involved in the response to cold stress. Further research revealed that IPA1 directly binds to the GTAC motif in the OsCBF3 promoter, promoting OsCBF3 expression and thereby improving rice cold tolerance (Jia et al., 2022). Furthermore, OsERF52 interacts with its partners, OsICE1, and IPA1 to synergistically promote the expression of OsCBFs, thereby enhancing rice tolerance to cold stress (Xu et al., 2024). However, the function and application of OsERF141 in rice cold response have not been reported. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of rice OsERF141 protein and its encoding gene in improving plant low temperature tolerance, and to provide a method for cultivating transgenic plants tolerant to low temperature stress, thereby providing a theoretical basis for the study of the molecular mechanism of plant cold resistance and the cultivation of new low temperature tolerant varieties.

[0005] To achieve the above objectives, the present invention provides the following technical solutions:

[0006] In one aspect, the present invention provides a use of a rice OsERF141 protein and a gene encoding the same for improving plant low temperature tolerance. The amino acid sequence of the rice OsERF141 protein is shown in SEQ ID No. 2.

[0007] The present invention constructs an overexpression vector and an OsERF141 gene editing vector for rice OsERF141, and obtains overexpression-positive plants and mutant-positive plants by infecting rice callus with Agrobacterium. Four mutant strains and four overexpression transgenic strains with varying degrees of upregulated expression folds are obtained. Low-temperature phenotype identification shows that all Oserf141 mutants exhibit a low-temperature-sensitive phenotype, with a survival rate significantly lower than that of the wild type; while all overexpression plants exhibit a cold-resistant phenotype, and the survival rate is positively correlated with the upregulated level of OsERF141 transcripts, indicating that OsERF141 is a positive regulatory factor in rice's response to low temperatures.

[0008] The "rice OsERF141 protein" in the present invention encompasses a fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein shown in SEQ ID No. 2, as well as a protein with the same function obtained by replacing and / or deleting and / or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID No. 2.

[0009] In one embodiment, the nucleotide sequence of the gene encoding the rice OsERF141 protein is shown as SEQ ID No. 1.

[0010] The present invention encompasses sequences having a similarity of 90% or more, preferably 95% or more, and more preferably 99% or more to the nucleotide sequence of SEQ ID No. 1 and having the same function. The present invention also encompasses sequences having one or more base substitutions, replacements, deletions, and / or additions to the nucleotide sequence of SEQ ID No. 1 and having the same function.

[0011] In another aspect, the present invention provides a use of a biological material comprising a gene encoding a rice OsERF141 protein for improving plant low temperature tolerance, wherein the biological material comprises:

[0012] (A) an expression cassette containing a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 1;

[0013] (B) a recombinant vector containing the expression cassette described in (A);

[0014] (C) a recombinant microorganism containing the expression cassette described in (A) or the recombinant vector described in (B);

[0015] (D) A recombinant cell containing the expression cassette described in (A) or the recombinant vector described in (B).

[0016] In one embodiment, the present invention comprises transgenic plants containing a gene encoding the rice OsERF141 protein. Such transgenic plants include seeds, callus tissue, whole plants, and cells. Such transgenic plants include not only first-generation transgenic plants obtained by transforming the gene into a target plant, but also progeny thereof.

[0017] In one embodiment, the recombinant vector is a recombinant expression vector, preferably an overexpression vector of the target gene (rice OsERF141 gene).

[0018] In one embodiment, the recombinant expression vector comprises a transcript that initiates transcription of the target gene. To achieve overexpression of the target gene, the promoters included in the recombinant expression vector include, but are not limited to, constitutive promoters; tissue-, organ-, and development-specific promoters; and inducible promoters.

[0019] In a preferred embodiment, the overexpression vector contains a Ubiquitin promoter or a CaMV 35S promoter; the nucleic acid molecule of the target gene is operably linked to the promoter.

[0020] In one embodiment, the recombinant expression vector comprises a suitable transcription terminator, including but not limited to: Agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, etc.

[0021] In one embodiment, the recombinant vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, such as but not limited to pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb vectors.

[0022] In one embodiment, the recombinant vector also includes a gene encoding an enzyme or luminescent compound that can produce color changes (GUS gene, luciferase gene, etc.) and an antibiotic marker gene (such as a gene that confers resistance to kanamycin and related antibiotics) to facilitate the identification and screening of transgenic plant cells or plants.

[0023] In one embodiment, the microorganism is Agrobacterium, preferably, the microorganism is Agrobacterium EHA105.

[0024] In one embodiment, the recombinant vector is Ubi-XX-3FLAG.

[0025] In one embodiment, the recombinant cell comprises an overexpression vector of the rice OsERF141 gene or an overexpression mutant of the rice OsERF141 gene.

[0026] In one embodiment, the application is to overexpress the rice OsERF141 protein or its encoding gene to improve the low temperature tolerance of the plant.

[0027] In another aspect, the present invention provides a method for cultivating transgenic plants tolerant to low temperature stress, wherein the expression level of the OsERF141 gene or the activity of the OsERF141 protein in the plant is increased by genetic engineering methods to obtain transgenic plants with improved low temperature tolerance.

[0028] In one embodiment, the method for increasing the expression level of the OsERF141 gene in the plant comprises introducing a gene encoding the OsERF141 protein into plant tissues or plant cells.

[0029] The present invention also provides a method for improving plant tolerance to low temperature stress, comprising: 1) constructing an expression vector containing the rice OsERF141 gene; 2) transforming the constructed expression vector into plants or plant cells; and 3) cultivating transgenic plants to obtain plants.

[0030] In the present invention, the expression vector can be introduced into plant cells by using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, electroporation and other conventional biotechnology methods; for example, it can be introduced into rice by infecting callus tissue.

[0031] In one embodiment, after the recombinant expression vector containing the target gene is infected with the target plant, positive plants are screened to obtain transgenic plants with enhanced cold resistance compared with normal plants.

[0032] In a specific embodiment, the improved cold resistance of the transgenic plant (introduced with the OsERF141 gene) is manifested as: the cold resistance of the transgenic plant is higher than that of non-transgenic plants (wild-type plants) or plants transfected with an empty vector without the target gene.

[0033] In one embodiment, the hosts transformed by the recombinant expression vector include various plants.

[0034] In one embodiment, the plant is a monocot or a dicot, including but not limited to Arabidopsis thaliana, rice, rapeseed, etc.

[0035] In a preferred embodiment, the plant is rice, preferably wild rice (Nipponbare).

[0036] Compared with the prior art, the present invention has the following outstanding beneficial effects:

[0037] The present invention clarifies that the OsERF141 gene is an important gene for rice cold tolerance. This gene can respond to low temperature stress and play a positive regulatory role in rice cold resistance. The rice gene OsERF141 or its encoded protein can be used to improve plant stress resistance (cold resistance).

[0038] The present invention provides applications for enhancing or improving plant cold tolerance by increasing the expression of the OsERF141 gene, thereby producing plants with improved cold resistance. This approach has high application value and lays a foundation for research into breeding cold-tolerant transgenic plants. Cultivating cold-stress-tolerant transgenic plants by overexpressing the rice OsERF141 protein or its encoding gene can improve plant survival rates in cold-stress environments, contributing to food security and sustainable agricultural development. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 Schematic diagram of the mutation sites of four mutants of rice OsERF141 gene;

[0040] Figure 2 is the transcription level of rice OsERF141 in wild type and different overexpression transgenic lines;

[0041] Figure 3 The results of the analysis of the rice Oserf141 mutant that reduces the cold resistance and survival rate of rice;

[0042] Figure 4 Analysis results of rice OsERF141 gene overexpression improving rice plant resistance to low temperature and survival rate. DETAILED DESCRIPTION

[0043] To make the objectives, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. The following embodiments and features of the embodiments may be combined with each other unless there is a conflict.

[0044] The detailed description of the embodiments of the present invention provided below is not intended to limit the scope of the claimed invention, but rather merely represents selected embodiments of the present invention. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are intended to fall within the scope of protection of the present invention.

[0045] In one embodiment, the present invention provides a use of rice OsERF141 protein or its encoding gene in improving plant drought tolerance, wherein the amino acid sequence of the rice OsERF141 protein is shown in SEQ ID No. 2; the nucleotide sequence of the gene encoding the rice OsERF141 protein is shown in SEQ ID No. 1.

[0046] ERF can specifically bind to GCC-box and / or DRE / CRT elements. It is by regulating the expression of related genes that ERF participates in the plant's salt resistance, drought resistance, cold resistance and other related biotic and abiotic stress processes. The plant's response to adversity is regulated by multiple signal pathways, which may promote or antagonize each other, thereby achieving adversity defense responses. At present, the functions of many ERF genes in rice are still unknown, and there are few research articles on the role of ERF transcription in rice drought resistance. In addition, plant drought resistance involves the participation of a large number of genes. Discovering and verifying more genes involved in plant drought resistance is of great significance for the research and cultivation of drought-resistant crop varieties. There is no literature report on the function of OsERF141 protein or its encoding gene in regulating rice cold resistance. The present invention proposes that the OsERF141 gene can improve the growth state of rice under low temperature conditions. Therefore, it can be used to cultivate transgenic plants that tolerate low temperature stress.

[0047] The present invention uses genetic engineering methods to study the expression characteristics of the gene and the phenotypic changes of the mutant strains; analyzes the relationship between the changes in gene expression and crop phenotype and survival rate, and finds that rice plants overexpressing the OsERF141 gene have significantly stronger cold tolerance than wild-type controls.

[0048] In another embodiment, the present invention provides a method for cultivating transgenic plants tolerant to low temperature stress, wherein the expression level of the OsERF141 gene or the activity of the OsERF141 protein in the plant is increased by genetic engineering methods to obtain transgenic plants with improved low temperature tolerance.

[0049] Under adverse stress, a series of responses will occur in plants, accompanied by many physiological, biochemical and developmental changes. Since plant stress tolerance is a complex trait regulated by multiple genes, clarifying the important genes in the stress resistance process is of great significance for the cold-resistant mechanism and the cultivation of cold-resistant crops. The present invention illustrates that up-regulation of the OsERF141 gene improves the tolerance of transgenic rice to low temperatures, and the loss of OsERF141 expression reduces the cold resistance of rice. The OsERF141 protein and its encoding gene can enhance the tolerance of plants to adversity, indicating that the gene has application value in crop drought resistance modification and stable yield. The present invention has important theoretical and practical significance for improving and enhancing rice stress resistance and accelerating the process of stress-resistant molecular breeding.

[0050] Definitions of terms used in this invention

[0051] The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides, and polymers thereof, in single- or double-stranded form.

[0052] The term "transcription factor" refers to a class of DNA-binding proteins that can specifically bind to cis-acting elements in the promoter region of eukaryotic genes, thereby activating or inhibiting the transcription and expression of downstream genes at a specific time and space.

[0053] In the present invention, the term "identity" or "similarity" refers to sequence similarity to a natural nucleic acid sequence. Identity or similarity can be evaluated with the aid of computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0054] As used herein, the term "expression" or "gene expression" refers to the transcription of a specific gene, genes, or gene construct into structural RNA (rRNA, tRNA) or mRNA, with or without subsequent translation of the RNA into protein. This process includes transcription of DNA and processing of the resulting mRNA product.

[0055] In the present invention, the term "increased expression / overexpression" refers to any form of expression that is increased relative to the original wild-type expression level. Methods for increasing the expression of genes or gene products have been described in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers.

[0056] In the present invention, the terms "increase", "improve" or "enhance" are interchangeable and shall in the applied sense mean at least 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%, preferably at least 15% or 20%, more preferably 25%, 30%, 35% or 40% more yield and / or growth and / or changes compared to control plants as defined herein.

[0057] In the present invention, the term "transformation" refers to a process by which a heterologous DNA sequence or a vector containing a DNA sequence is introduced into a host cell or organism.

[0058] In the present invention, the term "recombinant expression vector" refers to one or more DNA vectors used to achieve plant transformation; these vectors are often referred to as binary vectors in the art.

[0059] In the present invention, the term "operably linked" refers to a functional connection between two or more elements, and the operably linked elements may be contiguous or non-contiguous.

[0060] In the present invention, the term "host cell" or "recombinant host cell strain" refers to a cell comprising a polynucleotide of the present invention, regardless of the method used to insert the polynucleotide to produce the recombinant host cell. The host cell can be a prokaryotic cell or a eukaryotic cell, and the host cell can also be a monocotyledonous or dicotyledonous plant cell.

[0061] Nucleotide sequence of the gene encoding the rice OsERF141 protein of the present invention

[0062]

[0063] Amino acid sequence of rice OsERF141 protein in the present invention

[0064] Example 1.

[0065] Cloning of the OsERF141 gene nucleotide sequence

[0066] RNA was extracted from rice using an Omega plant extraction kit. First-strand cDNA was synthesized using 1 μg of RNA as a template according to the cDNA synthesis kit (Yeasen).

[0067] The complete ORF of OsERF141 was obtained from the website (http: / / rice.plantbiology.msu.edu / expression.shtml), and specific primers were designed: forward primer 5'-ATGGACGCGGTGGACAGA-3' (SEQ ID No. 3); reverse primer 5'-TTAGTCCTTCCATGCGAA-3' (SEQ ID No. 4) for PCR amplification reaction.

[0068] The PCR reaction system was as follows: 2×PhantaMaxMasterMix 25 μL, 1 μL each of 10 μM forward / reverse primers, 5 μL of template (cDNA), and sterile water to make up to 50 μL.

[0069] The PCR reaction procedure was as follows: 36 cycles of initial denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 20 s, followed by a final extension at 72°C for 5 min. The resulting amplified full-length cDNA sequence of 822 bp for OsERF141 (shown in SEQ ID No. 1) encoding 473 amino acids (shown in SEQ ID No. 2) was obtained.

[0070] Example 2

[0071] Construction of OsERF141 gene mutant and overexpression vector

[0072] The CRISPR / Cas9 genome editing system was used to edit the OsERF141 gene in wild-type rice (Nipponbare) to obtain mutant plants.

[0073] (1) The exon sequence of the target gene OsERF141 was analyzed using the CRISPR-GE website (http: / / skl.scau.edu.cn / ), and two specific target sequences were selected: target site 1 (Cas9-1) 5'-GGCACGGGCGGAGATGGAA GGG-3' (SEQ ID No. 5) and target site 2 (Cas9-2) 5'-CAGCAGCAGCCGCAGTTTG GGG-3' (SEQ ID No. 6).

[0074] (2) Synthesize specific primer sgRNA-1: (5'-TGTGTGGGCACGGGCGGAGATGG

[0075] AA-3') (SEQ ID No. 11) and sgRNA-2: (5'-TGTGTGCAGCAGCAGCCGCAGTTTG-3') (SEQ ID No. 12), and the editing vector psgR-CAS9-Os was digested with BsaI.

[0076] (3) First anneal the primers. The annealing reaction system is:

[0077] Target site 1, 10 μL F (5'-TGTGTGGGCACGGGCGGAGATGGAA-3') (SEQ ID No. 11) + 10 μL R (5'-AAACCAAACTGCGGCTGCTGCTGCA-3') (SEQ ID No. 13) + 80 μL ddH2O and mix well;

[0078] Target site 2, 10 μL F (5′-TGTGTGCAGCAGCAGCCGCAGTTTG-3′) (SEQ ID No. 12) + 10 μL R (5′-AAACCAAACTGCGGCTGCTGCTGCA-3′) (SEQ ID No. 13) + 80 μL ddH 2 O and mix well.

[0079] After the reaction system was mixed, annealed at 95 °C for 10 min;

[0080] Then it was connected with the enzyme-digested vector psgR-CAS9-Os. The connection system was as follows: 2 μL annealing product (containing sgRNA) + 2 μL recovered enzyme-digested vector + 0.5 μL 10×T4 buffer + 0.5 μL T4 ligase, and connected at room temperature for 15 minutes to obtain the psgR-CAS9-OsERF141 vector containing the OsERF141 specific target.

[0081] The psgR-CAS9-OsERF141 vector containing the OsERF141-specific target was transformed into the competent Escherichia coli DH5α. The transformation system was as follows: 5 μL of the ligation product was added to the competent Escherichia coli, incubated on ice for 30 minutes, heat-shocked at 42°C for 90 seconds, incubated on ice for 2 minutes, added with 400 μL of antibody-free LB, revived at 37°C for 1 hour, centrifuged at 5000 rpm for 1 minute, most of the supernatant was aspirated, and 100 μL of the liquid was retained for mixing. The mixture was spread on an LB plate (50 mg / L Kan) and cultured at 37°C overnight.

[0082] After the plasmids were extracted from the positive clones, they were sent to the company for sequencing. The psgR-CAS9-OsERF141 plasmid with the correct sequence alignment was selected and the mutant plants were obtained by infecting rice callus with Agrobacterium.

[0083] To construct Ubi:OsERF141-3FLAG, a full-length PCR product (1419 bp) of the target gene, excluding the stop codon, was amplified and purified using the Omega gel extraction kit. The recovered product was then ligated with the HindIII-digested vector Ubi-XX-3FLAG by homologous recombination. The reaction mixture consisted of 2 μL of linearized vector, 3 μL of insert, 4 μL of 5× Cell Buffer, 2 μL of Exnase II, and a final volume of 20 μL with sterile water. The reaction conditions were 37°C for 30 minutes. The ligation product was transformed into DH5α competent cells and cultured overnight at 37°C (for kanamycin resistance). Positive single colonies were isolated the next day for sequencing.

[0084] Example 3

[0085] Construction of OsERF141 gene mutants and overexpressing plants

[0086] Wild-type Nipponbare callus was transformed with Agrobacterium.

[0087] The OsERF141 gene mutant vector and plant overexpression vector obtained in Example 2 were transformed into Agrobacterium tumefaciens EHA105. The Agrobacterium-mediated rice genetic transformation system was primarily based on the method reported by Hiei et al. (Agrobacterium-mediated transformation of rice using immature embryos or calliinduce from mature seeds, 2008, Nature protocol. Doi: 10.1038 / nprot.2008.46).

[0088] The expression of the target gene in wild-type and transgenic plants was detected by hygromycin screening and qRT-PCR, and overexpression-positive plants were preliminarily screened; for mutant-positive plants, gDNA of the T0 generation plants needed to be extracted, PCR identified and sequenced.

[0089] Identification of Oserf141 mutants: Genomic DNA from leaves of T0 transgenic plants was extracted and used as a template. Based on the OsERF141 target site information, specific primers F (5'-CGTCTCGCTCGTTTTACC-3'; SEQ ID No. 7) and R (5'-GCGGGAACAGAGGAGACG-3'; SEQ ID No. 8) were designed for PCR amplification. A single, clear amplification product of interest (665 bp in positive plants) was recovered and sent to the company for sequencing to screen for mutant lines. T0 generation plants were continuously self-pollinated to obtain the T2 generation. T2 generation plants were again screened with hygromycin and identified by PCR to identify independent lines that were vector-free and homozygous for the mutation.

[0090] like Figure 1 As shown, four mutant strains were ultimately obtained, named Oserf141-1, Oserf141-2, Oserf141-3, and Oserf141-4. Two mutations occurred at target sequence 1: Oserf141-1, which had a two-base deletion (-GG), and Oserf141-2, which had a one-base deletion (-G). Two mutations also occurred at target sequence 2: Oserf141-3, which had a one-base insertion (+T), and Oserf141-4, which had a two-base insertion (+TT). All four mutants resulted in frameshift mutations in the encoded protein.

[0091] Identification of OsERF141 overexpressing plants: RNA extraction was carried out according to the instructions of the RNA plant extraction kit of Yeasen. RNA was extracted from 14-day-old wild-type and transgenic rice seedlings. 1 μg of RNA was used as a template and the first-strand cDNA was synthesized according to the operating instructions of the cDNA synthesis kit (Yeasen). Specific quantitative PCR primers were designed based on the OsERF141 gene cDNA (F is 5'-TGAACCGAAGCCTCTGCTCTTC-3', SEQ ID No. 9; R is 5'-TTCAGCTCGGGTTCTGGAATGC-3', SEQ ID No. 10). The expression of the OsERF141 gene in the wild-type and overexpressing transgenic lines was detected by qRT-PCR. The expression folds of #4, #5, #17 and #18, which were all upregulated and had different folds of upregulation, were selected for the following experiments. Figure 2 As shown, the horizontal axis represents the selected different transgenic lines, and the vertical axis represents the expression level of OsERF141.

[0092] Example 4

[0093] Functional characterization of OsERF141 mutants and overexpressing plants

[0094] 1. Analysis of cold tolerance of OsERF141 mutants and overexpressing plants

[0095] After soaking the seeds of wild type (Nipponbare), Oserf141 mutant and Ubi:OsERF141 in the dark at 37°C for germination, each plant was planted in nutrient soil and then placed in the same plastic pot to ensure consistent water supply. After the plants grew for about 3 weeks, they were treated with low temperature for 3 to 5 days (depending on the degree of leaf curling) and then recovered. The phenotypes were observed and the survival rate was calculated. The results showed that the Oser141 mutant showed a low temperature sensitive phenotype, and the survival rate was significantly lower than that of the wild type ( Figure 3 ); while the overexpressing plants all showed a cold-resistant phenotype, and the survival rate was positively correlated with the up-regulation level of OsERF141 transcripts ( Figure 4 These results indicate that OsERF141 is a positive regulator of low temperature response in rice.

[0096] Finally, it should be noted that the scope of protection of the present invention is not limited to the above embodiments. Ordinary technicians in this field should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents; and these modifications or replacements will fall within the scope of protection of the present invention.

Claims

1. Application of rice OsERF141 protein and its encoding gene in improving plant low temperature tolerance, characterized in that: The amino acid sequence of the rice OsERF141 protein is shown in SEQ ID No.

2.

2. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding the rice OsERF141 protein is shown in SEQ ID No.

1.

3. Use of a biomaterial comprising a gene encoding rice OsERF141 protein for improving plant low temperature tolerance, characterized in that: The biomaterial includes any one or more of (A)-(D): (A) an expression cassette containing a nucleic acid molecule having a nucleotide sequence as shown in SEQ ID No. 1; (B) a recombinant vector containing the expression cassette described in (A); (C) a recombinant microorganism containing the expression cassette described in (A) or the recombinant vector described in (B); (D) A recombinant cell containing the expression cassette described in (A) or the recombinant vector described in (B).

4. The use according to claim 3, characterized in that The recombinant vector is an overexpression vector of the rice OsERF141 gene.

5. The use according to claim 4, characterized in that The overexpression vector contains Ubiquitin promoter or CaMV 35S promoter.

6. The use according to claim 3, characterized in that The recombinant cell contains an overexpression vector of the rice OsERF141 gene or an overexpression mutant of the rice OsERF141 gene.

7. The use according to any one of claims 1 to 6, characterized in that The application is to over-express rice OsERF141 protein or its coding gene to improve the low temperature tolerance of plants.

8. A method for cultivating transgenic plants tolerant to low temperature stress, characterized in that: The expression level of the OsERF141 gene or the activity of the OsERF141 protein in the plant is improved by a genetic engineering method, thereby obtaining a transgenic plant with improved low temperature tolerance.

9. The method according to claim 8, characterized in that The method for increasing the expression level of the OsERF141 gene in the plant comprises introducing the gene encoding the OsERF141 protein into plant tissues or plant cells.

10. The method according to claim 8 or 9, characterized in that The plant is rice.

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