Application of plant RNA methyltransferase METTL4 in enhancing high temperature adaptability of crops
By isolating and cloning the Arabidopsis METTL4 gene, regulating alternative splicing of the BR signaling pathway, and promoting thermomorphogenesis, the function of METTL4 in plant high-temperature adaptation was unclear, and a new gene resource for enhancing hypocotyl elongation and breeding crops for high-temperature tolerance was realized.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- FUDAN UNIVERSITY
- Filing Date
- 2026-05-15
- Publication Date
- 2026-07-24
AI Technical Summary
In the existing technology, the specific function and mechanism of METTL4 in plant thermomorphogenesis are still unclear, which affects the adaptability and growth of plants under high temperature conditions.
The Arabidopsis METTL4 gene was isolated and cloned. By regulating alternative splicing of the BR signaling pathway, thermomorphogenesis was promoted. The METTL4 protein or its homologs were used to improve heat adaptation in plants. Specific methods included introducing genes encoding the METTL4 protein to enhance the elongation of hypocotyls, petioles, or roots.
It significantly enhances the hypocotyl elongation ability of plants under high temperature conditions, provides new genetic resources for high temperature tolerance breeding of crops, and improves the growth performance of plants under high temperature conditions.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of bioengineering technology, specifically relating to the application of Arabidopsis RNA methyltransferase METTL4 in regulating plant thermomorphogenesis. Background Technology
[0002] Global warming-induced heat stress has become one of the major environmental factors limiting crop yields. Plants, as sessile organisms, cannot actively avoid high temperatures; instead, they adapt to warmer environments through a series of morphological and physiological changes—a process called thermomorphogenesis. Typical manifestations include hypocotyl and petiole elongation, leaf elevation, and earlier flowering. These changes help plants dissipate heat and mitigate heat damage (Plant Biol, 2019, 70: 321-46). Therefore, identifying and utilizing key genes regulating thermomorphogenesis is of great significance for breeding heat-resistant, high-yielding crops.
[0003] Thermomorphogenesis is regulated by multiple signaling pathways, among which transcription factor PIF4, plant hormone brassinolide, alternative splicing, and epigenetic modifications all play important roles (Plant Biol, 2019, 70: 321-46; Mol Plant, 2023, 16(10): 1612-34; Cell Rep, 2024, 43(2): 113726). Due to their sessile growth characteristics, plants inevitably face numerous adverse environmental influences during their growth. To survive, plants have evolved a variety of environmental response regulatory mechanisms that are distinct from those of animals. After sensing adverse environmental stress, plants will change a series of metabolic pathways and the expression of stress-response genes, such as regulating plant hormones, especially endogenous hormones such as auxin and brassinolide, participating in various transcription factor and kinase cascade amplification reactions to activate or inhibit the expression of various downstream stress genes (cell, 2016, 167(2):313-324). A growing body of research demonstrates that some epigenetic proteins also play crucial roles in plant heat responses. Epigenetics primarily studies the mechanisms by which gene expression differs despite genotype invariance. Chromatin structure and function are regulated by various epigenetic mechanisms, including histone modifications, DNA methylation, ATP-dependent chromatin remodeling, histone variant replacement, and regulation by non-coding RNA. In recent years, RNA modifications (such as m...) have become increasingly important. 6 A、m 6 The functions of aminotransferases (Am) and their methyltransferases in plant environmental responses are receiving increasing attention. METTL4 is a conserved RNA methyltransferase that catalyzes the methylation of U2 snRNA in both animals and plants. 6Am modification is involved in the regulation of precursor mRNA splicing (Cell Res, 2020, 30(6): 544-7; NucleicAcids Res, 2020, 48(16): 9250-61; Nat Commun, 2022, 13(1): 5636). However, the specific function and mechanism of action of METTL4 in plant thermal morphogenesis remain unclear. Summary of the Invention
[0004] The purpose of this invention is to provide the application of plant RNA methyltransferase METTL4 and its encoding gene in enhancing plant high-temperature adaptability and improving growth under high temperatures.
[0005] This invention isolated and cloned the full-length cDNA of the Arabidopsis METTL4 gene. Experiments showed that this gene endows the plant with the ability to elongate the hypocotyl under high temperature (28℃) conditions and participates in the regulation of alternative splicing of brassinolide signaling pathway genes.
[0006] This invention reveals for the first time that Arabidopsis thaliana's METTL4 promotes thermomorphogenesis by regulating alternative splicing of key genes in the BR signaling pathway. The mutant mettl4-1 exhibits a heat-induced hypocotyl elongation inhibition phenotype, which can be reversed by introducing the METTL4 protein into the mutant, providing a new gene resource for heat-tolerant crop breeding.
[0007] Therefore, this invention proposes the application of plant RNA methyltransferase METTL4 or its homologous protein in improving plant high-temperature adaptability and / or promoting plant organ elongation under high temperatures. Specifically, the gene encoding the METTL4 protein is introduced into plants to obtain transgenic plants with enhanced hypocotyl, petiole, or root elongation under high temperatures; the METTL4 protein is derived from Arabidopsis thaliana and is a protein with one of the following amino acid residue sequences:
[0008] (1) SEQ ID No:1;
[0009] (2) A protein that has a regulatory effect on plant growth and development by substituting and / or deleting and / or adding one to fifty amino acid residues in the amino acid sequence of SEQ ID No:1.
[0010] In this invention, the plants include monocotyledonous plants and dicotyledonous plants.
[0011] SEQ ID No:1 consists of 414 amino acid residues.
[0012] The gene METTL4, which encodes the RNA methyltransferase METTL4 of the present invention, is the nucleotide sequence of SEQ ID No:2 in the sequence listing. It consists of 1245 bases, and its coding frame is from the 5' end, from the 1st to the 1245th base, encoding a protein having the amino acid residue sequence of SEQ ID No:1 in the sequence listing.
[0013] This invention also includes the mechanism of action of the METTL4 gene in regulating the thermomorphogenesis of plantoids and its specific applications.
[0014] This invention also includes the mechanism and specific effects of the METTL4 gene in improving agronomic traits such as plant heat sensitivity.
[0015] In practical applications, knocking out the METTL4 gene can eliminate the excessive growth phenomenon in Arabidopsis thaliana under moderate to mild high temperatures. The hypocotyl length of this plant was significantly lower than that of the wild type under high temperature conditions of 28℃, and its sensitivity to brassinolide signaling decreased.
[0016] Therefore, by knocking out the METTL4 gene, this invention can significantly reduce hypocotyl elongation in plants under high temperature conditions, and can be applied to high-temperature tolerance breeding of crops.
[0017] This invention provides new gene resources and technical solutions for heat-resistant plant breeding, and has important agricultural application value. Attached Figure Description
[0018] Figure 1 Phenotypic results of wild-type Arabidopsis thaliana (Col-0) and the T-DNA insertion mutant plant of Arabidopsis thaliana RNA methyltransferase gene METTL4 (mettl4-1) under normal conditions, high temperature treatment at 28℃, drought stress treatment, salt stress treatment and manganese toxicity test.
[0019] Figure 2 Phenotypic results of wild-type Arabidopsis thaliana (Col-0), T-DNA insertion mutant of Arabidopsis thaliana RNA methyltransferase gene METTL4 (mettl4-1), and transgenic line HA-METTL4 / mettl4 under normal (22℃) and high-temperature (28℃) conditions.
[0020] Figure 3 Phenotypic results of wild-type Arabidopsis thaliana (Col-0) and the T-DNA insertion mutant of Arabidopsis thaliana RNA methyl transfer gene METTL4 (mettl4-1) under normal (22℃) and high-temperature (28℃) conditions after treatment with different levels of brassinosteroids.
[0021] Figure 4The transcriptional levels of the METTL4 gene in wild-type Arabidopsis thaliana (Col-0) and the T-DNA insertion mutant of Arabidopsis thaliana RNA methyltransferase gene METTL4 (mettl4-1) were measured under normal (22℃) and high-temperature (28℃) conditions. Detailed Implementation
[0022] The present invention will be further described below with reference to the embodiments and accompanying drawings.
[0023] Unless otherwise specified, the methods described in the following examples are conventional methods. The primers and sequencing were performed by Beijing Qingke Biotechnology Co., Ltd.
[0024] Example 1. Obtaining the Arabidopsis RNA methyltransferase METTL4 gene.
[0025] The AT1G19340 gene sequence was obtained from the Arabidopsis database tair (https: / / www.arabidopsis.org / ). A pair of primers was designed based on the 5' and 3' end sequences. The primer sequences are as follows:
[0026] 5'-GAGCCGTTAAGAGCTCGGTACCATGTACCCATACGATGTT-3' (SEQ ID No: 3) and
[0027] 5'-GACTCTAGAGGATCCCCGCTAAACTTTCAAAAAGTATCTCG-3' (SEQ ID No: 4).
[0028] Total RNA was extracted from Arabidopsis thaliana seedlings (Promega, SV total RNA isolation system). Using the total RNA as a template, cDNA was synthesized using AMV reverse transcriptase (TaKaRa) (according to the user manual of Plant RT-PCR Kit 2.01 (TaKaRa)). Using the cDNA as a template, the full-length cDNA sequence of the Arabidopsis thaliana RNA methyltransferase gene METTL4 was amplified by PCR. The 50 μL PCR reaction mixture contained: 2 μL template, 1 μL high-fidelity enzyme KOD plus (TOYOBO), 5 μL 10× buffer, 8 μL 2.5 μM dNTPs, 1 μL each of 20 μM 5' and 3' primers, and 32 μL pure water. The reaction conditions were: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, and 68℃ extension for 1.5 min, for a total of 30 cycles. After the reaction, the PCR product was detected by 0.8% agarose gel electrophoresis. The amplified fragment of about 1245 bp was recovered and purified and cloned into the vector pCAMBIA1300 to obtain a recombinant plasmid containing the recovered fragment. Sequencing showed that the full-length cDNA of Arabidopsis RNA methyltransferase gene METTL4 has the nucleotide sequence of SEQ ID No:2 in the sequence listing. SEQ ID No:2 in the sequence listing consists of 1245 bases, and its coding frame is from the 5' end, from the 1st to the 1245th base, which encodes a protein with the amino acid residue sequence of SEQ ID No:1 in the sequence listing. The encoded protein was named METTL4.
[0029] Example 2. Obtaining HA-METTL4 / mettl4 transgenic plants
[0030] I. Construction of METTL4 self-reply expression vector
[0031] To analyze the function of the METTL4 gene, a self-reverting expression vector for the METTL4 gene was constructed, targeting the METTL4 gene. The promoter region sequence of the AT1G19340 gene (approximately 1700 bp upstream of the METTL4 gene coding sequence promoter) was obtained from the Arabidopsis database tair (https: / / www.arabidopsis.org / ). A pair of primers was designed based on the 5' and 3' end sequences. The primer sequences are as follows:
[0032] 5'-CATGATTACGAATTCCAGCCAATGCCAACCACC-3' (SEQ ID No: 5), and
[0033] 5'- CATGGTACCGAGCTCTTAACGGCTCCAACACTCTCTAGTC-3' (SEQ ID No: 6). The 1700 bp amplified fragment was amplified, purified, and recovered as described in Example 1. Using the pCAMBIA1300-METTL4 recombinant plasmid obtained in Example 1 and the amplified promoter sequence PCR fragment as substrates, the products were digested with EcoRI and KpnI and recovered. Ligation was performed using T4 ligase to obtain pCAMBIA1300-P. METTL4 :: METTL4 recombinant vector.
[0034] II. Transformation of Arabidopsis plants with METTL4 self-reply expression vector
[0035] Arabidopsis thaliana was transformed using the flower-dip method described by Clough et al. (Plant Journal, 1998, 16: 735-743). The plasmid pCAMBIA1300-P was used to transform the plasmid. METTL4 :: METTL4 was transfected into Agrobacterium GV301 via electroporation, and positive clones were obtained through screening. Agrobacterium GV301 carrying the plasmid was inoculated into 5 mL of YEB liquid medium containing 100 mg / L kanamycin and cultured at 28°C in a shaker until the late logarithmic growth phase. The culture was then scaled up 1:100 until the OD 600 was approximately 0.5. The bacterial culture was suspended in transfection medium. Arabidopsis inflorescences were soaked and infected using standard methods, cultured in the dark for three days, and then cultured normally until maturity to obtain T0 generation seeds. To further confirm the stable inheritance of this transgenic trait, multiple generations of self-pollination were performed, and positive homozygous plants were selected using Hygromycin B (Roche) until a sufficient number of seeds with stable traits were obtained.
[0036] Example 3. Identification of stress-related phenotypes in mettl4-1 mutant plants
[0037] To determine the function of METTL4 in the stress response of Arabidopsis thaliana, we transferred vernalized culture plates to a plant incubator and conducted normal culture or subsequent stress treatments according to the required experimental conditions:
[0038] (1) Normal culture conditions (for propagation and transplanting): 22 ℃, 60% humidity, white light long-day (16 h light, 8 h darkness), light intensity 150 μmol / m 2 • The flat plate is placed horizontally;
[0039] (2) Heat treatment conditions: 22 ℃, 60% humidity, white light full sun irradiation (24 h light irradiation), light intensity 150 μmol / m 2• After four days of horizontal incubation on plates, the control group continued incubation under the same conditions for three days, while the experimental group was incubated at 28 °C for three days, with other conditions remaining unchanged.
[0040] (3) Other stress treatments: 22 ℃, 60% humidity, full sunlight under white light, light intensity 150 μmol / m 2 • After four days of vertical culture on plates, Arabidopsis seedlings were transplanted into plates containing 100 mM mannitol (drought stress treatment), 100 mM NaCl (salt stress treatment), and 1.5 mM MnCl2 / MnSO4 (heavy metal toxicity test) in a clean bench. The control group was transplanted onto new 1 / 2 MS plates. All other conditions remained unchanged, and the seedlings were cultured for another three days.
[0041] like Figure 1 As shown, under normal conditions, the mettl4-1 mutant seedlings did not show significant phenotypic differences compared to wild-type Col-0 seedlings. Figure 1 A). Adding 100 mM mannitol to the culture medium simulates drought conditions ( Figure 1 C) or salt treatment with 100 mM NaCl ( Figure 1 After D), the growth status of the mettl4-1 mutant seedlings was basically the same as that of the Col-0 control group, with similar phenotypes and no obvious differences between the two. However, after adding 1.5 mM MnCl2 to the culture medium... Figure 1 E) or 1.5mM MnSO4 ( Figure 1 In the manganese toxicity test (F), after treatment with heavy metal manganese ion salts, the root length of the mettl4-1 mutant was significantly shorter than that of Col-0, and the leaf shrinkage of the mutant was also more severe than that of the wild type. It is noteworthy that even more significant phenotypic differences occurred in the heat response test to medium-high temperatures; after treatment at high temperature (28°C)... Figure 1 Following B), the mettl4-1 mutant exhibited a highly significant defect in thermomorphogenesis. Specifically, compared to the wild-type Col-0 plant treated with the same high-temperature conditions, the hypocotyl elongation induced by high temperature was significantly inhibited in the mettl4-1 mutant. Based on these results, we conclude that METTL4 positively regulates thermomorphogenesis in Arabidopsis thaliana.
[0042] Example 4. Identification of the thermal response phenotype of METTL4-related mutant plants
[0043] To more accurately determine the role of METTL4 in Arabidopsis thermomorphogenesis, such as Figure 2As shown, we conducted the heat response experiment in Example 3 on Col-0, mettl4-1, and HA-METTL4 / mettl4 plants. Under high temperature conditions (28 °C), hypocotyl elongation was significantly inhibited in the mettl4-1 mutant plants compared to the wild-type Col-0. However, in the HA-METTL4 / mettl4 transgenic plants, these defective phenotypes were rescued, and the hypocotyl length was restored to the Col-0 level. These results indicate that METTL4 regulates thermomorphogenesis in Arabidopsis thaliana.
[0044] Example 5. Identification of the sensitivity of METTL4 and MRG1 / 2 related mutant plants to brassinolide signals
[0045] To determine the brassinosteroid signaling levels during thermomorphogenesis in wild-type and mettl4-1 mutants, we conducted brassinosteroid-induced hypocotyl elongation experiments on Col-0 and mettl4-1 plant materials under normal growth temperature (22℃) and high temperature (28℃) treatments, respectively. The results are as follows: Figure 3 As shown, increasing the concentration of exogenously applied brassinosteroids significantly promoted hypocotyl elongation in all four seedling types. However, compared with Col-0, the mettl4-1 mutant showed significantly reduced sensitivity to brassinosteroids. In other words, after treatment with the same level of brassinosteroids, the hypocotyl elongation of the mettl4-1 mutant plants was lower than that of the wild type. Furthermore, this insensitivity to brassinosteroids due to the absence of METTL4 was more pronounced under high temperature conditions of 28℃, indicating that METTL4 can regulate thermomorphogenesis by affecting the brassinosteroid signaling pathway.
[0046] Example 6. RNA-seq detection of METTL4 expression in METTL4 mutant plants
[0047] Comparative analysis of the transcriptional levels of METTL4 in Col-0 and mettl4-1 mutant materials revealed that METTL4 lacked complete transcription in the mettl4-1 mutant. (See [link to relevant documentation]). Figure 4 As shown.
Claims
1. The application of plant RNA methyltransferase METTL4 or its homologous protein in improving plant high-temperature adaptability and / or promoting plant organ elongation under high temperatures, characterized in that, The gene encoding the METTL4 protein was introduced into plants to obtain transgenic plants with enhanced hypocotyl, petiole, or root elongation under high temperatures; wherein the METTL4 protein is derived from Arabidopsis thaliana and is a protein with one of the following amino acid residue sequences: (1) SEQ ID No:1; (2) A protein whose amino acid residue sequence of SEQ ID No:1 has been replaced, deleted or added by one or more amino acid residues and still has high temperature regulation function.
2. The application according to claim 1, characterized in that, The gene encoding METTL4 has the nucleotide sequence shown in SEQ ID No:
2.
3. The application according to claim 2, characterized in that, The plants mentioned include monocotyledonous plants and dicotyledonous plants.
4. The application according to claim 3, characterized in that, By knocking out the METTL4 gene, the elongation of the hypocotyl in plants under high-temperature conditions can be reduced, thus enabling high-temperature tolerance breeding of crops.