Use of otu4 gene or protein coded thereby in improving plant resistance to high temperature stress

Abstract

The application discloses application of OTU4 genes or proteins coded by the genes in improving the high-temperature stress resistance of plants. The application discloses for the first time the influence of a new plant peroxisome protein coded by the OTU4 gene on the heat resistance of plants, and the mutation of the OTU4 gene causes the root growth defect of plants under high temperature. The application discloses for the first time the deubiquitinating enzyme in the peroxisome, and provides a theoretical basis and material support for the ubiquitination research in the peroxisome. The application provides a new gene resource and theoretical guidance for deepening the peroxisome function research and the research and application of the heat resistance of plants and the like.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, and in particular to the application of the OTU4 gene or the protein it encodes in improving the ability of plants to resist high temperature stress. Background Technology

[0002] Over the past decade, the rate of warming has far exceeded the long-term trend. Rising global temperatures have significantly shortened the growing season of crops and reduced their growth rate, making high temperatures one of the main factors affecting global food supply. Although scientists have conducted extensive and systematic research on the molecular mechanisms of heat stress signal transduction and heat tolerance formation in plant cytoplasm, research on how peroxisomes participate in plant heat tolerance is relatively limited.

[0003] Peroxisomes are small, widespread organelles in eukaryotic cells, composed of a single membrane. Despite their small size and simple structure, peroxisomes exhibit high diversity in morphology, protein content, and metabolism. They can respond to various signals and participate in a wide range of physiological functions, including plant hormone biosynthesis, lipid catabolism, and reactive oxygen species metabolism. Therefore, peroxisomes play an important role in plant stress resistance.

[0004] Ubiquitination is an important post-translational modification of proteins. It involves the attachment of small ubiquitin molecules to lysine residues of specific proteins via an E1, E2, E3 cascade pathway, participating in many biological processes in eukaryotic cells, such as cell cycle regulation, DNA damage response, transcriptional regulation, and signal transduction. It plays a crucial role in the rapid response of plants to various abiotic stresses.

[0005] Ubiquitination is considered a dynamic process, thus deubiquitinating enzymes play a crucial role. Among them, deubiquitinating enzymes with OTU domains play important roles in various stages of plant disease resistance, drought resistance, salt tolerance, cold resistance, and growth and development. For example, OTU1a has been shown to regulate plant salt stress tolerance by participating in the degradation of endoplasmic reticulum-associated proteins (Zang, Yuepeng, et al., Arabidopsis OTU1, a linkage-specific deubiquitinase, is required for endoplasmic reticulum-associated protein degradation, The Plant Journal, 2020, 101(1)-141~155). OTU5 is involved in the regulation of the epigenome (Jwaha, B, A.Mry, and B.Pyca.″Epigenomic regulation of OTU5 in Arabidopsis thaliana-ScienceDirect.″Genomics112.5(2020):3549-3559.). Histone deubiquitinase OTU1b epigenetically regulates DA1 and DA2 to control Arabidopsis seed and organ size (Keren, I., et al. "Histone Deubiquitinase OTU1 Epigenetically Regulates DA1 and DA2, Which Control Arabidopsis Seed and Organ Size." iScience 23.3(2020):100948.).

[0006] Ubiquitination extensively affects protein states in the cytoplasm; however, its influence on protein states within peroxisomes remains unclear, and the effects of heat stress on protein states within peroxisomes are even less understood. Identifying and analyzing genes involved in protein ubiquitination within peroxisomes, and understanding how peroxisome proteins contribute to plant heat stress tolerance, will deepen our understanding of peroxisomes, enhance our knowledge of eukaryotic cell biology and metabolism, and ultimately improve crop quality and yield under heat stress. Summary of the Invention

[0007] This invention has discovered the effect of a novel plant peroxisome protein encoded by the OTU4 gene on plant heat tolerance. This protein can be used to improve crop quality and provides new gene resources and theoretical guidance for deepening research on peroxisome function and how plants resist high-temperature stress.

[0008] This invention first provides the application of the OTU4 gene or its encoded protein in improving the ability of plants to withstand high-temperature stress. Preferably, the nucleotide sequence of the OTU4 gene is as shown in SEQ ID NO.1 or SEQ ID NO.2. Preferably, the plant is a dicotyledonous plant. More preferably, the plant is Arabidopsis thaliana.

[0009] This invention further provides the application of the OTU4 gene in screening plants with high resistance to high-temperature stress. By detecting the expression level of the OTU4 gene in the plants to be screened, plants with high OTU4 gene expression levels are selected. Preferably, the nucleotide sequence of the OTU4 gene is shown in SEQ ID NO.1 or SEQ ID NO.2.

[0010] This invention also provides a method for improving plant resistance to heat stress by transferring the OTU4 gene into plants to obtain transgenic plants with high OTU4 gene expression. Preferably, the plant is a plant with poor resistance to heat stress, and the transgenic plant obtained after transferring the OTU4 gene has normal or higher resistance to heat stress. Preferably, the nucleotide sequence of the OTU4 gene is as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0011] The nucleotide sequence shown in SEQ ID NO.1 is the full-length genome sequence of the OTU4 gene, with a sequence length of 2200 bp; the nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the coding region of the OTU4 gene, with a sequence length of 954 bp; the encoded amino acid sequence is 317 aaa (SEQ ID NO.3).

[0012] This invention, through evolutionary analysis of the OUT family, identified a gene localized to the peroxisome, named OTU4. ​​We discovered that OTU4 possesses a highly conserved peroxisome localization signal in plants. This was verified by injecting tobacco leaves, confirming OTU4's localization to the peroxisome. Subsequently, we found that the OTU4 gene specifically responds to heat stress under various abiotic conditions. In Arabidopsis, T-DNA insertion-induced OTU4 gene mutants exhibited abnormal root growth under high-temperature stress, with shorter roots compared to the wild-type. These results indicate that the OTU4 gene encodes a novel plant peroxisome protein that influences plant resistance to high-temperature stress.

[0013] The aforementioned proteins can help plants resist high-temperature stress, while reduced or lost protein activity will affect the plant's resistance to high-temperature stress, leading to abnormal root development.

[0014] The OTU4 gene provided by this invention has potential application value in the study of peroxisome ubiquitination levels and crop breeding.

[0015] This invention also provides a method for constructing a heat-intolerant plant model, comprising the following steps: using gene mutation, gene knockout, gene interference, or gene silencing techniques to induce the deletion or reduced expression of the OTU4 gene in the plant, thereby constructing a heat-intolerant plant model. Preferably, the plant is Arabidopsis thaliana.

[0016] In scientific research, it is necessary to construct model plants with abnormal ubiquitination levels in peroxisomes. Based on the fact that OTU4 is a conserved peroxisome deubiquitinizing enzyme, and its loss of function leads to abnormal plant development under high-temperature stress, this invention provides the application of this gene in constructing heat-intolerant plant models. Specifically, genetic engineering techniques are used to reduce or eliminate the expression of the OTU4 gene in plants.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) This invention discloses for the first time the effect of a novel plant peroxisome protein encoded by the OTU4 gene on plant heat resistance. Mutations in the OTU4 gene cause root growth defects in plants under high temperatures.

[0019] (2) This invention is the first to report deubiquitination enzymes in peroxisomes, providing a theoretical basis and material support for the study of ubiquitination in peroxisomes.

[0020] (3) This invention provides new gene resources and theoretical guidance for deepening the study of peroxisomal function and the study and application of plant heat resistance and other traits. Attached Figure Description

[0021] Figure 1 This is an analysis diagram of the OTU4 family, where each square represents a conserved sequence and the triangles represent peroxisome localization signals.

[0022] Figure 2 35S-YFP-OTU4 spectrum

[0023] Figure 3 This is a transient subcellular localization map of tobacco expression, where CFP-PTS1 is a peroxisome marker, and YFP-OTU4 is the OTU4 major spliceosome protein fused to the C-terminus of YFP fluorescent protein; Bar = 30 μM.

[0024] Figure 4 This refers to OTU4's response to various adversities.

[0025] Figure 5 This diagram illustrates the insertion sites of mutant T-DNA, where white boxes represent UTR regions, black boxes represent exon regions, and black lines represent intron regions.

[0026] Figure 6 The image shows the results of the heat intolerance test for mutants OTU4-1 and OTU4-2.

[0027] Figure 7 35S-OTU4 spectrum

[0028] Figure 8 Phenotypic diagram of heat-intolerant mutants that compensate for 35S-driven OTU4 expression. Detailed Implementation

[0029] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions are performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0030] Example 1

[0031] (1) Prediction and evolutionary analysis of conserved sites in the OUT family

[0032] The Arabidopsis OTU family sequences were downloaded from tair (https: / / www.arabidopsis.org / ), protein structures were predicted using SWISS-MODEL (https: / / swissmodel.expasy.org / ), and evolutionary analysis was performed using MEGA6 software with all parameters set to default. The results are shown below. Figure 1 The results showed that only OTU4 had a peroxisome localization signal.

[0033] (2) Construction of OTU4 gene subcellular localization vector

[0034] Primers were designed based on the CDS sequence of the major spliceosome of the OTU4 gene (as shown in SEQ ID NO.2). Using wild-type Arabidopsis cDNA as a template, the CDS sequence was amplified. The amplified fragment was then ligated to the C-terminus of YFP (yellow fluorescent protein). The primers used were Y-OTU4-F and Y-OTU4-R.

[0035] The primer sequences are:

[0036] Y-OTU4-F: 5'-GCGTCGACTCGAGTGTCTAGAATGATGATTTGTTACTCTCCAATTACAACC-3';

[0037] Y-OTU4-R: 5'-GGTCTTAATTAACTCTCTAGACTAAAGTTTAGATTTTGGAATCGAAGC-3';

[0038] The PCR reaction conditions are as follows:

[0039] Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 2 min, 40 cycles; extension at 72℃ for 5 min.

[0040] This fragment was recovered by agarose gel electrophoresis.

[0041] For vectors with YFP fusion at the C-terminus of the gene, the pEarleyGate 101 vector was digested with XbaI, and the target fragment and the linearized vector were ligated using homologous recombination to obtain the 35S-YFP-OTU4 recombinant vector. (See diagram below.) Figure 2 .

[0042] Take 1 μl of the recombinant product and transform it into E. coli DH5α using the freeze-thaw method. Spread the transformation product onto LB medium containing kanamycin (50 μg / ml). Incubate overnight at 37°C, and select single colonies for plasmid extraction. After sequencing verification, the constructed vector is stored for later use.

[0043] (3) Subcellular localization experiment

[0044] The fusion expression vector was injected into tobacco leaves using the Agrobacterium permeation method, and the fluorescence signal was observed using a laser confocal microscope 48 hours later.

[0045] The experimental results are shown in Figure 3 The YFP protein fusion was located at the N-terminus of the major spliceosomal protein (the peroxisome localization signal peptide PTS1 of the OTU4 protein was not blocked), thus localizing to the peroxisome; the results indicate that OTU4 is a peroxisome protein.

[0046] Example 2

[0047] (1) OTU4's response to various adversities

[0048] The response data of the OTU4 gene to various stresses were analyzed from the BAR database (http: / / bar.utoronto.ca), and heatmaps were generated using TBTOOLS software, such as... Figure 4 As shown, OTU4 exhibits a specific response to high-temperature stress.

[0049] (2) Identification of T-DNA insertion mutants

[0050] Two types of OTU4 mutants caused by T-DNA insertion were purchased from the Arabian Biological Resource Center (ABRC, https: / / abrc.osu.edu / ). Genotyping of the mutants was performed using a three-primer method. Primers were designed using the T-DNA primer design website (http: / / signal.salk.edu / tdnaprimers.2.html). The primers designed on the website are designated as OTU4-1-LP and OTU4-1-RP, and OTU4-2-LP and OTU4-2-RP, respectively. The LB primer was LBb1.3, provided by the website.

[0051] The primer sequences are:

[0052] OTU4-1-LP: 5'-CTTAGGAGCAACAGTTGGTCG-3';

[0053] OTU4-1-RP: 5'-GCTGTTCGAGACTATTGCCAG-3';

[0054] OTU4-2-LP: 5'-TGCTTGTCCACCTTGGTAATC-3';

[0055] OTU4-2-RP: 5'-GGTACGTAATTCGTCAGCGAG-3';

[0056] LBb1.3: 5'-ATTTTGCCGATTTCGGAAC-3';

[0057] The PCR reaction conditions are as follows:

[0058] 95℃, 3 min; 95℃, 15 sec; 58℃, 15 min; 72℃, 15 sec, 32 cycles; 72℃, 5 min.

[0059] The purchased homozygous mutants were designated as otu4-1 and otu4-2, respectively. The T-DNA insertion sites are shown below. Figure 5 .

[0060] (3) Phenotypic analysis of T-DNA insertion mutants

[0061] 1-2 MS medium containing 1% sucrose was prepared. T-DNA was inserted into the mutant and the corresponding wild-type seeds and cultured on the medium. The mixture was incubated at 4°C for 72 hours. Subsequently, the seeds were placed in plant growth chambers under normal and high-temperature conditions, respectively. The normal growth chamber environment was: light / dark 16h / 8h, temperature 22°C; the high-temperature plant growth chamber conditions were: light / dark 16h / 8h, temperature 30°C, normal 22°C. After seven days of growth, the plants were photographed. The results are as follows: Figure 6 As shown, the roots of the otu4-1 and otu4-2 mutants are significantly shorter than those of the wild type at high temperatures.

[0062] Example 3

[0063] (1) OTU4 complementation vector construction and genetic transformation experiments

[0064] To further verify that the heat intolerance phenotype of the T-DNA mutant lines is caused by the OTU4 gene mutation, a 35S-driven OTU4 vector was constructed for complementation.

[0065] Primers were designed based on the CDS sequence of the major spliceosome of the OTU4 gene (as shown in SEQ ID NO.2), and the CDS sequence was amplified using Arabidopsis cDNA as a template. The primers used were OTU4-OE-F and OTU4-OE-R.

[0066] OTU4-OE-F: 5'-TTTGGAGAGGACACGCTCGAGATGATGATTTGTTACTCTCCAATIACAAC-3';

[0067] OTU4-OE-R: 5'-GGTCTTAATTAACTCTCTAGACTAAAGTTTAGATTTTGGAATCGAAGC-3';

[0068] The PCR reaction conditions are as follows:

[0069] Pre-denaturation at 95℃ for 3 min; denaturation at 95℃ for 15 sec, annealing at 55℃ for 15 sec, extension at 72℃ for 2 min, 40 cycles; extension at 72℃ for 5 min.

[0070] This fragment was recovered by agarose gel electrophoresis.

[0071] For the vector driving OTU4 expression under 35S, the pEarleyGate100-YFP vector was digested with XhoI and XbaI, and the target fragment and the linearized vector were ligated using homologous recombination to obtain the 35S-OTU4 recombinant vector. (See diagram below.) Figure 7

[0072] Take 1 μl of the recombinant product and transform it into E. coli DH5α using the freeze-thaw method. Spread the transformation product onto LB medium containing kanamycin (50 μg / ml). Incubate overnight at 37°C, and select single colonies for plasmid extraction. After sequencing verification, the constructed vector is stored for later use.

[0073] Transform the correctly sequenced vector into Agrobacterium using the instructions in the kit.

[0074] Agrobacterium was transferred into the OTU4-1 mutant using the inflorescence infection method to obtain T0 generation transgenic seeds. Transgenic plants with the 35S-OTU4 insertion were then identified. The identified transgenic seeds were then propagated.

[0075] (2) Experiment on 35S-OTU4 compensating for the phenotype of heat-intolerant mutants

[0076] A 1-2 MS medium containing 1% sucrose was prepared. Transgenic plants, T-DNA-inserted mutants, and corresponding wild-type seeds were placed on the medium and incubated at 4°C for 72 hours. Subsequently, the seeds were placed in plant growth chambers under normal and high-temperature conditions, respectively. The normal growth chamber environment was: 16h / 8h light / dark, temperature 22°C; the high-temperature plant growth chamber conditions were: 16h / 8h light / dark, temperature 30°C. After seven days of growth, the plants were photographed. The results are as follows: Figure 8 As shown, introducing 35S-OTU4 into plants can significantly restore the root development defect phenotype of heat-intolerant mutants.

Claims

1. OTU4 The use of the gene in screening plants with high resistance to high temperature stress, characterized in that, By detecting the plants to be screened OTU4 Gene expression levels, screening OTU4 Plants with high gene expression levels The nucleotide sequence of the gene is as shown in SEQ ID NO. 1 or SEQ ID NO.

2. OTU4 The nucleotide sequence of the gene is as shown in SEQ ID NO. 1 or SEQ ID NO.

2. The plant is Arabidopsis thaliana.

2. A method for improving the ability of a plant to withstand high temperature stress, characterized in that, The OTU4 transformation of plants with genes OTU4 transgenic plants with high expression of genes The plant is a plant with poor ability to resist high temperature stress, and the method comprises the following steps: OTU4 The transgenic plant obtained after the gene is transferred has normal ability to resist high temperature stress. The OTU4 nucleotide sequence of the gene is as shown in SEQ ID NO. 1 or SEQ ID NO. 2; The plant is Arabidopsis thaliana.

3. A method of constructing a model of a thermolabile plant, characterized by, comprising the following steps: causing the deletion or reduction of the expression of the gene in the plant by means of genetic mutation, gene knockout, gene interference or gene silencing technology, so as to construct a heat-intolerant plant model; OTU4 comprising the following steps: causing the deletion or reduction of the expression of the gene in the plant by means of genetic mutation, gene knockout, gene interference or gene silencing technology, so as to construct a heat-intolerant plant model; The OTU4 The nucleotide sequence of the gene is shown in SEQ ID NO. 1 or SEQ ID NO. 2; The plant is Arabidopsis thaliana.

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