Transcription factor for improving cold resistance of cassava and application

Abstract

The invention provides an application of a substance, the substance is selected from one or more of MeICE1, MeETC1, MeDFR and MePAL2 genes, or a coding protein thereof, or an accelerant or an inhibitor thereof, and the resistance of cassava to low temperature is regulated through genetic improvement, so that the planting area of cassava is widened.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and botany, and more particularly to a method and application of an R3-MYB transcription factor in improving cold resistance of cassava. Background Art

[0002] Cassava is heat-resistant, drought-resistant, and tolerant to infertility, and is widely cultivated in tropical Asia, America, and Africa (Prochnik et al., 2012). Cassava is not only a staple feed in my country but also a raw material for industrial starch and fuel ethanol. "Northward migration of cassava" has become an effective way to increase cassava production without competing with staple crops for land. However, cassava is vulnerable to low temperatures and is susceptible to chilling damage. The cold snaps of 2008 and 2016 severely impacted the cassava industry, necessitating the development of new cold-resistant germplasm.

[0003] The main goal and direction of current plant cold tolerance research is to evaluate plant cold tolerance by measuring physiological and biochemical indicators related to plant cold tolerance, and to further investigate the physiological mechanisms of cold tolerance. Domestic research on cassava cold tolerance has been conducted, with the consensus that physiological indicators such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), malondialdehyde (MDA), proline, relative conductivity, and chlorophyll content can serve as important indicators for evaluating cassava cold tolerance (Jia et al., 2013).

[0004] There are various approaches to cultivating cold-resistant germplasm, including screening existing varieties for cold resistance, traditional hybridization, and molecular breeding. However, due to the relatively narrow genetic base of current cassava varieties and their inherent self-incompatibility, low flowering, and low fertility, the first two approaches have hindered the development of cassava breeding. With the advancement of molecular biology and biotechnology, genetic engineering is considered an effective method for genetic improvement. Therefore, this study aims to improve cassava's cold tolerance through genetic improvement, thereby expanding the cassava planting area and alleviating the supply-demand imbalance in the cassava industry (Yan Huabing et al., 2015). To provide insights for genetically engineered cold-resistance breeding in cassava and guide other crops, we aim to understand the underlying molecular mechanisms of plant cold regulation by exploring plant physiological responses to cold stress and analyzing the effects of stress metabolites on plant resistance, thereby applying these insights to genetic improvement in cassava.

[0005] The molecular regulatory pathways of cassava under low temperature stress are complex and sophisticated. Exploring and verifying the functions of its key regulatory factors will not only help to analyze the physiological characteristics of cassava's intolerance to low temperatures, but also provide ideal target genes for rapid genetic engineering breeding, thereby cultivating new cassava germplasm with cold-resistant qualities and providing ideal materials for the cassava northward migration strategy. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a gene that can improve the low temperature resistance of plants and its application.

[0007] In a first aspect, the present invention provides a method for regulating a plant's low-temperature tolerance. The method comprises: administering to a plant a substance selected from the group consisting of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes, or their encoded proteins, or promoters or inhibitors thereof. In one or more embodiments, the low temperature is 0-10°C. Preferably, the low temperature is 4°C.

[0008] In one or more embodiments, the substance is one or more of MeICE1, MeETC1, MeDFR, MePAL2 genes or their encoded proteins, or promoters thereof, and regulating the low temperature resistance of the plant includes improving the low temperature resistance of the plant.

[0009] In one or more embodiments, the substance is an inhibitor of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes, and regulating the low-temperature resistance of the plant includes reducing the low-temperature resistance of the plant.

[0010] In one or more embodiments, the promoter is selected from the group consisting of a small molecule compound, a nucleic acid molecule, or a combination thereof. In one or more embodiments, the promoter is a coding sequence of the gene; the coding sequence is DNA or RNA.

[0011] In one or more embodiments, the inhibitor is an inhibitory molecule that specifically interferes with the transcription and / or expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes.

[0012] In one or more embodiments, the inhibitory molecule targets one or more of the MeICE1, MeETC1, MeDFR, MePAL2 genes or their transcripts.

[0013] In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a dsRNA, a sgRNA, an antibody, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1).

[0014] In one or more embodiments, the inhibitory molecule has the sequence shown in SEQ ID NO:59.

[0015] In one or more embodiments, the inhibitor further comprises a Cas enzyme (eg, Cas9), a coding sequence thereof, and / or a nucleic acid construct expressing the Cas enzyme.

[0016] In one or more embodiments, the amino acid sequence of MeICE1 is shown as SEQ ID NO: 54; the amino acid sequence of MeETC1 is shown as SEQ ID NO: 55; the amino acid sequence of MeDFR is shown as SEQ ID NO: 56; and the amino acid sequence of MePAL2 is shown as SEQ ID NO: 57.

[0017] In one or more embodiments, the plant is a woody plant.

[0018] In one or more embodiments, the woody plant is a plant of the Euphorbiaceae family.

[0019] In one or more embodiments, the Euphorbiaceae plant includes cassava.

[0020] In one or more embodiments, the MeICE1, MeETC1, MeDFR, MePAL2 gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0021] A second aspect of the present invention provides a method for regulating plant resistance to low temperatures, comprising regulating the expression or activity of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes in the plant, thereby regulating the plant's resistance to low temperatures.

[0022] In one or more embodiments, the plant is a woody plant.

[0023] In one or more embodiments, the woody plant is a plant of the Euphorbiaceae family.

[0024] In one or more embodiments, the Euphorbiaceae plant includes cassava.

[0025] In a preferred embodiment, the method for regulating the low temperature resistance of a plant comprises: upregulating the expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes in the plant; thereby enhancing the low temperature resistance of the plant.

[0026] In one or more embodiments, upregulating the expression of one or more of MeICE1, MeETC1, MeDFR, and MePAL2 genes in the plant comprises: transferring one or more of MeICE1, MeETC1, MeDFR, and MePAL2 genes into the plant to obtain a transformed plant.

[0027] In one or more embodiments, the method enhances the low temperature resistance of a plant, comprising the step of upregulating the expression or activity of the MeICE1 gene in the plant, comprising:

[0028] (1) providing Agrobacterium carrying a nucleic acid construct containing the MeICE1 gene,

[0029] (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0030] In one or more embodiments, the method enhances the low temperature resistance of a plant, comprising the step of upregulating the expression or activity of the MeETC1 gene in the plant, comprising:

[0031] (1) providing Agrobacterium carrying a nucleic acid construct containing MeICE1 and / or MeETC1 gene,

[0032] (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0033] In one or more embodiments, the method enhances the low temperature resistance of a plant, comprising the step of upregulating the expression or activity of a MeDFR gene in the plant, comprising:

[0034] (1) providing Agrobacterium carrying a nucleic acid construct containing one or more of the MeICE1, MeETC1, and MeDFR genes,

[0035] (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0036] In one or more embodiments, the method enhances the low temperature resistance of a plant, the method comprising the step of upregulating the expression or activity of the MePAL2 gene in the plant, comprising:

[0037] (1) providing Agrobacterium carrying a nucleic acid construct containing one or more of the MeICE1, MeETC1, and MePAL2 genes,

[0038] (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0039] In one or more embodiments, the nucleic acid construct is a vector, such as an expression vector or a recombinant vector.

[0040] In one or more embodiments, the nucleic acid construct includes a MeETC1 gene promoter, a MeETC1 gene, and a terminator from the 5' end to the 3' end.

[0041] In one or more embodiments, the method of upregulating the expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes in a plant further comprises:

[0042] (3) selecting plant tissues, organs or seeds into which the corresponding gene has been transferred; and

[0043] (4) Regenerating the plant tissue, organ or seed in step (3) into a plant.

[0044] In another preferred embodiment, the method for regulating the low temperature resistance of plants comprises: down-regulating the expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes in plants; thereby reducing the low temperature resistance of plants.

[0045] In one or more embodiments, downregulating the expression of one or more of MeICE1, MeETC1, MeDFR, and MePAL2 genes in the plant comprises introducing an inhibitor that downregulates MeETC1 gene transcription, protein expression, or protein activity into the plant.

[0046] In one or more embodiments, the inhibitor is an inhibitory molecule that specifically interferes with the transcription and / or expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes.

[0047] In one or more embodiments, the inhibitory molecule targets one or more of the MeICE1, MeETC1, MeDFR, MePAL2 genes or their transcripts.

[0048] In one or more embodiments, the inhibitory molecule is selected from the group consisting of: (1) a small molecule compound, an antisense nucleic acid, a microRNA, a siRNA, an RNAi, a dsRNA, a sgRNA, an antibody, or a combination thereof, and (2) a nucleic acid construct capable of expressing or forming (1).

[0049] In one or more embodiments, the inhibitory molecule has the sequence shown in SEQ ID NO:59.

[0050] In one or more embodiments, the inhibitor further comprises a Cas enzyme (eg, Cas9), a coding sequence thereof, and / or a nucleic acid construct expressing the Cas enzyme.

[0051] In one or more embodiments, the method of downregulating the expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes in a plant comprises:

[0052] (i) providing Agrobacterium carrying a nucleic acid construct capable of interfering with gene expression, wherein the nucleic acid construct contains or produces the inhibitor;

[0053] (ii) contacting plant cells, tissues or organs with the Agrobacterium in step (i), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0054] In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector.

[0055] In one or more embodiments, the method of downregulating the expression of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes in a plant further comprises:

[0056] (iii) selecting plant tissues, organs or seeds into which the nucleic acid construct has been introduced; and

[0057] (iv) regenerating the plant tissue, organ or seed of step (iii) into a plant.

[0058] The present invention also provides a method for obtaining a plant with improved low temperature resistance, comprising:

[0059] (1) providing Agrobacterium carrying a nucleic acid construct containing one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes,

[0060] (2) contacting plant cells, tissues or organs with the Agrobacterium in step (1), thereby transferring the nucleic acid construct into the plant tissues or organs.

[0061] (3) selecting plant tissues, organs or seeds into which one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes have been introduced; and

[0062] (4) Regenerating the plant tissue, organ or seed in step (3) into a plant.

[0063] Another aspect of the present invention provides a use of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes as molecular markers for identifying the low temperature resistance of plants.

[0064] In one or more embodiments, the plant is a woody plant.

[0065] In one or more embodiments, the woody plant is a plant of the Euphorbiaceae family.

[0066] In one or more embodiments, the Euphorbiaceae plant includes cassava.

[0067] In one or more embodiments, the MeICE1, MeETC1, MeDFR, MePAL2 gene comprises a cDNA sequence, a genomic sequence, or a combination thereof.

[0068] In one or more embodiments, the MeICE1, MeETC1, MeDFR, MePAL2 genes are from Euphorbiaceae plants, preferably from cassava.

[0069] The present invention also provides an expression cassette for expressing the MeETC1 gene, wherein the expression cassette comprises the following elements from 5' to 3': a promoter, an ORF sequence of the MeETC1 gene, and a terminator. In one or more embodiments, the promoter is the MeETC1 gene promoter or the 35S promoter.

[0070] The present invention also provides a nucleic acid construct comprising the expression cassette described herein or a complementary sequence thereof.

[0071] In one or more embodiments, the nucleic acid construct is an expression vector or a recombinant vector.

[0072] The present invention also provides a host cell, which (1) comprises a nucleic acid construct comprising the expression cassette described herein or its complementary sequence, or (2) has the expression cassette described herein integrated into its chromosome.

[0073] The present invention also provides a use of the expression cassette described herein for improving the cold resistance of plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic diagram of the ProMeETC1::GUS gene and its expression in various cassava plant parts. A is a schematic diagram of the MeETC1 promoter::GUS and its expression in various cassava plant parts. Young leaf: young leaf; Old leaf: mature leaf. B is the relative expression level of MeETC1 transcripts in different cassava organs. AB: terminal bud; L: leaf; S: stem; P: petiole; FR: fibrous root; DR: developing storage root; MR: storage root.

[0075] Figure 2 is the subcellular localization of MeETC1.

[0076] Figure 3MeETC1 expression is induced by low temperature. A is the real-time PCR expression of MeETC1, and B is the expression of MeETC1 after 6 hours of cold treatment.

[0077] Figure 4 MeETC1 positively regulates cassava's cold stress resistance. A shows the phenotype of MeETC1 transgenic plants before and after cold treatment (4°C for 6 hours); B shows the drooping angle of the leaves of MeETC1 transgenic plants after 4°C cold treatment for 6 hours.

[0078] Figure 5 Physiological indicators of MeETC1 transgenic plants before and after low temperature treatment were measured. A represents the oxidative function of MeETC1-interfered transgenic plants, B represents the degree of leaf damage in MeETC1-interfered transgenic plants during low temperature treatment, C represents the leaf angle of MeETC1-interfered plants, and D represents the chlorophyll content in the leaves of the interfered plants.

[0079] Figure 6 MeETC1 promotes the clearance of reactive oxygen species at low temperatures.

[0080] Figure 7 MeETC1 promotes anthocyanin accumulation at low temperatures. A shows the anthocyanin accumulation phenotype of MeETC1 transgenic plants during natural cooling in the field. B shows anthocyanin staining of MeETC1 transgenic plants. CD shows anthocyanin content determination in MeETC1 transgenic plants. E shows the relative expression levels of anthocyanin synthesis-related genes at different temperature ranges; PAL: phenylalanine ammonia lyase; DFR: dihydroflavonol-4-reductase; ANS: anthocyanidin synthase; CHS: naringin-chalcone synthase; F3'H: flavanone-3-hydroxylase.

[0081] Figure 8 MeETC1 positively regulates cassava's cold stress tolerance. A shows the leaf phenotype of MeETC1 transgenic plants during a natural field temperature drop (10-18°C). B shows the leaf phenotype of MeETC1 transgenic plants during a natural field temperature drop (3-11°C).

[0082] Figure 9 The chlorophyll and anthocyanin content of transgenic plants in the field was measured during a natural cooling process. A and B show the chlorophyll content of transgenic cassava leaves when the field temperature was reduced to 10-18°C and 3-11°C, respectively. C and D show the anthocyanin content of transgenic cassava shoots when the field temperature was reduced to 10-18°C and 3-11°C, respectively.

[0083] Figure 10MeETC1 can directly bind to the MePAL2 promoter and activate its expression. A shows a tobacco transactivation assay verifying that MeETC1 can directly bind to the MePAL2 promoter; MePAL2pro:LUC: a luciferase reporter gene driven by the MePAL2 promoter; 35S-MeETC1: a MeETC1 gene driven by the strong 35S promoter. B shows a dual-luciferase assay verifying that MeETC1 activates MePAL2 expression; ETC1-62sk: a 62sk vector ligated with the CDS of the MeETC1 gene; MePAL2pro: an 0800 vector ligated with the MePAL2 promoter sequence; 62sk-EV: an empty 62sk vector. C is a DNA gel retardation experiment demonstrating that MeETC1 binds to the MePAL2 promoter through the MBS binding element; MBS probe: a digoxigenin-labeled probe of the MBS sequence on the MePAL2 promoter; Competitor: a competitor probe not labeled with digoxigenin; MeETC1-GST: a purified MeETC1 protein tagged with GST.

[0084] Figure 11 MeETC1 can directly bind to the MeDFR promoter and activate its expression. A shows a tobacco transactivation assay verifying that MeETC1 can directly bind to the MeDFR promoter; MeDFRpro:LUC: a luciferase reporter gene driven by the DFR promoter; 35S-MeETC1: a MeETC1 gene driven by the strong 35S promoter. B shows a dual-luciferase assay verifying that MeETC1 activates MeDFR expression; MeETC1-62sk: a 62sk vector ligated with the MeETC1 gene CDS; MeDFRp: an 0800 vector ligated with the MeDFR promoter sequence; 62sk-EV: an empty 62sk vector. C is a DNA gel retardation experiment demonstrating that MeETC1 binds to the MeDFR promoter through the MBS binding element; MBS probe: a digoxigenin-labeled probe of the MBS sequence on the MeDFR promoter; Competitor: a competitor probe not labeled with digoxigenin; MeETC1-GST: a purified MeETC1 protein tagged with GST.

[0085] Figure 12MeICE1 directly binds to the MeETC1 promoter and activates its expression. A is a yeast one-hybrid assay demonstrating that MeICE1 can bind to the MeETC1 promoter; Prey: prey vector containing MeICE1; Bait: bait vector containing the MeETC1 promoter. B is a dual-fluorescein assay confirming that MeICE1 can activate MeETC1 expression; ICE1-62sk: 62sk vector containing the ICE1 CDS sequence; 62sk EV: empty 62sk vector; ETC1p: 0800 vector containing the MeETC1 promoter. C is a DNA gel retardation assay demonstrating that MeICE1 can bind to the MYC element in the MeETC1 promoter; MYC probe: digoxigenin-labeled probe targeting the MYC element in the MeETC1 promoter; Competitor: unlabeled probe; MeICE1-GST: purified GST-tagged ICE1 protein.

[0086] Figure 13 Figure 5 represents the field pilot phenotype of MeETC1 transgenic cassava. A represents the field pilot phenotype of the aboveground and belowground parts of MeETC1 transgenic cassava. All transgenic plants were grown in the field for five months. WT: wild type; OE-10 and OE-20: MeETC1-OE transgenic plants; Ri-4 and Ri-5: MeETC1 RNA interference transgenic plants. BE represents the statistical data of field parameters such as root length, root diameter, total root biomass, and root number of MeETC1 transgenic plants. DETAILED DESCRIPTION

[0087] The inventors revealed for the first time that by targeted regulation of the expression level of the MeETC1 gene in plants, the plant's resistance to low temperatures can be significantly adjusted, thereby cultivating new plant germplasm with cold-resistant qualities.

[0088] As used herein, "woody plant" refers to a plant with a woody stem. Preferably, the woody plant is a Euphorbiaceae plant; more preferably, the Euphorbiaceae plant is cassava.

[0089] In a first aspect, the present invention provides a use of a substance in regulating the low temperature resistance of a plant, wherein the substance is selected from the following group: MeETC1 gene or its encoded protein, or a promoter or inhibitor thereof.

[0090] "MeETC1-encoded protein" refers to a polypeptide having MeETC1 activity, including but not limited to variant forms of the polypeptide. "Variants" include (but are not limited to): deletion, insertion, and / or substitution of several amino acids (usually 1-50, preferably 1-30, 1-20, 1-10, 1-8, 1-5), and addition or deletion of one or several amino acids (usually within 20, preferably within 10, and more preferably within 5) at the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids with similar or similar properties generally does not alter the function of the protein. In the art, amino acids with similar properties often refer to a family of amino acids with similar side chains, which has been clearly defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with non-polar side chains (e.g., alanine, valine, leucine, isoleucine, lactic acid, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). For another example, adding one or more amino acids to the amino and / or carboxyl termini generally does not alter the function of a polypeptide or protein. Conservative amino acid substitutions for many common, known non-genetically encoded amino acids are known in the art. Conservative substitutions for other non-encoded amino acids can be determined based on a comparison of their physical properties with the properties of the genetically encoded amino acids.

[0091] The variant forms of polypeptides include: homologous sequences, conservative variants, allelic variants, natural mutants, and induced mutants.

[0092] The present invention also relates to polynucleotide sequences encoding MeETC1 or variants thereof. The polynucleotides may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or synthetic DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0093] The present invention also relates to variants of the above-mentioned polynucleotides, which encode fragments, analogs and derivatives of polypeptides having the same amino acid sequence as the present invention. Variants of this polynucleotide may be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide, which may be a substitution, deletion or insertion of one or more nucleotides, but will not substantially change the function of the polypeptide it encodes. A "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include a polynucleotide further comprising additional coding and / or non-coding sequences.

[0094] The full-length MeETC1 nucleotide sequence of the present invention or its fragments can generally be obtained by PCR amplification, recombination, or artificial synthesis. For PCR amplification, primers can be designed based on the relevant nucleotide sequences disclosed in the present invention, particularly the open reading frame sequences, and a commercially available DNA library or a cDNA library prepared by conventional methods known to those skilled in the art is used as a template to amplify the relevant sequence. When the sequence is long, two or more PCR amplifications are often required, and then the fragments amplified in each step are spliced together in the correct order. Once the relevant sequence is obtained, the relevant sequence can be obtained in large quantities by recombination. Typically, it is cloned into a vector, then transferred into cells, and then the relevant sequence is isolated from the proliferated host cells by conventional methods.

[0095] In addition, artificial synthesis methods can also be used to synthesize relevant sequences, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then connecting them, very long fragments of sequence can be obtained. At present, DNA sequences encoding proteins of the present invention (or their fragments, or their derivatives) can be obtained completely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

[0096] The present invention also provides a recombinant vector comprising the gene of the present invention. As a preferred embodiment, the promoter downstream of the recombinant vector comprises a multiple cloning site or at least one restriction enzyme site. When it is necessary to express the target gene of the present invention, the target gene is connected to a suitable multiple cloning site or restriction enzyme site, thereby operably connecting the target gene to the promoter. As another preferred embodiment, the recombinant vector comprises (from 5' to 3' direction): a promoter, a target gene, and a terminator. If necessary, the recombinant vector may further comprise an element selected from the following group: a 3' polynucleotide signal; a non-translated nucleic acid sequence; a transport and targeting nucleic acid sequence; a resistance selection marker (dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green fluorescent protein, etc.); an enhancer; or an operator.

[0097] Methods for preparing recombinant vectors are well known to those of ordinary skill in the art. Expression vectors can be bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses, or other vectors. In short, any plasmid or vector can be used as long as it can replicate and be stable in the host.

[0098] Those skilled in the art can construct expression vectors containing the genes of the present invention using well-known methods. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like. When constructing recombinant expression vectors using the genes of the present invention, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before the transcription initiation nucleotide.

[0099] Vectors containing the genes, expression cassettes, or proteins of the present invention can be used to transform appropriate host cells to allow the host to express the protein. The host cells can be prokaryotes, such as Escherichia coli, Streptomyces, or Agrobacterium; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as plant cells, preferably Euphorbiaceae plant cells, and more preferably cassava cells. Those skilled in the art will appreciate how to select appropriate vectors and host cells. Transformation of host cells with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. When the host is a prokaryotic organism (such as Escherichia coli), CaCl2 treatment or electroporation can also be used. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods (such as microinjection, electroporation, liposome packaging, etc.). Plants can also be transformed using methods such as Agrobacterium transformation or gene gun transformation, for example, the leaf disc method, the embryonic embryo transformation method, the flower bud immersion method, etc. Transformed plant cells, tissues, or organs can be regenerated into plants using conventional methods to obtain transgenic plants. When the polynucleotide is expressed in higher eukaryotic cells, transcription will be enhanced if an enhancer sequence is inserted into the vector. Enhancers are cis-acting factors of DNA, usually about 10 to 300 base pairs, that act on the promoter to enhance gene transcription.

[0100] It is clear to those skilled in the art how to select appropriate vectors, promoters, enhancers and host cells.

[0101] The polypeptides described herein can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include (but are not limited to): conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic shock, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0102] Transformation of hosts with recombinant DNA can be performed using conventional techniques familiar to those skilled in the art. Plants can be transformed using methods such as Agrobacterium transformation or gene gun transformation, for example, spraying, leaf disc, and rice embryo transformation. Transformed plant tissues or organs can be regenerated into plants using conventional methods to obtain plants with altered traits.

[0103] The present invention provides a use of the MeETC1 gene for regulating the low-temperature resistance of plants. As a preferred embodiment, the MeETC1 gene can be used to increase the low-temperature resistance of plants.

[0104] The present invention also relates to MeETC1 upregulators or inhibitors and their uses. Since MeETC1 upregulators or inhibitors can regulate the expression and / or activity of MeETC1, the upregulators or inhibitors can also regulate the low temperature resistance of plants by affecting MeETC1, thereby achieving the purpose of improving plants.

[0105] In one aspect, any substance that can increase the activity of MeETC1, improve its stability, promote its expression, prolong its effective duration, or promote its gene transcription and translation can be used in the present invention as a "promoter" of the MeETC1 gene to regulate plant cold tolerance. For example, an expression vector that increases the transcription, expression, or activity of the MeETC1 gene.

[0106] On the other hand, any substance that can reduce the activity of MeETC1, reduce its stability, inhibit its expression, reduce its effective action time, or reduce its transcription and translation can be used in the present invention as a down-regulator, antagonist or inhibitor of MeETC1, such as an interfering molecule that interferes with the expression of the MeETC1 gene (such as an interfering molecule that can form microRNA). The inhibitor, antagonist or inhibitor can be used to regulate plant agronomic traits. After knowing the target sequence, methods for preparing interfering molecules that interfere with the expression of specific genes are well known to those skilled in the art.

[0107] Furthermore, to downregulate MeETC1 gene expression or activity, a gene knockout vector can be introduced into the cell, and / or the gene can be edited using gene editing technologies such as ZFN, TALEN, or CRISPR / Cas9. ZFN, TALEN, and CRISPR / Cas9 technologies suitable for use in the present invention are well known in the art. Each technology achieves target gene knockout through the combined action of a DNA recognition domain and an endonuclease.

[0108] The present invention also relates to a method for regulating agronomic traits of a plant, which comprises regulating the expression of the MeETC1 gene in the plant.

[0109] In one aspect, the present invention provides a method for regulating a plant's cold tolerance, comprising: causing the plant to overexpress the MeETC1 gene, thereby increasing the plant's cold tolerance. Once the purpose of the MeETC1 gene is known, various methods well known to those skilled in the art can be used to regulate the expression of the MeETC1 gene. For example, an expression unit (such as an expression vector or virus) carrying the MeETC1 gene can be delivered to a target site using methods known to those skilled in the art to cause the site to express active MeETC1.

[0110] In one embodiment of the present invention, the MeETC1 gene is cloned into an appropriate vector using conventional methods, and the recombinant vector carrying the exogenous gene is introduced into plant tissues or organs to cause the plant to express the MeETC1 gene. Plants overexpressing the MeETC1 gene can be obtained by regenerating the plant tissues or organs into plants.

[0111] On the other hand, the present invention provides another method for regulating the low temperature resistance of a plant, comprising: reducing the expression of the MeETC1 gene in the plant (including preventing the MeETC1 gene from being expressed or causing it to be expressed at a low level); thereby reducing the low temperature resistance.

[0112] Various methods well known to those skilled in the art can be used to reduce or eliminate the expression of the MeETC1 gene, such as delivering an expression unit (such as an expression vector or virus) carrying the antisense MeETC1 gene to the target site, so that the cell or plant tissue does not express or reduces the expression of MeETC1. Alternatively, the MeETC1 gene can be knocked out by methods known to those skilled in the art, and / or gene editing technologies such as ZFN, TALEN, or CRISPR / Cas9 can be used to knock out or knock down the MeETC1 gene.

[0113] The present invention cloned this gene for the first time, and the gene was significantly upregulated by low temperature induction. By subjecting the obtained ETC1 overexpression and RNAi interference plants to simulated low temperature treatment, it was found that the leaves of the overexpression plants were firm and green, and indicators such as MDA, chlorophyll content, leaf angle and ROS content further demonstrated that the overexpression plants had improved low temperature resistance. During the natural cooling process in the field, the overexpression plants enriched more anthocyanins and had greatly enhanced ROS scavenging ability. Both simulated low temperature and field cooling conditions confirmed that the overexpression plants had significantly improved low temperature resistance. In addition, through a series of biochemical experiments such as EMSA, Y1H and dual luciferase, the downstream regulatory genes MePAL2 and MeDFR of ETC1 and the upstream regulatory factor ICE1 were identified, the low temperature resistance mechanism of ETC1 was analyzed, and a complete network was established. The present invention not only obtains new low temperature-resistant varieties, but also provides a theoretical basis for the study of low temperature resistance of cassava, with very significant effects and extremely strong application value.

[0114] Other aspects of the present invention will be apparent to those skilled in the art from the disclosure herein. The present invention will be further described below with reference to the specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental procedures in the following examples, for which specific conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0115] Example

[0116] (1) Experimental materials

[0117] 1. Plant materials:

[0118] Wild-type cassava TMS60444, from our laboratory; MeETC1 transgenic cassava (overexpression line and RNAi line).

[0119] Wild-type Nicotiana benthamiana, from our laboratory.

[0120] 2. Strains

[0121] Escherichia coli: DH5α, TOP10;

[0122] Agrobacterium tumefaciens: LBA4404 (RifrChlr), GV3101 (RifrTetr);

[0123] Yeast AH109.

[0124] 3. Plasmid

[0125] pMD18-T, pA7-GFP, p1300-GFP, pCAMBIA1300-GUS, pCAMBIA1301s (pCAMBIA1301 backbone, Kanr), pBSRNAi (pBluescriptSK backbone, Ampr), p35RNAi (pCAMBIA1300 backbone, Kanr), pLL00r-BAR and pGEX-4T were preserved in our laboratory; pGreen-0800 and pGreen-62sk were gifts from Li Laigeng's laboratory; pGADT7, pGBKT7 and pAbAi were purchased from Ouyi Company.

[0126] (2) Experimental steps

[0127] 1. Construct a MeETC1 overexpression and RNAi vector (MeETC1 siRNA sequence: SEQ ID NO: 59) and stably transform cassava callus using Agrobacterium tumefaciens to obtain MeETC1 overexpression and RNAi transgenic cassava. The primers used included MeETC1-F and MeETC1-R, as shown in Table 1.

[0128] Table 1: Primer and probe sequences used in this article

[0129]

[0130]

[0131]

[0132] 2. Conduct field trials on wild-type and MeETC1 transgenic cassava and observe their phenotypes.

[0133] 3. Use molecular biology techniques to explore the molecular mechanism by which MeETC1 regulates cassava's low temperature resistance.

[0134] (3) Experimental results (effects):

[0135] 1. Tissue expression pattern of MeETC1

[0136] To investigate the specific localization of the ETC1 transcription factor in cassava, the MeETC1 gene promoter sequence (shown in SEQ ID NO: 58) was ligated to the pCAMBIA1300-GUS vector and transformed into cassava calli to obtain transgenic plants. Primers used included 1300-GUS-ETC1Pro F and 1300-GUS-ETC1Pro R (shown in SEQ ID NO: 33-34). GUS staining showed that the MeETC1 gene was primarily expressed in leaves, with a small amount of expression also in the root vascular tissue ( Figure 1 , A).

[0137] Using wild-type cassava grown in the field for about 6 months, the expression level of MeETC1 was detected in the terminal buds, stems, leaves, petioles, fibrous roots, developing roots and storage roots. The results showed that the expression level of MeETC1 was highest in the cassava tips and young leaves ( Figure 1 , B).

[0138] 2. MeETC1 protein is localized in the cell nucleus

[0139] To understand the localization of ETC1 protein in cassava cells, the cassava ETC1 protein was fused with GFP fluorescence and transformed into cassava leaf protoplasts to observe the localization of ETC1 in cassava cells. Compared with the 35S-activated GFP protein expressed in the entire cell, ETC1 was expressed in the nucleus, indicating that ETC1 is a nuclear-localized protein in cassava ( Figure 2 Ds-Red: chloroplast autofluorescence; eGFP: fluorescence field; FPMT: bright field; Merge: overlay field. 35S:GFP: GFP protein; 35S:ETC1-GFP: ETC1 and GFP fusion protein. Scale bar: 5 μm. Primers used include: ETC1-PA7-F and ETC1-PA7-R (shown in SEQ ID NOs: 35-36).

[0140] 3.MeETC1 expression is induced by low temperature

[0141] Since MeETC1 was a transcription factor screened from cassava low-temperature-related microarray results, to further analyze its expression profile under low temperature, wild cassava grown in a greenhouse for 2 months was cold-treated (4°C). Gene expression was detected at different time points, including 0, 1, 2, 3, 6, 9, and 12 hours. Real-time PCR showed that MeETC1 could be significantly upregulated by low temperature, and its expression level reached a peak after 6 hours of treatment at 4°C ( Figure 3 , A). GUS staining was performed on GUS seedlings driven by the MeETC1 gene promoter after 6 h of cold treatment (4°C). The results showed that MeETC1 expression was significantly upregulated after 6 h of cold treatment ( Figure 3, B). GUS staining results at different time points indicate that cassava MeETC1 is a cold-induced early response gene. Primers used include: MeACTIN-qF, MeACTIN-qR, MeETC1-qF, and MeETC1-qR (shown in SEQ ID NOs: 37-40).

[0142] 4. Potted transgenic plants overexpressing MeETC1 have enhanced resistance to low temperatures

[0143] (1) Analysis of low-temperature phenotypes of potted MeETC1 transgenic plants

[0144] The expression of MeETC1 is induced by low temperature ( Figure 3 ), we used transgenic plants to study the role of MeETC1 in cassava under low-temperature stress. Cassava plants grown in a greenhouse for two months were treated in a 4°C incubator. Six hours after treatment, compared with wild-type cassava, the leaves of MeETC1-knockout transgenic seedlings drooped significantly, with a larger leaf angle and a chlorotic phenotype. The leaves of MeETC1-overexpressing transgenic seedlings were firm and greener. Figure 4 ).

[0145] (2) Physiological index determination of MeETC1 transgenic plants in pots treated with low temperature

[0146] When treated at 4°C, the phenotypes of wild-type and transgenic plants differed significantly. We measured cold-related physiological indicators such as electrical conductivity and malondialdehyde content to reflect the differences in cold response between wild-type and transgenic plants. After low-temperature treatment, the malondialdehyde (MDA) content of MeETC1-interference transgenic plants was significantly higher than that of wild-type plants, while the MDA content of MeETC1-overexpressing transgenic plants was lower than that of wild-type plants, indicating that the oxidative function of MeETC1-interference transgenic plants was damaged ( Figure 5 , A). The electrical conductivity of MeETC1 knockout transgenic plants was significantly higher than that of the wild type, while that of overexpressed transgenic plants was significantly lower than that of the wild type, indicating that the degree of damage to the leaves of MeETC1 knockout transgenic plants was higher than that of the wild type under low temperature treatment ( Figure 5 , B). There is a significant difference in the drooping angle of the leaves before and after cold treatment ( Figure 4 ), we measured the leaf angles of wild-type and transgenic plants and found that the leaf angles of MeETC1-interference plants were significantly increased, while the leaf angles of overexpression plants were significantly smaller than those of wild-type and interference plants ( Figure 5 , C). The chlorophyll content results were consistent with the phenotype. The chlorophyll content of the leaves of the interference plants was significantly reduced, while that of the overexpression plants was significantly increased ( Figure 5, D). Physiological index analysis showed that MeETC1-interference transgenic plants, after being exposed to low temperature, showed increased cell membrane lipid peroxidation, increased cell membrane permeability, and increased electrolyte permeability, ultimately leading to leaf wilting and necrosis. The above MDA detection was carried out using the thiobarbituric acid (TBA) method (An JP, Li R, Qu FJ, et al. R2R3-MYB transcriptionfactor MdMYB23 is involved in the cold tolerance and proanthocyanidin accumulation in apple. Plant Journal, 2018, 963: 562-577.); the conductivity detection referred to the Plant Physiology Experiment Tutorial (An JP, Wang XF, Zhang XW, et al. An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation. Plant Biotechnology Journal, 2020, 182: 337-353. Ronchi A, Farina G, Gozzo F, et al. Effects of a triazolic fungicide on maize plant metadata: modifications of transcript abundance in resistance-related pathways. Plant Science, 1997, 1301: 51-62.).

[0147] 5. Determination of ROS Content in MeETC1 Transgenic Plants Treated with Low Temperature

[0148] Plants will accumulate reactive oxygen species after being damaged by low temperatures, and excessive accumulation of reactive oxygen species will cause peroxidation of cell membrane lipids, and in severe cases, even damage to the plants. By performing DAB and NBT staining on leaves treated with low temperatures, it was found that a large amount of ROS accumulated in the leaves of RNA interference plants after cold treatment, while overexpression plants effectively cleared the excessive ROS generated by low temperatures ( Figure 6 The above DAB staining reagents were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd. with the catalog number D7304-1SET; NBT staining reagents were purchased from Shanghai Saiyi Biotechnology Co., Ltd. with the catalog number MB4693_1G.

[0149] 6. Phenotypes of MeETC1 transgenic plants during moderate cooling

[0150] To detect the accumulation of anthocyanins during the cassava growth period, samples were taken from the tops of cassava plants at moderate temperatures. Phenotypic observations showed that as the field temperature gradually decreased, anthocyanins accumulated in the leaves of both wild-type and transgenic plants, but the accumulation of anthocyanins in the leaves of MeETC1-overexpressing plants was more pronounced ( Figure 7 , A). The results of anthocyanin content determination further confirmed that as the temperature decreased, the overexpression transgenic plants accumulated more anthocyanins ( Figure 7 , BD).

[0151] Referring to the biosynthesis pathway of anthocyanins, a flavonoid substance, real-time PCR detection was performed on the key genes for anthocyanin synthesis. It was found that as the temperature decreased, the expression levels of genes such as MePAL2, MeDFR, MeANS, MeCHS, and MeF3'H were gradually upregulated in the overexpression strains ( Figure 7 , E). The primers used above are listed in Table 1: MePAL2-qF, MePAL2-qR, MeDFR-qF, MeDFR-qR, MeCHS-qF, MeCHS-qR, MeF3'H-qF, MeF3'H-qR, MeANS-qF, and MeANS-qR (shown in SEQ ID NOs: 41-50).

[0152] The above results indicate that MeETC1 can activate the expression of anthocyanin biosynthesis genes and promote the accumulation of anthocyanins in overexpressing plants. As an antioxidant, it can effectively remove excessive reactive oxygen species accumulated in plants and further improve the cold resistance of plants.

[0153] 7. Phenotype of MeETC1 transgenic plants during low-temperature cooling

[0154] By observing the phenotype of cassava during low-temperature cooling, we found that as the temperature dropped, the leaf morphology of the plant changed significantly, especially at the top, where the structure became more compact and the leaves became smaller. Compared with the wild type, the MeETC1 overexpressing transgenic tops significantly accumulated more anthocyanins, while the MeETC1-interfered transgenic tops had no obvious anthocyanin accumulation; the leaves of the overexpressing plants were greener than those of the wild type, while the leaves of the RNA-interferenced plants were significantly chlorotic. As the temperature dropped, the leaf morphology of the wild-type cassava remained intact, but obvious brown spots appeared; the RNA-interferenced transgenic plants showed a phenotype of wilting, partial chlorosis, and even necrosis, while the leaf morphology of the overexpressing plants was intact, and no chlorosis or necrosis occurred ( Figure 8 ).

[0155] The above experimental results show that MeETC1 overexpression plants have significantly higher tolerance to low temperature than the wild type, while RNA interference plants have significantly lower tolerance to low temperature than the wild type. MeETC1 has a positive regulatory effect on cassava's low temperature tolerance.

[0156] 8. Physiological indicators of MeETC1 transgenic cassava seedlings under low temperature

[0157] Based on the natural cooling phenotype in the field, we measured the chlorophyll and anthocyanin contents in the shoots and leaves. The results showed that the shoots and leaves of the overexpressing plants accumulated more anthocyanin and chlorophyll; the anthocyanin and chlorophyll contents in the shoots and leaves of the RNA interference plants were significantly lower than those of the wild type ( Figure 9 Under low-temperature stress, anthocyanins in leaves can reduce oxidative damage and improve photosynthetic efficiency. Based on the accumulation of anthocyanins in plants, we can predict abiotic resistance. The chlorophyll content was determined using the acid ethanol method. Fresh plant leaves were chopped and added to a mixture of 95% ethanol and 0.1% hydrochloric acid. The mixture was incubated at 4°C overnight, centrifuged, and the supernatant was measured for absorbance at wavelengths of 663 nm and 646 nm. Anthocyanin content was determined using the reference method (Ronchi et al., 1997).

[0158] 9. MeETC1 regulates the expression of anthocyanin biosynthesis pathway genes MePAL2 and MeDFR

[0159] During moderate and moderate cooling in the field, a large amount of anthocyanins accumulated in the leaves of cassava plants overexpressing MeETC1. We speculate that MeETC1 may be directly or indirectly involved in the regulation of anthocyanin biosynthesis. To verify the regulatory relationship, we obtained the upstream 2kb promoter sequence of the cassava anthocyanin biosynthesis gene through sequence alignment and constructed the vector pLL00r-BAR for subsequent verification. Tobacco transactivation experiments showed that MeETC1 can directly bind to MePAL2 ( Figure 10 , A) and MeDFR( Figure 11 , A) promoter; Dual luciferase assay results showed that MeETC1 could activate MePAL2 ( Figure 10 , B) and MeDFR( Figure 11 , B); EMSA was further used to prove that MeETC1 can bind to the MYB binding site (MBS) binding element ( Figure 10 , C, Figure 11, C). These results indicate that MeETC1 can directly bind to the MBS element of the MePAL2 and MeDFR promoters to activate their expression. The tobacco transactivation assay described above was performed using the same protocol as (An et al., 2018). The dual-luciferase assay was performed using the Dual Luciferase Kit, purchased from Shanghai Weihuan Biotechnology Co., Ltd., catalog number K1136-10. The primers used were MeETC1-62SK-F, MeETC1-62SK-R, MePAL2P-0800-F, MePAL2P-0800-R, MeDFRP-0800-F, and MeDFRP-0800-R. The EMSA assay was performed using the same protocol as (An et al., 2020), using the PAL2-MBS and DFR-MBS primers.

[0160] 10. MeICE1 regulates the expression of MeETC1

[0161] MeETC1 can promote the synthesis of anthocyanins by regulating the expression of downstream plant anthocyanin synthesis genes, thereby improving the plant's resistance to low temperatures. In order to construct a complete regulatory network, we analyzed the upstream regulatory factors of ETC1. Through analysis of the MeETC1 promoter, we found the presence of MYC elements, which are recognized as binding elements of MeICE1. In order to verify the existence of the binding relationship, we first used yeast one-hybrid assay to analyze and proved that MeICE1 can indeed bind to the MeETC1 promoter ( Figure 12 , A); Dual luciferase assay further demonstrated that MeICE1 could activate the expression of MeETC1 ( Figure 12 , B); EMSA experiments demonstrated that MeICE1 could bind to the MYC element of the MeETC1 promoter ( Figure 12 , C). Therefore, these results confirm that MeICE1 can bind to the MYC element in the MeETC1 promoter and activate its expression. In the yeast one-hybrid assay, the target promoter fragment was linked to the pAbai vector, the transcription factor CDS was linked to the pGADT7 vector, and the yeast strain used was Y1H. The primers used included ETC1P2000-PAbai-F, ETC1P-PAbai-R, ETC1-pGADT7-F, and ETC1-pGADT7-R. The dual-fluorescein assay was performed using the dual-fluorescein kit, with the primers MeICE1-62SK-F, MeICE1-62SK-R, MeETC1P-0800-F, and MeETC1P-0800-R. EMSA experiments were performed according to (An et al., 2020), using the MYC primer (shown in SEQ ID NO: 53).

[0162] 11. Field pilot test of genetically modified cassava

[0163] We plant MeETC1 transgenic cassava in Wushe Farm, Songjiang District, Shanghai, every May. We harvest and observe phenotypes and measure yield in November. The main yield indicators include plant height, root diameter, root length, root number, and total root biomass.

[0164] Field test phenotypes showed that there was no significant difference in plant height between overexpression and wild-type plants, but the overexpression plants had significantly more branches above ground than the wild-type plants; RNA interference transgenic plants were significantly shorter than the wild-type plants ( Figure 13 , A).

[0165] Statistical results showed that the overexpression plants had no significant differences in root length, total root biomass, and root number compared to the wild type. However, the storage roots of the overexpression plants were significantly thicker than those of the wild type cassava. Moreover, the overexpression transgenic plants mostly had mature roots and produced fewer fibrous roots. Compared with the wild type cassava, the RNA interference transgenic plants showed fewer roots, shorter roots, and lower total root biomass. This suggests that the MeETC1 gene is likely to affect the development of cassava root shape ( Figure 13 , BE).

[0166] (IV) Conclusion

[0167] In summary, MeICE1 is a transcription factor highly correlated with low temperatures, acting upstream of the MeETC1 gene by binding to its promoter and directly activating its expression. Inducing MeETC1 to activate the expression of MePAL2 and MeDFR promotes anthocyanin accumulation, helps scavenge ROS, and improves cassava's cold tolerance without affecting yield.

Claims

1. Use of a substance in regulating the low-temperature resistance of plants, wherein the substance is selected from the group consisting of one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2, or their encoded proteins, or their promoters or inhibitors, Preferably, the low temperature is 0-10°C, Preferably, the promoter is selected from the group consisting of small molecule compounds, nucleic acid molecules, or combinations thereof; the inhibitor is an inhibitory molecule that specifically interferes with the transcription and / or expression of one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2, Preferably, the inhibitory molecule is selected from the group consisting of: (1) small molecule compounds, antisense nucleic acids, microRNAs, siRNAs, RNAi, dsRNAs, sgRNAs, antibodies, or combinations thereof, and (2) nucleic acid constructs capable of expressing or forming (1), More preferably, the inhibitor further comprises a Cas enzyme, its coding sequence, and / or a nucleic acid construct expressing the Cas enzyme.

2. The use according to claim 1, wherein, the amino acid sequences of MeICE1, MeETC1, MeDFR, and MePAL2 are shown in SEQ ID NO:54-57 respectively, Preferably, the plant is a woody plant, more preferably, the woody plant is a Euphorbiaceae plant, and further preferably, the Euphorbiaceae plant includes cassava.

3. A method for regulating the low-temperature resistance of plants, the method comprises: regulating the expression or activity of one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2 in plants, Preferably, the method for regulating the low-temperature resistance of plants comprises: upregulating the expression of one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2 in plants.

4. The method according to claim 3, wherein, the method comprises: (1) providing Agrobacterium carrying a nucleic acid construct containing one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2, and (2) contacting the cells or tissues or organs of the plant with the Agrobacterium in step (1), so that the nucleic acid construct is transferred into the plant tissue or organ, Preferably, the nucleic acid construct is a vector, Preferably, the method further comprises: (3) selecting plant tissues, organs, or seeds into which one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2 have been transferred; and (4) regenerating the plant tissues, organs, or seeds in step (3) into plants.

5. A method for obtaining plants with improved low-temperature resistance, comprises: (1) providing Agrobacterium carrying a nucleic acid construct containing one or more of the genes MeICE1, MeETC1, MeDFR, MePAL2, (2) contacting the cells or tissues or organs of the plant with the Agrobacterium in step (1), so that the nucleic acid construct is transferred into the plant tissue or organ, (3) Select plant tissues, organs or seeds into which one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes have been transferred; and (4) Regenerate plants from the plant tissues, organs or seeds in step (3).

6. Use of one or more of the MeICE1, MeETC1, MeDFR, and MePAL2 genes for use as molecular markers for identifying the low-temperature resistance of plants, Preferably, the MeICE1, MeETC1, MeDFR, and MePAL2 genes include cDNA sequences, genomic sequences, or combinations thereof.

7. An expression cassette expressing the MeETC1 gene, the expression cassette having the following elements in sequence from 5' to 3': a promoter, the ORF sequence of the MeETC1 gene, and a terminator, Preferably, the promoter is the MeETC1 gene promoter or the 35S promoter.

8. A nucleic acid construct comprising the expression cassette according to claim 7 or its complementary sequence.

9. A host cell which (1) contains a nucleic acid construct comprising the expression cassette according to claim 7 or its complementary sequence, or (2) has the expression cassette integrated into its chromosome.

10. Use of the expression cassette according to claim 7 for improving the low-temperature resistance of plants.

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