Sweet potato low-temperature-resistant related protein IbbHLH129 and related biological material and application thereof

Abstract

The invention discloses a sweet potato low-temperature-resistant related protein IbbHLH129 as well as a related biological material and application thereof, and relates to the field of genetic engineering. In order to improve the cold resistance of sweet potatoes, the invention provides an application of IbbHLH129 protein and a coding gene thereof in regulation and control of the cold resistance of the sweet potatoes, and a sweet potato plant with a transformed IbbHLH129 gene is obtained by regulating and controlling the content or expression of the IbbHLH129 protein in the sweet potatoes. Compared with a wild type sweet potato plant, the wilting degree of the sweet potato plant with the overexpressed IbbHLH129 gene under cold treatment is reduced; therefore, the IbbHLH129 protein and the coding gene play an important role in regulating the cold resistance of the plant. The IbbHLH129 protein and the coding gene thereof provided by the invention have important application value in regulation and control of plant cold tolerance, and have wide application space and market prospect in the agricultural field.

Description

Technical Field

[0001] This application relates to the field of genetic engineering, specifically to a sweet potato low-temperature resistance-related protein IbbHLH129 and its related biomaterials and applications. Background Technology

[0002] Low temperatures severely inhibit plant growth. They reduce cellular enzyme activity, slow down photosynthesis and respiration, and hinder nutrient synthesis and absorption. A sudden drop in temperature can cause ice to form in intercellular spaces, damaging cell membrane structure and leading to cell dehydration and necrosis. For crops, low temperatures can delay germination, damage seedlings, and may cause abnormal flowering and pollination, resulting in flower and fruit drop. Furthermore, low temperatures weaken plant resistance, making them more susceptible to pests and diseases, ultimately leading to reduced crop yields, lower quality, and even the death of the entire plant.

[0003] sweet potato( Ipomoea batatas Sweet potatoes (L.) Lam. are an important food and economic crop and a new energy crop widely cultivated globally, playing a significant role in ensuring food security. Sweet potatoes thrive in warm temperatures and are sensitive to cold; low temperatures easily cause frost damage to seedlings, stunted tuber growth, and in severe cases, rot and yield reduction. Frequent spring frosts and autumn frosts in many parts of my country restrict the planting area and yield of sweet potatoes. Conducting research on cold tolerance in sweet potatoes, cultivating cold-resistant varieties, and improving their stress resistance to ensure their growth and yield in low-temperature environments are of great practical significance for stabilizing sweet potato production and expanding planting areas.

[0004] However, the transcriptional regulatory mechanisms of cold tolerance in sweet potatoes are not well understood. Identifying the genes involved in cold tolerance regulation, conducting in-depth research on cold resistance mechanisms, and regulating plant cold tolerance through plant genetic engineering are crucial. Summary of the Invention

[0005] The technical problem this application aims to solve is: how to regulate the cold resistance of sweet potatoes. More specifically, the technical problem this application aims to solve is: how to improve the cold resistance of sweet potatoes. To solve this technical problem, this application provides the following technical solution:

[0006] This application provides the use of the IbbHLH129 protein, or a substance that regulates the expression of the gene encoding the IbbHLH129 protein, or a substance that regulates the activity or content of the IbbHLH129 protein, in any of the following situations. A1) Its application in regulating plant cold tolerance; A2) Application in the preparation of products that regulate plant cold tolerance; A3) Application in plant cold tolerance breeding or assisted breeding; A4) Application in the preparation of products for cold-resistant plant breeding or assisted breeding; The IbbHLH129 protein is any of the following proteins: a1) Proteins with amino acid sequences as shown in SEQ ID NO:1; a2) Proteins obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence shown in a1). a3) and a1) are proteins with more than 70% amino acid sequence identity and the same function; a4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of at least one of the proteins described in a1) to a3).

[0007] In this application, the regulation may be to increase, promote, or adjust.

[0008] In this application, the regulation may also be a reduction, suppression, or downregulation.

[0009] In this application, the indicators for plant breeding include cold tolerance.

[0010] In this application, the purpose of plant breeding includes cultivating plants with altered cold tolerance. Specifically, the purpose of plant breeding includes cultivating plants with enhanced cold tolerance.

[0011] In this application, the protein may be derived from sweet potato.

[0012] The proteins mentioned above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] In the above-mentioned proteins, the connection may be that adjacent amino acids (or amino acid residues) are linked by peptide bonds.

[0014] The protein tag refers to a polypeptide or protein fused with a target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag protein tag, His protein tag, MBP protein tag, HA protein tag, myc protein tag, GST protein tag, and / or SUMO protein tag, etc.

[0015] In this application, the substance regulating the expression of the IbbHLH129 protein-encoding gene or the substance regulating the activity or content of the IbbHLH129 protein is a biological material, and the biological material is any one of the following: B1) The nucleic acid molecule encoding the IbbHLH129 protein mentioned above; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Transgenic plant cells containing the nucleic acid molecules described in B1), or transgenic plant cells containing the expression cassette described in B2), or transgenic plant cells containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3).

[0016] In this application, the nucleic acid molecule described in B1) is the DNA molecule described in g1) or g2) below: g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:2; g2) is a DNA molecule that has more than 70% identity with the DNA molecule described in g1) and encodes the aforementioned IbbHLH129 protein.

[0017] In this application, the recombinant microorganism may specifically be yeast, bacteria, algae, or / and fungi.

[0018] In this application, the plant tissue may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos, and / or anthers.

[0019] In this application, the genetically modified plant organs may be the roots, stems, leaves, flowers, fruits, and / or seeds of the genetically modified plant.

[0020] In this application, the transgenic plant cells, transgenic plant tissues, and transgenic plant organs may or may not include propagation material.

[0021] This application also provides a method for regulating plant cold tolerance, the method comprising regulating the expression level of the encoding gene of the IbbHLH129 protein in the recipient plant and / or regulating the activity or content of the IbbHLH129 protein in the recipient plant to regulate the cold tolerance of the recipient plant.

[0022] Furthermore, the method includes increasing the expression level of the gene encoding the IbbHLH129 protein in the recipient plant and / or increasing the activity or content of the IBBHLH129 protein in the recipient plant to improve the cold tolerance of the recipient plant.

[0023] This application also provides a method for obtaining a target plant with altered cold tolerance, the method comprising obtaining a target plant with altered cold tolerance by regulating the expression level of the gene encoding the IbbHLH129 protein in the recipient plant and / or regulating the activity or content of the IbbHLH129 protein in the recipient plant.

[0024] Furthermore, the method includes obtaining a target plant with improved cold tolerance by increasing the expression level of the gene encoding the IbbHLH129 protein in the recipient plant and / or regulating the activity or content of the IbbHLH129 protein in the recipient plant.

[0025] In this application, the increase in the expression level of the gene encoding the IbbHLH129 protein in the recipient plant and / or the increase in the activity or content of the IbbHLH129 protein in the recipient plant are achieved by introducing the gene encoding the IbbHLH129 protein into the recipient plant.

[0026] This application also provides a cold-resistant plant, which is a plant containing the above-mentioned biological material.

[0027] In some embodiments, the cold-resistant plant is obtained by the method described above for obtaining a target plant with altered cold resistance.

[0028] In some embodiments, the cold-resistant plant is obtained by introducing the gene encoding the IbbHLH129 protein into the recipient plant.

[0029] In some specific embodiments, the cold-resistant plant is a genetically modified plant.

[0030] The transgenic plant refers to transgenic plant cells, plant tissues, plant organs, and / or the whole plant.

[0031] "Transgenic plant" refers to a plant whose genome has been altered by integrating or inserting recombinant DNA molecules, constructs, cassettes, or sequences for the expression of non-coding RNA molecules, mRNA, and / or proteins. Transgenic plants include R0 generation plants that develop or regenerate from initially transformed plant cells and their offspring, or plants obtained by hybridization with R0 generation transgenic plants containing recombinant DNA molecules, constructs, cassettes, or sequences.

[0032] "Transgenic plant cells" are biological cells of transgenic plants, which are taken from transgenic plants or derived from cultures obtained by culturing cells taken from transgenic plants.

[0033] "Transgenic plant cell" refers to any plant cell transformed with a stably integrated recombinant DNA molecule, construct, cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0034] The plants described in this application can be viable, non-viable, renewable, and / or non-renewable. The plants described in this application include propagules or propagation material. "Propagules or propagation material" can include any plant part that can grow into a whole plant. "Plant part" can refer to any organ or tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof.

[0035] The aforementioned IbbHLH129 protein and the aforementioned biological materials are also the technical solutions to be protected in this application.

[0036] In this application, the plant is a dicotyledonous plant.

[0037] In this application, the dicotyledonous plant is a plant of the order Tubulariaceae.

[0038] In this application, the plants described in the order Tubularflorales are plants of the family Convolvulaceae.

[0039] In this application, the Convolvulaceae plant is a plant of the Ipomoea genus.

[0040] In this application, the plant referred to as Ipomoea is sweet potato.

[0041] In this application, the improved cold resistance manifests itself in at least one of the following ways: D1) The degree of wilting in plants was significantly reduced under low temperature stress; D2) Under low temperature stress, the plants showed changes in at least one of the following indicators: increased SOD enzyme activity, increased POD enzyme activity, decreased MDA (malondialdehyde) content, increased proline content, increased GA content, or increased IAA content. D3) Increased expression of at least one of the following genes associated with low-temperature stress: IbSOD , IbPOD , IbCAT , IbP5CS, IbP5CR , IbGA2ox2 , IbARF1 or IbIAA17 ; D4) The following are related to low temperature stress IbP5CDH Gene expression levels are downregulated.

[0042] The beneficial technical effects achieved by this application are as follows: This application provides an IbbHLH129 protein and its encoding gene, the functions of which are currently unknown, and their uses. Transformation can be achieved by regulating the activity, content, or expression of this IbbHLH129 protein in sweet potato. IbbHLH129 Experiments have shown that sweet potato plants overexpressing the gene have significantly higher yields compared to wild-type sweet potato plants. IbbHLH129 The sweet potato plants with the gene showed reduced wilting under cold treatment, indicating that the IbbHLH129 protein and its encoding gene play an important role in regulating plant cold tolerance. The IbbHLH129 protein and its encoding gene provided in this application have significant application value in regulating plant cold tolerance, and have considerable application potential and market prospects in the agricultural field. Attached Figure Description

[0043] Figure 1 PCR identification results and expression level analysis of transgenic sweet potato plants; where A represents overexpression. IbbHLH129 PCR identification results of gene-positive sweet potato plants. M represents the DNA molecular marker, WT represents the genomic DNA of wild-type sweet potato plants, H represents negative control water, and P represents the positive control plasmid pCAMBIA1302. IbbHLH129 - mgfp OE-B1 to OE-B11 are overexpressed IbbHLH129 Genomic DNA of gene-positive sweet potato plants; B is IbbHLH129 Genes are being transferred IbbHLH129 Analysis of gene expression levels in positive and wild-type sweet potato plants. WT represents wild-type sweet potato plants, and OE-B1 to OE-B11 represent overexpression. IbbHLH129 Genetically modified sweet potato plants.

[0044] Figure 2 Overexpression under low temperature treatment IbbHLH129 Phenotypic identification of gene-positive sweet potato plants and wild-type sweet potato plants; among them, WT represents wild-type sweet potato plants, and OE-B6, OE-B8, and OE-B9 represent overexpressed genes. IbbHLH129 Gene-positive sweet potato plants.

[0045] Figure 3 Overexpression under low temperature treatment IbbHLH129Cold tolerance indices were determined in gene-positive and wild-type sweet potato plants. A represents SOD enzyme activity, B represents POD enzyme activity, C represents MDA content, D represents proline content, E represents GA content, F represents JA content, G represents ABA content, and H represents IAA content. WT represents wild-type sweet potato plants, and OE-B6, OE-B8, and OE-B9 represent overexpressing genes. IbbHLH129 Gene-positive sweet potato plants.

[0046] Figure 4 Overexpression under low temperature treatment IbbHLH129 Expression analysis of cold tolerance-related genes in gene-positive and wild-type sweet potato plants; where A represents... IbSOD B is IbPOD C is IbCAT D is IbP5CS E is [[ID= F is ​ G is ​ H is ​ I is ​ Among them, WT represents wild-type sweet potato plants, while OE-B6, OE-B8, and OE-B9 are overexpressed varieties. ​ Gene-positive sweet potato plants. Detailed Implementation

[0047] I. Terms used in this application: Examples of resources describing many of the molecular biology-related terms used in this article can be found in the following literature: Alberts et al., Molecular Biology of The Cell, 5th ed., Garland Science Publishing, Inc.: New York, 2007; Rieger et al., Glossary of Genetics: Classical and Molecular, 5th ed., Springer-Verlag: New York, 1991; King et al., A Dictionary of Genetics, 6th ed., Oxford University Press: New York, 2002; and Lewin, GenesIX, Oxford University Press: New York, 2007.

[0048] Any references cited in this article, including, for example, all patents, published patent applications and non-patent publications, are incorporated in their entirety by reference.

[0049] For ease of understanding this application, several terms and abbreviations used herein are defined as follows: In this application, "identity" refers to the similarity of amino acid or nucleotide sequences. The similarity of amino acid sequences (or nucleotide sequences) can be determined using homology search sites on the Internet, such as the BLAST page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, and setting the Gap existence cost, Perresidue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the similarity of a pair of amino acid sequences, the similarity value (%) can be obtained.

[0050] Specifically, the consistency of 70% or more can be 75% or more. Specifically, the consistency of 75% or more can be 80% or more. Specifically, the consistency of 80% or more can be 85% or more. Specifically, the consistency of 85% or more can be 90% or more. Specifically, the consistency of 90% or more can be 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. More specifically, the consistency of 70% or more can be at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% consistency.

[0051] When used in a list of two or more items, the term "and / or" means that any of the listed items can be used alone or in combination with any one or more of the listed items. For example, the expression "A and / or B" is intended to mean either or both of A and B, i.e., A alone, B alone, or a combination of A and B. The expression "A, B and / or C" means A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C.

[0052] The term "comprising" is not intended to be restrictive, but rather inclusive and implies the presence of other elements besides those listed, and can be interpreted as "including but not limited to". The term "comprising" also encompasses the terms "consisting of" and "substantially consisting of". In this document, the terms "including" and "comprise" are used interchangeably.

[0053] The terms “protein,” “peptide,” and “polypeptide” are used interchangeably herein and refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, proteins, peptides, or polypeptides are at least 3 amino acids in length. Proteins, peptides, or polypeptides can refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide can be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications. Proteins, peptides, or polypeptides can also be single molecules or can be multi-molecular complexes. Proteins, peptides, or polypeptides can simply be fragments of naturally occurring proteins or peptides. Proteins, peptides, or polypeptides can be naturally occurring, recombinant, or synthetic, or any combination thereof. Any protein provided herein can be produced by any method known in the art. For example, the proteins provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins containing peptide linkers.

[0054] As used in this article, the term "fusion protein" refers to a hybrid polypeptide containing protein domains from at least two different proteins. One protein may be located at the N-terminal (N-terminal) portion or the C-terminal (C-terminal) portion of the fusion protein, thus forming an "N-terminal fusion protein" or a "C-terminal fusion protein," respectively.

[0055] The term "biomaterial" refers to any material that carries genetic information and is capable of self-replication or replication within a biological system, such as genes, plasmids, microorganisms, animals, and plants.

[0056] As used in this article, "plant" includes explants, plant parts, seedlings, plantlets, or whole plants at any stage of regeneration or development.

[0057] As used herein, "plant part" can refer to any organ or intact tissue of a plant, such as meristematic tissue, bud organs / structures (e.g., leaves, stems, or nodes), roots, flowers or floral organs / structures (e.g., flowers, bracts, sepals, petals, stamens, carpels, anthers, and ovules), seeds (e.g., embryo, endosperm, and seed coat), fruits (e.g., mature ovaries), propagules, or other plant tissues (e.g., vascular tissue, dermal tissue, ground tissue, etc.) or any part thereof. The plant part in this application can be viable, non-viable, renewable, and / or non-renewable. "Propagule" can include any plant part that can grow into a whole plant.

[0058] Plant cells are biological cells of plants, derived from plants or derived from cultures obtained by culturing cells taken from plants. As used herein, “transgenic plant cell” means any plant cell transformed with a stably integrated recombinant DNA molecule, construct, expression cassette, or sequence. Transgenic plant cells can include original transformed plant cells, transgenic plant cells regenerated or developed from R0 generation transgenic plant cells, transgenic plant cells cultured from another transgenic plant cell, or transgenic plant cells from any progeny or offspring of a transformed R0 generation plant, including cells of plant seeds or embryos, or cultured plant cells, callus cells, etc.

[0059] As is commonly understood in the art, the term "promoter" generally refers to a DNA containing an RNA polymerase binding site, a transcription start site, and / or a TATA box that assists or promotes the transcription of transcribed DNA. Promoters can be artificially synthesized, modified, or derived from known or naturally occurring promoters. Promoters can also include chimeric promoters comprising combinations of two or more heterologous sequences. Therefore, the promoters of this application may include variants of promoter sequences that are compositionally similar but not identical to other promoter sequences provided herein.

[0060] Promoters can be classified according to various criteria related to the expression patterns of the associated coding or transcribed sequences or genes (including transgenes) operably linked to them, such as constitutive, developmental, tissue-specific, and inducible promoters. A promoter that drives expression in all or most tissues of a plant is called a "constitutive" promoter. A promoter that drives expression at certain times or stages of development is called a "developmental" promoter. A promoter that drives enhanced expression in certain tissues of a plant relative to other tissues is called a "tissue-enhancing" or "tissue-preferred" promoter. Therefore, a "tissue-preferred" promoter elicits relatively high or preferential expression in a specific tissue of the plant, but lower expression levels in other tissues. A promoter that is expressed in a specific tissue of the plant but rarely or not expressed in other tissues is called a "tissue-specific" promoter. An "inducible" promoter is a promoter that initiates transcription in response to environmental stimuli (e.g., cold, drought, or light) or other stimuli (e.g., injury or chemical application). Promoters can also be classified according to their origin, such as heterologous, homologous, chimeric, synthetic, etc.

[0061] The term "transcribed DNA" refers to DNA that can be transcribed into RNA molecules.

[0062] The term "operationally ligated" can refer to a functional connection between a promoter and transcribed DNA, enabling the promoter to function and initiate transcription of the transcribed DNA. The term "operationally ligated" can also refer to a functional connection between other regulatory elements and a target gene to regulate the transcription and / or expression of the target gene.

[0063] The term "construct" refers to any recombinant DNA or recombinant RNA molecule. Recombinant DNA molecules can be plasmids, granules, viruses, bacteriophages, or linear or circular DNA. Constructs typically include one or more expression cassettes.

[0064] As used herein, an "expression cassette" refers to a cassette containing at least transcribed DNA operatively linked to one or more regulatory elements, typically at least a promoter and a 3' UTR (such as a terminator).

[0065] As used herein, the term "vector" refers to any construct that can be used for transformation purposes, i.e., to introduce heterologous DNA into a host cell. Examples include plasmids, granules, viruses, bacteriophages, or linear or circular DNA.

[0066] II. Implementation Examples The present application will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the present application and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the present application in any way.

[0067] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0068] In the following examples, the sweet potato varieties “Xushu 33” and “Yanshu 25” were preserved in our laboratory. The sweet potato variety “Xushu 33” is described in the following literature (i.e., X33 in the literature): “Li, Z., Pan, J., Liu, S., Yang, Z., Zhang, H., Yu, T., & He, S. (2025). Integrated transcriptome and metabolome analysis provides insights into the low-temperature response in sweet potato (…)” ​ ​L.). Genes (Basel). 2025 Jul 28;16(8):899. doi: 10.3390 / genes16080899.” The public can obtain it from the Potato Research Laboratory of the Crop Research Institute of Liaoning Academy of Agricultural Sciences to repeat this experiment, but it should not be used for other purposes.

[0069] The cloning vector pCE3 Blunt Vector is a product of Nanjing Novizan Biotechnology Co., Ltd., with product catalog number C603.

[0070] vector pCAMBIA1302- ​ Synthesized by Beijing Qingke Biotechnology Co., Ltd., this is a circular DNA molecule with the nucleotide sequence shown in SEQ ID NO:3. Specifically, positions 8086 to 8431 of SEQ ID NO:3 represent the nucleotide sequence of the CaMV 35S promoter, and positions 8446 to 8451 represent... ​ The nucleotide sequence of the I restriction enzyme recognition site, positions 8453 to 8458 are... ​ The nucleotide sequence of the II restriction enzyme recognition site is as follows: positions 8463 to 9173 are the coding gene for mgfp, positions 9180 to 9197 are the coding gene for the 6×His tag, positions 9201 to 9203 are the stop codon, and positions 9229 to 9481 are the nucleotide sequence of the NOS terminator.

[0071] The plant total RNA extraction kit is the RNA Easy Fast Plant Tissue Rapid Extraction Kit, a product of Tiangen Biotech (Beijing) Co., Ltd., with product catalog number DP452.

[0072] The cDNA synthesis reagent is a product of Takara Bio Engineering (Dalian) Co., Ltd., with product catalog number 6210A.

[0073] qRT-PCR reagent SYBR ​ Ta is a product of Takara Bio Engineering (Dalian) Co., Ltd., with product catalog number DRR041A.

[0074] Unless otherwise specified, the quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0075] The experimental results in the following examples are expressed as mean ± standard deviation, using Student's... t -test test, This indicates a significant difference. P <0.05), This indicates a highly significant difference ( P<0.01); different lowercase letters indicate ANOVA-based representations. ​ Tukey's test, in P There is a statistically significant difference when <0.05.

[0076] Example 1 ​ Acquisition of genes 1. Obtaining cDNA template Fresh leaves of sweet potato 33 were aliquoted into 2 mL pre-cooled centrifuge tubes, frozen in liquid nitrogen, and then ground into powder using a high-throughput tissue homogenizer. Total RNA was extracted from the sweet potato using the RNA Easy Fast Plant Tissue Rapid Extraction Kit, and cDNA first strand was synthesized using a cDNA synthesis reagent.

[0077] 2. Obtain from transcriptome data ​ The transcript sequence was obtained by performing a BLAST scan on NCBI (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). ​ CDS sequence.

[0078] 3. Design and artificially synthesize primers ​ -F and ​ -R, using the cDNA obtained in step 1 as a template, PCR amplification was performed to obtain a PCR amplification product of approximately 1197 bp. This product was then ligated to the cloning vector pCE3 Blunt Vector to obtain a recombinant vector, which was then sequenced. The primer sequences are as follows: ​ -F:5'-ATGTTTAGCTCAGAAGCTCCGA -3'; ​ -R:5'-TTATTGCATTCCAACCTTTTGAG -3'.

[0079] The results showed that the nucleotide sequence of the PCR amplification product was as shown in SEQ ID NO:2, and the gene represented by this sequence was named... ​ Gene, ​ The coding sequence of the gene is shown in SEQ ID NO:2, and the protein it encodes is named IbbHLH129 protein or protein IbbHLH129. The amino acid sequence of IbbHLH129 protein is shown in SEQ ID NO:1.

[0080] The protein shown in SEQ ID NO:1 consists of 398 residues, and its specific amino acid sequence is shown below: MFSSEAPISRELTSGSNNSSFLFPSANSAYKNSSVMQNNNNNELLKSKEVMGSDLYQNQSSGLMRYRSAPSSFFAGVMDSAAANFSAANDAAADSFMVNDGSSSSDSETMFSSILNCGGSESRDLKNSVQFLKPEMAVESHKRSSEAQQMVFEAPEMGSYGVEMEMQAQMRQQQFRNPSNLIRQSSSPAGFFSGFDVMREGGNYNKGSDGANRAAASLSSNGFNNHINYSSAQSSSSNYMPSIAENESWNDSSFNCLKRSRDGDFKILSALNGMEAQSGGEPRNCTPGLTHHLSLPSSVEIEKYLHFQQDSVPCKIRAKRGCATHPRSIAERNRRTRISERMKKLQELFPKMDKQTSTADMLDWAVEHIKELQKQVEILTDKKAKCTCSSEAQKVGMQ。

[0081] The nucleotide sequence of SEQ ID NO:2 is shown below:

[0082] Example 2: Application of IbbHLH129 protein in improving plant cold tolerance I. Recombinant plasmid pCAMBIA1302- ​ - ​ The acquisition The result obtained in Example 1 ​ Using the gene CDS sequence as a reference, the gene was synthesized by Beijing Qingke Biotechnology Co., Ltd., and then integrated into the backbone vector pCAMBIA1302- via seamless cloning. ​ Seamless cloning site is ​ I and ​ II. The recombinant plasmid obtained was named pCAMBIA1302- ​ - ​ For recombinant plasmid pCAMBIA1302- ​ - ​ The structure is described as follows: The backbone vector pCAMBIA1302- is replaced with a foreign gene DNA molecule whose nucleotide sequence is SEQ ID No:2, positions 1 to 1194. ​ Restriction endonucleases ​ I and ​ II identifies small fragments between sequences, maintaining pCAMBIA1302- ​ The other nucleotide sequences remained unchanged, resulting in the recombinant expression vector pCAMBIA1302- ​ - ​ Recombinant expression vector pCAMBIA1302- ​ - ​ The exogenous gene shown in SEQ ID No:2, driven by the CaMV 35S promoter, is transcribed and expressed together with the mgfp tag and 6×His tag on the backbone vector to form the IbbHLH129-mgfp fusion protein shown in SEQ ID NO:4. Specifically, positions 1 to 398 of SEQ ID NO:4 represent the amino acid sequence of the IbbHLH129 protein, positions 402 to 638 represent the amino acid sequence of mgfp, and positions 641 to 646 represent the amino acid sequence of the 6×His tag.

[0083] The amino acid sequence of the IbbHLH129-mgfp fusion protein, SEQ ID NO:4: .

[0084] II. Obtaining Transgenic Sweet Potato Plants 1. Overexpression ​ Obtaining transgenic sweet potato plants: 1) The constructed pCMBIA1302- ​ - ​ The vector was transformed into Agrobacterium rhizogenes K599 to obtain recombinant Agrobacterium, which was named K599 / pCAMBIA1302- ​ - ​ .

[0085] 2) Agrobacterium culture: Add 2 μg of correctly sequenced plasmid to thawed Agrobacterium competent cells (100 μL per tube), gently mix, and incubate on ice for 5 min. Remove the competent cells from the ice and flash freeze in liquid nitrogen for 1 min, then incubate at 37°C for 5 min. Add 600 μL of LB liquid medium to the tube and incubate at 28°C with a shaker at 200 rpm for 4 h. Spread 200 μL of the competent cell culture onto a plate containing 50 mg L... -1 Kan and 100 mg L -1 Rif LB solid culture plates. After incubation at 28℃ for 2 days, single clones were picked for colony PCR identification. Those successfully transformed into pCMBIA1302- were retained. ​ - ​ Positive bacterial suspension. Agrobacterium rhizogenes was propagated in a shaker at 28°C until the OD600 was between 0.8 and 1.0.

[0086] 3) Pretreatment of sweet potato variety Yanshu 25: First, material screening was carried out, prioritizing fresh-looking, healthy, and disease-free Yanshu 25 sweet potato stem segments. The selected stem segments should include the top apical structure and have 4 to 10 lateral branches below the apical structure to ensure good physiological activity. Next, wound preparation was carried out by pruning the qualified stem segments with a sterile blade, focusing on removing the lateral branches in the middle and lower parts of the stem segments to form natural wounds. After pruning, only 4 to 6 lateral branches below the apical structure were retained. Finally, wound creation was carried out by puncturing the stem nodes with a sterile needle. The puncture area covered the stem node itself and an area 1 cm above and below it, with 6 to 8 holes evenly punctured in each area to provide a contact channel for subsequent infection by Agrobacterium rhizogenes.

[0087] 4) Infect sweet potato stem segments with Agrobacterium rhizogenes bacterial solution: Soak the pretreated stem segments in the pretreated bacterial solution, ensuring that the wound area is completely immersed in the bacterial solution, and let stand at 28°C in the dark for 12 hours.

[0088] 5) Planting: Ridges are prepared in the field, and the infected stem segments are removed from the bacterial solution and planted in the soil. Overexpression was obtained. ​ Transgenic sweet potato plant tubers.

[0089] 3. Identification of transgenic plants: A combination of PCR and qRT-PCR methods was used.

[0090] 1) Detection of DNA levels using PCR: Extracting wild-type sweet potatoes and transgenic sweet potatoes ​ DNA from the genetically modified sweet potato lines was used for PCR identification. pCAMBIA1302- ​ - ​ The recombinant plasmid served as a positive control, while water and wild-type WT served as negative controls. Primers are as follows: ​ -JDF:5'-GATGTGATATCTCCACTGACGT -3'; ​ -JDR:5'-TTGCATACCAACCTTCTGAGC -3'.

[0091] The amplified PCR products were separated by electrophoresis on a 1% agarose gel. PCR-positive plants should have a specific 1311 bp electrophoretic band.

[0092] The results are as follows ​ As shown in Figure A, overexpression ​ Electrophoretic bands appeared around 1311 bp in the positive control and transgenic sweet potato plants OE-B1 to OE-B11. Wild-type sweet potatoes and the negative control did not show any bands, preliminarily confirming that this application obtained transgenic sweet potato positive plants OE-B1 to OE-B11.

[0093] 2) Detection of RNA levels using qRT-PCR: RNA was extracted from positive sweet potato plants, reverse transcribed to obtain cDNA, and then subjected to qRT-PCR, with wild-type plants as a control. Sweet potato ​ Genes as internal references: ​ -F: 5'-AGCAGCATGAAGATTAAGGTTGTAGCACT-3'; ​ -R: 5'-GGAAAATTAGAAGCACTTCCTGTGAAC-3'.

[0094] ​ The qRT-PCR primer sequences are: ​ -F:5'- CCAGAGAGTTGACCAGTGGCAG -3'; ​ -R:5'-CGTTGGCGGCTGAGAAATTGG -3'.

[0095] The results are as follows ​ As shown in Figure B, the results indicate that ​ Gene expression was significantly upregulated in overexpressing transgenic sweet potato plants. Three overexpressing transgenic sweet potato plants (OE-B6, OE-B8, and OE-B9) were selected for asexual propagation to obtain... ​ Transgenic sweet potato lines OE-B6, OE-B8, and OE-B9 were used for phenotypic verification.

[0096] III. Low-temperature treatment ​Phenotypic identification of transgenic sweet potato plants and wild-type sweet potato plants WT is wild-type sweet potato 25, and genetically modified sweet potato is... ​ Transgenic sweet potato lines OE-B6, OE-B8, and OE-B9.

[0097] The 4-week-old wild-type sweet potato 25 and the asexually propagated sweet potato obtained in step two were used. ​ Transgenic sweet potato lines OE-B6, OE-B8, and OE-B9 were planted in nutrient soil in 13cm diameter pots, with one plant per pot and three replicates per line. After three days, once the plants had stabilized, they were treated at 4℃ for 48 hours, followed by a 25℃ recovery period of 24 hours. During this period, samples were taken at different time points, and photographs were taken to observe the plant growth.

[0098] The results are as follows ​ As shown, the results indicate that under normal conditions ​ The overexpression lines showed no significant difference from the wild-type Yanshu 25, but after 24 hours of stress at 4℃, ​ The overexpression lines showed less wilting than the wild-type Yanshu 25.4℃ after continuous treatment for 48 h. ​ The overexpression lines and wild-type Yanshu 25 showed increased wilting, but ​ The overexpression lines still showed less wilting than the wild type. After 24 hours of recovery at 25°C, compared to the wild-type Yanshu 25, ​ Overexpression lines recovered more quickly.

[0099] The above results indicate that overexpression ​ It improved the cold resistance of sweet potato plants.

[0100] Example 3, Low-temperature treatment ​ Cold tolerance indicators were determined for transgenic sweet potato plants and whole wild-type sweet potato plants. The 4-week-old wild-type sweet potato 25 and the asexually propagated sweet potato obtained in Example 1 were used. ​ Transgenic sweet potato lines OE-B6, OE-B8, and OE-B9 were planted in nutrient soil in 13cm diameter pots, with one plant per pot. After 3 days, once the plants were stable, they were treated at 4℃ for 12 hours, and then whole plant samples were taken. The samples before the low-temperature treatment (25℃) served as a control. Each line was replicated in 3 pots, and the average value of the results was taken.

[0101] I. Assay for SOD (Superoxide Dismutase) Activity The SOD enzyme activity was determined according to the operating procedures in the product instruction manual of the Plant SOD Enzyme Activity Detection Kit (manufacturer: Suzhou Keming, product number: SOD-2-Y).

[0102] II. POD enzyme (peroxidase) activity assay The POD enzyme activity was determined according to the operating procedures in the product instruction manual of the Plant POD Enzyme Activity Detection Kit (manufacturer: Suzhou Keming, product number: POD-2-Y).

[0103] III. Determination of MDA (malondialdehyde) content The MDA content was determined according to the operating procedures in the product instruction manual of the Plant MDA Quantitative Detection Kit (manufacturer: Suzhou Keming, product number: MDA-2-Y).

[0104] IV. Determination of Proline Content The proline content was determined according to the operating procedures in the instruction manual of the Plant Proline Quantitative Detection Kit (manufacturer: Suzhou Keming, product number: PRO-2-Y).

[0105] V. Gibberellin Content Determination The gibberellin content was determined according to the operating procedures of the ELISA kit for quantitative detection of plant gibberellins (manufacturer: Wuhan Jilide Biotechnology, product number: J38882).

[0106] VI. Determination of Jasmonic Acid Content The jasmonic acid content was determined according to the operating procedures of the Plant Jasmonic Acid (JA) Quantitative Detection Kit (ELISA) (manufacturer: Suzhou Keming, product number: JA-4-Q).

[0107] VII. Determination of Auxin Content The auxin content was determined according to the operating procedures of the Plant Growth Hormone (IAA) Quantitative Detection Kit (ELISA) (manufacturer: Wuhan Jilide Biotechnology, product number: J38986).

[0108] VIII. Determination of Abscisic Acid Content The abscisic acid content was determined according to the operating procedures of the Plant Abscisic Acid Quantitative Detection Kit (ELISA) (manufacturer: Wuhan Jilide Biotechnology, product number: J38924).

[0109] The results are as follows ​ As shown, the results indicate that overexpression under low temperature stress ​ SOD activity, POD activity, and proline content in sweet potato plant leaves were significantly higher than in wild-type plants, while MDA content was significantly lower. Regarding hormone content, overexpression was observed under low-temperature stress. ​ The contents of GA and IAA in the leaves of sweet potato plants were significantly increased, while the contents of JA and ABA showed no significant difference, indicating that... ​ It may positively regulate the cold tolerance of sweet potatoes through the ROS, GA and IAA hormone pathways.

[0110] Example 4: Overexpression under low temperature treatment​ Analysis of cold tolerance-related gene expression in gene-positive and wild-type sweet potato plants Wild-type sweet potato 25 with uniform growth and overexpression at 4 weeks of age were used. ​ Sweet potato plants were subjected to a 4°C low-temperature treatment, and whole-plant samples were collected after 3 hours for expression analysis of cold-resistance-related genes. A 25°C normal treatment condition was used as a control. RNA extraction, cDNA first-strand synthesis, and RT-qPCR analysis were performed according to Examples 1 and 2. Wild-type plants were used as a control. ​ The gene was used as an internal control, and the primer sequences for other cold-resistance-related genes are as follows. Three replicates were set up for each line.

[0111] ​ -F: 5'-AGCAGCATGAAGATTAAGGTTGTAGCACT-3'; ​ -R: 5'-GGAAAATTAGAAGCACTTCCTGTGAAC-3'.

[0112] ​ -F:5'-TCCTGGACCTCATGGATTTC-3'; ​ -R:5'-GCCACTATGTTTCCCAGGTC-3'.

[0113] ​ -F:5'-TTCACGACTGCTTCGTTGA-3'; ​ -R:5'-TTCTCAACCGCGGTCTTAA-3'.

[0114] ​ -F:5'-ACGCAATTCCCGGACGTGAT-3'; ​ -R: 5'-AAGCCTTCCATGTGGCGGTA-3'.

[0115] ​ -F:5'-GCCTGATGCACTTGTTCAGA-3'; ​ -R:5'-TTGAGCAATTCAGGGACCTC-3'.

[0116] ​ -F: 5'-ATAGAGGCATTGGCTGATGG-3'; ​-R: 5'-GGTAGTCCCACCTGGTGATG-3'.

[0117] ​ -F:5'-TCAGTTACAAGCGTTCACACAGT-3'; ​ -R:5'-TGCAACTTTAATAAATGACTCCCCA-3'.

[0118] ​ -F: 5'-CGGAAAGAAGGTTTAGTGGG-3'; ​ -R:5'-TTGTGGAGGTTGGGGATTT-3'.

[0119] ​ -F:5'-GACTTGGAGGCGTTTCACA-3'; ​ -R:5'-TCCATCTTTGTCTTCGTAGGTT-3'.

[0120] ​ -F: 5'-ATGCCGTGCCGTTGATAG-3'; ​ -R: 5'-AGGGACGCCGTGGTTG-3'.

[0121] The results are as follows ​ As shown, the results indicate that for ​ RT-qPCR analysis was performed on the ROS signaling pathway, proline biosynthesis pathway, GA synthesis, and IAA synthesis-related genes in the overexpressed line and wild-type Yanshu 25. The results showed that after low-temperature treatment... ​ ROS signaling pathway genes in overexpressed strains ​ , ​ and ​ Significantly upregulated expression compared to wild type; proline biosynthesis-related genes IbP5CS and IbP5CR Significantly upregulated compared to wild type, and proline degradation pathway-related genes IbP5CDH Significantly downregulated expression; GA synthesis-related genes IbGA2ox2 and genes related to IAA signal transduction IbARF1 and IbIAA17 Both showed significant upregulation of expression.

[0122] In this application, the nucleotide sequence shown in SEQ ID NO:3 is as follows:

[0123] The present application has been described in detail above. Those skilled in the art will recognize that the present application can be implemented in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments are given in this application, it should be understood that further modifications can be made to the present application. In summary, in accordance with the principles of this application, this application is intended to include any changes, uses, or improvements to the present application, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The use of the IbbHLH129 protein, or a substance regulating the expression of the gene encoding the IbbHLH129 protein, or a substance regulating the activity or content of the IbbHLH129 protein, in any of the following: A1) Its application in regulating plant cold tolerance; A2) Application in the preparation of products that regulate plant cold tolerance; A3) Application in plant cold tolerance breeding or assisted breeding; A4) Application in the preparation of products for cold-resistant plant breeding or assisted breeding; The IbbHLH129 protein is any of the following proteins: a1) Proteins with amino acid sequences as shown in SEQ ID NO:1; a2) Proteins obtained by substituting and / or deleting and / or adding amino acid residues in the amino acid sequence shown in a1). a3) and a1) are proteins with more than 70% amino acid sequence identity and the same function; a4) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of at least one of the proteins described in a1) to a3).

2. The application according to claim 1, characterized in that, The substance regulating the expression of the IbbHLH129 protein-encoding gene or the substance regulating the activity or content of the IbbHLH129 protein is a biological material, and the biological material is any one of the following: B1) A nucleic acid molecule encoding the IbbHLH129 protein as described in claim 1; B2), an expression cassette containing the nucleic acid molecule described in B1); B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) Recombinant microorganisms containing the nucleic acid molecules described in B1), or recombinant microorganisms containing the expression cassette described in B2), or recombinant microorganisms containing the recombinant vector described in B3); B5) Transgenic plant cells containing the nucleic acid molecules described in B1), or transgenic plant cells containing the expression cassette described in B2), or transgenic plant cells containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecules described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) Transgenic plant organs containing the nucleic acid molecules described in B1), or transgenic plant organs containing the expression cassette described in B2), or transgenic plant organs containing the recombinant vector described in B3).

3. The application according to claim 2, characterized in that, B1) The nucleic acid molecule is the DNA molecule described in g1) or g2) below: g1) The coding sequence of the coding strand is the DNA molecule of SEQ ID NO:2; g2) has more than 70% identity with the DNA molecule described in g1) and encodes the IbbHLH129 protein described in claim 1.

4. A method for regulating plant cold tolerance, characterized in that, The method includes regulating the expression level of the gene encoding the IbbHLH129 protein as described in claim 1 in the recipient plant and / or regulating the activity or content of the aforementioned IbbHLH129 protein in the recipient plant to regulate the cold tolerance of the recipient plant.

5. A method for obtaining a target plant with altered cold tolerance, characterized in that, The method includes obtaining a target plant with altered cold tolerance by regulating the expression level of the gene encoding the IbbHLH129 protein as described in claim 1 in the recipient plant and / or regulating the activity or content of the IbbHLH129 protein in the recipient plant.

6. The method according to claim 4 or 5, characterized in that, The method includes improving the cold tolerance of the recipient plant and / or obtaining a target plant with improved cold tolerance by increasing the expression level of the gene encoding the IbbHLH129 protein of claim 1 in the recipient plant and / or regulating the activity or content of the IbbHLH129 protein in the recipient plant.

7. The method according to claim 6, characterized in that, By introducing the gene encoding the IbbHLH129 protein as described in claim 1 into the recipient plant, the expression level of the gene encoding the IbbHLH129 protein in the recipient plant and / or the activity or content of the IbbHLH129 protein in the recipient plant can be increased.

8. A cold-resistant plant, comprising the biological material described in claim 2 or 3.

9. The method according to any one of claims 1 to 8, characterized in that, The plant in question is a dicotyledonous plant.

10. The IbbHLH129 protein of claim 1 and the biomaterial of claim 2 or 3.

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