Wheat heat-resistant protein taht3, gene encoding the same and application

Abstract

The present application discloses a wheat heat-resistant protein TaHT3, a coding gene and application thereof. The technical problem is solved by transgenic technology to improve the heat resistance of wheat. The specific disclosure is the use of the substance for up-regulating or enhancing or improving the expression of the coding gene of the protein or the substance for up-regulating or enhancing or improving the activity or content of the protein in improving the heat resistance of wheat. The protein is the protein with the amino acid sequence shown in sequence 2, and variants and fusion proteins thereof. The heat resistance of the plant with overexpressed TaHT3 protein coding gene is obviously higher than that of the wild type plant, and can be used for industrial production and plant breeding.

Description

Technical Field

[0001] This invention specifically relates to the wheat heat-resistant protein TaHT3, its encoding gene, and its applications. Background Technology

[0002] Wheat is one of the most widely cultivated crops globally and a crucial staple food crop in my country. Sustained and stable wheat production is of paramount importance for maintaining national food security. In recent years, global warming and rising annual temperatures have severely damaged wheat production. Studies indicate that global temperatures are projected to rise by 1.5°C-2.0°C by 2040, further threatening global food security and potentially reducing global grain production by 30%-40%. Therefore, identifying superior heat-resistant genes in wheat and elucidating their molecular mechanisms and genetic pathways regulating heat tolerance is crucial for breeding stress-resistant and high-yielding new wheat varieties. Summary of the Invention

[0003] This invention provides a method for improving the heat tolerance of wheat plants based on the TaHT3 protein. The heat tolerance of wheat plants includes at least one of biomass and survival rate.

[0004] In a first aspect, the present invention provides the use of substances that upregulate or enhance or increase the expression of protein-coding genes or substances that upregulate or enhance or increase the activity or content of proteins in improving the heat resistance of wheat plants.

[0005] The protein is any one of the following:

[0006] B1) The amino acid sequence of the protein is shown in sequence 2;

[0007] B2) A protein having more than 80% identity and the same function as the protein shown in B1) obtained by substituting and / or deleting and / or adding amino acid residues.

[0008] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0009] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the 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 tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0010] In the above-mentioned proteins, identity refers to the identity of the amino acid sequences. The identity of amino acid 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, using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing an identity search on a pair of amino acid sequences to calculate the identity value (%), then the identity value can be obtained.

[0011] In the aforementioned proteins, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0012] Of the proteins described above, sequence 2 (SEQ ID No. 2) consists of 348 amino acid residues. It is named TaHT3-A protein. Its encoding gene is the TaHT3-A gene (TaHT3 gene).

[0013] In this application, substances that upregulate, enhance, or increase the expression of the gene encoding the protein, or the activity or content of the protein, can enhance plant heat tolerance. In the foregoing, the heat tolerance index may be survival rate and / or biomass.

[0014] In the above applications, the protein is derived from wheat.

[0015] Secondly, the present invention provides a product for improving the heat resistance of wheat plants, the product comprising the aforementioned substances.

[0016] The wheat mentioned above may be the wheat variety Fielder.

[0017] In the above text, the substance regulating gene expression can be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcriptional level of the gene; 2) post-transcriptional regulation of the gene (i.e., regulation of splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (i.e., regulation of mRNA transport of the gene from the nucleus to the cytoplasm); 4) regulation of translation of the gene; 5) regulation of mRNA degradation of the gene; and 6) post-translational regulation of the gene (i.e., regulation of the activity of the protein translated from the gene).

[0018] In the above-described uses or products, the uses include increasing the biomass and / or survival rate of the wheat plants.

[0019] In the above text, the stress temperature for the heat resistance is 42°C. The stress time for the heat resistance is 3 days.

[0020] In the above-described uses, products, or other uses or products, the substance that upregulates, enhances, or increases the expression of the protein-coding gene, or the substance that upregulates, enhances, or increases the activity or content of the protein, or the substance is any one of the following:

[0021] B1) Nucleic acid molecules that encode the above proteins;

[0022] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0023] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0024] 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);

[0025] B5), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0026] B6) Transgenic plant tissue containing the nucleic acid molecule 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);

[0027] B7) A transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3).

[0028] In the nucleic acid molecule described in B1), those skilled in the art can easily mutate the nucleotide sequence encoding the protein TaHT3-A of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more identity with the nucleotide sequence of the protein TaHT3-A isolated in the present invention, as long as they encode and function the protein TaHT3-A, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0029] The aforementioned 80% or higher identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.

[0030] In this article, identity refers to the similarity of amino acid or nucleotide sequences. The identity of amino acid 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, using blastp as the procedure, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively, and performing a search to calculate the identity of amino acid sequences, then the identity value (%) can be obtained.

[0031] In this document, the vectors described are known to those skilled in the art and include, but are not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phage), granules (i.e., Cosmids), Ti plasmids, or viral vectors. Specifically, it may be the pWMB110 vector;

[0032] In the aforementioned biological materials, the expression cassette described in B2) refers to DNA capable of expressing the gene in a host cell. This DNA may include not only promoters that initiate gene transcription but also terminators that terminate gene transcription. Furthermore, the expression cassette may also include enhancer sequences. Promoters that can be used in this invention include, but are not limited to: constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); chemically inducible promoters from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); tomato protease inhibitor II promoter (PIN2) or LAP promoter (both can be induced by jasmonic acid methyl ester); heat shock promoter (US Patent 5,187,267); tetracycline inducible promoter (US Patent 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of beta-conglycin, napin, oleosin and soybean beta-conglycin (Beachy et al. (1985) EMBOJ.4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited here are cited in full. Suitable transcription terminators include, but are not limited to: Agrobacterium carmine synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator, and carmine and octopine synthase terminators (see, for example: Odell et al. (1985) Nature 313:810; Rosenberg et al. (1987) Gene, 56:125; Guerineau et al. (1991) Mol. Gen. Genet, 262:141; Proudfoot (1991) Cell, 64:671; Sanfacon et al. Genes Dev., 5:141; Mogen et al. (1990) Plant Cell, 2:1261; Munroe et al. (1990) Gene, 91:151; Ballad et al. (1989) Nucleic Acids Res. 17:7891; Joshi et al. (1987) Nucleic Acid Res., 15:9627.

[0033] In B3) above, the recombinant vector can be a recombinant expression vector containing the gene expression cassette constructed using a plant expression vector. The plant expression vector can be a Gateway system vector or a binary Agrobacterium vector, such as pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pMDC85, or pCAMBIA1391-Xb. When constructing a recombinant expression vector using TaHT1-A, any enhancing, constitutive, tissue-specific, or inducible promoter can be added before its transcription initiation nucleotide, such as the cauliflower mosaic virus (CAMV) 35S promoter, the ubiquitin gene Ubiqutin promoter (pUbi), etc., which can be used alone or in combination with other plant promoters. Furthermore, when constructing a plant expression vector using the gene of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and start codons are wide-ranging; they can be natural or synthetic. The translation initiation region can originate from the transcription initiation region or structural genes. As a specific embodiment, this application uses the pWMB110 vector as the expression vector.

[0034] In the above applications, the nucleic acid molecule described in B1) is a DNA molecule with the nucleotide sequence shown in Sequence 1.

[0035] In the above text, the ORF of the gene encoding the protein shown in Sequence 2 may be as shown in Sequence 1.

[0036] In the above text, the sequence of the gene encoding the protein shown in Sequence 2 is Sequence 1.

[0037] Thirdly, the present invention provides a method for cultivating heat-prone plants.

[0038] The method includes upregulating or enhancing or increasing the expression level of the gene encoding the above-mentioned protein in the target wheat species, and / or, the activity and / or content of the protein to obtain a heat-resistant wheat species, wherein the heat resistance of the heat-resistant wheat species is higher than that of the target wheat species.

[0039] Fourthly, the present invention provides a method for increasing the biomass and / or survival rate of wheat plants under high temperature stress.

[0040] The method includes increasing the biomass and / or survival rate of wheat plants under high-temperature stress by upregulating or enhancing or increasing the expression of genes encoding the aforementioned proteins in wheat plants, and / or, the activity of the aforementioned proteins.

[0041] In this application, the wheat species may be wheat. The wheat may be the wheat variety Fielder.

[0042] In the above method, the upregulation, enhancement, or increase of the expression of the coding gene of the above protein in wheat plants is achieved by expressing the coding gene of the above protein in the target wheat plant.

[0043] In the above text, the overexpression of the coding gene of the above-mentioned protein in the target wheat plant includes introducing the nucleic acid molecule described in B1), the expression cassette described in B2), or the recombinant vector described in B3) into the target plant.

[0044] In the above text, the nucleic acid molecule may be the nucleic acid molecule described in Sequence 1.

[0045] In the above-mentioned uses, products, or methods, the wheat plant is wheat.

[0046] In the above-mentioned uses, products, or methods, the wheat plant is a seedling wheat plant.

[0047] The wheat mentioned above includes, but is not limited to, Yannong 15, Lumai 15, Jimai 38, Zhengmai 379, Jimai 44, Chuanmai 104, Xinong 511, Zhongmai 578, Weilong 169, Zhengmai 1860, Bainong 207, Luyuan 502, Huaimai 33, Bainong 4199, Xinmai 26, Malan 1, Yangmai 25, Yumai 14 (superior variety), Xinmai 19, Fengdecunmai 20, Zhongxinmai 998, Zhengmai 136, Bainong 307, Yannong 1212, Zhoumai 16, Zhoumai 22, or Fielder.

[0048] In this application, the wheat may be the wheat variety Fielder.

[0049] In the above text, the heat resistance index may be survival rate and / or biomass.

[0050] In the above text, the biomass refers to the weight of the aboveground parts.

[0051] In the above text, the survival rate is defined as the number of surviving seedlings divided by the total number of seedlings.

[0052] In the above text, high heat resistance refers to higher survival rate and biomass under high temperature stress conditions.

[0053] The seedling stage mentioned above refers to a growth period of 7-10 days. Specifically, it can be 7 or 10 days.

[0054] The 7-10 days of growth mentioned above refers to the period from sowing. Specifically, it can be 7 or 10 days.

[0055] In this application, the high-temperature pressing condition is a heat treatment at 42°C for 3 days.

[0056] Beneficial effects

[0057] This invention discloses the wheat heat-resistant protein TaHT3, its encoding gene, and its applications. The technical problem solved is to improve the heat resistance of wheat through transgenic technology. Specifically, it discloses the use of substances that upregulate, enhance, or increase the expression of the gene encoding the TaHT3 protein, or substances that upregulate, enhance, or increase the activity or content of the TaHT3 protein, in improving the heat resistance of wheat plants; the protein is a protein with the amino acid sequence shown in Sequence 2, its variants, and fusion proteins. Plants overexpressing the TaHT3 protein encoding gene exhibit significantly higher heat resistance than wild-type plants and can be used for industrial production and plant breeding. Attached Figure Description

[0058] Figure 1 This is a transcriptional map of the TaHT3 gene in wild-type wheat Fielder, OE-1, and OE-2.

[0059] Figure 2 Phenotypic diagrams of wild-type wheat Fielder, OE-1, and OE-2 before and after heat stress.

[0060] Figure 3 Biomass figures for wild-type wheat Fielder, OE-1, and OE-2 under heat stress and normal conditions.

[0061] Figure 4 Survival rates of wild-type wheat Fielder, OE-1, and OE-2 under heat stress and normal conditions. Detailed Implementation

[0062] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and 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 invention in any way.

[0063] 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.

[0064] The following examples used SPSS 11.5 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA was used. P < 0.05 (*) indicates a significant difference, P < 0.01 (**) indicates a highly significant difference, and P < 0.001 (***) indicates a highly significant difference.

[0065] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0066] In this invention, the products include, but are not limited to, reagents, kits, or devices.

[0067] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear, cyclic, or branched, may contain modified amino acids, particularly conserved modified amino acids, and may be interrupted by non-amino acid components. The term also includes modified amino acid polymers, such as those modified by sulfation, glycosylation, esterification, acetylation, phosphorylation, iodination, methylation, oxidation, proteolytic processing, isopreneation, racemization, selenoylation, transfer-RNA-mediated amino addition such as arginination, ubiquitination, or any other manipulation such as conjugation with a labeled component. As used herein, the term “amino acid” refers to natural and / or non-natural or synthetic amino acids, including glycine and its D or L optical isomers, as well as amino acid analogs and peptide mimics. “Derived from” a specified protein refers to the source of the polypeptide. The term also includes polypeptides expressed by specified nucleic acid sequences.

[0068] The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as subsequently modified amino acids such as hydroxyproline, γ-carboxyglutamic acid, and O-phosphoserine. In this document, amino acids are represented using the commonly used three-letter or single-letter codes recommended by the IUPAC-IUB Committee on Biochemistry Nomenclature. Similarly, nucleotides are represented using their generally accepted single-letter codes.

[0069] The term "Triticum" refers to plants belonging to the genus *Triticum*, a monocotyledonous plant within the family Poaceae. These are annual or biennial herbs. Their culms are erect, tufted, with 6-7 nodes, 60-100 cm tall and 5-7 mm in diameter. Leaf sheaths loosely enclose the stem, the lower sheaths longer than the upper ones but shorter than the internodes; the ligule is membranous, about 1 mm long; the leaf blade is lanceolate. The spike-like inflorescence is erect, 5-10 cm long (excluding the awn), 1-1.5 cm wide; spikelets contain 3-9 florets, the upper ones undeveloped; glumes are ovate, 6-8 mm long, the midrib ridged on the upper back, extending to a tooth about 1 mm long at the apex; the ridges and apical teeth of the lateral veins are indistinct; the lemma is oblong-lanceolate, 8-10 mm long, with or without an awn at the apex; the palea is nearly equal in length to the lemma.

[0070] The term "wheat" refers to wheat of the genus *Triticum*. This includes, but is not limited to, varieties such as Yannong 15, Lumai 15, Jimai 38, Zhengmai 379, Jimai 44, Chuanmai 104, Xinong 511, Zhongmai 578, Weilong 169, Zhengmai 1860, Bainong 207, Luyuan 502, Huaimai 33, Bainong 4199, Xinmai 26, Malan 1, Yangmai 25, Yumai 14 (superior variety), Xinmai 19, Fengdecunmai 20, Zhongxinmai 998, Zhengmai 136, Bainong 307, Yannong 1212, Zhoumai 16, Zhoumai 22, or Fielder.

[0071] The term "heat tolerance" refers to a plant's ability to adapt to and resist high-temperature stress. Specifically, it can be reflected in the effects of high-temperature stress on various indicators and / or traits, including but not limited to photosynthesis, respiration, transpiration, osmotic regulation content, antioxidant systems, plant hormones, cell ultrastructure, biomass, and / or survival rate.

[0072] In this invention, the uses of improving the heat resistance of wheat plants include, but are not limited to, preparing products that improve the heat resistance of wheat plants, improving the heat resistance of wheat plants, and / or wheat plant breeding.

[0073] The terms "high temperature stress" and "high temperature stress environment" have the same meaning, referring to high temperatures that have a harmful effect on plants.

[0074] The term "expression cassette" refers to DNA capable of expressing a target protein or nucleic acid in a host cell. The expression cassette may also include a single-stranded or double-stranded nucleic acid molecule containing all the regulatory sequences necessary for the expression of any of the aforementioned proteins. These regulatory sequences, under compatible conditions, guide the coding sequence to express any of the aforementioned proteins in a suitable host cell. The regulatory sequences include, but are not limited to, leader sequences, polyadenylated sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences must include a promoter and termination signals for transcription and translation. To introduce specific restriction enzyme sites into the vector for linking the regulatory sequences to the coding region of the nucleic acid sequence encoding the protein, a regulator-linked regulatory sequence may be provided. The regulatory sequence may be a suitable promoter sequence, i.e., a nucleic acid sequence that can be recognized by the host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates protein expression. The promoter may be any nucleic acid sequence that is transcriptionally active in the selected host cell, including mutated, truncated, and heterozygous promoters, and may be derived from genes encoding extracellular or intracellular proteins that are homologous or heterologous to those of the host cell. The regulatory sequence can also be a suitable transcription termination sequence, i.e., a sequence that can be recognized by the host cell and thus terminate transcription. The termination sequence is operatively attached to the 3' end of the nucleic acid sequence encoding the protein. Any terminator that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, i.e., an untranslated region of mRNA that is crucial for translation in the host cell. The leader sequence is operatively attached to the 5' end of the nucleic acid sequence encoding the protein. Any leader sequence that can function in the selected host cell can be used in this invention. The regulatory sequence can also be a signal peptide coding region, which encodes an amino acid sequence attached to the amino terminus of a protein that guides the encoded protein into the cellular secretory pathway. Signal peptide coding regions that guide the expressed protein into the secretory pathway of the host cell can be used in this invention. Adding a regulatory sequence that can regulate protein expression according to the growth status of the host cell may also be necessary. Examples of regulatory systems are those that respond to chemical or physical stimuli (including in the presence of regulatory compounds), thereby turning gene expression on or off. Other examples of regulatory sequences are those that can amplify genes. In these examples, the nucleic acid sequence encoding the protein should be operatively linked to the regulatory sequence.

[0075] The term "vector" generally refers to a vector capable of delivering exogenous DNA or a target gene into host cells for amplification and / or expression. This vector can be a cloning vector or an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material they carry to be amplified and / or expressed within the host cells. Those skilled in the art can select appropriate vectors based on the purpose of genetic engineering and the properties of the recipient cells. The vectors include, but are not limited to: plasmids, phages (such as λ phage or M13 phage), cosmids (i.e., Cosmids), phagemids, shuttle vectors (such as yeast expression vectors), Ti plasmids, artificial chromosomes (such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), P1 artificial chromosomes (PAC), or Ti plasmid artificial chromosomes (TAC)), and viral vectors (such as baculovirus vectors, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, papillomaviruses (such as SV40), and herpesviruses (such as herpes simplex virus)). A vector may contain multiple elements controlling expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may also contain a replication initiation site.

[0076] The term "microorganism" typically includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria mentioned can be from genera such as *Escherichia* sp. (e.g., *Escherichia coli*), *Erwinia* sp., *Agrobacterium* sp. (e.g., *Agrobacterium tumefaciens*), *Flavobacterium* sp., *Alcaligenes* sp., *Pseudomonas* sp., and *Bacillus* sp. (e.g., *Bacillus*). The viruses mentioned can include rotaviruses, baculoviruses, retroviruses (e.g., lentiviruses), adenoviruses, adeno-associated viruses, poxviruses, papillomaviruses, influenza viruses, papillomaviruses (e.g., SV40), and herpesviruses (e.g., herpes simplex virus). The fungi may originate from genera such as *Saccharomyces* sp. (e.g., *Saccharomyces cerevisiae*, *Methanolac*, *Pichia pastoris*), *Fusarium* sp., *Rhizoctonia* sp., *Verticillium* sp., *Penicillium* sp., *Aspergillus* sp., and *Cephalosporium* sp. The actinomycetes may originate from genera such as *Streptomyces* sp. The algae may originate from phyla such as *Cyanophyta* (e.g., cyanobacteria), genera such as *Fucus* sp., *Achnanthes* sp., *Amphiprora* sp., *Amphora* sp., *Ankistrodesmus* sp., *Asteromonas* sp., and *Boekelovia* sp. .

[0077] The term "host cell," also known as the recipient cell, generally refers to any type of cell that can be used to introduce a vector, such as plant and animal cells. The term "host cell" can be understood not only to the specific recipient cell but also to its offspring, which, due to natural, accidental, or intentional mutations and / or alterations, may not necessarily be identical to the original parent cell but are still included within the scope of the host cell. Suitable host cells are those known in the art, including: plant cells such as Arabidopsis thaliana, tobacco (Nicotiana tabacum), maize (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum), etc., but not limited to these; animal cells such as mammalian cells (e.g., Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells or NK cells, etc.), avian cells (e.g., chicken or duck cells), and amphibian cells (e.g., Xenopus laevis cells or Andrias davidianus cells). These include, but are not limited to, davidianus cells, fish cells (e.g., grass carp, carp, rainbow trout, or catfish cells), insect cells (e.g., Sf21 cells, Sf-9 cells, or Hi-5 cells).

[0078] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by ligating a foreign target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell.

[0079] The goal is to provide the recipient cells with the ability to replicate, integrate, amplify, and / or express the exogenous target gene.

[0080] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by linking a foreign target gene to a vector in vitro. It can be constructed in any suitable way, as long as the constructed recombinant vector can carry the foreign target gene into the recipient cell and provide the foreign target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0081] The term "recombinant microorganism" generally refers to a recombinant microorganism whose genes have been manipulated and modified to obtain a functionally altered microorganism. This can be achieved by introducing a foreign target gene or recombinant vector into the target microorganism, or by directly editing the endogenous genes of the target microorganism.

[0082] The term "recombinant host cell" generally refers to a recombinant host cell whose genes have been manipulated and modified to obtain a recombinant host cell with altered function. This can include introducing a foreign target gene or recombinant vector into the host cell, or directly editing the host cell's endogenous genes.

[0083] The term "overexpression" refers to the process of increasing the expression level of a target gene beyond its normal physiological state through certain means. Overexpression involves constructing the coding region of the target gene into a plasmid or viral vector, and then introducing it into the target cell, plant, organ, or organ to increase the expression level of the target gene.

[0084] The terms “comprising, comprises, and comprised of” as used herein are synonymous with “including and includes” or “containing and contains”, and are inclusive or open-ended, and do not exclude additional, unstated members, elements, or method steps. The terms “comprising, comprises, and comprised of” also include the term “composed of”.

[0085] The term "identity" is used to describe the percentage of identical amino acids or nucleotides between two amino acid sequences or nucleic acid sequences relative to a reference sequence, determined by conventional methods, for example, see Ausubel et al., eds. (1995), Current Protocols in Molecule & Lar Biology, Chapter 19 (Greene Publishing and Wiley-Interscience, New York); and the ALIGN program (Dayhoff (1978), Atlas of Protein Sequence and Structure 5: Suppl. 3 (National Biomedical Research Foundation, Washington, DC). There are many algorithms for aligning sequences and determining sequence identity, including the homology alignment algorithm of Needleman et al. (1970) J. Mol. Biol. 48: 443; the local homology algorithm of Smith et al. (1981) Adv. Appl. Math. 2: 482; and the local homology algorithm of Pearson et al. (1988) P… Similarity search methods are described in roc. Natl. Acad. Sci. 85: 2444; the Smith-Waterman algorithm (Meth. Mol. Biol. 70: 173-187 (1997); and the BLASTP, BLASTN, and BLASTX algorithms (see AltschμL et al. (1990) J. Mol. Biol. 215: 403-410). Computer programs utilizing these algorithms are also available, including but not limited to: ALIGN or Megalign (DNASTAR) software, or WU-BLAS. T-2 (AltschμL et al., Meth. Enzym., 266:460-480 (1996)); or GAP, BESTFIT, BLASTAltschμL et al., above, FASTA, and TFASTA, available in Genetics Computing Group (GCG) package, version 8, Madison, Wisconsin, USA; and CLUSTAL in the PC / Gene program provided by Intelligenetics, MountainView, California.

[0086] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0087] When referring to measurable values ​​such as quantities, temporary durations, etc., the term “about” means a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0088] Example 1: Obtaining and Identifying Transgenic Plants

[0089] I. Construction of Recombinant Plasmids

[0090] 1. Primers (TaHT3-F and TaHT3-R) were designed to amplify the ORF fragment of TaHT3-A with the restriction enzyme linker from the cDNA of wheat Fielder seedling leaves, and the restriction enzyme site (BamHI) was introduced. PCR amplification was performed using primers TaHT3-F and TaHT3-R to obtain the ORF fragment of TaHT3-A with the restriction enzyme linker.

[0091] The ORF fragment sequence of TaHT3-A is shown in Sequence 1.

[0092] Sequence 1 is as follows:

[0093] ATGGTCTCCAGCGCCCGTAGTTCCCGGCGGCATGGCTCACTCGGCTTCCAACAGCGCCG

[0094] GAAGCGCCGGTTCCCTCTCCGGCGGCATCGGGCCCGGCAAGGGGGTTGCGGCGGCGGC

[0095] CGCCACGACCTCGGCGTCAGCGTCCACGCCGGCCAGCGGGAGCACCGTTGCCCGCCGC

[0096] CTCAATGATCTCGAAATTCAGGGAGATGACCCCGCCATCGTCGCAGCCCGTTGCTAGCA

[0097] AGAAGAAAAAGAAAGGAACACGCGTAGTAGGTCCCGACAAAGGTAACCGTGGATTGC

[0098] GCCAATTCAGTATGAAAGTCTGTGAGAAAGTTGAAGGCAAAGGGAGAACAACCTACA

[0099] ATGAGGTGGCAGACGAACTTGTAGCTGAGTTTGCAGATCCAAATAGTAATATTGGGTC

[0100] ACCAGATCCTGATAATCCCAACACACAACAATATGATGAGAAAAATATACGAAGAAG

[0101] GGTTTACGATGCACTAAACGTCCTGATGGCTATGGATATTATATCTAAAGATAAAAAG

[0102] GAAATTCAGTGGAAGGGCTTACCCCGAACAAGTTTGAGTGATATTGATAAATTGAAGA

[0103] CTGAGGTCATTGGGCTGAAAGGTAGAATTGACAAGAAAAGTGCATATCTGCAGGAATT

[0104] ACAAGATCAATATGCGGGCCTCCAAAATTTGGTAGAGCGAAATGAGCAGCTATATGGT

[0105] TCGGGAGATGCTCCATCTGGCGGAGTGGCCCTGCCATTCATATTGGTTCAGACACGTCC

[0106] TCATGCAACTGTCGAAGTGGAGATATCAGAAGATATGCAGTTGGTGCATTTTGATTTCA

[0107] ATAGCACTCCGTTTGAGTTGCACGATGATTCCTTTGTATTGAAAGCAATGGGGTTCTCT

[0108] GGTAAAGAAGAAACTGACGGTACAGTGGCTCTGGTTGCAAATGCGGTTGAATGCTCAA

[0109] GTGCATCAAATGTTTATGGGCGTCGATCACCACAACCTGCAAGGCCAAATGGAATTAG

[0110] GCTACGAACCTCACCTCCTATTCCAGGGATACTGAAAGGGCGTGTCAAGCATGAACAC

[0111] TAG

[0112] The protein sequence encoded by TaHT3-A is shown in Sequence 2 as follows:

[0113] MVSSARSSGGMAHSASNSAGSAGSPSGGIGPGKGVAAAAATTSASASTPASGSTVARRLNDLEIQGDDPPSSQPVASKKKKKGTRVVGPDKGNRGLRQFSMKVCEKVEGKGRTTYNEVADELVAEFADPNSNIGSPDPDNPNTQQYDEKNIRRRVYDALNVLMAMDIISKDKKEIQWKGLPRTSLSDIDKLKTEVIGLKGRIDKKSAYLQELQDQYAGLQNLVERNEQLYGSGDAPSGGVALPFILVQTRPHATVEVEISEDMQLVHFDFNSTPFELHDDSFVLKAMGFSGKEETDGTVALVANAVECSSASNVYGRRSPQPARPNGIRLRTSPPIPGILKGRVKHEH*

[0114]

[0115] TaHT3-F: 5'- GGATCC ATGGTCTCCAGCGCCCGTAG-3'; The underlined part is the BamHI restriction enzyme recognition site;

[0116] TaHT3-R: 5'- GGATCC CTAGTGTTCATGCTTGACAC3'; the underlined part is the BamHI restriction enzyme recognition site.

[0117] 2. The product fragment with the enzyme restriction site adapter (the ORF fragment of TaHT3-A with the enzyme restriction adapter) obtained by amplification is recovered and purified to obtain the PCR amplification product.

[0118] 3. Digest the PCR amplification product from step 2 with the restriction endonuclease BamHI, recover the digested product, and obtain the PCR digested product.

[0119] 4. Digest the pWMB110 vector (p110 vector is described in the following literature: Wang, K., Shi, L., Liang, X. et al. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nat. Plants 8, 110–117 (2022). https: / / doi.org / 10.1038 / s41477-021-01085-8, in which it is named pWMB110) with the restriction endonuclease BamHI for later use, to obtain the digested pWMB110 vector.

[0120] 5. Using T4 ligase, the PCR digestion product from step 3 and the digested pWMB110 vector from step 4 are ligated to obtain the pWMB110-TaHT3-A recombinant plasmid.

[0121] Sequencing results showed that the pWMB110-TaHT3-A recombinant plasmid was obtained by replacing the sequence between the restriction endonuclease BamHI recognition sites of the pWMB110 vector with sequence 1, while maintaining the correct expression of TaHT3-A (sequence 1) and keeping the other nucleotides of the pWMB110 vector unchanged. This recombinant plasmid was named pWMB110-TaHT3-A recombinant plasmid.

[0122] II. Obtaining TaHT3 overexpression transgenic wheat

[0123] 1. Using Fielder's cDNA as a template, the ORF fragment of TaHT3 containing the BamHI vector adapter with restriction site was amplified using TaHT3-p110-F and TaHT3-p110-R primers to construct an overexpression vector.

[0124] 2. After recovering the amplified DNA fragment by agarose gel, TaHT3 was constructed into the p110 vector using homologous recombination to form the TaHT3 overexpression vector (pWMB110-TaHT3-A recombinant plasmid).

[0125] 3. Transform the pWMB110-TaHT3-A recombinant plasmid into E. coli to obtain E. coli Mach1-T1 / pWMB110-TaHT3-A recombinant plasmid. The transformed E. coli Mach1-T1 / pWMB110-TaHT3-A recombinant plasmid is E. coli containing the pWMB110-TaHT3-A recombinant plasmid.

[0126] 4. Positive clones were screened by PCR reaction and sequenced. The clones with the correct sequence were named pWMB110-TaHT3-A recombinant plasmid. At the same time, pWMB110-TaHT3-A recombinant plasmid was extracted and transformed into Agrobacterium, resulting in Agrobacterium / pWMB110-TaHT3-A recombinant plasmid transformation and Agrobacterium EHA105 / pWMB110-TaHT3-A recombinant plasmid transformation. These Agrobacterium clones containing pWMB110-TaHT3-A recombinant plasmid were sent to the wheat transgenic platform for wheat (recipient is Fielder) genetic transformation, resulting in 8 TaHT3-A overexpressing T0 generation transgenic wheat lines. These were then self-crossed to obtain 2 TaHT3-A overexpressing T2 generation homozygous transgenic wheat lines, named OE1 and OE2.

[0127] 5. PCR identification was performed on the obtained TaHT3-A overexpression T2 generation homozygous transgenic wheat (OE1 and OE2).

[0128] The expression level was determined as follows:

[0129] T3 generation seeds (OE1 T3 homozygous seeds and OE2 T3 homozygous seeds) and control group seeds (wheat variety Fielder) were planted. When the leaves were at the three-leaf stage, RNA was extracted and reverse transcribed into cDNA using a reverse transcription kit (VazymeBiotech, R223-01). The expression level was monitored using quantitative primers. (Expression level identification is a complete process and requires the following information: sampling time for Fielder, OE1, and OE2, specific primers and internal control primers, quantitative fluorescence kit and specific conditions).

[0130] Detection primers: TaHT3-qF: ACAAGATCAATATGCGGGC; TaHT3-qR: AAAGGAATCATCGTGCAACTC. TaACTIN is used as an internal control; internal control primers: TaACTIN-F: GGAATCCATGAGACCACCTAC; TaACTIN-R: GACCCAGACAACTCGCAAC.

[0131] The results are as follows Figure 1 ( Figure 1 In the figure, Fielder is the control, and OE1 and OE2 are homozygous seedlings of the T3 generation of OE1 and OE2, respectively. As shown, the expression level of TaHT3 gene was significantly increased in the T3 generation homozygous seedlings overexpressing TaHT3 compared with Fielder seedlings.

[0132] 6. After harvesting the identified positive lines, the plants were multiplied in a greenhouse, and the T3 generation plants were identified positively by PCR.

[0133] Example 2: Identification of heat tolerance phenotype in transgenic plants

[0134] 1. TaHT3 overexpression wheat heat stress experiment

[0135] Seeds from two lines, N-OE1 and N-OE2, which were identified as positive overexpression lines (OE1 homozygous T3 generation and OE2 homozygous T3 generation), as well as seeds of wild-type Fielder, were selected, disinfected with 1% hydrogen peroxide for 10 min, washed 3-4 times with distilled water, placed in petri dishes with two layers of filter paper, a small amount of distilled water added, and placed at room temperature for 48 h.

[0136] Seedlings with uniform germination were selected and transplanted into culture boxes containing nutrient soil (the culture substrate in the culture box was obtained by uniformly mixing vermiculite and nutrient soil in a volume ratio of 2:1). Each pot was divided into two parts: one part containing control seeds (seeds of wild-type Fielder) and the other part containing seeds of overexpression lines (OE1 and OE2), with a total of four replicates. The experiment was divided into a heat tolerance experimental group and a control group.

[0137] The heat resistance test group performed the following operations:

[0138] After being planted in the culture box, the seedlings were grown for 7 days under the following conditions: 22℃-18℃, 16 hours of light / 8 hours of darkness, and 60%-70% humidity. Afterwards, their heat tolerance at 42℃ was assessed.

[0139] Fielder and TaHT3 overexpressing plants (OE1 homozygous T3 generation and OE2 homozygous T3 generation) were heat-treated at 42℃ for 3 days, and then recovered for 7 days under culture conditions of 22℃-18℃, 16 hours of light / 8 hours of darkness, and 60%-70% humidity. Phenotypic data were recorded by photographing, and survival rate and biomass before and after stress were statistically analyzed.

[0140] The control group underwent the following procedures:

[0141] After being planted in the culture box, the entire process was carried out under the culture conditions of 22℃-18℃, 16 hours of light / 8 hours of darkness, and 60%-70% humidity (under normal conditions). After the heat-resistant experimental group recovered for 7 days, they were photographed together, and the survival rate and biomass before and after stress were statistically analyzed.

[0142] Survival rate = Number of surviving seedlings / Total number of seedlings.

[0143] The criteria for survival are: the stem remains green, the leaves are partially or completely green again, or new leaves have unfolded.

[0144] The criteria for determining death are: leaves turning yellow and dying, and the stem completely dying with no signs of regrowth.

[0145] Biomass before and after stress was measured by cutting aboveground wheat seedlings from the stem base and weighing and recording the weight.

[0146] Phenotypic results are as follows Figure 2 As shown ( Figure 2 The left side of the image shows wild-type wheat Fielder, the middle section shows OE1 homozygous T3 generation, and the left side shows OE2 homozygous T3 generation. Normal conditioning served as the control group, and HS treatment (42℃) was the heat tolerance experimental group. Under normal conditions, there was no significant difference between overexpressing plants and wild-type plants. After heat treatment, the overexpressing lines showed significantly stronger growth than the wild-type lines.

[0147] Biomass results as follows Figure 3 As shown ( Figure 3 In the left bar chart, the biomass bar chart is shown. The Normal comdition is the control group, and the HS treatment (42℃) is the heat tolerance experimental group. Fielder represents wild-type wheat Fielder, OE-1 is the OE1 homozygous T3 generation, and OE-2 is the OE2 homozygous T3 generation. The ordinate represents biomass. After 7 days of recovery, there were significant differences in the dry and fresh weights of the different lines, with the overexpression lines having a higher fresh weight than the wild-type plants. In addition, the dry weight of the overexpression lines after recovery was slightly higher than that of the wild-type lines. Figure 4 ).

[0148] Survival results as follows Figure 4 As shown ( Figure 4 Bar chart of survival rates in heat-resistant experimental groups; Fielder is wild-type wheat Fielder, OE-1 is OE1 homozygous T3 generation, OE-2 is OE2 homozygous T3 generation, and the vertical axis is Survival Rate (%). The results show that the survival rates of the overexpression lines (OE1 homozygous T3 generation and OE2 homozygous T3 generation) were significantly lower than those of the wild-type plants after recovery.

[0149] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced 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 have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. The use of substances that upregulate, enhance, or increase the expression of protein-coding genes or substances that upregulate, enhance, or increase protein content in improving the heat resistance of wheat plants; The protein is any one of the following: A1) The amino acid sequence is that of the protein shown in sequence 2; A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1); The substance that upregulates, enhances, or increases the expression of the protein-coding gene, or the substance that upregulates, enhances, or increases protein content, or the substance is any one of the following: B1) Nucleic acid molecules encoding the protein; 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), a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) Transgenic plant tissue containing the nucleic acid molecule 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); The plant in question is wheat.

2. The use according to claim 1, characterized in that, The intended use is to increase the biomass and / or survival rate of wheat plants under high-temperature stress.

3. The use according to claim 1 or 2, characterized in that, The wheat species in question are wheat seedlings.

4. A method for cultivating highly heat-resistant plants, characterized in that, This includes upregulating or enhancing or increasing the expression level of the gene encoding the protein described in claim 1 or 2 in a target wheat plant, and / or, the content of said protein results in a highly heat-resistant wheat plant, wherein the heat resistance of said highly heat-resistant wheat plant is higher than that of the target wheat plant; the wheat plant is wheat.

5. A method for increasing the biomass and / or survival rate of wheat plants under high-temperature stress, characterized in that, This includes increasing the biomass and / or survival rate of wheat plants under high-temperature stress by upregulating or enhancing or increasing the expression of the gene encoding the protein described in claim 1 or 2 in wheat plants, and / or increasing the content of the protein described in claim 1 or 2; wherein the wheat plant is wheat.

6. The method according to claim 4 or 5, characterized in that, The upregulation, enhancement, or increase of the expression of the gene encoding the protein of claim 1 or 2 in the target wheat plant is achieved by expressing the gene encoding the protein of claim 1 or 2 in the target wheat plant.

7. The method according to claim 6, characterized in that, The wheat species in question are wheat seedlings.

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