Wheat heat-resistant protein taht1, its coding gene and application

Abstract

The present application discloses a wheat heat-resistant protein TaHT1, a coding gene and application thereof. Specifically disclosed are the application of the protein, a substance for regulating the expression of the coding gene of the protein or a substance for regulating the activity or content of the protein in any one of the following: A1) improving the heat resistance of plants and / or preparing a product for improving the heat resistance of plants; A2) improving the survival rate of plants under heat stress and / or preparing a product for improving the survival rate of plants under heat stress; A3) improving the biomass of plants under salt stress and / or preparing a product for improving the biomass of plants under salt stress. The plant overexpressing the TaHT1 protein coding gene has a significantly higher heat resistance than the wild type plant, and can be used for industrial production and plant breeding.

Description

TECHNICAL FIELD

[0001] The present application relates to a wheat heat-resistant protein TaHT1, a coding gene thereof and an application thereof. BACKGROUND

[0002] Wheat is one of the most widely planted crops in the world, providing about 20% of the protein and carbohydrate intake of humans. As a cool crop, the optimum temperature for the growth and development of wheat is 15-25℃. However, the global temperature rise due to climate warming has caused great damage to the agricultural production of wheat. Studies have shown that the global temperature is expected to rise by 1.5-2.0℃ by 2040, which will further threaten global food security and may reduce the global grain yield by 30-40%. Therefore, it is of great significance to mine excellent heat-resistant genes in wheat, analyze the molecular mechanism and genetic mechanism of heat resistance regulation, and cultivate new wheat varieties with resistance and stable yield. SUMMARY

[0003] The technical problem solved by the present application is how to regulate the heat resistance of wheat, especially to improve the heat resistance of wheat.

[0004] In order to solve the above problems, the present application provides the following applications.

[0005] The application of a protein, a substance for up-regulating or enhancing or improving the expression of a gene encoding the protein, or a substance for up-regulating or enhancing or improving the activity or content of the protein in any of the following:

[0006] A1) the application in improving the heat resistance of plants and / or the application in preparing a product for improving the heat resistance of plants;

[0007] A2) the application in improving the survival rate of plants under heat stress and / or the application in preparing a product for improving the survival rate of plants under heat stress;

[0008] A3) the application in improving the biomass of plants under salt stress and / or the application in preparing a product for improving the biomass of plants under salt stress;

[0009] The protein is any of the following:

[0010] B1) the protein with the amino acid sequence shown in SEQ ID NO. 2;

[0011] B2) a protein obtained by substitution, deletion and / or addition of amino acid residues of the protein of B1) and having 80% or more identity with the protein of B1) and having the same function;

[0012] B3) a fusion protein obtained by connecting the N-terminal or / and C-terminal of B1) or B2) with a protein tag.

[0013] In the above protein, the protein-tag refers to a polypeptide or protein fused and expressed with the target protein by using DNA in vitro recombination technology, so as to facilitate the expression, detection, tracking and / or purification of the target protein. The protein-tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0014] In the above protein, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined by using the homology search site on the Internet, such as the BLAST webpage of the NCBI homepage website. For example, the identity (%) of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and performing a search in the advanced BLAST 2.1.

[0015] In the above protein, the identity of more than 80% can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100%.

[0016] In the above protein, sequence 2 (SEQ ID No. 2) consists of 874 amino acid residues. It is named as TaHT1-A protein. Its encoding gene is TaHT1-A gene.

[0017] In the present application, the regulation can be over-regulation or enhancement or increase, and / or knockout or decrease or reduction.

[0018] In the present application, the substance that up-regulates or enhances or increases the expression of the encoding gene of the protein or the activity or content of the protein can enhance the heat tolerance of the plant. The substance that knocks out or decreases or reduces the expression of the encoding gene of the protein or the activity or content of the protein can reduce the heat tolerance of the plant.

[0019] In the above, the amino acid sequence of the protein can be sequence 2, sequence 4 and / or sequence 6.

[0020] In the above, the heat tolerance index can be survival rate and / or biomass.

[0021] In the above application, the protein is derived from wheat.

[0022] The wheat mentioned above may be the wheat variety Fielder.

[0023] 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).

[0024] In the above applications, the substance that upregulates, enhances, or increases the expression of the protein-coding gene is any one of the following:

[0025] B1) Nucleic acid molecules that encode the above proteins;

[0026] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0027] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0028] 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);

[0029] 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);

[0030] 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);

[0031] 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).

[0032] B1) or B8), the person of ordinary skill in the art can easily mutate the nucleotide sequence encoding the protein TaHT1-A of the present application using known methods, such as methods of directed evolution or point mutation. Those artificially modified nucleotides having 80% or more identity with the nucleotide sequence of the protein TaHT1-A isolated from the present application are derived from the nucleotide sequence of the present application and equivalent to the sequence of the present application, as long as they encode the protein TaHT1-A and have the function of the protein TaHT1-A.

[0033] The above-mentioned 80% or more identity can be 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0034] Herein, the identity refers to the identity of the amino acid sequence or the nucleotide sequence. The identity of the amino acid sequence can be determined using the homology search site on the Internet, such as the BLAST page of the NCBI homepage. For example, the value of the identity (%) can be obtained by calculating the identity of the amino acid sequence using blastp as the program in Advanced BLAST 2.1, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values), respectively, and performing a search.

[0035] Herein, the vector is well known to those skilled in the art, including but not limited to: a plasmid, a bacteriophage (such as lambda phage or M13 filamentous phage, etc.), a cosmid (i.e., a cos plasmid), a Ti plasmid or a viral vector. Specifically, it can be a pWMB110 vector;

[0036] Among the above-mentioned biomaterials, the expression cassette of B2) and B10) refers to DNA capable of expressing the gene in a host cell, which can include not only a promoter that initiates transcription of the gene, but also a terminator that terminates transcription of the gene. Further, the expression cassette can also include an enhancer sequence. The promoters that can be used in the present application include, but are not limited to, constitutive promoters, tissue-, organ-, and development-specific promoters, and inducible promoters. Examples of the promoters include, but are not limited to, the constitutive promoter 35S of the cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al. (1999) Plant Physiol 120:979-992); the chemically-inducible promoter from tobacco, pathogenesis-related (PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-thiohydroxy acid S-methyl ester)); the tomato protease inhibitor II promoter (PIN2) or the LAP promoter (both of which can be induced by jasmonate acid methyl ester); the heat shock promoter (U.S. Patent 5,187,267); the tetracycline-inducible promoter (U.S. Patent 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al. (1985) EMBO J. 4:3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and opine synthase terminators (see, e.g., 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).

[0037] In the above B3) and B11), the recombinant vector can be a recombinant expression vector containing the gene expression cassette constructed with 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 the recombinant expression vector is constructed using TaHT1-A, any one of the enhanced, constitutive, tissue-specific or inducible promoters can be added before the 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; in addition, when the plant expression vector is constructed using the gene of the present application, enhancers, including translation enhancers or transcription enhancers, can also be used, and these enhancer regions can be the ATG start codon or the adjacent region start codon, etc., but must be the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the start codon is wide, which can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene. As a specific example, the present application uses the pWMB110 vector as the expression vector.

[0038] In the above application, the nucleic acid molecule of B1) is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1, 1st-2622nd position.

[0039] In the above application, characterized in that the nucleic acid molecule of B9) is a gene encoding the protein shown in SEQ ID NO: 2.

[0040] In the above, the ORF of the gene encoding the protein shown in SEQ ID NO: 2 can be shown in SEQ ID NO: 1.

[0041] In order to solve the above problems, the present application also provides a method for cultivating a high heat salt-tolerant plant.

[0042] The method comprises up-regulating or enhancing or increasing the expression amount of the gene encoding the above-mentioned protein in the target plant, and / or the activity and / or content of the protein, so as to obtain a high heat salt-tolerant plant, wherein the heat tolerance of the high heat salt-tolerant plant is higher than that of the target plant.

[0043] In order to solve the above problems, the present application also provides a method for improving the heat tolerance of a plant.

[0044] The method comprises increasing the heat tolerance of a plant by up-regulating or enhancing or increasing the expression of a gene encoding the above-mentioned protein in the plant, and / or the activity and / or content of the above-mentioned protein.

[0045] In the present application, the plant can be wheat. The wheat can be wheat variety Fielder.

[0046] In the above-mentioned method, the up-regulation or enhancement or increase of the expression of a gene encoding the above-mentioned protein in the plant comprises introducing the nucleic acid molecule of B1), the expression cassette of B2), or the recombinant vector of B3) into the plant of interest.

[0047] In the above-mentioned method, the nucleic acid molecule of B1) is a DNA molecule whose nucleotide sequence is shown in SEQ ID NO: 1, positions 1-2622.

[0048] In the above-mentioned method, the nucleic acid molecule can be the nucleic acid molecule of SEQ ID NO: 1.

[0049] To solve the above-mentioned problems, the present application further provides a method for breeding a plant with low heat tolerance.

[0050] The method comprises knocking out or reducing or decreasing the expression of a gene encoding the above-mentioned protein in the plant of interest, and / or the activity and / or content of the protein, to obtain a plant with low heat tolerance, wherein the heat tolerance of the plant with low heat tolerance is lower than that of the plant of interest.

[0051] The use according to any one of the above or the method according to any one of the above.

[0052] The plant is any one of the following:

[0053] J1) a plant of the family Poaceae;

[0054] J2) a plant of the genus Triticum;

[0055] J3) wheat.

[0056] In the present application, the wheat can be wheat variety Fielder.

[0057] In the above-mentioned method, the heat tolerance indicator can be survival rate and / or biomass.

[0058] In the above-mentioned method, high heat tolerance means higher survival rate and biomass under heat stress conditions.

[0059] In the above-mentioned method, low heat tolerance means lower survival rate and biomass under heat stress conditions.

[0060] In the present application, the heat stress conditions are 42℃ heat treatment for 3 days.

[0061] Advantages

[0062] The present application discloses a wheat heat-resistant protein TaHT1, a coding gene thereof and an application thereof. Specifically disclosed are an application of a protein, a substance for regulating expression of a coding gene of the protein, or a substance for regulating activity or content of the protein in any one of the following: A1) improving heat resistance of a plant and / or preparing a product for improving heat resistance of a plant; A2) improving survival rate of a plant under heat stress and / or preparing a product for improving survival rate of a plant under heat stress; A3) improving biomass of a plant under salt stress and / or preparing a product for improving biomass of a plant under salt stress. A plant overexpressing a TaHT1 protein coding gene has a significantly higher heat resistance than a wild-type plant, and can be used for industrial production and plant breeding. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 Fig. 1 is a phenotype of wild-type wheat Fielder, overexpression line OE-1 and overexpression line OE-2 under normal culture conditions and heat stress culture conditions.

[0064] Figure 2 Fig. 2 is a statistical result of biomass of wild-type wheat Fielder, overexpression line OE-1 and overexpression line OE-2 under normal culture conditions and heat stress culture conditions, and a statistical result of survival rate of wild-type wheat Fielder, overexpression line OE-1 and overexpression line OE-2 under heat stress culture conditions.

[0065] Figure 3 Fig. 3 is a detection graph of overexpression plant quantity, in which wheat variety Fielder is a control, OE1 and OE2 are T3 generation homozygous seedlings of TaHT1 overexpression. DETAILED DESCRIPTION

[0066] The present application will be further described in details in connection with the specific embodiments. The examples given are only for illustrating the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those skilled in the art, and do not constitute any limitation on the present application in any way.

[0067] In the following examples, the experimental methods are all conventional methods, and are performed according to the techniques or conditions described in the literature in the art or according to the product instructions, unless otherwise specified. The materials, reagents and the like used in the following examples can be obtained from commercial channels, unless otherwise specified.

[0068] The following examples use SPSS 11.5 statistical software to process data, the experimental results are expressed as mean ± standard deviation, using One-way ANOVA test, P<0.05 (*) indicates a significant difference, P<0.01 (**) indicates a highly significant difference, P<0.001 (***) indicates a highly significant difference.

[0069] Example 1, obtaining and identifying of transgenic plants

[0070] I. Construction of recombinant plasmid

[0071] 1. Design primers (TaHT1-F and TaHT1-R) to amplify TaHT1-A ORF fragment with enzyme cutting adapter from wheat Fielder seedling stage leaf cDNA, and introduce enzyme cutting site (BamH I). Use primers TaHT1-F and TaHT1-R to perform PCR amplification to obtain TaHT1-A ORF fragment with enzyme cutting adapter.

[0072] The sequence of TaHT1-A ORF fragment is shown in SEQ ID NO. 1.

[0073] SEQ ID NO. 1 is as follows:

[0074]

[0075] The sequence of the protein encoded by TaHT1 -A is shown in SEQ ID NO: 2, as follows:

[0076] MGGGRKRGRSQRRHFKQERENVWKDNPRRPPASAGEGGEGNGWQPFATENLAFEAYYK

[0077] GQQIVPEEEWDAFMSMLRKPLPAAFRINVSSQFCQDICSQLENDFRKSLETEVNDDHEKEAI

[0078] RPLAWYPGNLAWHLNFSRMQLRRNQALESFHEFLKQENEVGNITRQEAVSMVPPLFLNVQ

[0079] PDHHILDMCAAPGSKTFQLLEMIHQSTKPGVLPTAMVVANDVDVQRCNLLIHQTKRMCTA

[0080] NLIVTNHEAQNFPGCSLAKFCPEAYIDESKPQRLEFDRILCDVPCSGDGTVRKAPDMWRTW

[0081] NIGMGNGLHRLQVEIAMRGIALLKVGGRMVYSTCSMNPVENEAVVGEILRRCGDSVELLD

[0082] VSNELPELIRRPGLSTWKVRDRASWLGSHKDVLHYRKNAILPSMFPSGKATTDSCTAGGSV

[0083] EVNIDAVDADMSESGGMVEGKQETKIATDDSNNGDNGKTEEIEQDESESVKVSRGSNEKT

[0084] DSTSIVTEHSNLPLHRCMRIIPHDQNSGAFFIAVLQKLSPLNESPVVEVMKGEHSTSKDKAL

[0085] KCSNGQGSDKVPAEEISVQQPGVDDSHVLVEQQNRDMDAEISKDRSSEEAKVIAGEVQND

[0086] QATRRDKRKTQNQGRWRGVDPVIFFKDEATIRSIVSFYGIKDSFTLEGHLVTRNPDTNHVK

[0087] RIYYVSKSVTEVLDLNVKVGERLKITSLGLKIFERQSSKEGSPCTFRLSSEGLPLLLPYITKQI

[0088] LYASAIDFQHLLQYRIIKFPDFVDAKFGEQASALLQGCCVVILREGHEDLESIGMDPSAIAV

[0089] VCWKGKTNLCVMVTPMDGRELLDRISFRFGLKIPKVDDGKPDLKSDDGSDEQPDGGAETVDPDCMPESKAAEDMDISDVKDAE.

[0090] The sequence of the ORF fragment of TaHT1-A with the enzyme cutting linker is sequence 3; sequence 3 is specifically as follows:

[0091] GGATCCGGATCC .

[0092] TaHT1-F: 5’- GGATCC ATGGGGGGCGGCAGGAAGC-3’; underlined is the restriction enzyme BamH I recognition site;

[0093] TaHT1-R: 5’- CCTAGG CTCGGCGTCCTTGACATCT-3’; underlined is the restriction enzyme BamH I recognition site.

[0094] 2. The product fragment with the enzyme cutting site adapter amplified (the ORF fragment of TaHT1-A with the enzyme cutting adapter) was recovered and purified to obtain the PCR amplification product.

[0095] 3. The PCR amplification product of step 2 was single-enzymatically cut with the restriction enzyme BamH I, and the enzyme cutting product was recovered to obtain the PCR enzyme cutting product.

[0096] 4. The pWMB110 vector (p110 vector is recorded 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, the name in the literature is pWMB110) was prepared by single-enzymatically cutting with the restriction enzyme BamH I to obtain the enzyme cutting pWMB110 vector.

[0097] 5. The PCR enzyme cutting product of step 3 and the enzyme cutting pWMB110 vector of step 4 were connected by using T4 ligase to obtain the pWMB110-TaHT1-A recombinant plasmid.

[0098] The sequencing result shows that the pWMB110-TaHT1-A recombinant plasmid is obtained by replacing the sequence between the restriction enzyme BamHI recognition sites of the pWMB110 vector with the sequence from the 7th to the 2628th in sequence 3, and keeping the sequence from the 7th to the 2628th in sequence 3 correctly expressed and other nucleotides of the pWMB110 vector unchanged, and the recombinant plasmid is named as the pWMB110-TaHT1-A recombinant plasmid.

[0099] II. Obtaining of TaHT1 overexpression transgenic wheat

[0100] 1. Using TaHT1-p110-F and TaHT1-p110-R primers to amplify the ORF fragment of TaHT1 containing the BamHI enzyme cutting site vector linker, using Fielder cDNA as template, for constructing the overexpression vector.

[0101] 2. After recovering the amplified DNA fragment by agarose gel, the method of homologous recombination is used to construct TaHT1 into p110 vector to form the overexpression vector of TaHT1 (pWMB110-TaHT1-A recombinant plasmid).

[0102] 3. The pWMB110-TaHT1-A recombinant plasmid is transformed into E. coli to obtain E. coli Mach1-T1 / pWMB110-TaHT1-A recombinant plasmid, which is E. coli containing pWMB110-TaHT1-A recombinant plasmid.

[0103] 4. The positive clones are screened by PCR reaction, sequencing, and the sequence-corrected pWMB110-TaHT1-A recombinant plasmid is named, and the pWMB110-TaHT1-A recombinant plasmid is extracted and transformed into Agrobacterium to obtain Agrobacterium / pWMB110-TaHT1-A recombinant plasmid, which is Agrobacterium containing pWMB110-TaHT1-A recombinant plasmid, and is sent to the wheat transgenic platform for wheat (recipient is Fielder) genetic transformation to obtain 8 strains of TaHT1-A overexpression T0 generation transgenic wheat, which are selfed to obtain 2 lines of TaHT1-A overexpression T2 generation homozygous transgenic wheat, which are named OE1 and OE2.

[0104] 5. The TaHT1-A overexpression T2 generation homozygous transgenic wheat (OE1 and OE2) is subjected to PCR identification.

[0105] The expression level identification is as follows:

[0106] The T3 generation seeds (OE1 T3 generation homozygous seeds and OE2 T3 generation homozygous seeds) and control group (wheat variety Fielder) seeds are sown, and at the three-leaf stage, the leaf parts are taken to extract RNA, and the reverse transcription kit (Vazyme Biotech, R223-01) is used for reverse transcription into cDNA, and the quantitative primers are used to monitor the expression level; (the expression level identification is a complete process, and the sampling time of Fielder, OE1 and OE2, the specific primers and internal reference primers, the fluorescence quantitative kit and specific conditions are required to be given).

[0107] Detection primers: TaHT1-qF: TCGTCTCCAAGTGGAAATAG; TaHT1-qR: AAGACGCTCTATCCCGTACC. TaACTIN is used as an internal control; internal control primers: TaACTIN-F: GGAATCCATGAGACCACCTAC; TaACTIN-R: GACCCAGACAACTCGCAAC.

[0108] The results are as follows Figure 3 ( Figure 3 In the study (with Fielder wheat variety as the control and OE1 and OE2 being homozygous T3 generation seedlings overexpressing TaHT1), the expression level of TaHT1 gene was significantly increased in the T3 generation homozygous seedlings overexpressing TaHT1 compared to Fielder seedlings.

[0109] 6. After harvesting the identified positive lines, the plants were multiplied in a greenhouse, and the T3 generation plants were identified positively by PCR.

[0110] III. TaHT1 overexpression wheat heat stress experiment

[0111] Seeds from two lines N-OE1 and N-OE2 (OE1 and OE2) that have been identified as positive overexpression lines (homozygous seeds of OE1 T3 generation and OE2 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 in the petri dishes, a small amount of distilled water added, and placed at room temperature for 48 h.

[0112] 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 contained control seeds (wild-type Fielder seeds), and the other part contained seeds of overexpression lines (OE1 and OE2) (OE1 T3 generation homozygous seeds and OE2 T3 generation homozygous seeds), with a total of four replicates. The experiment was divided into a heat tolerance experimental group and a control group.

[0113] The heat resistance test group performed the following operations:

[0114] 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.

[0115] Fielder and TaHT1 knockout plants 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 results were recorded by photographing, and survival rate and biomass before and after stress were statistically analyzed.

[0116] The control group underwent the following procedures:

[0117] 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.

[0118] Survival rate = Number of surviving seedlings / Total number of seedlings.

[0119] The criteria for survival are: the stem remains green, the leaves are partially or completely green again, or new leaves have unfolded.

[0120] The criteria for determining death are: leaves turning yellow and dying, and the stem completely dying with no signs of regrowth.

[0121] Biomass before and after stress was measured by cutting aboveground wheat seedlings from the stem base and weighing and recording the weight.

[0122] Phenotypic results are as follows Figure 1 As shown ( Figure 1 In this study, Fielder was the wild-type wheat variety, TaHT1-OE#1 was the progeny of OE-1, TaHT1-OE#2 was the progeny of OE-2, Normal comdition was 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 growth of the overexpressing lines was significantly stronger than that of the wild-type lines.

[0123] Quantity results as follows Figure 2 As shown ( Figure 2 In the diagram, the left bar chart represents biomass, with Normal comdition representing the control group and HS treatment (42℃) representing the heat tolerance experimental group; Fielder represents wild-type wheat Fielder, TaHT1-OE#1 represents the progeny of OE-1, and TaHT1-OE#2 represents the progeny of OE-2; 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. Furthermore, the dry weight of the overexpression lines after recovery was slightly higher than that of the wild-type lines. Figure 2 ).

[0124] Survival results as follows Figure 2 As shown ( Figure 2In the above, the right column chart is the survival rate column chart of the heat resistance experiment group; Fielder is the wild type wheat Fielder, TaHT1-OE#1 is the offspring of OE-1, TaHT1-OE#2 is the offspring of OE-2, and the vertical coordinate Survival Rate (%) is the survival rate. The results show that the survival rate of the overexpression lines (offspring of OE-1 and OE-2) is significantly lower than that of the wild type plants after recovery.

[0125] The application has been described in detail above. For those skilled in the art, the application can be implemented in a wider range under equivalent parameters, concentrations and conditions without departing from the purpose and scope of the application and without unnecessary experiments. Although the application gives a special example, it should be understood that the application can be further improved. In summary, according to the principle of the application, the present application intends to include any change, use or improvement of the application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. Use of a protein, a substance that up-regulates or enhances or increases expression of a gene encoding the protein, or a substance that up-regulates or enhances or increases content of the protein, in any one of the following: A1) use in increasing heat tolerance of a plant and / or use in the manufacture of a product for increasing heat tolerance of a plant; A2) use in increasing survival rate of a plant under heat stress and / or use in the manufacture of a product for increasing survival rate of a plant under heat stress; A3) use in increasing biomass of a plant under heat stress and / or use in the manufacture of a product for increasing biomass of a plant under heat stress; the protein is any one of: P1) a protein whose amino acid sequence is set forth in SEQ ID NO: 2; P2) a fusion protein obtained by linking a protein tag to the N-terminus or / and C-terminus of B1); the substance is any one of: B1), a nucleic acid molecule encoding the protein; B2), an expression cassette comprising the nucleic acid molecule of B1); B3), a recombinant vector comprising the nucleic acid molecule of B1), or a recombinant vector comprising the expression cassette of B2); B4), a recombinant microorganism comprising the nucleic acid molecule of B1), or a recombinant microorganism comprising the expression cassette of B2), or a recombinant microorganism comprising the recombinant vector of B3); B5), a transgenic plant cell line comprising the nucleic acid molecule of B1), or a transgenic plant cell line comprising the expression cassette of B2), or a transgenic plant cell line comprising the recombinant vector of B3); B6), a transgenic plant tissue comprising the nucleic acid molecule of B1), or a transgenic plant tissue comprising the expression cassette of B2), or a transgenic plant tissue comprising the recombinant vector of B3); B7), a transgenic plant organ comprising the nucleic acid molecule of B1), or a transgenic plant organ comprising the expression cassette of B2), or a transgenic plant organ comprising the recombinant vector of B3); the plant is wheat.

2. Use according to claim 1, characterized in that, the protein is derived from wheat.

3. Use according to claim 2, characterized in that, the nucleic acid molecule of B1) is a DNA molecule whose nucleotide sequence is set forth in SEQ ID NO: 1 from position 1 to position 2622.

4. A method of breeding a plant with high heat tolerance, characterized by, A high heat tolerance plant having higher heat tolerance than a plant of interest, wherein the high heat tolerance is derived from up-regulating or enhancing or increasing expression of a gene encoding the protein of claim 1 or 2, or content of the protein of claim 1 or 2, in the plant of interest; the plant is wheat.

5. A method for increasing heat tolerance in plants, characterized by, A plant having increased heat tolerance, wherein the increased heat tolerance is derived from up-regulating or enhancing or increasing expression of a gene encoding the protein of claim 1 or 2, or content of the protein of claim 1 or 2, in the plant; the plant is wheat.

6. The method of claim 4 or 5, wherein, The up-regulating or enhancing or increasing expression of a gene encoding the protein of claim 1 or 2 in a plant comprises introducing into the plant of interest the nucleic acid molecule of B1), the expression cassette of B2), or the recombinant vector of B3) of claim 1.

7. The method of claim 6, wherein, the nucleic acid molecule of B1) is a DNA molecule whose nucleotide sequence is set forth in SEQ ID NO: 1 from position 1 to position 2622.

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