Application of wheat protein and its related biomaterials in improving plant heat tolerance

Abstract

The present invention discloses the application of wheat protein and its related biological materials in improving plant heat tolerance. One technical solution to be protected by the present invention is the application of a protein derived from wheat in improving plant heat tolerance or high temperature stress tolerance. The name of this protein is TaOEP24, and it can be a protein with an amino acid sequence as sequence 1 in the sequence listing. The present invention cloned the wheat gene TaOEP24 from the wheat cultivar Chinese Spring and overexpressed it in the embryos of the wheat cultivar Fielder. The obtained TaOEP24 overexpressing plants have a stronger ability to tolerate high temperature at 42 °C than the wild type. Therefore, the wheat TaOEP24 protein has the function of regulating wheat heat tolerance and can be applied to the breeding of heat-tolerant wheat.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of wheat protein and its related biological materials in improving plant heat tolerance. Background Art

[0002] Wheat is a crop that prefers cool climate and is vulnerable to dry hot winds during flowering and filling periods, resulting in shortened filling periods, chlorosis of leaves, and reduced yields. According to statistics, for every 1°C increase in global temperature, the yield of food crops decreases by about 6% (Zhao et al., 2017). Therefore, exploring wheat heat-resistant genes and investigating the wheat heat-resistant mechanism provide a strong theoretical basis for food production.

[0003] During the growth and development process of plants, they are continuously affected by the external environment. The perception and response process of chloroplasts to stress environments is very complex. Heat stress can damage the plant cell membrane, disrupt the photosynthetic system, hinder leaf photosynthesis, cause plastid gene expression disorders, resulting in reduced protein and enzyme activities in cells and accumulation of toxic substances such as ROS. These factors will cause chloroplast dysfunction, and plant growth and development will stagnate or even die. At the same time, there are also some nuclear-encoded proteins in plants that can enter chloroplasts and regulate the plant's tolerance to heat stress. Therefore, exploring plastid heat-resistant related genes and their functions in plants can not only help to understand the relationship between plant nuclear-cytoplasmic regulation more deeply, providing a scientific idea for exploring more plant stress-tolerant genes; by observing the agronomic traits of transgenic plants and investigating their stress resistance, new evidence can be added to the stress resistance research of chloroplasts, and new beneficial genes can be explored for future food production. Summary of the Invention

[0004] The technical problem to be solved by the present invention is how to improve plant heat tolerance or high-temperature stress tolerance and / or how to obtain heat-resistant plants or high-temperature stress-tolerant plants.

[0005] To solve the above technical problems, the present invention first provides any one of the following applications of a protein or a substance that regulates protein expression or a substance that regulates protein activity:

[0006] P1. Application in regulating plant heat tolerance or high-temperature stress tolerance,

[0007] P2. Application in improving plant heat tolerance or high-temperature stress tolerance,

[0008] P3. Application in plant heat-resistant or high-temperature stress-tolerant breeding,

[0009] P4. Application in improving the quality of plant heat-resistant or high-temperature stress-tolerant plants;

[0010] The protein may be a protein of any of the following A1), A2), or A3):

[0011] A1) A protein with an amino acid sequence that is Sequence 1 in the sequence listing;

[0012] A2) A protein derived from A1) or having more than 80% identity with the protein shown in A1) and having the same function, which is obtained by substitution and / or deletion and / or addition of amino acid residues to the amino acid sequence shown in Sequence 1 in the sequence listing;

[0013] A3) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of A1) or A2).

[0014] Furthermore, in the above application, the indicators for breeding may include heat tolerance (high temperature stress tolerance).

[0015] Furthermore, in the above application, the purpose of breeding may include cultivating plants with high heat tolerance (high temperature stress tolerance) (heat tolerance or high temperature stress tolerance higher than that of the parent).

[0016] In the above application, the protein may be derived from wheat.

[0017] The above protein can be artificially synthesized, or its coding gene can be synthesized first and then expressed biologically.

[0018] In the above protein, the protein-tag refers to a polypeptide or protein that is fused and expressed with the target protein by using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing, and / or purification of the target protein. The protein-tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0019] In the above protein, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI homepage website. 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, 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 a pair of amino acid sequences, and then the identity value (%) can be obtained.

[0020] In the above protein, the above 80% identity can be at least 81%, 82%, 85%, 86%, 88%, 90%, 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

[0021] In the above application, the plant can be any one of the following:

[0022] D1) Dicotyledonous plants;

[0023] E1) Monocotyledonous plants,

[0024] E2) Plants of the order Poales,

[0025] E3) Gramineae plants,

[0026] E4) Triticum;

[0027] E5) Wheat.

[0028] To solve the above technical problems, the present invention also provides any one of the following applications of the biological material related to the protein described above:

[0029] Q1. Application of the biological material in regulating plant heat tolerance or high temperature stress;

[0030] Q2. Application of the biological material in improving plant heat tolerance or high temperature stress;

[0031] Q3. Application of the biological material in breeding plants for heat tolerance or high temperature stress;

[0032] Q4. Application of the biological material in improving the quality of plants for heat tolerance or high temperature stress;

[0033] The biological material is any one of the following B1) to B9):

[0034] B1) A nucleic acid molecule encoding the protein described above;

[0035] B2) An expression cassette containing the nucleic acid molecule described in B1);

[0036] B3) A recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0037] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0038] 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);

[0039] B6) A transgenic plant tissue containing the nucleic acid molecule described in B1), or a transgenic plant tissue containing the expression cassette described in B2);

[0040] 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);

[0041] B8) A nucleic acid molecule that promotes or enhances the gene expression of the protein described above;

[0042] B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line containing the nucleic acid molecule described in B8).

[0043] Furthermore, in the above applications, the breeding indicators may include heat tolerance (high temperature stress tolerance).

[0044] Furthermore, in the above applications, the breeding objective may include cultivating plants with high heat tolerance (high temperature stress tolerance) (heat tolerance or high temperature stress tolerance higher than that of the parent).

[0045] In the above applications, the nucleic acid molecule described in B1) may be the coding gene of the protein shown in the following b1) or b2):

[0046] b1) A DNA molecule whose coding sequence of the coding strand is the nucleotide of sequence 2 in the sequence listing;

[0047] b2) A cDNA molecule or DNA molecule that hybridizes with the defined cDNA or DNA molecule and encodes a protein with the same function.

[0048] In the above applications, the plant may be any of the following:

[0049] D1) Dicotyledonous plants;

[0050] E1) Monocotyledonous plants,

[0051] E2) Plants of the order Poales,

[0052] E3) Gramineous plants,

[0053] E4) Genus Triticum;

[0054] E5) Wheat.

[0055] In the above biological materials, the expression cassette containing the nucleic acid molecule described in B2) refers to DNA that can express the protein described in the above applications in a host cell. This DNA may not only include a promoter that initiates the transcription of the protein-coding gene, but also include a terminator that terminates the transcription of the protein-coding gene. Further, the expression cassette may also include an enhancer sequence. Promoters that can be used in the present invention include, but are not limited to: constitutive promoters, tissue-, organ- and development-specific promoters, and inducible promoters.

[0056] An existing plant expression vector can be used to construct a recombinant expression vector containing the expression cassette of the protein-coding gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc., such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (from CAMBIA), etc. The plant expression vector may further contain the 3'-untranslated region of the foreign gene, that is, it contains a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylate to the 3' end of the mRNA precursor. For example, the nopaline synthase gene Nos of the Agrobacterium tumefaciens Ti plasmid gene and the 3'-untranslated region transcribed from plant genes (such as soybean storage protein genes) have similar functions. When constructing a plant expression vector using the gene of the present invention, enhancers can also be used, including translational enhancers or transcriptional enhancers. These enhancer regions can be the ATG start codon or the start codon in the adjacent region, etc., but must be in the same reading frame as the coding sequence to ensure the correct translation of the entire sequence.

[0057] Among the above biological materials, the recombinant microorganism can specifically be yeast, bacteria, algae and fungi.

[0058] To solve the above technical problems, the present invention also provides a method for improving the heat tolerance or high temperature stress resistance of plants, including enhancing or increasing the activity of the protein described above in the target plant and / or the expression level of the coding gene of the protein described above, thereby improving the heat tolerance or high temperature stress resistance of the plant.

[0059] In the above method, enhancing or increasing the activity of the protein described above in the target plant and / or the expression level of the coding gene of the protein described above is achieved by introducing the coding gene of the protein described above into the target plant.

[0060] In the above method, the plant and / or the target plant is any one of the following:

[0061] D1) Dicotyledonous plants;

[0062] E1) Monocotyledonous plants,

[0063] E2) Plants of the order Poales,

[0064] E3) Gramineous plants,

[0065] E4) Genus Triticum;

[0066] E5) Wheat.

[0067] In the above method, the coding gene of the protein can be first modified as follows and then introduced into the target plant to achieve better expression effects:

[0068] 1) It is connected to promoters expressed in various plants to facilitate its expression in plants; the promoters may include constitutive, inducible, temporal regulation, developmental regulation, chemical regulation, tissue-preferred and tissue-specific promoters; the selection of promoters will vary with the needs of expression time and space and also depends on the target species; for example, tissue- or organ-specific expression promoters depend on at what stage of development the receptor is needed; although many promoters derived from dicotyledonous plants have been proven to be functional in monocotyledonous plants and vice versa, ideally, dicotyledonous plant promoters are selected for expression in dicotyledonous plants and monocotyledonous plant promoters for expression in monocotyledonous plants;

[0069] 2) It is connected to a suitable transcription terminator, which can also improve the expression efficiency of the gene of the present invention; for example, tml derived from CaMV and E9 derived from rbcS; any available terminator known to be functional in plants can be connected to the gene of the present invention;

[0070] 3) Enhancer sequences are introduced, such as intron sequences (e.g., derived from Adhl and bronzel) and viral leader sequences (e.g., derived from TMV, MCMV, and AMV).

[0071] In the above method, the stress-sensitive plants can be transgenic plants or plants obtained by conventional breeding techniques such as hybridization.

[0072] In the above method, the transgenic plants are understood to include not only the first-generation to second-generation transgenic plants but also their progeny. For transgenic plants, the gene can be propagated in this species, or the gene can be transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, whole plants, and cells.

[0073] The proteins and / or biomaterials described above also fall within the protection scope of the present invention.

[0074] The above heat resistance or high-temperature stress tolerance can be a tolerance to a temperature of 38°C - 42°C. Further, the high temperature can be 42°C.

[0075] The present invention discloses a wheat channel protein capable of regulating the heat tolerance mechanism of plants, its coding gene and application. The technical solution to be protected by the present invention is the application of a protein derived from wheat in regulating heat tolerance in plants under heat stress. The name of this protein is TaOEP24, and its encoded amino acid sequence is the protein of Sequence 1 in the sequence listing. The present invention cloned the wheat gene TaOEP24 from the wheat cultivar Chinese Spring, and overexpressed it in the embryos of the wheat cultivar Fielder to obtain transgenic TaOEP24 overexpressing plants. The transgenic TaOEP24 overexpressing plants have stronger heat tolerance than the wild-type Fielder under heat stress. Therefore, the TaOEP24 protein has the function of regulating wheat heat tolerance and can be used as an important indicator for future plant breeding and selection of excellent varieties.

[0076] The role of the TaOEP24 gene in the process of wheat responding to heat stress. The present invention is of great significance for breeding heat-tolerant wheat. Brief Description of the Drawings

[0077] Figure 1 Detection of the relative transcriptional level of TaOEP24 in T2 generation homozygous transgenic plants. The vertical axis represents the relative transcriptional level of TaOEP24.

[0078] Figure 2 Detection of the protein expression level of TaOEP24 in T2 generation homozygous transgenic overexpressing plants. The upper figure shows the detection result of the expression of TaOEP24-GFP protein, and the lower figure shows the detection result of the expression of the reference Actin protein.

[0079] Figure 3 Observation results of the high-temperature heat stress treatment and recovery phenotypes of T2 generation homozygous transgenic plants. In the figure, from left to right are the Fielder control wheat, TaOEP24 overexpressing lines OE1, OE2, OE6, and OE7.

[0080] Figure 4 Statistics of the survival rate of T2 generation homozygous transgenic overexpressing plants after high-temperature heat stress treatment. The vertical axis represents the survival rate. ** represents p≤0.01; *** represents p≤0.001.

[0081] Figure 5 Schematic diagram of the pWMB111 vector.

[0082] Figure 6 Schematic diagram of the recombinant pWMB111 vector after inserting the TaOEP24 and GFP gene sequences. Detailed Embodiments

[0083] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention. The following embodiments can be used as a guide for those of ordinary skill in the art to make further improvements, and do not limit the present invention in any way.

[0084] The experimental methods in the following embodiments are all conventional methods unless otherwise specified, and are carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0085] Example 1: Cloning of TaOEP24 gene

[0086] The material selected in this example is the wheat variety Chinese Spring, which is the first wheat variety to complete sequencing and assembly. The wheat cultivar Chinese Spring was germinated on germination paper for 7 days to obtain wheat seedlings. After sampling the wheat seedlings, they were frozen in liquid nitrogen and ground into samples. RNA was extracted by the Trizol method, and cDNA was obtained by reverse transcription using RNA as a template.

[0087] Using the cDNA obtained in the above step as a template, PCR amplification was carried out with primers TaOEP24-F: 5’-ATGAAGGCGACGGTCAAGGGC-3’; TaOEP24-R: 5’-GATCTCATAGTTCCACGTGCT-3’. The PCR product was ligated onto a blunt-ended T vector and sequenced to obtain the T-TaOEP24 vector.

[0088] The sequencing results showed that the nucleotide sequence of the PCR amplification product was Sequence 2 in the sequence listing. The gene with the CDS sequence of Sequence 2 in the sequence listing was named TaOEP24. Among them, the 1st to 729th nucleotides from the 5’ end of Sequence 1 were the ORF, encoding a protein composed of 242 amino acid residues. This protein was named TaOEP24, and the amino acid sequence of the TaOEP24 protein was Sequence 1 in the sequence listing.

[0089] The sequence of Sequence 1 is as follows:

[0090] MKATVKGRYEGDKATAAATLALAAAGDLRLRASATDAAFAAGPSLDGLTLTLEKPGAFLLDLKPHNQDVRFQFMNSATVLDKRVSLTYTHSTSLAPAAPKPAIPAPAAGAAPAPPPKGPPPGRTALDLSIAFDPANKVSVSHALGGGGCRVKYTYAHGAGRLTTIEPVYDTAKNAWEFAVARKFDAGDVVRGTYQASTKQLGLEWTRSSSIGGSFKVATTFDLSDQSKAPKLVAESTWNYEI。

[0091] The sequence of Sequence 2 (CDS) is as follows (5'-3'):

[0092] ATGAAGGCGACGGTCAAGGGCCGCTACGAGGGCGACAAGGCCACCGCGGCCGCCACGCTCGCGCTCGCCGCCGCCGGCGACCTCCGCCTCCGCGCCTCCGCCACCGACGCCGCCTTCGCCGCCGGCCCCTCCCTCGACGGCCTCACCCTCACCCTCGAGAAGCCCGGCGCCTTCCTCCTCGACCTCAAGCCCCACAACCAGGATGTGCGCTTCCAGTTCATGAACTCGGCGACGGTGCTCGACAAGCGGGTCAGCCTCACCTACACGCACTCCACCTCCCTCGCGCCCGCCGCGCCCAAGCCCGCCATCCCGGCCCCGGCCGCCGGCGCCGCGCCCGCGCCCCCGCCTAAGGGGCCGCCTCCCGGCCGCACGGCGCTCGACCTCTCCATCGCCTTCGACCCCGCCAACAAGGTCAGCGTCTCCCACGCGCTCGGCGGGGGCGGCTGCCGGGTCAAGTACACCTACGCGCACGGCGCCGGCCGCCTCACCACCATCGAGCCCGTCTACGACACCGCCAAGAACGCCTGGGAGTTCGCCGTCGCCAGGAAGTTCGACGCCGGGGACGTCGTCAGGGGCACCTACCAGGCCTCCACCAAGCAGCTCGGGCTCGAGTGGACCAGGAGCTCCAGCATCGGCGGCTCCTTCAAGGTTGCGACGACGTTCGATCTGTCCGATCAAAGCAAAGCACCGAAGCTCGTAGCGGAGAGCACGTGGAACTATGAGATCTGA。

[0093] Example 2: Obtaining TaOEP24 Gene Overexpression Plants and Verifying Their Functions

[0094] The TaOEP24 gene overexpression vector used in this example is the recombinant plasmid pWMB111-TaOEP24-EGFP( Figure 6 ). pWMB111-TaOEP24-EGFP is based on the pWMB111 vector( Figure 5, donated by the research group of Teacher Ye Xingguo from the Institute of Crop Sciences, Chinese Academy of Agricultural Sciences. Related literature: Riaz B, Chen HQ et al. Overexpression of Maize ZmC1 and ZmR Transcription Factors in Wheat Regulates Anthocyanin Biosynthesis in a Tissue-Specific Manner[J]. International Journal of Molecular Sciences, 2019, 20, 5806. Ke, Wang, Huiyun, Liu et al. Generation of marker-free transgenic hexaploid wheat via an Agrobacterium-mediated co-transformation strategy in commercial Chinese wheat varieties[J]. Plant Biotechnology Journal, 2017. DOI: 10.1111 / pbi.12660..) The nucleotide sequence inserted at the EcoRI site is TaOEP24 (OEP24 protein) of Sequence 2 in the sequence listing, and the recombinant vector is obtained by keeping other nucleotide sequences of the pWMB111 vector unchanged. The specific construction method of pWMB111-TaOEP24-EGFP is as follows.

[0095] 1. Obtaining of TaOEP24 transgenic plants

[0096] 1.1 Construction of recombinant expression vector pWMB111-TaOEP24-EGFP

[0097] Using the T-TaOEP24 vector with correct sequencing in Example 1 as a template, and primers pWMB111-TaOEP24-F: 5’- TCCCCGGGTACCGAGCTC ATGAAGGCGACGGTCAAGGGC-3’ and pWMB111-TaOEP24-R: 5’- TCGGGGAA ATTCGAGCTCG GATCTCATAGTTCCACGTGCT-3’ for PCR amplification (the underlined parts of the primers are the sequences for vector ligation). After amplification, the product is recovered and purified by gel (Zhuangmeng Microcolumn Concentrated DNA Gel Recovery Kit ZPV202) to obtain the purified PCR product for subsequent ligation;

[0098] The recombinant vector pWMB111-EGFP (a recombinant vector obtained by adding the EGFP fusion tag gene to the unique overexpression transformation vector pWMB111 in wheat transformation) was digested with the restriction endonuclease EcoRI to linearize it, and the linearized vector was recovered and purified to obtain the linearized vector. Then, the linearized vector was ligated recombinantly with the purified PCR product (recombinant kit Novoprotein ClonExpress II One Step Cloning Kit C112-1). The recombinant product was transformed into Escherichia coli competent cells (FastT1, Vazyme, no. C505-02), and then cultured overnight at 37 °C in an inverted manner on a solid LB medium containing kanamycin (Kan). The next day, monoclonal colonies were selected and subjected to colony PCR detection and sequencing in an LB liquid medium containing kanamycin (Kan). The verification primers were the universal primers of the pWMB111 vector ubi-F: 5’-TTTAGCCCTGCCTTCATACGC-3’, Tnos-R: 5’-AGACCGGCAACAGGATTCAATC-3’. The size of the PCR amplification product fragment was 1629 bp. The positive transformant single colonies with correct sequencing results were cultured in a shaker and the plasmids were extracted (high-purity plasmid miniprep and midiprep rapid extraction kit DP110-1) to obtain the recombinant vector plasmid pWMB111-TaOEP24-EGFP. The recombinant vector plasmid pWMB111-TaOEP24-EGFP contains the CDS sequence of the cDNA of TaOEP24 shown in Sequence 2 in the Sequence Listing, and can express the recombinant protein TaOEP24-EGFP whose coding sequence is Sequence 3 in the Sequence Listing.

[0099] The sequence of Sequence 3 is as follows (5’-3’):

[0100]

[0101] The recombinant vector plasmid pMWpWMB111-TaOEP24-EGFP was transformed into the competent cells of Agrobacterium tumefaciens GV3101, and then transferred to YEP medium (containing 50 mg / L kanamycin + rifampicin antibiotic) and cultured upside down at 28 °C for two days. Then, positive clones were selected and identified by PCR using the primers ubi-F: 5’-TTTAGCCCTGCCTTCATACGC-3’ and Tnos-R: 5’-AGACCGGCAACAGGATTCAATC-3’ in the above text (the product size is 1629 bp). The positive bacterial solution obtained by PCR identification was named recombinant Agrobacterium GV3101 / pMWpWMB111-TaOEP24-EGFP and stored at -80 °C.

[0102] 1.2 Obtaining of TaOEP24 gene-transformed plants

[0103] Take immature wheat ears of Fielder and wash them 3 times with 5% sodium hypochlorite for 20 min each time in a laminar flow hood, and wash them thoroughly 4-5 times with sterile ddH2O. Then, take the intact embryos of the seeds in the laminar flow hood and put them into the corresponding liquid medium;

[0104] The recombinant Agrobacterium GV3101 / pMWpWMB111-TaOEP24-EGFP obtained in 1.1 was streaked on YEP solid medium supplemented with Kan. The grown single colonies were inoculated into 10 mL of YEP liquid medium (containing Kan) and cultured with shaking until OD 600nm= 0.6 - 0.8; Centrifuge the bacteria at 5000 rpm for 10 min to enrich the bacteria, and add an appropriate amount of resuspension solution to resuspend to obtain a resuspended bacterial solution; The wheat transformation system refers to the method in the reference, related literature: Wang K, Shi L, Liang X, Zhao P, Wang W, Liu J, Chang Y, Hiei Y, Yanagihara C, Du L, Ishida Y, Ye X. The gene TaWOX5 overcomes genotype dependency in wheat genetic transformation. Nat Plants. 2022 Feb;8(2):110 - 117. doi:10.1038 / s41477 - 021 - 01085 - 8. Epub 2022 Jan 13. Erratum in: Nat Plants. 2022 Jun;8(6):717 - 720. At 14 days after anthesis in wheat, take the wheat ears with immature grains, surface - sterilize them with 70% ethanol for 1 min and 5% sodium hypochlorite for 15 min under sterile conditions, and rinse them 5 times with sterile water. Isolate the wheat embryos and obtain transgenic plants according to the Agrobacterium - mediated transformation method. The specific steps are as follows:

[0105] Co - culture the embryos and Agrobacterium in WLS1 liquid medium (1 / 10 Linsmaier and Skoog (LS) salts, 1 / 10 Murashige and Skoog (MS) vitamins, glucose 10 g l -1 , 2 - (N - morpholino)ethanesulfonic acid (MES) 0.5 g l-1 and acetosyringone (AS) 100 μM, pH 5.8) at room temperature for 5 minutes, and then transfer them to a solid co - culture medium supplemented with vitamins and culture for 2 days (WLS liquid medium plus silver nitrate 0.85 mg l -1 , CuSO4·5H2O 1.25 mg l -1 and agarose 8 g l -1), with the scutellum facing upwards, under dark conditions at 25°C. After co-culture, the embryonic axis was removed with a scalpel, and the remaining scales were transferred to plates containing callus induction medium (LS salts, MS vitamins, 2,4-D 0.5 mg / l, picloram 2.2 mg / l, AgNO3 0.85 mg / l, ascorbic acid 100 mg / l, carbenicillin 250 mg / l, cefotaxime 100 mg / l, MES 1.95 g / l, and agarose 5 g / l) and cultured for 5 days under the same conditions. Then, the tissue was cultured on selection medium (callus induction medium + phosphatidylcholine (PPT, Sigma, no. 45520). 5 mg / l, without cefotaxime) to further induce callus. After 2 weeks, the callus was placed on selection medium containing 10 mg / l PPT and cultured for 3 weeks under dark conditions to induce embryonic callus. At 25°C, 100 μmol m -2 s -1 Under light conditions, embryogenic callus was differentiated on 1 / 2 MS medium without zeatin containing 5 mg / l PPT. The regenerated shoots were transplanted into cups filled with rooting medium + 5 mg / l PPT for elongation and rooting. Plants with well-developed roots were transplanted into flower pots and cultured in a growth chamber to obtain T0 generation transgenic seedlings.

[0106] For the T0 generation transgenic seedlings, positive plants detected by PCR amplification using primers pMWpWMB111-TaOEP24-F: 5’- TCCCCGGGTACCGAGCTC ATGAAGGCGACGGTCAAGGGC-3’ and Tnos-R: 5’-AGACCGGCAACAGGATTCAATC-3’ were individually harvested for T1 generation seeds, which were then planted again to obtain T1 generation seedlings; the leaves of T1 generation seedlings at the seedling stage were taken for PCR detection again. Plants with all individual plants being positive were planted again, and 8 homozygous T2 generation transgenic seeds (OE1 - OE8) were harvested for subsequent phenotypic experiments.

[0107] After planting the T2 generation seeds, T2 generation homozygous overexpression line plants were obtained. RNA (by Trizol method) and proteins were extracted from the leaves of the T2 generation overexpression line plants at the seedling stage. RT-qPCR was performed using qRT-TaOEP24-F: 5’-CTTCCTCCTCGACCTCAAGC-3’ and qRT-TaOEP24-R: 5’-ATCGGACAGATCGAACGTCG-3’. The internal reference was the Actin gene, and the detection primers for the internal reference gene were β-ACTIN-F: 5’-GACCGTATGAGCAAGGAGAT-3’, β-ACTIN-R: 5’-CAATCGCTGGACCTGACTC-3’.

[0108] The protein extraction method is as follows:

[0109] Take 0.5 g of seedling leaves and extract plant RNA using the Trizol (Invitrogen TRIzol, Thermo Fisher Scientific, no. 15596026) method. First, grind the sample, add 1 ml of Trizol and let it stand for 2 - 3 min, then centrifuge at 12000 rpm for 3 min; pipette the supernatant into a new centrifuge tube and add 300 μl of pre-cooled chloroform, mix well by shaking, and centrifuge at 12000 rpm for 15 min; transfer the supernatant to a new EP tube and add the corresponding isopropanol mentioned, keep it at -20 °C for more than 30 min, and centrifuge at 12000 rpm for 15 min; wash the precipitate twice with 1 ml of 75% ethanol (prepared with DEPC water); after air-drying, dissolve it in DEPC water. The dissolved RNA sample was reverse transcribed (HiScript II Q RT SuperMix for qPCR (+gDNA wiper), Vazyme, no. R223-01) for RT-qPCR to detect the transcriptional level;

[0110] For protein extraction, take 0.5 g of wheat seedling leaves, add SDS-loading buffer (5x SDS-PAGE protein loading buffer containing bromophenol blue (SDS-loading buffer, Beyotime, P0015) + DTT and 20 μl / ml of mercaptoethanol -1 ) and boil for 15 min, centrifuge at 12000 g for 5 min, for Western Blot detection, and detect with an EGFP antibody (TransGen Anti-GFP Mouse Monoclonal Antibody, no. HT801-01). The internal reference is APX3. After detection, the relative transcriptional levels of TaOEP24 in 4 TaOEP24 transgenic overexpression lines (OE1, OE2, OE6, and OE7)Figure 1 ) and the TaOEP24 protein expression level ( Figure 2 the upper figure in the middle) were both higher than those of the wild type. Therefore, the TaOEP24 overexpression lines OE1, OE2, OE6, and OE7 were used for subsequent phenotypic identification.

[0111] 1.3 Heat tolerance phenotypic identification

[0112] The TaOEP24 overexpression lines OE1, OE2, OE6, and OE7 and the wild type Fielder were germinated simultaneously. After being cultured at 20 °C in a plant incubator for one week, they were transferred to a 42 °C incubator for 6 days and then transferred back to a 20 °C incubator for one week of recovery. Then, the survival rate was counted. The results showed that after the 42 °C high-temperature treatment, compared with the wild type Fielder, the TaOEP24 overexpression OE materials had more upright plants, less wilting, fewer wilted and withered leaves on the plants compared with Fielder, and the stems were more upright than those of the wild type Fielder ( Figure 3 ), and the plant survival rate was higher ( Figure 4 ), showing a better heat tolerance phenotype.

[0113] Therefore, the TaOEP24 protein has the function of improving the heat stress resistance of wheat. Overexpression of the TaOEP24 protein can significantly improve the heat tolerance of plants and can be applied to plant heat tolerance breeding and variety improvement.

[0114] The above details the present invention. For those skilled in the art, without departing from the purpose and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations, and conditions. Although the present invention gives specific embodiments, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to include any changes, uses, or improvements to the present invention, including changes made using conventional techniques known in the art that are outside the scope disclosed in this application.

Claims

1. Any of the following applications of proteins or substances that regulate protein expression or substances that regulate protein activity: P1. Application in regulating wheat heat tolerance or high temperature stress resistance. P2. Application in improving wheat heat tolerance or high temperature stress resistance. P3. Application in wheat breeding for heat resistance or high temperature stress resistance. P4. Application in improving the quality of wheat with heat or high temperature stress tolerance; The protein is the following protein: A1) The amino acid sequence is the protein of sequence 1 in the sequence listing; A2) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of A1).

2. Any of the following uses of the biological material related to the protein of claim 1: Q1. Application of the biological material in regulating heat tolerance or high temperature stress resistance of wheat. Q2. Application of the biological material in improving the heat resistance or high temperature stress resistance of wheat. Q3. Application of the biological material in breeding wheat for heat resistance or high temperature stress resistance. Q4. Application of the biological material in improving the quality of wheat resistant to heat or high temperature stress; The biological material is any of the following: B1) a nucleic acid molecule encoding the protein according to 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) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) A nucleic acid molecule that promotes or increases the gene expression of the protein according to claim 1; B6) An expression cassette, a recombinant vector or a recombinant microorganism containing the nucleic acid molecule described in B8).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is a gene encoding the protein shown in b1) or b2): b1) The coding sequence of the coding strand is a DNA molecule of nucleotides in sequence 2 in the sequence list; b2) A cDNA molecule or DNA molecule that hybridizes with a defined cDNA or DNA molecule and encodes a protein having the same function.

4. A method for improving the heat tolerance or high temperature stress resistance of wheat, comprising enhancing or increasing the activity of the protein described in claim 1 or / and the expression level of the gene encoding the protein described in claim 1 in the target wheat, thereby improving the heat tolerance or high temperature stress resistance of wheat.

5. The method according to claim 4, characterized in that: The enhancement or increase of the activity of the protein described in claim 1 or / and the expression level of the gene encoding the protein described in claim 1 in the target wheat is achieved by introducing the gene encoding the protein described in claim 1 into the target wheat.

Images