wheat heat-resistant protein TaNAC034 and its encoding gene and applications

Abstract

This invention discloses the wheat heat-resistant protein TaNAC034, its encoding gene, and its applications, addressing the technical problem of regulating plant heat resistance. Specifically, it discloses the application of the protein, a substance regulating the expression of the gene encoding the protein, or a substance regulating the activity or content of the protein in any of the following: A1) application in regulating plant heat resistance and / or application in preparing products that regulate plant heat resistance; A2) application in regulating plant dry weight under heat stress and / or application in preparing products that regulate plant dry weight under heat stress; A3) application in regulating plant fresh weight under heat stress and / or application in preparing products that regulate plant fresh weight under heat stress; the protein is any of the following: B1) a protein with the amino acid sequence shown in Sequence 2; B2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in B1) that has more than 80% identity and the same function as the protein shown in B1); B3) a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2). By regulating the expression of the TaNAC034 gene in plants, heat tolerance can be controlled, which can be used in industrial production.

Description

Technical Field

[0001] This invention specifically relates to a gene for identifying heat resistance in wheat and its application. Background Technology

[0002] In major production areas of crops such as corn, rice, and wheat worldwide, high-temperature stress not only occurs frequently but is also concentrated primarily during the critical growth cycles of these crops. Models predict that, assuming unchanged application of nitrogen fertilizers, wheat yields will decrease by 6% for every 1°C increase in global average temperature. Even more worrying is that the global average surface temperature has already risen by 0.8°C in the last 30 years, a rate far faster than the previous 30 years.

[0003] In actual production, wheat is highly susceptible to high-temperature stress when it enters the spikelet development stage. This effect is mainly reflected in the reduction of the total number of spikelets per plant during the spikelet differentiation process (from the two-ridge stage to the end of spikelet differentiation, i.e., the terminal spikelet formation stage). As a cool-season crop, wheat requires a high degree of coordination among multiple organs and tissues to complete its floret fertilization process, and high-temperature stress during the reproductive growth stage can easily lead to yield loss. Successful pollination and fertilization are closely related to the normal development of stamens, the flowering sequence of stamens and pistils, pollen germination, and pollen tube elongation. High temperatures can affect the lemma between the lemma and the base of the ovary in wheat florets, preventing them from opening and thus hindering spikelet opening. High temperatures can also damage anther tapetum differentiation and microspore formation, accelerating the PCD process in anther cells, thereby affecting anther dehiscence and leading to male sterility. Furthermore, because pollen tube growth in the style / filament and ovary (third and fourth stages) transport tissues is highly sensitive to temperature, prolonged high-temperature stress during the critical pollen shedding period severely impacts pollen germination, pollen tube growth, and fertilization, increasing the proportion of aborted grains. Finally, abnormal diurnal temperature variations can cause abnormal pistil development, leading to early or late flowering, which also results in a large number of aborted grains. After fertilization, high-temperature stress severely affects embryo and endosperm development, leading to zygotic abortion. Therefore, high-temperature stress is one of the key factors affecting the final yield of wheat. Improving the heat resistance of wheat is a technical problem faced by researchers in this field. Summary of the Invention

[0004] The technical problem solved by this invention is how to improve the heat resistance of wheat.

[0005] To address the above problems, the present invention provides the following applications.

[0006] The use of a protein, a substance that regulates the expression of the gene encoding the protein, or a substance that regulates the activity or content of the protein in any of the following;

[0007] A1) Applications in regulating plant heat tolerance and / or applications in the preparation of products that regulate plant heat tolerance;

[0008] A2) Application in regulating plant dry weight under heat stress and / or application in the preparation of products that regulate plant dry weight under heat stress;

[0009] A3) Application in regulating plant fresh weight under heat stress and / or application in the preparation of products that regulate plant fresh weight under heat stress;

[0010] The protein is any one of the following:

[0011] B1) The amino acid sequence of the protein is shown in sequence 2;

[0012] 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.

[0013] B3) A fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2).

[0014] 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.

[0015] 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.

[0016] 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.

[0017] Of the proteins described above, sequence 2 (SEQ ID No. 2) consists of 389 amino acid residues. It is named the TaNAC034 protein. Its encoding gene is the TaNAC034 gene.

[0018] In this application, the regulation may be over-regulation, enhancement, or increase, and / or knockout, reduction, or decrease.

[0019] 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 resistance.

[0020] Knocking out or reducing the expression of the gene encoding the protein, or reducing the activity or content of the protein, can decrease plant heat tolerance.

[0021] In the above text, the heat resistance indicators can be dry weight, fresh weight, glyoxalase activity, and / or glyoxal content. Dry weight, fresh weight, and glyoxalase activity are positively correlated with heat resistance. Glyoxal content is negatively correlated with heat resistance.

[0022] In the above text, the glyoxalase may be glyoxalase I.

[0023] In the above applications, the protein is derived from wheat.

[0024] The wheat mentioned above may be the wheat variety Fielder.

[0025] 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).

[0026] In the above applications, the substance regulating the expression of the protein-coding gene is any one of the following:

[0027] B1) Nucleic acid molecules that encode the above proteins;

[0028] B2), an expression cassette containing the nucleic acid molecule described in B1);

[0029] B3), a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0030] 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);

[0031] 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);

[0032] 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);

[0033] 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);

[0034] B8) Nucleic acid molecules that inhibit, reduce, or downregulate the expression of the gene encoding the protein of claim 1 or 2, or inhibit, reduce, or downregulate the activity or content of the protein;

[0035] B9) The gene encoding the nucleic acid molecule described in B8);

[0036] B10), an expression cassette containing the gene described in B9);

[0037] B11), a recombinant vector containing the gene described in B9), or a recombinant vector containing the expression cassette described in B10;

[0038] B12) recombinant microorganisms containing the gene described in B9), or recombinant microorganisms containing the expression cassette described in B10), or recombinant microorganisms containing the recombinant vector described in B11);

[0039] B13), a transgenic plant cell line containing the gene described in B9), or a transgenic plant cell line containing the expression cassette described in B10), or a transgenic plant cell line containing the recombinant vector described in B11;

[0040] B14), transgenic plant tissue containing the gene described in B9), or transgenic plant tissue containing the expression cassette described in B10), or transgenic plant tissue containing the recombinant vector described in B11;

[0041] B15), a transgenic plant organ containing the gene described in B9), or a transgenic plant organ containing the expression cassette described in B10), or a transgenic plant organ containing the recombinant vector described in B11).

[0042] In the nucleic acid molecules described in B1) or B8), those skilled in the art can easily mutate the nucleotide sequence encoding the protein TaHT1-A of the present invention using known methods, such as directed evolution or point mutation. Those artificially modified nucleotides that have 80% or more of the nucleotide sequence identity with the protein TaHT1-A isolated in the present invention, as long as they encode and function the protein TaHT1-A, are all derived from and equivalent to the nucleotide sequence of the present invention.

[0043] 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%.

[0044] 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.

[0045] 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;

[0046] In the aforementioned biological materials, the expression cassettes described in B2) and B10) refer 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) EMBO J.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.

[0047] In B3) and B11) 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 or the pBUE411 vector as the expression vector.

[0048] In the above applications, the nucleic acid molecule described in B1) is a DNA molecule with the nucleotide sequence shown in Sequence 1.

[0049] In the above applications, the nucleic acid target gene described in B9) is the gene encoding the protein shown in sequence 2.

[0050] To address the aforementioned problems, the present invention also provides a method for cultivating highly heat- and salt-tolerant plants.

[0051] The method includes upregulating or enhancing or increasing the expression level of the coding gene of the above-mentioned protein in the target plant, and / or, the activity and / or content of the protein to obtain a heat-resistant plant, wherein the heat resistance of the heat-resistant plant is higher than that of the target plant.

[0052] To address the aforementioned problems, the present invention also provides a method for improving the heat resistance of plants.

[0053] The method includes improving plant heat tolerance by upregulating or enhancing or increasing the expression of genes encoding the aforementioned proteins in plants, and / or the activity and / or content of the aforementioned proteins.

[0054] In this application, the plant may be a plant of the genus *Triticum*. The *Triticum* plant may be wheat. The wheat may be the wheat variety Fielder.

[0055] In the above method, downregulating or weakening or reducing the expression of the gene encoding the protein in the 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.

[0056] In the above text, the nucleic acid molecule may be the nucleic acid molecule described in Sequence 1.

[0057] To address the aforementioned problems, the present invention also provides a method for cultivating plants with low heat tolerance.

[0058] The method includes knocking out, reducing, or decreasing the expression level of the gene encoding the aforementioned protein in the target plant, and / or, the activity and / or content of the protein to obtain a low-heat-tolerant plant, the low-heat-tolerant plant having lower heat tolerance than the target plant.

[0059] In any of the applications described above or in any of the methods described above, the plant is any of the following:

[0060] J1) Grasses (Poaceae family);

[0061] J2) Plants of the Triticum genus;

[0062] J3) Wheat.

[0063] In this application, the wheat may be the wheat variety Fielder.

[0064] In the above text, the heat resistance indicators can be dry weight, fresh weight, glyoxalase activity, and / or glyoxal content. Dry weight, fresh weight, and glyoxalase activity are positively correlated with heat resistance. Glyoxal content is negatively correlated with heat resistance.

[0065] In this application, the heat stress condition is a heat treatment at 42°C. The heat treatment time can be 14 days or 6 hours.

[0066] Beneficial effects

[0067] This invention discloses the wheat heat-resistant protein TaNAC034, its encoding gene, and its applications, addressing the technical problem of regulating plant heat resistance. Specifically, it discloses the application of the protein, a substance regulating the expression of the gene encoding the protein, or a substance regulating the activity or content of the protein in any of the following: A1) application in regulating plant heat resistance and / or application in preparing products that regulate plant heat resistance; A2) application in regulating plant dry weight under heat stress and / or application in preparing products that regulate plant dry weight under heat stress; A3) application in regulating plant fresh weight under heat stress and / or application in preparing products that regulate plant fresh weight under heat stress; the protein is any of the following: B1) a protein with the amino acid sequence shown in Sequence 2; B2) a protein obtained by substituting and / or deleting and / or adding amino acid residues of the protein in B1) that has more than 80% identity and the same function as the protein shown in B1); B3) a fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of B1) or B2). By regulating the expression of the TaNAC034 gene in plants, heat tolerance can be controlled, which can be used for industrial production.

[0068] 1. This invention discloses the wheat gene TaNAC034. The wheat gene TaNAC034 is reported for the first time in wheat. It is a drought and heat resistance-related gene obtained through homologous cloning. Overexpression experiments in wheat show that this gene significantly improves heat tolerance in wheat seedlings, and this gene family has not been reported in current mainstream crop stress research. Therefore, exploring this gene resource can provide important molecular evidence for wheat stress resistance breeding.

[0069] 2. This invention discloses that the cytotoxic substance methylglyoxal may be involved in the wheat response to heat stress. Specifically, the wheat gene TaNAC034 significantly enhances the activity of glyoxalase I and reduces the content of the substrate methylglyoxal under heat stress, thereby enhancing the heat tolerance of wheat seedlings.

[0070] 3. The TaNAC034 gene of this invention was used to obtain a three-copy mutant of the three homologous genes using CRISPR technology. Phenotypic identification showed that the mutant exhibited significantly reduced heat tolerance compared to the wild-type Fielder. This further demonstrates that this gene is closely related to wheat stress resistance and has operational value in improving crop agronomic traits through gene editing. Attached Figure Description

[0071] Figure 1 This is a phylogenetic tree analysis of NAC034 in different species.

[0072] Figure 2 This involves the analysis of the functional domains of the TaNAC034 protein.

[0073] Figure 3 This is an amino acid sequence analysis of TaNAC034.

[0074] Figure 4 This is a graph showing the expression level of TaNAC034 overexpression transgenic wheat.

[0075] Figure 5 This is the sequence information of the TaNAC034 knockout strain.

[0076] Figure 6 This is a diagram illustrating the heat stress phenotype identification of TaNAC034 overexpression transgenic wheat.

[0077] Figure 7 This is a statistical chart of the dry and fresh weight of TaNAC034 overexpressing transgenic wheat before and after heat stress.

[0078] Figure 8 This is a diagram illustrating the heat stress phenotype identification of transgenic wheat strains with the TaNAC034 knockout line.

[0079] Figure 9 This is a statistical chart of the dry and fresh weights of transgenic wheat from the TaNAC034 knockout strain before and after heat stress.

[0080] Figure 10 This study investigated the activity of glyoxalase I and the content of methylglyoxal in different transgenic lines of TaNAC034 under heat stress. Detailed Implementation

[0081] 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.

[0082] 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.

[0083] 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.

[0084] Example 1: Evolution and sequence analysis of wheat heat tolerance gene TaNAC034 and its encoding gene

[0085] Using the amino acid sequence of Arabidopsis thaliana AtNAC034 as the query sequence, BLASTN search, comparison, and collection were performed against sequences in the wheat gene annotation database. Subsequently, gene sequence information of NAC034 family members from wheat, barley, rice, *Triticum aureum*, *Brachypoges davidiana*, and Arabidopsis thaliana was collected, and a phylogenetic tree was generated. Figure 1 We can clearly see that the TaNAC034 gene is most closely related to emmer wheat, followed by barley, and then to bryophyte, rice, and Arabidopsis thaliana.

[0086] According to the SMART protein domain prediction website, TaNAC034 contains a typical NAC domain PF02365, which is expected of a NAC transcription factor. Figure 2 Simultaneously, the protein sequences of NAC034 were compared in several monocotyledonous plants, including wheat, barley, emmer wheat, and Brachypodium distichum. Figure 3 We can clearly see that there are no SNP differences in the functional domain of the NAC transcription factor in different species, so we speculate that the function of this gene is very important and conserved.

[0087] Example 2: Obtaining and Identifying the TaNAC034 Transgenic Wheat Line

[0088] I. Construction of TaNAC034 Overexpression Recombinant Plasmid

[0089] 1. To clone the TaNAC34 gene from wheat, we first used the wheat variety *Chinese Spring* as a template to extract RNA and then reverse transcribed it to obtain cDNA. Based on the predicted TaNAC34 ORF sequence from the ensemble plant website, we designed specific primers (TaNAC34-F and TaNAC34-R). We then added specific adapters containing BamHI restriction sites to the pWMB110 vector to the primers to amplify the target gene and obtain the PCR product.

[0090] The specific amplification primers are as follows:

[0091] TaNAC34-F:5'-AGGTCGACTCTAGA GGATCC ATGAGCAGAGACGTCGACGA-3';

[0092] In TaNAC34--F, the underlined part is the BamHI restriction site.

[0093] TaNAC34-R: 5'-AGGTCGACTCTAGA GGATCC TTTAAAACTAGAACTGCCGT-3';

[0094] In TaNAC34-R, the underlined part is the BamHI restriction site.

[0095] 2. After digesting the pWMB110 vector with the restriction endonuclease BamHI at 37℃ for 1 hour, store it at -20℃ to obtain the digested vector fragment.

[0096] 3. Using the seamless ligation method of the seamless ligation kit (Bomaide, catalog number: CL116-01), the PCR product obtained in step 1 was ligated to the restriction vector fragment of step 2 (pWMB110 vector is described in the following literature: CRISPR / Cas9editing of wheat TaQ genes alters spike morphogenesis and grainthreshability, named Plasmid pWMB110 in the literature, which can also be purchased through conventional commercial channels), transformed into E. coli, and plasmids were extracted from positive colonies and sequenced. The plasmid with correct sequencing was named pWMB110-TaNAC034 recombinant plasmid.

[0097] The pWMB110-TaNAC034 recombinant plasmid is obtained by replacing the fragment between the 5'-AGGTCGACTCTAGAGGATCC-3' and 5'-GGATCCCCGGGTACCGAGCT-3' sequences of the pWMB110 vector with the complete CDS sequence of TaNAC034 (Sequence 1), while keeping the other sequences of the pWMB110 vector unchanged. The resulting recombinant vector is named pWMB110-TaNAC034.

[0098] Sequence 1 is as follows:

[0099]

[0100] Sequence 1 is the CDS sequence of the wheat variety TaNAC034 from China Spring, which encodes the protein shown in Sequence 2.

[0101] Sequence 2 is as follows:

[0102] MSRDVDEGSVSAATAGAGGGGEVGGEAAAGVSDEAAVDSHENDLVMPGFRFHPTEEELIEFYLRRKVEGRRFNVELITFLDLYRFDPWELPAMAVIGEKEWFFYVPRDRKYRNGDRPNRVTASGYWKATGADRMIRGENSRPIGLKKTLVFYSGKAPKGVRSSWIMNEYRLPPPTTDADLFYKSEISLCRVYKRS GIDDGHGRPSSSNVQASSSARPGTSRTIIPPAGQQVSSPLSTPMSPTQQPSFHGILGQGECSPAPLPAIMDQATAQLHQPPPPPPPRPSAFASAMSSTMSVAPAARSCTYSLMALADAPMMGSSSTPGDELSRLVGHSQAYPNHPAVGSHFLPSPSSSQIPPHGEMPVSPADKLWDWIHPDTTGSRDYGSSSFK.

[0103] The recombinant vector was transformed into Escherichia coli.

[0104] 4. Positive clones were screened by PCR reaction and sequenced. At the same time, the pWMB110-TaNAC034 recombinant plasmid was extracted and transformed into Agrobacterium GV3101 to obtain Agrobacterium EHA105 of pWMB110-TaNAC034. This was then sent to the wheat transgenic platform for wheat genetic transformation (the recipient plant was the wheat variety Fielder).

[0105] 5. Perform PCR identification on the obtained T0 generation plants.

[0106] 6. After harvesting the identified positive lines, the plants were multiplied in a greenhouse, and the positive results of the T1 generation were identified by PCR.

[0107] Take T0 generation seeds, transfer positive plants to germination paper on a culture medium for germination, and after uniform germination, transfer them to soil. Cultivate the plants until they bear fruit, and detect the expression level of the NAC34 gene during plant growth. Harvest seeds from T1 generation positive plants. Plant T1 generation seeds in the same way, then plant them in soil. Harvest individual plants after maturity to obtain T2 seeds for further observation and testing. Take T2 generation seeds and screen positive plants according to the above method. For a certain T1 generation plant, if all its T2 generation plants are positive, the T1 generation plant and its self-pollinated offspring constitute a homozygous transgenic line, obtaining two TaNAC034 overexpressing T2 generation homozygous lines, namely OE#1 T2 generation homozygous line and OE#2 T2 generation homozygous line. Self-pollinate the T2 generation plants to obtain T3 generation seeds, namely OE#1 T3 generation homozygous seeds and OE#2 T3 generation homozygous seeds.

[0108] II. Positive Identification of TaNAC034 Overexpression Lines

[0109] Seeds of the transgenic lines to be tested (OE#1T3 homozygous lines (also known as OE#1T3 homozygous seeds) and OE#2T3 homozygous lines (also known as OE#2T3 homozygous seeds)) and WT 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 (Vazyme Biotech, R223-01). The expression level was monitored using quantitative primers. The amplification program was set as follows: pre-denaturation 95℃ for 3 min (95℃ for 15 s, 60℃ for 15 s, 72℃ for 20 s) 40 cycles 65℃-94℃. The melting curve of the reaction was calculated and plotted. Detection primers: TaActin was generally selected as the internal reference gene for gene expression analysis. The average of three replicates was used to represent the relative expression level of the target gene. The significance was tested using a two-tailed t-test with equal variances ('*' represents P<0.05, '**' represents P<0.01).

[0110] Detection primers: TaNAC034-qF: CCTGGCAGACGCACCCA; TaNAC034-qR: GGTGGATCCAATCCCAAAGCT. TaACTIN was used as an internal control, with the following primers: TaACTIN-F: GGAATCCATGAGACCACCTAC; TaACTIN-R: GACCCAGACAACTCGCAAC.

[0111] The results are as follows Figure 4 ( Figure 4In the figure, WT is the wheat variety Fielder, OE#1 is the homozygous seedling of OE#1 T3 generation, and OE#2 is the homozygous seedling of OE#2 T3 generation. As shown, the expression level of TaNAC034 gene was significantly increased in the T3 generation homozygous seedlings (OE#1 and OE#2) overexpressing TaNAC034 compared with WT seedlings, proving that these two lines are indeed positive overexpression lines.

[0112] IV. Obtaining TaNAC034 transgenic knockout wheat

[0113] 1. TaNAC034 knockout primers:

[0114] TaNAC034-p411-F: 5'-ATATATGGTCTCTGGCGGGTGATTGGGGAGAAGGAGTT-3';

[0115] TaNAC034-p411-F0: 5'-TGGGTGATTGGGGAGAAGGAG GTTTTAGAGCTAGAAATAGC-3';

[0116] TaNAC034-p411-R0: 5'-AACCGGATCATCCTGTCGGCCCCCGCTTCTTGGTGCC-3';

[0117] TaNAC034-p411-R: 5'-ATTATTGGTCTCTAAACCGGATCATCCTGTCGGCCCCC-3'.

[0118] 2. Using pMT1T2 plasmid as a template, PCR was performed using the above 4 primers to obtain DNA fragments containing vector adapters and target sites, which were then purified by gel extraction to obtain gel-recovered fragments.

[0119] The pMT1T2 plasmid (pMT1T2 vector) is described in the following literature: "A CRISPR / Cas9 toolkit for multiplex genome editing in plants," where it is named pCBC-MT1T2. It is also a conventional vector that can be purchased through regular channels. The vector used in this application was donated by Professor Chen Qijun of the College of Biological Sciences, China Agricultural University.

[0120] 3. The pBUE411 restriction vector was digested with BsaI to obtain the pBUE411 restriction fragment. The pBUE411 restriction fragment was then ligated with the gel-recovered fragment (containing the vector adapter and target DNA fragment) using T4 ligase overnight and transformed into E. coli. Plasmids were extracted from positive bacteria and sequenced. The correctly sequenced recombinant plasmid was named the pBUE411-TaNAC034 knockout vector.

[0121] Vector pBUE411 was purchased from AddGene, catalog number 62200. https: / / www.addgene.org / 62200 / )

[0122] The pBUE411-TaNAC034 knockout vector is obtained by replacing the sequence between fragment 1 (5'-TGCAGATGATCCGTGGC-3') and fragment 2 (5'-ATTTCTAGCTCTAAAAC-3') of the pBUE411 vector with a DNA fragment (sequence 3) containing the target adapter sequence MT1T2, while keeping the other sequences of the pBUE411 vector unchanged. The resulting recombinant vector is named pBUE411-TaNAC034 knockout vector. After plasmid extraction, it is transformed into Agrobacterium GV3101 and sent to a wheat transgenic platform for genetic transformation of wheat (recipient plant is the wheat Fielder variety).

[0123] Sequence 3 is as follows:

[0124] GGTGATTGGGGAGAAGGAGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTTTCGTTTTGCATTGAGTTTTCTCCGTCGCATGTTTGCAGTTTTATTTTCCGTTTTGCATTGAAATTTCTCCGTCTCATGTTTGCAGCGTGTTCAAAAAGTACGCAGCTGTATTTCACTTATTTACGGCGCCACATTTTC ATGCCGTTTGTGCCAACTATCCCGAGCTAGTGAATACAGCTTGGCTTCACACAACACTGGTGACCCGCTGACCTGCTCGTACCTCGTACCGTCGTACGGCACAGCATTTGGAATTAAAGGGTGTGATCGATACTGCTTGCTGCTCATGAATCCAAACCACACGGAGTTCAAATTCCCACAGATTAAGGCTCGTCCGTCGCACAAGGTAATGTGTGAATATTAT ATCTGTCGTGCAAAATTGCCTGGCCTGCACAATTGCTGTTATAGTTGGCGGCAGGGAGAGTTTTAACATTGACTAGCGTGCTGATAATTTGTGAGAAAATAATAATTGACAAGTAGATACTGACATTTGAGAAGAGCTTCTGAACTGTTATTAGTAACAAAAATGGAAAGCTGATGCACGGAAAAAGGAAAGAAAAAGCCATACTTTTTTTTAGGTAGGAAAAG AAAAAGCCATACGAGACTGATGTCTCTCAGATGGGCCGGGATCTGTCTATCTAGCAGGCAGCAGCCCACCAACCTCACGGGCCAGCAATTACGAGTCCTTCTAAAAGCTCCCGCCGAGGGGCTGGCGCTGCTGTGCAGCAGCACGTCTAACATTAGTCCCACCTCGCCAGTTTACAGGGAGCAGAACCAGCTTATAAGCGGAGGCGCGGCACCAAGAAGCG CGGATCATCCTGTCGGCCCC The underlined sequence represents the target sequence.

[0125] 4. Detection of target editing in TaNAC034 transgenic knockout wheat

[0126] DNA was extracted from T0 generation plants. Three subgenome-specific primers were designed near the target site, and amplification was performed for first-generation sequencing to determine whether the area near the target site had been edited. The detection primers are:

[0127] TaNAC034-A-CRI-F:TCGACTCGCACGAGAACGACT

[0128] TaNAC034-A-CRI-R:AACAGCATGAACAACGGCTGG

[0129] TaNAC034-B-CRI-F: CGGCGCAAGGTGGAGGGCCGA

[0130] TaNAC034-B-CRI-R: TGAAGTGAGCGCTACCTGTAAC

[0131] TaNAC034-D-CRI-F:TGGGAGCTCCCCGGTACATAGT

[0132] TaNAC034-D-CRI-R:AGAGATGAGGAATTTCCGGGG

[0133] After harvesting, the identified positive lines were subcultured in a greenhouse, and the T1 generation plants were identified positively using PCR sequencing. Further edited T1 generation plants were selected, self-pollinated, and identified, yielding two TaNAC034 knockout T2 generation homozygous lines: KO#2T2 and KO#5. The T2 generation plants were then self-pollinated to obtain T3 generation seeds (KO#2T3 homozygous seeds and KO#5 homozygous seeds).

[0134] The edit type for KO#2 is:

[0135] In subgenome A, 107 oxyribonucleotide residues are deleted between positions 667 and 773 of sequence 4, resulting in a frameshift mutation in the TaNAC034-A gene. This causes the encoded protein (sequence 2) to be mistranslated at amino acid 99, encoding 203 amino acids (SPRREQPAHRPQEDARLLLRQGPQGRPQQLDHERVPPPAAHHRRRSLLQVRDLALPRVQALRHRR RPRAALLQQRPSVVLGEAGHLTHHYSAGWPAGVITVVHADVTDAAAQLPRHTRPGRVLAGAAASHHGPGHRAAASAS ASSSSEAERLCVGDELNDVGCAGREELHVLAHGPGRRTHDGQQFHAGGRAEPAGGPQPGLP), which leads to the knockout of the original protein domain of the TaNAC034-A protein.

[0136] In the B subgenome, 107 oxyribonucleotide residues are deleted between positions 666 and 772 of sequence 4, resulting in a frameshift mutation in the TaNAC034-B gene. This causes the encoded protein (sequence 2) to be mistranslated at amino acid 99, encoding 203 amino acids (SPRREQPAHRPQEDARLLLRQGPQGRPQQLDHERVPPPAAHHRRRSLLQVRDLALPRVQALRHRR RPRAALLQQRPSVVLGEAGHLTHHYSAGWPAGVITVVHADVTDAAAQLPRHTRPGRVLAGAAASHHGPGHRAAASAS ASSSSEAERLCVGDELNDVGCAGREELHVLAHGPGRRTHDGQQFHAGGRAEPAGGPQPGLP), which leads to the knockout of the original protein domain of the TaNAC034-B protein.

[0137] In the D subgenome, three oxyribonucleotide residues are missing between positions 666 and 668 of sequence 4, and seven oxyribonucleotide residues (TGATCCG) are missing between positions 771 and 777 of sequence 1. This results in a frameshift mutation in the TaNAC034-D gene, causing the deletion of glutamate at position 100 of the protein encoded by sequence 2. Furthermore, the protein is prematurely terminated after 30 amino acids mistranslated starting at amino acid position 132 (TARTAGPSASRRRSSSTPARPPRASAAAGS), resulting in the knockout of the original protein domain of the TaNAC034-D protein.

[0138] The edit type for KO#5 is:

[0139] In subgenome A, the 668th nucleotide residue of sequence 4 is missing, and a single base insertion (T) occurs between the 774th and 775th nucleotide residues of sequence 1, resulting in a complete translation error of the encoded protein (sequence 2) from amino acid 100 to amino acid 135, thus knocking out the TaNAC034-A gene;

[0140] In the B subgenome, 111 oxyribonucleotide residues are deleted between positions 666 and 776 of sequence 4, resulting in a frameshift mutation in the TaNAC034-B gene. This causes the 99th amino acid of the encoded protein (sequence 2) to be mistranslated as a serine and the amino acids between positions 100 and 136 to be deleted, thereby knocking out the TaNAC034-B gene.

[0141] In the D subgenome, 107 oxyribonucleotide residues are deleted between positions 667 and 773 of sequence 4, resulting in a frameshift mutation in the TaNAC034-D gene. This causes the encoded protein (sequence 2) to prematurely terminate at amino acid 99 after encoding 203 amino acids (SPRREQPAHRPQEDARLLLRQGPQGRPQQLDHERVPPPAAHHRRRSLLQVRDLALPRVQALRHRRRPRAALLQQRPSVVLGEAGHLTHHYSAGWPAGVITVVHADVTDAAAQLPRHTRPGRVLAGAAASHHGPGHRAAASASASSSSEA ERLCVGDELNDVGCAGREELHVLAHGPGRRTHDGQQFHAGGRAEPAGGPQPGLP), thus knocking out the original protein domain of the TaNAC034-D protein.

[0142] The specific sequence of sequence 4 is as follows:

[0143] TAGCCAGTGCTTAGTAGCCACTACAGTAGCTACTAGCCATTAATTATTACCAAGTTAATCATCGGTTCAATCAAAA

[0144] CTAACCAGGATGATTAAGCAAAGCCTCCACTCCAGTATATATCGATGGTGCAAACTCCACTCTTATCCTCTCTCCTCCC

[0145] ACCTACTTGCGCACCCCCAAATTGCTTGCCTTTCCTCAGCCATCTGGTTGCCCTAGCTAGCTGGCTCGCCGGCCTGCTGC

[0146] TACCGTGCGCACACGCGCGCTATGCGGGCAATAGGCACGGCGAGGGAGAGCCGGCAGCCATGAGCAGAGACGTCGACGAA

[0147] GGAAGCGTTTCGGCGGCGACAGCAGGAGCAGGAGGAGGAGGGGAAGTGGGTGGCGAGGCGGCGGCCGGCGTCAGCGACGA

[0148] GGCGGCAGTCGACTCGCATGAGAACGACCTGGTGATGCCGGGATTCCGGTTCCACCCGACGGAGGAGGAGCTGATCGAGT

[0149] TCTACCTCCGGCGCAAGGTGGAGGGCCGGCGCTTCAACGTCGAGCTCATCACCTTCCTCGACCTCTACCGCTTCGACCCA

[0150] TGGGAGCTCCCCGGTACATAGTCATACACACACATACAGCACCGCTTAACTCAAATTGGGGTCTTGATTAATTTCTTGGT

[0151] GTTGGCAGCAATGGCGGTGATTGGGGAGAAGGAGTGGTTCTTCTACGTGCCCCGGGACCGCAAGTACCGGAACGGCGACC

[0152] GGCCGAACCGGGTGACGGCGTCGGGGTACTGGAAGGCGACGGGGGCCGACAGGATGATCCGCGGCGAGAACAGCCGGCCC

[0153] ATCGGCCTCAAGAAGACGCTCGTCTTCTACTCCGGCAAGGCCCCCAAGGGCGTCCGCAGCAGCTGGATCATGAACGAGTA

[0154] CCGCCTCCCGCCGCCCACCACCGACGCCGATCTCTTCTACAAGGTAATTGCTCCGATCGAGCGAGCGAAATTCGACTAGG

[0155] GTTTTGGAATTTGGAGGCCAGACTTAAGACCCCGGAAATTCCTCATCTCTTGATTGGTTTTTGTTTTGCAAATTCTTGGG

[0156] AAGCTAGCTATATATCAGCCGTTGTTCATGCTGTTAATCATTGATTTGATCTGCTTTTTGTTCACTGCTAGCTTTTTTGT

[0157] TGGTTGGCATGATTTACATGAACATGTGACATGCCTTTCCTCTCTTTCTTTTCAGATCTAAGAGCTTAGGGTTCGCTTTT

[0158] TGCTCGTTCTTTTTGGCATACATATGGACATGTACAAATGCTACCATGTTGAATTCAGAATTCCTCTTATAGCCAGCTAG

[0159] ATACCACTTGCATGTCCTCCAGATTCTTCAAATTAACTATTTGCATGTCCAAGCTAAACAGTCGATTCAGTTCCCCTTTG

[0160] CTCAAATATTCAGATCCGAACTTAATTGAAGCATTTAATTCCATCTCACAGCTAGCACTCACTTCATATGAAAAGCATGC

[0161] ACCCTGTTCCTCAGTCCTCACTCAATTTACTGCAACCTATGCCACCTAGCTAGTATCATGTACGTACTCGTGTAAGATCT

[0162] TTGGTATTGGCAGTAATTTCTTTAGCTTTGACATGTTGCAGTAAATTTTGAATTCCTAATTTGATCGTTCTTGGTTTTTC

[0163] GACAAAGGGTGGATTTGATCGTGCTTGCTTATGTAGTTTATCCGAAGTTTCACAAAATGTCTCATGCATGCAGTCCGAGA

[0164] TCTCGCTCTGCCGCGTGTACAAGCGCTCCGGCATCGACGACGGCCACGGGCGGCCCTCCTCCAGCAACGTCCAAGCGTCG

[0165] TCCTCGGCGAGGCCGGGCACCTCACGCACCATTATTCCGCCGGCTGGCCAGCAGGTGTCATCACCGTTGTCCACGCCGAT

[0166] GTCACCGACGCAGCAGCCCAGCTTCCACGGCATACTCGGCCAGGGCGAGTGCTCGCCGGCGCCGCTGCCAGCCATCATGG

[0167] ACCAGGCCACCGCGCAGCTGCATCAGCCTCCGCCTCCTCCTCCTCCGAGGCCGAGCGCCTTTGCGTCGGCGATGAGCTCA

[0168] ACGATGTCGGTTGCGCCGGCCGCGAGGAGCTGCACGTACTCGCTCATGGCCCTGGCAGACGCACCCATGATGGGCAGCAG

[0169] TTCCACGCCGGGGGACGAGCTGAGCCGGCTGGTGGGCCACAGCCAGGCCTACCCTAACCACCCGGCCGTCGGCAGCCACT

[0170] TCCTCCCCTCACCATCGTCGTCGCAAATTCCGCCCCACGGAGAGATGCCGGTGTCGCCGGCCGACAAGCTTTGGGATTGG

[0171] ATCCACCCTGACACCACAGGCAGCAGGGACTACGGCAGTTCTAGTTTTAAATGACTCCAAAACCAAGTAAATGCAGTCTT

[0172] GTTGACAGGAGGTTGCATTGCATATGCCAGTGGGCAGTGGCACTGGGCATAATTCGTAGGTTTTCACAGC。

[0173] Example 3 Identification of the phenotype of wheat TaNAC034 transgenic lines under heat stress at the seedling stage

[0174] Two F3 homozygous lines, KO#2 and KO#5, and two F3 homozygous lines, OE#1 and OE#2, as well as seeds of wild-type Fielder (seeds of OE#1 T3 homozygous line, OE#2 T3 homozygous line, KO#2 T3 homozygous line, KO#5 homozygous line, and wild-type Fielder) were selected as phenotypic identification subjects.

[0175] The seeds were disinfected with 1% hydrogen peroxide for 10 minutes, rinsed 3-4 times with distilled water, placed in petri dishes lined with two layers of filter paper, and a small amount of distilled water was added. The dishes were then incubated at room temperature for 48 hours. Seedlings with uniform germination were selected and placed in germination boxes. Different transgenic lines and corresponding wild-type wheat strains were placed in the same germination box, i.e., the experiment was divided into 4 groups: overexpression control group, overexpression heat treatment group, gene editing control group, and gene editing heat treatment group. The overexpression control group and overexpression heat treatment group were planted with wild-type Fielder, OE#1T3 homozygous lines, and OE#2T3 homozygous lines, respectively. The gene editing control group and gene editing heat treatment group were planted with wild-type Fielder, KO#2T3 homozygous lines, and KO#5 homozygous lines, respectively. Each line contained 16 seeds.

[0176] The experiment was divided into a control group and a heat treatment group, with four biological replicates in each group.

[0177] After growing the above germination boxes for 5 days under the conditions of 22℃-18℃, 16 hours of light / 8 hours of darkness, and 60%-70% humidity, the following experiment was conducted:

[0178] Heat Treatment Group:

[0179] After 5 days of germination, the germination boxes were placed in the constant temperature and light greenhouse of the West Campus of China Agricultural University and cultured for 14 days under the conditions of 42℃, 16 hours of light / 8 hours of darkness, and 60%-70% humidity (heat stress). Then, they were cultured for 3 days under the conditions of 22℃ / 18℃, 16 hours of light / 8 hours of darkness, and 60%-70% humidity (recovery). After that, photos were taken, and the fresh and dry weights and dry weights before and after the stress were recorded.

[0180] Fresh weight = weight of the aboveground part of a single plant after 3 days of culture at normal temperature following heat stress, with at least 10 plants measured per group.

[0181] Dry weight = weight of the aboveground part of a single plant after 3 days of cultivation at normal temperature following heat stress, and after drying in a 65℃ oven for 2 days. At least 10 plants were measured in each group.

[0182] Control group:

[0183] The only difference was that no heat stress was performed (while the heat-treated group underwent heat stress culture, the control group underwent normal culture (22℃ / 18℃, 16 hours of light / 8 hours of darkness, 60%-70% humidity culture)). All other tests and operations were the same as those in the heat-treated group.

[0184] Phenotypic results are as follows Figure 6 and 8 ( Figure 6 WT is a wild-type Fielder, OE#1 is a homozygous OE#1T3 line, and OE#2 is a homozygous OE#2T3 line. Figure 8 WT represents wild-type Fielder, KO#1 is a homozygous KO#1 T3 generation line, KO#2 is a homozygous KO#2 T3 generation line, and the control group and heat-treated group correspond to the above control group and heat-treated group, respectively. Fresh weight and dry weight results are shown below. Figure 7 and Figure 9 ( Figure 7 WT is a wild-type Fielder, OE#1 is a homozygous OE#1T3 line, and OE#2 is a homozygous OE#2T3 line. Figure 9 WT represents wild-type Fielder, KO#1 represents the KO#1 T3 generation homozygous line, KO#2 represents the KO#2 T3 generation homozygous line, and the control group and heat-treated group correspond to the above control group and heat-treated group, respectively.

[0185] The results showed that, under normal conditions, there was no significant difference in aboveground growth among wild-type wheat, OE, and KO lines. However, after heat stress treatment and subsequent recovery to room temperature for 5 days, the heat tolerance of TaNAC034 overexpressing lines was significantly increased, while the knockout lines became more sensitive to heat stress. Further analysis of dry and fresh weight before and after stress revealed that before heat stress treatment, there was no significant difference in dry and fresh weight among different lines, but the dry and fresh weight of TaNAC034 overexpressing lines increased significantly after stress, while the knockout lines showed the opposite. Figure 6 , 8 ).

[0186] Example 4: Determination of glyoxalase I activity in wheat TaNAC034 transgenic line

[0187] Seeds from two F3 homozygous lines KO#2 and KO#5 (identified as pure lines) of knockout wheat material, two F3 homozygous lines OE#1 and OE#2, and wild-type Fielder control seeds (OE#1 T3 homozygous, OE#2 T3 homozygous, KO#2 T3 homozygous, KO#5 homozygous, and wild-type Fielder seeds), as well as wild-type Fielder control seeds, were disinfected with 1% hydrogen peroxide for 10 minutes, rinsed 3-4 times with distilled water, placed in petri dishes lined with two layers of filter paper, and a small amount of distilled water was added. The dishes were then incubated at room temperature for 48 hours. Seedlings with uniform germination were 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 2:1 volume ratio). After 7 days of growth under conditions of 22℃ / 18℃, 16 hours light / 8 hours dark, and 60%-70% humidity, 7-day-old seedlings were obtained.

[0188] Seven-day-old seedlings (wild-type Fielder seedlings, OE#1T3 generation homozygous seedlings, OE#2T3 generation homozygous seedlings, KO#2T3 generation homozygous seedlings, and KO#5 homozygous seedlings) were selected for the following experiments:

[0189] Heat Treatment Group:

[0190] The cells were cultured at 42℃ for 16 hours under light / 8 hours in darkness and 60%-70% humidity for 6 hours. Glyoxalase I (GLO1) activity was measured using a glyoxalase I (GLO1) assay kit (Merck; MAK114). The specific steps are as follows:

[0191] 1. Cut off the aerial leaves of each group of samples and add them to a protein extraction buffer (pre-cooled at 4°C) containing 0.1M phosphate-buffered saline (pH=7.4) (Merck; P3563), 0.1% Triton (Merck; X100-5ML), and a 1X Halt protease inhibitor mixture (ThermoFisher, CA, USA). Add steel balls and grind the samples under liquid nitrogen conditions to obtain the ground samples.

[0192] 2. Subsequently, the ground sample was homogenized using the FastDNA Soil Kit (MP Biochemical, CA, USA) with plant tissue lysis (frozen to -20°C before use) at a frequency of 30 s / s and 40 s per cycle for 5 cycles.

[0193] 3. After centrifuging at 14,000g at 4℃ for 10 minutes, collect the supernatant containing protein and determine the protein concentration.

[0194] 4. Using the glyoxalase I activity assay kit (Mock, Merck), the absorbance at OD 240nm was recorded every 20 seconds at 26°C, with orbital vibrations at intervals (2 seconds between each reading).

[0195] 5. Calculate enzyme activity using absorbance (ΔOD) at different time points within the linear range. Specific formula: Activity = 350 × [(A240)final – (A240)initial] units / L (see kit instructions for details). Perform 10 replicates of the above experiment.

[0196] Control group:

[0197] The only difference was that no heat stress was performed (while the heat-treated group underwent heat stress culture, the control group underwent normal culture (22℃ / 18℃ 16 hours light / 8 hours dark, 60%-70% humidity culture)). All other tests and operations were the same as those in the heat-treated group.

[0198] The results showed that by measuring the glyoxalase I activity of the TaNAC034 transgenic lines under normal and heat stress conditions, we found that in the two overexpression lines of this gene under normal conditions, there was no significant difference in glyoxalase I activity in wheat plants compared to the wild type. However, in the two homozygous triple knockout lines, glyoxalase I activity was slightly decreased. It is noteworthy that under heat stress conditions, overexpression of this gene significantly enhanced glyoxalase I activity in wheat, while the knockout lines showed the opposite result. Figure 10 The left bar chart shows that WT represents wild-type Fielder, OE#1 represents the OE#1 T3 generation homozygous line, and OE#2 represents the OE#2 T3 generation homozygous line; WT represents wild-type Fielder, KO#1 represents the KO#1 T3 generation homozygous line, and KO#2 represents the KO#2 T3 generation homozygous line. The control group and heat-treated group correspond to the above control group and heat-treated group, respectively.

[0199] Example 5: Determination of methylglyoxal content in the wheat TaNAC034 transgenic line

[0200] Seeds from two F3 homozygous lines KO#2 and KO#5 (identified as pure lines) of knockout wheat material, two F3 homozygous lines OE#1 and OE#2, and wild-type Fielder control seeds (OE#1 T3 homozygous, OE#2 T3 homozygous, KO#2 T3 homozygous, KO#5 homozygous, and wild-type Fielder), as well as wild-type Fielder control seeds, were disinfected with 1% hydrogen peroxide for 10 minutes, rinsed 3-4 times with distilled water, placed in petri dishes lined with two layers of filter paper, and a small amount of distilled water was added. The petri dishes were incubated at room temperature for 48 hours. Seedlings with uniform germination were 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 2:1 volume ratio). After 7 days of growth under conditions of 22℃ / 18℃, 16 hours light / 8 hours darkness, and 60%-70% humidity, 7-day-old seedlings were obtained.

[0201] Seven-day-old seedlings (wild-type Fielder seedlings, OE#1T3 generation homozygous seedlings, OE#2T3 generation homozygous seedlings, KO#2T3 generation homozygous seedlings, and KO#5 homozygous seedlings) were selected for the following experiments:

[0202] Heat Treatment Group:

[0203] The cells were cultured at 42℃ for 16 hours under light / 8 hours in darkness and 60%-70% humidity for 6 hours, and then tested using a glyoxalase I (GLO1) assay kit (Merck; MAK114). The specific steps are as follows:

[0204] 1. After thoroughly grinding each sample (above-ground plant tissue) with liquid nitrogen, weigh 0.1g and add it to a 1.5mL centrifuge tube.

[0205] 2. Then add 1 mL of 5% HClO4 (Merck; 1.00518) to each tube and mix well by pipetting. Centrifuge at 14000 rpm and 4℃ for 10 min.

[0206] 3. Take an appropriate amount of supernatant according to the ratio, and use the Abcam Methylglyoxal Assay Kit (ab241006) to detect the 535 / 587nm ratio in each wheat sample according to the procedure. Refer to the standard curve y = 0.3844x - 0.0085, where y is the absorbance value (as described in the kit instructions) to calculate the content of methylglyoxal (note that if the final absorbance measurement result is too high, it needs to be diluted 2-3 times).

[0207] Control group:

[0208] The only difference was that no heat stress was performed (while the heat-treated group underwent heat stress culture, the control group underwent normal culture (22℃ / 18℃ 16 hours light / 8 hours dark, 60%-70% humidity culture)). All other tests and operations were the same as those in the heat-treated group.

[0209] By measuring the methylglyoxal (MG) content in TaNAC034 transgenic lines under normal and heat stress conditions, we found no significant difference between gene overexpression lines, gene knockout lines, and wild-type under normal conditions. Notably, under heat stress, gene overexpression, compared to wild-type materials, significantly enhanced glyoxalase I activity, thereby reducing the excessive accumulation of MG. Conversely, gene knockout lines showed a more significant abnormal accumulation of MG under heat stress. These results suggest that the TaNAC034 gene likely improves wheat heat resistance by regulating glyoxalase I activity, thereby degrading excessive MG produced under heat stress. Figure 10 , Figure 10 The right-hand bar chart shows that WT represents wild-type Fielder, OE#1 represents the OE#1 T3 generation homozygous line, and OE#2 represents the OE#2 T3 generation homozygous line; WT represents wild-type Fielder, KO#1 represents the KO#1 T3 generation homozygous line, and KO#2 represents the KO#2 T3 generation homozygous line. The control group and heat-treated group correspond to the above control group and heat-treated group, respectively.

[0210] 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 a protein or a substance regulating the expression of a gene encoding said protein in any of the following; A1) Applications in improving plant heat resistance and / or applications in the preparation of products that improve plant heat resistance; A2) Application in improving the dry weight of plants under heat stress and / or application in the preparation of products that improve the dry weight of plants under heat stress; A3) Application in increasing the fresh weight of plants under heat stress and / or application in the preparation of products that increase the fresh weight of plants under heat stress; The protein is any one of the following: b1) The amino acid sequence is that of the protein shown in sequence 2; b2) The fusion protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of b1); The substance that regulates the expression of the gene encoding the protein 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. Use according to claim 1, characterized in that, The protein is derived from wheat.

3. Use according to claim 1 or 2, characterized in that, The nucleic acid molecule described in B1) is a DNA molecule with the nucleotide sequence shown in Sequence 1.

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 and / or the content of the protein in the target plant to obtain a heat-resistant plant, wherein the heat resistance of the heat-resistant plant is higher than that of the target plant; the plant is wheat.

5. A method for improving the heat resistance of plants, characterized in that, This includes improving heat tolerance of plants by upregulating or enhancing or increasing the expression of genes encoding the proteins described in claim 1 or 2 and / or the content of the proteins described in claim 1 or 2 in the target plant; the plant being wheat.

6. The method as described in 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 plant comprises introducing into the target plant the nucleic acid molecule of claim 1(B1), the expression cassette of claim 1(B2), or the recombinant vector of claim 1(B3).

Images