TaPHL2 protein and related biological materials in regulating wheat growth and drought resistance

Abstract

The application discloses TaPHL2 protein and application of related biological materials thereof in regulating growth and development and drought resistance of wheat. Experiments prove that, after TaPHL2 gene is introduced into wheat to be overexpressed, the transgenic wheat has the advantages that the number of lateral roots, yield and drought resistance of the transgenic wheat are reduced compared with a receptor wheat Fielder, and the height of the transgenic wheat is increased; and the RNAi interference wheat has the advantages that the number of lateral roots and drought resistance of the RNAi interference wheat are increased compared with the receptor wheat Fielder, and the height of the RNAi interference wheat is reduced. The protein and the gene provided by the application provide a basis for artificially controlling the root structure, plant type, yield and improving the drought resistance of wheat, and will play an important role in cultivating plants with enhanced stress resistance.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of TaPHL2 protein and related biomaterials in regulating wheat growth, development, and drought resistance. Background Technology

[0002] Food security is a fundamental guarantee for the development of human society. With global climate change, extreme weather events such as drought and high temperatures have become major factors affecting food security. Wheat is an important food crop, providing the main energy intake for 35-40% of the world's population. With the continuous increase in the world's population, the demand for wheat is projected to increase by 60% by 2050, while drought severely threatens wheat yield and food security. Improving the drought resistance of wheat is a key issue in current wheat research. Discovering the functional genes that maintain the vitality and final yield of wheat under drought stress, and designing breeding strategies to improve crop survival based on this, can provide a better solution for future food security.

[0003] During evolution, plants have gradually developed complex drought resistance and avoidance mechanisms, including rapid growth before drought stress and advancing the reproductive stage to avoid the effects of drought; increasing the internal water content and water absorption capacity of plants to maintain plant moisture content and avoid tissue damage during drought periods; enhancing plant drought tolerance; and enhancing the tolerance and antioxidant capacity of plant cells in low water conditions during drought environments. Currently, research on plant stress resistance has gradually penetrated to the cellular and molecular levels, combining with genetics and genetic engineering research to explore the use of biotechnology to improve plant growth characteristics and thereby enhance plants' adaptability to adversity.

[0004] Roots are vital organs for plants to absorb nutrients and water, and are closely related to drought resistance and growth. Under drought stress, plants inhibit the growth of above-ground parts to reduce water loss and simultaneously promote root development to obtain more water and nutrients from the soil in order to maintain normal growth and development. Understanding the regulatory mechanisms of plant drought resistance and discovering effective drought-regulating genes are key issues in breeding drought-resistant crops. Improving plant stress resistance through gene transformation and knockout techniques provides feasible solutions for cultivating drought-resistant plants. Summary of the Invention

[0005] The purpose of this invention is to provide the application of TaPHL2 protein and related biomaterials in regulating wheat growth, development and drought resistance.

[0006] To achieve the above objectives, the present invention first provides new uses for TaPHL2 protein or related biomaterials.

[0007] This invention provides the use of TaPHL2 protein or related biomaterials in any of the following 1)-5):

[0008] 1) Regulate the number of lateral roots in wheat;

[0009] 2) Regulate wheat plant height;

[0010] 3) Regulate wheat yield;

[0011] 4) Regulate wheat drought resistance;

[0012] 5) Breed transgenic wheat with altered lateral root number and / or plant height and / or yield and / or drought resistance;

[0013] The TaPHL2 protein is any one of the proteins described in (a1)-(a4) below:

[0014] (a1) The protein shown in sequence 2;

[0015] (a2) A fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of the protein described in (a1);

[0016] (a3) Proteins obtained by substituting and / or deleting and / or adding one or more amino acid residues of (a1) that are related to the number of lateral roots and / or plant height and / or yield and / or drought resistance in wheat.

[0017] (a4) is a protein that shares more than 98% identity with (a1) and is associated with the number of lateral roots and / or plant height and / or yield and / or drought resistance in wheat.

[0018] In the protein described in (a2) above, the 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 tag may be a Flag tag, His tag, MBP tag, HA tag, MYC tag, GST tag, and / or SUMO tag, etc.

[0019] In the protein described in (a3) ​​above, the substitution and / or deletion and / or addition of one or more amino acid residues is as follows: substitution and / or deletion and / or addition of no more than 10 amino acid residues, or substitution and / or deletion and / or addition of no more than 9 amino acid residues, or substitution and / or deletion and / or addition of no more than 8 amino acid residues, or substitution and / or deletion and / or addition of no more than 7 amino acid residues, or substitution and / or deletion and / or addition of no more than 6 amino acid residues, or substitution and / or deletion and / or addition of no more than 5 amino acid residues, or substitution and / or deletion and / or addition of no more than 4 amino acid residues, or substitution and / or deletion and / or addition of no more than 3 amino acid residues, or substitution and / or deletion and / or addition of no more than 2 amino acid residues, or substitution and / or deletion and / or addition of no more than 1 amino acid residue.

[0020] In the protein described in (a4) above, the 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 page on the NCBI homepage. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as the matrix, setting the Gap existence cost, Per residue gap cost, and Lambdaratio to 11, 1, and 0.85 (default values) respectively, and performing a search for the identity of a pair of amino acid sequences to calculate the identity value (%), the result can be obtained.

[0021] In the above applications, the relevant biological material is a nucleic acid molecule encoding the TaPHL2 protein or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule.

[0022] Furthermore, the nucleic acid molecule encoding the TaPHL2 protein is a DNA molecule as described in either (A1) or (A2):

[0023] (A1) The DNA molecule shown in sequence 1;

[0024] (A2) is a DNA molecule that has more than 75% identity with (A1) and encodes the TaPHL2 protein.

[0025] Those skilled in the art can readily mutate the nucleotide sequence encoding the TaPHL2 protein of this invention using known methods, such as directed evolution and point mutation. Artificially modified nucleotides that have 75% or higher identity with the TaPHL2 nucleotide sequence isolated according to this invention, as long as they encode the TaPHL2 protein and have the same function, are derived from and are equivalent to the nucleotide sequence of this invention.

[0026] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences that have 75% or higher, 80% or higher, 85% or higher, 90% or higher, or 95% or higher identity with the nucleotide sequence of a protein constituting the amino acid sequence shown in Sequence 2 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0027] Furthermore, the expression cassette refers to DNA capable of expressing the TaPHL2 protein in a host cell. This DNA may include not only a promoter to initiate TaPHL2 transcription but also a terminator to terminate TaPHL2 transcription. Additionally, the expression cassette may also include an enhancer sequence.

[0028] The vector may be a plasmid, granule, bacteriophage, or viral vector.

[0029] The microorganism may be yeast, bacteria, algae or fungi; the bacteria may be Agrobacterium.

[0030] To achieve the above objectives, the present invention provides a new use for substances that inhibit the TaPHL2 protein.

[0031] This invention provides the use of a substance that inhibits the TaPHL2 protein in any of the following (d1)-(d4):

[0032] (d1) Regulating the number of lateral roots in wheat;

[0033] (d2) Regulating wheat plant height;

[0034] (d3) Regulates wheat drought resistance;

[0035] (d4) Breed transgenic wheat with altered lateral root number and / or plant height and / or drought resistance.

[0036] Furthermore, the substance that inhibits the TaPHL2 protein includes substances that inhibit the activity of the TaPHL2 protein or substances that reduce the content of the TaPHL2 protein.

[0037] The substance that inhibits the activity of the TaPHL2 protein can be a protein, polypeptide, or small molecule compound that inhibits the function of the TaPHL2 protein.

[0038] The substance that reduces the content of TaPHL2 protein can be a substance that inhibits TaPHL2 protein synthesis, promotes TaPHL2 protein degradation, or knocks down or eliminates the TaPHL2 gene.

[0039] Furthermore, the substance that knocks down the TaPHL2 gene can be any substance that can prevent the gene encoding the TaPHL2 protein from being expressed, such as miRNA, siRNA, dsRNA, shRNA, etc.

[0040] The substance used to knock out the TaPHL2 gene can be any substance that prevents the host cell from producing the functional protein product of that gene. Specific methods include removing all or part of the coding gene sequence, introducing mutations to prevent the production of the functional protein, removing or altering regulatory components (e.g., promoter editing) to prevent transcription of the coding gene sequence, or blocking translation by binding to mRNA. Typically, knockout is performed at the genomic DNA level, ensuring that the cell's offspring permanently carry the knockout.

[0041] In a specific embodiment of the present invention, the substance that inhibits the TaPHL2 protein is an RNAi fragment that interferes with the expression of the TaPHL2 gene or a vector containing the RNAi fragment. Specifically, the RNAi fragment is the DNA molecule shown in sequence 3.

[0042] In any of the above applications, the regulation can be increased or decreased. The yield can be the grain weight per plant.

[0043] To achieve the above objectives, the present invention also provides a method for breeding transgenic wheat with altered lateral root number and / or plant height and / or yield and / or drought resistance.

[0044] The method for cultivating transgenic wheat with altered lateral root number and / or plant height and / or yield and / or drought resistance provided by the present invention includes the step of increasing the activity and / or content of TaPHL2 protein in recipient wheat to obtain transgenic wheat; wherein the transgenic wheat has a lower lateral root number and / or yield and / or drought resistance than the recipient wheat, and the transgenic wheat has a higher plant height than the recipient wheat.

[0045] Furthermore, the lower yield of the genetically modified wheat compared to the recipient wheat is reflected in the lower grain weight per plant of the genetically modified wheat compared to the recipient wheat.

[0046] The lower drought resistance of the genetically modified wheat compared to the recipient wheat is manifested in any one of the following (x1)-(x7):

[0047] (x1) Under controlled water treatment (e.g., in soil with a moisture content of 250%, 350%, or 450%), the aboveground fresh weight of the transgenic wheat is lower than that of the recipient wheat;

[0048] (x2) Under controlled water treatment (e.g., in soil with a water content of 250%, 350%, or 450%), the catalase content of the transgenic wheat is lower than that of the recipient wheat.

[0049] (x3) Under controlled water treatment (e.g., in soil with a water content of 250%, 350%, or 450%), the proline content of the transgenic wheat is lower than that of the recipient wheat;

[0050] (x4) Under controlled water treatment (e.g., in soil with a moisture content of 250%, 350%, or 450%), the malondialdehyde content of the transgenic wheat is higher than that of the recipient wheat;

[0051] (x5) Under drought stress (e.g., in soil with a moisture content of 30%), the plant height of the transgenic wheat is lower than that of the recipient wheat;

[0052] (x6) Under drought stress (e.g., in soil with a moisture content of 30%), the effective tiller number of the transgenic wheat is lower than that of the recipient wheat;

[0053] (x7) Under drought stress (e.g., in soil with a moisture content of 30%), the grain weight per plant of the transgenic wheat is lower than that of the recipient wheat.

[0054] The method for increasing the activity and / or content of TaPHL2 protein in recipient wheat is to overexpress TaPHL2 protein in recipient wheat.

[0055] Furthermore, the overexpression method involves introducing the gene encoding the TaPHL2 protein into recipient wheat.

[0056] To achieve the above objectives, the present invention finally provides a method for breeding transgenic wheat with altered lateral root number and / or plant height and / or drought resistance.

[0057] The method for cultivating transgenic wheat with altered lateral root number and / or plant height and / or drought resistance provided by the present invention includes the step of reducing the activity and / or content of TaPHL2 protein in the recipient plant to obtain transgenic plants; wherein the transgenic wheat has a higher lateral root number and / or drought resistance than the recipient wheat, and the transgenic wheat has a lower plant height than the recipient wheat.

[0058] Furthermore, the higher drought resistance of the genetically modified wheat compared to the recipient wheat is manifested in any one of the following (y1)-(y7):

[0059] (y1) Under controlled water treatment (e.g., in soil with a moisture content of 250%, 350%, or 450%), the aboveground fresh weight of the transgenic wheat is higher than that of the recipient wheat;

[0060] (y2) Under controlled water treatment (e.g., in soil with a water content of 250%, 350%, or 450%), the catalase content of the transgenic wheat is higher than that of the recipient wheat.

[0061] (y3) Under controlled water treatment (e.g., in soil with a water content of 250%, 350%, or 450%), the transgenic wheat has a higher proline content than the recipient wheat;

[0062] (y4) Under controlled water treatment (e.g., in soil with a moisture content of 250%, 350%, or 450%), the malondialdehyde content of the transgenic wheat is lower than that of the recipient wheat;

[0063] (y5) Under drought stress (e.g., in soil with a moisture content of 30%), the plant height of the transgenic wheat was greater than that of the recipient wheat;

[0064] (y6) Under drought stress (e.g., in soil with a moisture content of 30%), the effective tiller number of the transgenic wheat is higher than that of the recipient wheat;

[0065] (y7) Under drought stress (e.g., in soil with a moisture content of 30%), the grain weight per plant of the transgenic wheat was higher than that of the recipient wheat.

[0066] Furthermore, the method for reducing the activity and / or content of TaPHL2 protein in the recipient plant is to introduce the aforementioned substance that inhibits TaPHL2 protein into the recipient wheat.

[0067] The substances that inhibit TaPHL2 protein mentioned above are also within the scope of protection of this invention.

[0068] In any of the above applications or methods, the wheat variety may specifically be Fielder.

[0069] This invention demonstrates through experiments that overexpression of the TaPHL2 gene in wheat results in transgenic wheat with reduced lateral root number, yield, and drought resistance, but increased plant height compared to the recipient wheat, Fielder. RNAi-interfered wheat, compared to Fielder wheat, shows increased lateral root number and drought resistance, but reduced plant height. The proteins and genes provided by this invention offer a foundation for artificially controlling wheat root structure, plant architecture, yield, and improving wheat drought resistance, and will play an important role in cultivating plants with enhanced stress resistance. Attached Figure Description

[0070] Figure 1A schematic diagram illustrating the construction of the TaPHL2 vector for overexpression and RNAi interference.

[0071] Figure 2 To detect positive PCR results for wheat overexpressing TaPHL2.

[0072] Figure 3 To detect the expression level of TaPHL2 overexpressing wheat by qRT-PCR.

[0073] Figure 4 This is a positive result for TaPHL2-RNAi wheat PCR detection.

[0074] Figure 5 To detect the expression level of TaPHL2-RNAi in wheat by qRT-PCR.

[0075] Figure 6 To identify the root structure phenotype and measure root length in wheat with TaPHL2 overexpression and TaPHL2 RNAi interference.

[0076] Figure 7 To identify the seedling phenotype and determine physiological indicators of wheat with TaPHL2 overexpression and TaPHL2 RNAi interference under controlled water conditions.

[0077] Figure 8 To identify plant architecture and analyze yield traits in wheat with TaPHL2 overexpression and TaPHL2 RNAi interference under normal and drought stress conditions.

[0078] Figure 9 Subcellular localization of TaPHL2. Detailed Implementation

[0079] The following examples are provided to better understand the present invention, but do not limit the invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were purchased from conventional biochemical reagent stores. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0080] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0081] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0082] In the quantitative experiments described below, three replicate experiments were conducted, and the average value of the results was taken.

[0083] Unless otherwise specified, all percentages in the following examples refer to mass percentages.

[0084] The common wheat (Triticum aestivum L.) variety Xiaobaimai used in the following examples is described in the literature “Sun Haitao et al., Screening of wheat TaDREB6 transcription factor interaction proteins, Chinese Journal of Agricultural Science, 2011, 44(22): 4740-4747.” It can be obtained by the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biological material is only used to repeat the relevant experiments of this invention and cannot be used for other purposes. It can also be obtained by the public from the National Germplasm Resource Bank (number ZM242).

[0085] The wheat variety Fielder described in the following examples is described in the literature “Kazuhiro Sato, Fumitaka Abe, Martin Mascher, Georg Haberer, Heidrun Gundlach, Manuel Spannagl, Kenta Shirasawa and Sachiko Isobe. Chromosome-scale genome assembly of the transformation-amenable common wheat cultivar 'Fielder'. DNA research: an international journal for rapid publication of reports on genes and genomes. 2021, 28(3), dsab008.”, which is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biological material is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0086] The pWMB110 vector in the following examples is described in the literature “Xiao-Yu Cui, Yuan Gao, Jun Guo, Tai-Fei Yu, Wei-Jun Zheng, Yong-Wei Liu, Jun Chen, Zhao-Shi Xu and You-Zhi Ma*. BES / BZR Transcription Factor TaBZR2 Positively Regulates Drought Responses by Activation of TaGST11. Plant Physiology. 2019, 180: 605-620”, which is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biomaterial is only for repeating the relevant experiments of this invention and should not be used for other purposes.

[0087] The solutions and their formulations involved in the following examples are shown in Tables 1-4.

[0088] Table 1. Enzyme hydrolysate

[0089] Components Content (20ml) Cellulase 0.2g pectinase 0.08g Mannitol 0.4M MES 20mM KCl 0.29g BSA 0.02g

[0090] Note: After mixing the above substances in water, cool to room temperature, and add CaCl2 to make the concentration of CaCl2 in the enzymatic hydrolysate 1M.

[0091] Table 2, W5 solution

[0092] Components Content (1L) NaCl 9.0g <![CDATA[CaCl2]]> 8.4g KCl 0.37g glucose 0.9g MES 0.3g

[0093] Note: Adjust the pH to 5.8 using KOH.

[0094] Table 3. PEG Solution

[0095] Components content PEG4000 1g 0.8M mannitol aqueous solution 0.625ml <![CDATA[1M aqueous CaCl2 solution]]> 0.25ml <![CDATA[H2O]]> 0.75ml

[0096] Table 4. MMG solution

[0097]

[0098]

[0099] Note: Adjust the pH to 5.6 using KOH.

[0100] Example 1: Cloning of TapHL2

[0101] I. Treatment of Plant Materials

[0102] The entire seedlings of white wheat (Triticum aestivum cv. Xiaobaimai) at the three-leaf stage, which had been grown hydroponically for about 10 days, were flash-frozen with liquid nitrogen and stored at -80℃ for later use.

[0103] II. Extraction of Total RNA

[0104] Total RNA was extracted from the leaves of the treated wheat seedlings obtained in step one using the Trizol method (TianGen).

[0105] III. Obtaining cDNA

[0106] cDNA was synthesized using the SMART method, with reverse transcriptase XL (AMV) used for the synthesis of the first-strand cDNA.

[0107] IV. PCR Amplification

[0108] Using the cDNA obtained in step three as a template, PCR amplification was performed using TaPHL2-F and TaPHL2-R to obtain the PCR product. The specific primer sequences for amplification are as follows:

[0109] TaPHL2-F: ATGTTCCCCCCTGGCCT;

[0110] TaPHL2-R: TCAGCAGGATGACTTGC.

[0111] V. Detection of PCR Products

[0112] The PCR product obtained in step four was detected by 1.0% agarose gel electrophoresis and then sequenced. The results showed that sequence 1 in the sequence listing was obtained by PCR amplification. The gene represented by sequence 1 in the sequence listing was named TaPHL2 gene, and the protein encoded by this gene was named TaPHL2 protein. The amino acid sequence of this protein is sequence 2 in the sequence listing, consisting of 255 amino acids.

[0113] The above sequence 1 can also be obtained through artificial synthesis.

[0114] Example 2: Obtaining TaPHL2 transgenic wheat and its stress tolerance analysis

[0115] I. Obtaining TaPHL2 wheat

[0116] 1. Construction of recombinant vectors overexpressing TaPHL2

[0117] (1) Amplification of the TaPHL2 gene

[0118] Using the PCR product obtained in step four of Example 1 as a template, amplification was performed using primers TaPHL2-110F and TaPHL2-110R to obtain the PCR product. The primer sequences are as follows (the primer ends are the upstream and downstream sequences of the BamHI restriction site on the vector, respectively, so that the TaPHL2 fragment can be ligated into the pWMB110 vector by homologous recombination method; the BamHI restriction site is underlined):

[0119] TaPHL2-110F:5'-CGACTCTAGA GGATCC ATGTTCCCCCCTGGCCT-3';

[0120] TaPHL2-110R: 5'-GGGTACCCGG GGATCC TCAGCAGGATGACTTGC-3'.

[0121] The PCR product was subjected to 1.2% agarose gel electrophoresis and then sequenced. The results showed that the PCR product contained the DNA sequence shown in Sequence 1 of the sequence listing, which is the TaPHL2 gene.

[0122] The PCR products were recovered and purified using the Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa, Code No: DV807A).

[0123] (2) The pWMB110 vector was digested with the restriction endonuclease BamHI, and the vector backbone was recovered and purified.

[0124] (3) The TaPHL2 gene PCR product and the vector backbone were ligated using the ClonExpressⅡOne Step Cloning Kit (Vazyme, Code No: C112) to obtain the ligation product.

[0125] (4) The ligation product obtained in step (3) was transformed into TOP10 strain (Tiangen, CB104-03) by heat shock, cultured overnight at 37°C, positive single clones were selected, plasmids were extracted and sequenced, and the plasmids with correct sequencing were named pWMB110-TaPHL2.

[0126] The TaPHL2 overexpression recombinant vector pWMB110-TaPHL2 is obtained by inserting the DNA molecule shown in Sequence 1 (the complete coding sequence of the TaPHL2 gene) into the BamHI restriction site of the pWMB110 vector. A schematic diagram of the structure of the TaPHL2 overexpression recombinant vector pWMB110-TaPHL2 is shown below. Figure 1 As shown in Figure A.

[0127] 2. Construction of the RNAi interference TaPHL2 recombinant vector

[0128] (1) The fragment shown in sequence 3 was artificially synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. The fragment includes, in sequence, a SmaI restriction site, a nucleotide sequence of position 180-442 of sequence 1 with a size of 262bp, a maize alcohol dehydrogenase (Adh) gene sequence of size 146bp (as an intron), the reverse complementary sequence of the nucleotide sequence of position 180-442 of sequence 1 with a size of 262bp, and a SacI restriction site.

[0129] (2) The pWMB110 vector was digested with SmaI and SacI enzymes. The digestion products were recovered using Agarose Gel DNA Purification Kit Ver. 2.0 (TaKaRa, Code No: DV807A). The artificially synthesized fragment (as shown in Sequence 3) in step (1) was inserted into the SmaI and SacI restriction sites of the pWMB110 vector to obtain the TaPHL2-pWMB110-RNAi interference vector.

[0130] The recombinant vector TaPHL2-pWMB110-RNAi, which interferes with TaPHL2, is obtained by replacing the DNA fragment between the SmaI and SacI restriction sites of the pWMB110 vector with the DNA molecule shown in Sequence 3. A schematic diagram of the structure of the recombinant vector TaPHL2-pWMB110-RNAi is shown below. Figure 1 As shown in B.

[0131] 3. Construction of recombinant bacteria

[0132] The recombinant plasmids pWMB110-TaPHL2 and TaPHL2-pWMB110-RNAi obtained in steps 1 and 2, respectively, were transformed into Agrobacterium EHA105 (Beijing Bairddi Biotechnology Co., Ltd.). After identification, recombinant bacteria EHA105 / pWMB110-TaPHL2 and EHA105 / TaPHL2-pWMB110-RNAi were obtained, respectively.

[0133] 4. Obtaining and detecting TaPHL2-transferred wheat

[0134] (1) Recombinant bacteria EHA105 / pWMB110-TaPHL2 and EHA105 / TaPHL2-pWMB110-RNAi were inoculated into YEP liquid medium and cultured at 28°C and 200 rpm for about 30 hours.

[0135] (2) Transfer the bacterial culture obtained in step (1) to YEP liquid medium (containing 50 μg / ml rifampicin and kanamycin) and incubate at 28°C and 200 rpm for about 14 hours (bacterial culture OD). 600 (Reaching 1.5-3.0).

[0136] (3) Collect the bacterial cells obtained in step (2), centrifuge at 4℃ and 4000g for 10 min, and dilute with 10% sucrose (containing 0.02% silwet) to OD. 600 It is approximately 1.0.

[0137] (4) In the clean bench, use a 1ml syringe needle to pick up Agrobacterium colonies and inject them into the Fielder wheat embryo. Then, culture the injected embryos under sterile and high humidity conditions until the tissue culture wheat seedlings grow. Continue to culture the wheat seedlings until the T0 generation of TaPHL2 overexpression and RNAi interference wheat seeds are harvested.

[0138] T0 generation wheat was subjected to TaPHL2 overexpression, RNAi interference, and sowing and self-pollination until T3 generation wheat was obtained.

[0139] (5) Use qRT-PCR and PCR to detect positive wheat plants with TaPHL2 overexpression and TaPHL2 RNAi interference.

[0140] The specific steps for PCR detection are as follows: DNA was extracted from the leaves of different wheat lines and recipient wheat plants that overexpress TaPHL2 and have TaPHL2 RNAi interference, and PCR amplification was performed using different primers with the extracted DNA as a template.

[0141] The primer sequences used for PCR detection of TaPHL2-overexpressing wheat lines are as follows:

[0142] 110-TaPHL2-Test-F: 5'-TTTTAGCCCTGCCTTCATACGC-3';

[0143] 110-TaPHL2-Test-R: 5'-AGGCGAACTGCTCCGAGACGA-3'.

[0144] The primer sequences used for PCR detection of TaPHL2 RNAi interference wheat lines are as follows:

[0145] RNAi-TaPHL2-Test-F: 5'-CCCTGTTGTTTGGTGTTACTTCTG-3';

[0146] RNAi-TaPHL2-Test-R: 5'-CCAAGGTATCTAATCAGCCATC-3'.

[0147] The results are as follows Figure 2 and Figure 4 As shown, the results indicate that specific bands were detected in all positive lines, but no specific bands were detected in the receptor Fielder, indicating that all of the above are positive lines.

[0148] The specific steps for qRT-PCR detection are as follows: RNA was extracted from the leaves of different wheat lines overexpressing TaPHL2 and those with TaPHL2 RNAi interference, as well as the recipient wheat, and reverse transcribed into cDNA. qRT-PCR detection was then performed using the following sequences as primers:

[0149] qRT-TaPHL2-F2: 5'-CCCAAGATGCGAAAGAAAG-3';

[0150] qRT-TaPHL2-R1: 5'-CTGGTGGAACACGGAGGAG-3'.

[0151] The results are as follows Figure 3 and Figure 5As shown, the results indicated that the expression levels of TaPHL2 in the overexpression lines were significantly higher than those in Fielder lines. Among them, TaPHL2 gene expression levels were highest in lines 2 and 4, which were named Ox-TaPHL2-1 and Ox-TaPHL2-2, respectively, for subsequent experimental analysis. The expression levels of TaPHL2 in interference lines 1 and 5 were significantly lower than those in Fielder lines, and the expression levels were similar. Figure 5 They were named RNAi-TaPHL2-1 and RNAi-TaPHL2-2 respectively for subsequent experimental analysis.

[0152] II. Identification of root structure and stress tolerance analysis of TaPHL2-transformed wheat

[0153] T3 generation wheat seeds overexpressing TaPHL2, designated Ox-TaPHL2-1 and Ox-TaPHL2-2, and TaPHL2 seeds designated RNAi-TaPHL2-1 and RNAi-TaPHL2-2 were respectively... Root structure of RNAi-interfered wheat seeds and Fielder seeds was observed after 7 and 10 days of normal growth in hydroponic trays, and the length of the taproot and the number of lateral roots were counted. Uniformly germinated wheat was planted in soils with different absolute moisture contents (250%, 350%, 450%) and grown for 20 and 25 days. Plant growth was observed after 20 and 25 days, and relevant physiological indicators (fresh weight of aboveground parts, CAT content, MDA content, and proline content) were measured after 25 days. Uniformly germinated wheat was planted in a greenhouse environment (16 hours light, 8 hours darkness, temperature 25℃) under normal water conditions with an average absolute moisture content of 70% and under drought stress conditions with an average absolute moisture content of 30%. After reaching full maturity (100 days), plant type (plant height, number of effective tillers) and yield traits (thousand-grain weight, single-plant grain weight) were identified. The specific detection methods for the physiological indicators are as follows:

[0154] CAT content determination: The CAT content was determined using a catalase (CAT) kit from Suzhou Keming Biotechnology Co., Ltd. (Comin, Code No: EC1.11.1.6). The specific steps are as follows: Weigh approximately 0.1g of tissue, add 1ml of extraction buffer, and homogenize on ice. Centrifuge at 8000g, 4℃ for 10min, collect the supernatant, and place on ice for analysis. Preheat the spectrophotometer for at least 30min, adjust the wavelength to 240nm, and zero the sample with distilled water. Before measurement, incubate the CAT detection working solution at 25℃ for 10min. Take 1mL of the CAT detection working solution into a 1mL quartz cuvette, add 35μL of sample, and mix for 5s. Immediately measure the initial absorbance A1 at 240nm and the absorbance A2 after 1min at room temperature, and calculate the CAT content.

[0155] MDA content determination: The MDA content was determined using a malondialdehyde (MDA) test kit from Suzhou Keming Biotechnology Co., Ltd. (Comin, Code No: MDA-2-Y). The specific steps are as follows: Weigh approximately 0.1g of tissue, add 1ml of extraction buffer, and homogenize in an ice bath. Centrifuge at 8000g, 4℃ for 10min, collect the supernatant, and place on ice for testing. Pipette 0.6ml of reagent I into a 1.5ml centrifuge tube, add 0.2ml of sample, and mix well. Incubate in a 95℃ water bath for 30min, cool in an ice bath, and centrifuge at 10000g, 25℃ for 10min. Pipette the supernatant into a 200μl glass cuvette, measure the absorbance at 532nm and 600nm, record them as A532 and A600, and calculate the MDA content.

[0156] Proline content determination: The proline content was determined using a proline (PRO) content test kit from Suzhou Keming Biotechnology Co., Ltd. (Comin, Code No: PRO-2-Y). Approximately 0.1g of tissue was weighed, and 1mL of extraction buffer was added. The mixture was homogenized in an ice bath. Afterward, it was extracted at 90℃ with shaking for 10min. The sample was centrifuged at 10000g and 25℃ for 10min. The supernatant was collected, cooled, and ready for analysis. The spectrophotometer was preheated for at least 30min, and the wavelength was adjusted to 520nm. The instrument was zeroed with distilled water. 0.5ml of sample + 0.5ml of reagent one + 0.5ml of reagent two were placed in a covered test tube and incubated in a boiling water bath for 30min, shaking every 10min. After cooling, 1ml of reagent three was added to the test tube, shaken for 30s, and allowed to stand for a moment to allow the pigment to transfer to reagent three. 0.8ml-1ml of the supernatant was transferred to a 1ml glass cuvette, and the absorbance was measured at 520nm. The absorbance value A was recorded, and the proline content was calculated.

[0157] Root structure identification results as follows Figure 6As shown: Under normal conditions, compared with Fielder plants, wheat plants overexpressing TaPHL2 had a significantly reduced number of lateral roots, while wheat plants with TaPHL2 RNAi interference had a significantly increased number of lateral roots. Specifically, after 7 days of normal growth, the average number of lateral roots in Fielder plants was 64.7, in T3 generation TaPHL2-overexpressing wheat plants (Ox-TaPHL2-1) it was 45.41, in T3 generation TaPHL2-overexpressing wheat plants (Ox-TaPHL2-2) it was 42.64, in TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) it was 99.23, and in TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) it was 99.23. The average number of lateral roots in wheat plants subjected to RNAi interference was 93.7. After 10 days of normal growth, the average number of lateral roots in Fielder plants was 99.52, the average number of lateral roots in T3 generation TaPHL2-expressing wheat plants (Ox-TaPHL2-1) was 57.11, the average number of lateral roots in T3 generation TaPHL2-expressing wheat plants (Ox-TaPHL2-2) was 61.35, the average number of lateral roots in TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 123.12, and the average number of lateral roots in TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 130.12.

[0158] The results of the stress resistance analysis are as follows Figure 7 As shown, under water-controlled conditions, wheat plants overexpressing TaPHL2 exhibited significantly inhibited growth compared to Fielder plants, with wilting leaves, partial plant death, and a significant decrease in aboveground fresh weight. Conversely, wheat plants interfering with TaPHL2 RNAi showed higher leaf vigor and significantly increased aboveground fresh weight compared to Fielder plants. Furthermore, the catalase (CAT) activity and proline content of the TaPHL2-overexpressing wheat lines were significantly lower than those of the Fielder material, while the malondialdehyde (MDA) content was significantly higher. This indicates that under water-controlled conditions, the TaPHL2-overexpressing wheat lines experienced increased cell damage and a significant decrease in antioxidant capacity. Conversely, the TaPHL2 RNAi-interfered wheat lines exhibited significantly higher catalase (CAT) activity and proline content than the Fielder material, while significantly lower MDA content. This indicates that under water-controlled conditions, the TaPHL2 RNAi-interfered wheat lines showed significantly reduced cell damage and a significantly improved antioxidant capacity.

[0159] Among them, under the condition of 250% moisture content, the average fresh weight of the aboveground parts of Fielder plants was 1.1g, the average fresh weight of the aboveground parts of wheat plants overexpressing TaPHL2 in the T3 generation (numbered Ox-TaPHL2-1) was 0.69g, the average fresh weight of the aboveground parts of wheat plants overexpressing TaPHL2 in the T3 generation (numbered Ox-TaPHL2-2) was 0.69g, the average fresh weight of the aboveground parts of TaPHL2 RNAi-interfered wheat plants (numbered RNAi-TaPHL2-1) was 1.59g, and the average fresh weight of the aboveground parts of TaPHL2 RNAi-interfered wheat plants (numbered RNAi-TaPHL2-2) was 1.56g. Under conditions of 350% moisture content, the average fresh weight of the aboveground parts of Fielder plants was 1.14 g, the average fresh weight of the aboveground parts of wheat plants overexpressing TaPHL2 in the T3 generation (Ox-TaPHL2-1) was 0.9 g, the average fresh weight of the aboveground parts of wheat plants overexpressing TaPHL2 in the T3 generation (Ox-TaPHL2-2) was 0.87 g, the average fresh weight of the aboveground parts of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 1.51 g, and the average fresh weight of the aboveground parts of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 1.56 g. Under conditions of 450% moisture content, the average fresh weight of the aboveground parts of Fielder plants was 1.79 g, the average fresh weight of the aboveground parts of wheat plants overexpressing TaPHL2 in the T3 generation (Ox-TaPHL2-1) was 1.45 g, the average fresh weight of the aboveground parts of wheat plants overexpressing TaPHL2 in the T3 generation (Ox-TaPHL2-2) was 1.4 g, the average fresh weight of the aboveground parts of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 2.25 g, and the average fresh weight of the aboveground parts of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 2.21 g.

[0160] Under conditions of 250% moisture content, the average CAT content of Fielder plants was 22.3, the average CAT content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 18.41, the average CAT content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 18.23, the average CAT content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 30.56, and the average CAT content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 29.33. Under conditions of 350% moisture content, the average CAT content of Fielder plants was 14.12, the average CAT content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 13.09, the average CAT content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 12.66, the average CAT content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 21.88, and the average CAT content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 22.35. Under conditions of 450% moisture content, the average CAT content of Fielder plants was 25.35, the average CAT content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 23.87, the average CAT content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 23.06, the average CAT content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 33.44, and the average CAT content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 32.78.

[0161] Under conditions of 250% moisture content, the average MDA content of Fielder plants was 10.31, the average MDA content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 13.57, the average MDA content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 13.22, the average MDA content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 8.84, and the average MDA content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 8.42. Under conditions of 350% moisture content, the average MDA content of Fielder plants was 11.26, the average MDA content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 14.35, the average MDA content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 14.19, the average MDA content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 10.93, and the average MDA content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 11.87. Under conditions of 450% moisture content, the average MDA content of Fielder plants was 8.13, the average MDA content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 12.88, the average MDA content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 12.09, the average MDA content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 8.07, and the average MDA content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 8.11.

[0162] Under conditions of 250% moisture content, the average proline content of Fielder plants was 522.31, the average proline content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 485.33, the average proline content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 468.42, the average proline content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 550.57, and the average proline content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 573.46. Under conditions of 350% moisture content, the average proline content of Fielder plants was 386.13, the average proline content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 361.25, the average proline content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 353.11, the average proline content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 521.88, and the average proline content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 512.47. Under conditions of 450% moisture content, the average proline content of Fielder plants was 575.43, the average proline content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 533.47, the average proline content of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 543.11, the average proline content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 684.15, and the average proline content of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 663.53.

[0163] The results of plant type identification and yield trait analysis under normal and drought stress conditions are as follows: Figure 8As shown: Under normal conditions, compared with Fielder plants, wheat plants overexpressing TaPHL2 had significantly higher plant height, while those with TaPHL2 RNAi interference had decreased plant height. Under drought stress, wheat plants overexpressing TaPHL2 had a sharp decrease in plant height, while those with TaPHL2 RNAi interference had only a slight decrease in plant height. Specifically, under normal growth conditions, the average plant height of Fielder plants was 79.03 cm, the average plant height of T3 generation TaPHL2-overexpressing wheat plants (Ox-TaPHL2-1) was 83.68 cm, the average plant height of T3 generation TaPHL2-overexpressing wheat plants (Ox-TaPHL2-2) was 83.12 cm, the average plant height of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 77.26 cm, and the average plant height of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was... The average plant height of wheat plants subjected to RNAi interference was 76.55 cm. Under drought stress, the average plant height of Fielder plants was 75.34 cm, the average plant height of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1) was 67.28 cm, the average plant height of T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2) was 69.69 cm, the average plant height of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 74.14 cm, and the average plant height of TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 77.26 cm.

[0164] Under normal conditions, there was no significant difference in the number of effective tillers among the different lines. However, under drought stress, compared with Fielder plants, the number of effective tillers in TaPHL2-overexpressing wheat plants was significantly reduced, while the number of effective tillers in TaPHL2 RNAi-interfered wheat plants was significantly increased. Specifically, under drought stress, the average number of effective tillers in Fielder plants was 8.33, the average number of effective tillers in T3 generation TaPHL2-overexpressing wheat plants (Ox-TaPHL2-1) was 6.57, the average number of effective tillers in T3 generation TaPHL2-overexpressing wheat plants (Ox-TaPHL2-2) was 6.29, the average number of effective tillers in TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-1) was 10.7, and the average number of effective tillers in TaPHL2 RNAi-interfered wheat plants (RNAi-TaPHL2-2) was 10.7.

[0165] Under normal and drought stress conditions, there was no significant difference in thousand-grain weight among the different strains.

[0166] Under normal conditions, the grain weight of wheat plants overexpressing TaPHL2 was significantly reduced, while the grain weight of wheat plants with TaPHL2 RNAi interference showed no significant difference compared to Fielder plants. Specifically, the average grain weight per Fielder plant was 9.78 g, the average grain weight per T3 generation overexpressing TaPHL2 (Ox-TaPHL2-1) was 9.13 g, the average grain weight per T3 generation overexpressing TaPHL2 (Ox-TaPHL2-2) was 8.81 g, the average grain weight per TaPHL2 RNAi interference wheat plant (RNAi-TaPHL2-1) was 10.16 g, and the average grain weight per TaPHL2 RNAi interference wheat plant (RNAi-TaPHL2-2) was 10.1 g. Under drought conditions, compared with Fielder plants, the grain weight of wheat plants overexpressing TaPHL2 decreased sharply, while the grain weight of wheat plants with TaPHL2 RNAi interference increased significantly. Specifically, the average grain weight per plant was 4.85 g for Fielder plants, 3.11 g for T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-1), 3.38 g for T3 generation TaPHL2 overexpressing wheat plants (Ox-TaPHL2-2), 8.27 g for TaPHL2 RNAi interference wheat plants (RNAi-TaPHL2-1), and 8.3 g for TaPHL2 RNAi interference wheat plants (RNAi-TaPHL2-2).

[0167] Example 3: Subcellular localization analysis of TaPHL2

[0168] I. Construction of the TaPHL2-GFP vector

[0169] 1. Using the PCR product obtained in step four of Example 1 as a template, PCR amplification was performed using TaPHL2-GFP-F and TaPHL2-GFP-R to obtain the PCR product. This product contains the full-length cDNA of TaPHL2 and carries the homologous sequence of the GFP vector. Specific primers are as follows:

[0170] TaPHL2-GFP-F: 5'-TATCTCTAGAGGATCCATGTTCCCCCCTGGCCT-3';

[0171] TaPHL2-GFP-R: 5'-TGCTCACCATGGATCCTCAGCAGGATGACTTGC-3'.

[0172] 2. The 16318hGFP vector was digested with BamHI enzyme, and the full-length cDNA of TaPHL2 was ligated into the 16318hGFP vector using homologous recombinase to obtain the recombinant plasmid TaPHL2-GFP.

[0173] II. Preparation of wheat protoplasts

[0174] 1. Prepare 15 mL of enzymatic hydrolysate (see Table 1 for the formula) and place it in a small beaker.

[0175] 2. Take 10 healthy wheat seedlings (about 1 week old), cut the part above the stem into thin strips, and place them in the enzyme hydrolysate.

[0176] 3. Wrap the small beaker with aluminum foil, evacuate it for 15 minutes, and then place it in the dark on a shaker at 25℃ and 50rpm for about 5 hours until the leaf cells are completely lysed.

[0177] 4. Filter the lysis solution through a sieve (100 mesh) to remove impurities. Dispense the filtered solution (the tip of the pipette should be cut off by about 3 cm) into 2.0 ml centrifuge tubes and centrifuge at 100 g, 4 °C for 2 min with an acceleration of 2.

[0178] 5. Discard the supernatant, gently mix the protoplasts with pre-cooled W5 solution, centrifuge at 100g, 4℃ for 1min, with acceleration set to 2.

[0179] 6. Discard the supernatant, gently mix the protoplasts with pre-cooled W5 solution, and let stand on ice for 30 minutes.

[0180] 7. Centrifuge at room temperature, 100g for 1 min, with acceleration set to 2, and discard the supernatant. Gently suspend each precipitate in 0.5 ml of MMg solution (this step and the following operations should be performed at 23°C).

[0181] 8. Add 20 μg of plasmid to 100 μl of protoplasts. Gently mix, then add 110 μl of PEG / Ca solution and mix gently. Place in a 25°C heating block and transform for 25 min in the dark. The plasmid used is the recombinant plasmid TaPHL2-GFP, which expresses the fusion protein of TaPHL2 and GFP, and is initiated by a strong 35S promoter and terminated by a strong NOS terminator.

[0182] 9. Add 440 μL of W5 solution to terminate the reaction. Centrifuge at 23°C, 100 g for 1 min, with an acceleration of 2.

[0183] 10. Discard the supernatant, add 1 mL of W5 solution and mix gently. Incubate in a 23°C incubator in the dark for 10-16 hours.

[0184] 11. Observe the green fluorescence under a confocal microscope.

[0185] Location results as follows Figure 9 As shown, the results indicate that TaPHL2-GFP is located in the cell nucleus.

[0186] 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. Application of inhibiting TaPHL2 protein-coding gene expression in any of the following (d1)-(d2): (d1) Improve wheat drought resistance; (d2) Breeding transgenic wheat with improved drought resistance; The amino acid sequence of the TaPHL2 protein is shown in Sequence 2; The method for inhibiting the expression of the TaPHL2 protein-coding gene is RNAi.

2. A method for breeding transgenic wheat with improved drought resistance, comprising the step of reducing the content of TaPHL2 protein in recipient wheat to obtain transgenic wheat; wherein the transgenic wheat has higher drought resistance than the recipient wheat; and the amino acid sequence of the TaPHL2 protein is shown in Sequence 2.

3. The method according to claim 2, characterized in that: RNAi was used to reduce the content of TaPHL2 protein in the recipient wheat.

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