Application of TaPHL2 protein and related biological materials thereof in regulation and control of growth and development and drought resistance of wheat

Abstract

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

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically relates to the application of TaPHL2 protein and its related biological materials in regulating the growth and development of wheat and drought resistance. Background Art

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

[0003] During the process of evolution, plants have gradually evolved a complex drought-resistant and damage-avoiding mechanism, including rapid growth before drought stress, advancing the reproductive stage to avoid the impact of drought; increasing the internal water content and water absorption capacity of plants to maintain the water content of plants during drought periods and avoid tissue damage; enhancing plant drought tolerance, and enhancing the tolerance and antioxidant capacity of plant cells during low-water periods in arid environments. At present, the research on plant stress resistance has gradually penetrated into the cellular and molecular levels, combined with genetics and genetic engineering research, to explore the use of biotechnology to improve the growth characteristics of plants and thereby improve the adaptability of plants to adversity.

[0004] The root system is an important organ for plants to absorb nutrients and water, and is closely related to the drought resistance and growth of plants. Under drought stress conditions, plants will inhibit the growth of above-ground parts to reduce water loss; at the same time, promote the growth of the root system structure to obtain more water and nutrients from the soil. Analyzing the regulatory mode of plant drought resistance and discovering effective drought-resistant regulatory genes are the key issues in cultivating drought-resistant crops. Improving the stress resistance of plants through gene transformation and knockout means provides a feasible solution for cultivating drought-resistant plants. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of TaPHL2 protein and its related biological materials in regulating the growth and development of wheat and drought resistance.

[0006] In order to achieve the above purpose, the present invention first provides a new use of TaPHL2 protein or its related biological materials.

[0007] The present invention provides the use of TaPHL2 protein or its related biological materials in any one of the following 1)-5):

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

[0009] 2) Regulating the plant height of wheat;

[0010] 3) Regulating the yield of wheat;

[0011] 4) Regulating the drought resistance of wheat;

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

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

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

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

[0016] (a3) A protein related to the number of lateral roots and / or plant height and / or yield and / or drought resistance of wheat obtained by substituting and / or deleting and / or adding one or several amino acid residues to (a1);

[0017] (a4) A protein having more than 98% identity with (a1) and related to the number of lateral roots and / or plant height and / or yield and / or drought resistance of wheat.

[0018] In the protein described in the above (a2), the tag refers to a polypeptide or protein that is fused and expressed together with the target protein by using in vitro DNA recombination technology for the purpose of facilitating the expression, detection, tracing, and / or purification of the target protein. The tag can 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 the one or several amino acid residues is the substitution and / or deletion and / or addition of no more than 10 amino acid residues or the substitution and / or deletion and / or addition of no more than 9 amino acid residues or the substitution and / or deletion and / or addition of no more than 8 amino acid residues or the substitution and / or deletion and / or addition of no more than 7 amino acid residues or the substitution and / or deletion and / or addition of no more than 6 amino acid residues or the substitution and / or deletion and / or addition of no more than 5 amino acid residues or the substitution and / or deletion and / or addition of no more than 4 amino acid residues or the substitution and / or deletion and / or addition of no more than 3 amino acid residues or the substitution and / or deletion and / or addition of no more than 2 amino acid residues or the 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 by using a homology search site on the Internet, such as the BLAST web page of the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, 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, the identity value (%) of a pair of amino acid sequences can be calculated.

[0021] In the above application, 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 any one of the following (A1) or (A2):

[0023] (A1) the DNA molecule shown in Sequence 1;

[0024] (A2) a DNA molecule having more than 75% identity with (A1) and encoding the TaPHL2 protein.

[0025] Those of ordinary skill in the art can easily use known methods, such as directed evolution and point mutation methods, to mutate the nucleotide sequence encoding the TaPHL2 protein of the present invention. Those artificially modified nucleotides having 75% or higher identity with the TaPHL2 nucleotide sequence isolated from the present invention, as long as they encode the TaPHL2 protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0026] As used herein, the term "identity" refers to sequence similarity with a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein consisting of the amino acid sequence shown in the coding sequence 2 of the present invention. Identity can be evaluated by the naked eye or by 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, which DNA may not only include a promoter that initiates TaPHL2 transcription, but also include a terminator that terminates TaPHL2 transcription. Further, the expression cassette may also include enhancer sequences.

[0028] The vector may be a plasmid, cosmid, phage or viral vector.

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

[0030] To achieve the above object, the present invention also provides a new use of a substance that inhibits the above TaPHL2 protein.

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

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

[0033] (d2) Regulating the plant height of wheat;

[0034] (d3) Regulating the drought resistance of wheat;

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

[0036] Furthermore, the substance that inhibits the above TaPHL2 protein includes a substance that inhibits the activity of the above TaPHL2 protein or a substance that reduces the content of the above TaPHL2 protein.

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

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

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

[0040] The substance that knocks out the TaPHL2 gene can be a substance that in any way enables the host cell not to produce the functional protein product of this gene. Specific ways include removing all or part of the coding gene sequence, introducing mutations so that no functional protein is produced, removing or altering regulatory components (such as promoter editing) so that the coding gene sequence is not transcribed, preventing translation by binding to mRNA, etc. Usually, knockout is carried out at the genomic DNA level, so that the offspring of the cell also permanently carry the knockout.

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

[0042] In any of the above applications, the regulation can be an increase or a decrease. The yield can be the grain weight per plant.

[0043] To achieve the above object, the present invention also provides a method for cultivating 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; the lateral root number and / or yield and / or drought resistance of the transgenic wheat are lower than those of the recipient wheat, and the plant height of the transgenic wheat is higher than that of the recipient wheat.

[0045] Furthermore, the fact that the yield of the transgenic wheat is lower than that of the recipient wheat is manifested as the grain weight per plant of the transgenic wheat being lower than that of the recipient wheat.

[0046] The fact that the drought resistance of the transgenic wheat is lower than that of the recipient wheat is manifested in any one of the following (x1)-(x7):

[0047] (x1) Under controlled water treatment (such as in soil with a water content of 250%, 350%, or 450%), the fresh weight of the above-ground part of the transgenic wheat is lower than that of the recipient wheat;

[0048] (x2) Under controlled water treatment (such as 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 (such as 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 (such as in soil with a water 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 (such as in soil with a water content of 30%), the plant height of the transgenic wheat is lower than that of the recipient wheat;

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

[0053] (x7) Under drought stress (such as in soil with a water 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 the recipient wheat is to overexpress TaPHL2 protein in the recipient wheat.

[0055] Furthermore, the method of overexpression is to introduce the coding gene of TaPHL2 protein into the recipient wheat.

[0056] To achieve the above object, the present invention finally provides a method for cultivating 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 a transgenic plant; the lateral root number and / or drought resistance of the transgenic wheat are higher than those of the recipient wheat, and the plant height of the transgenic wheat is lower than that of the recipient wheat.

[0058] Further, the higher drought resistance of the transgenic wheat than that of the recipient wheat is reflected in any one of the following (y1)-(y7):

[0059] (y1) Under controlled water treatment (such as in soil with water content of 250%, 350% or 450%), the fresh weight of the above-ground part of the transgenic wheat is higher than that of the recipient wheat;

[0060] (y2) Under controlled water treatment (such as in soil with 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 (such as in soil with water content of 250%, 350% or 450%), the proline content of the transgenic wheat is higher than that of the recipient wheat;

[0062] (y4) Under controlled water treatment (such as in soil with water 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 (such as in soil with water content of 30%), the plant height of the transgenic wheat is higher than that of the recipient wheat;

[0064] (y6) Under drought stress (such as in soil with water content of 30%), the effective tiller number of the transgenic wheat is higher than that of the recipient wheat;

[0065] (y7) Under drought stress (such as in soil with water content of 30%), the grain weight per plant of the transgenic wheat is higher than that of the recipient wheat.

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

[0067] The above-mentioned substance that inhibits TaPHL2 protein also belongs to the protection scope of the present invention.

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

[0069] The present invention proves through experiments that by introducing the TaPHL2 gene into wheat for overexpression, the obtained transgenic wheat has a reduced number of lateral roots, reduced yield and drought resistance, and increased plant height compared with the recipient wheat Fielder; the RNAi-interfered wheat has an increased number of lateral roots and drought resistance and a reduced plant height compared with the recipient wheat Fielder. The protein and gene provided by the present invention provide a basis for artificially controlling the root system structure, plant type, yield of wheat, and improving the drought resistance of wheat, and will play an important role in cultivating plants with enhanced stress resistance. Brief Description of the Drawings

[0070] Figure 1Schematic diagram for constructing overexpression and RNAi interference vectors of TaPHL2.

[0071] Figure 2 PCR positive detection of overexpressed TaPHL2 in wheat.

[0072] Figure 3 qRT-PCR expression level detection of overexpressed TaPHL2 in wheat.

[0073] Figure 4 PCR positive detection of TaPHL2-RNAi wheat.

[0074] Figure 5 qRT-PCR expression level detection of TaPHL2-RNAi wheat.

[0075] Figure 6 Phenotype identification of root architecture and root length measurement of TaPHL2 overexpressed and TaPHL2 RNAi interfered wheat.

[0076] Figure 7 Phenotype identification at seedling stage and physiological index measurement of TaPHL2 overexpressed and TaPHL2 RNAi interfered wheat under controlled water conditions.

[0077] Figure 8 Plant type identification and yield trait analysis of TaPHL2 overexpressed and TaPHL2 RNAi interfered wheat under normal and drought stress conditions.

[0078] Figure 9 Subcellular localization of TaPHL2. Specific implementation manners

[0079] The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all obtained from regular biochemical reagent stores unless otherwise specified. In the following examples, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0080] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0081] The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0082] In the following examples, all quantitative tests are set with three repeated experiments, and the results are averaged.

[0083] In the following examples, %, unless otherwise specified, are all mass percentages.

[0084] The common wheat (Triticum aestivum L.) variety Xiaobaimai in the following examples is recorded in the literature "Sun Haitao et al., Screening of Interaction Proteins of Wheat TaDREB6 Transcription Factor, Scientia Agricultura Sinica, 2011, 44(22): 4740-4747.", and the public can obtain it from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes; the public can also obtain it from the National Genebank (accession number ZM242).

[0085] The wheat variety Fielder in the following examples is recorded 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.", and the public can obtain it from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0086] The pWMB110 vector in the following examples is recorded 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", and the public can obtain it from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot 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, Enzymatic Hydrolysate

[0089] Component Content (20 ml) Cellulase 0.2g Pectinase 0.08g Mannitol 0.4M MES 20 mM 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] Component Content (1 L) NaCl 9.0g <![CDATA[Calcium chloride]]> 8.4g KCl 0.37g Glucose 0.9g MES 0.3g

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

[0094] Table 3, PEG Solution

[0095] Component Content PEG4000 1g 0.8 M Aqueous Mannitol 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 with KOH.

[0100] Example 1, Cloning of TaPHL2

[0101] I. Treatment of Plant Materials

[0102] The whole three-leaf-stage seedlings of common wheat (Triticum aestivum cv. Xiaobaimai) grown hydroponically for about 10 days were quickly frozen with liquid nitrogen and stored at -80°C for later use.

[0103] II. Extraction of Total RNA

[0104] The total RNA of the leaves of the treated common wheat seedlings obtained in Step I was extracted using the Trizol method (TianGen).

[0105] III. Obtaining of cDNA

[0106] cDNA was synthesized using the SMART method, and the first-strand cDNA was synthesized using reverse transcriptase XL (AMV).

[0107] IV. PCR Amplification

[0108] Using the cDNA obtained in Step III as a template, PCR amplification was performed with TaPHL2-F and TaPHL2-R to obtain a 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 products obtained in Step 4 were subjected to 1.0% agarose gel electrophoresis and the PCR products were sequenced. The results showed that: Sequence 1 in the sequence listing was obtained by PCR amplification. The gene shown in Sequence 1 in the sequence listing was named TaPHL2 gene, the protein encoded by this gene was named TaPHL2 protein, and the amino acid sequence of this protein was Sequence 2 in the sequence listing, which consisted of 255 amino acids.

[0113] The above-mentioned Sequence 1 can also be obtained by artificial synthesis.

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

[0115] I. Obtaining of TaPHL2 transgenic wheat

[0116] 1. Construction of overexpression TaPHL2 recombinant vector

[0117] (1) Amplification of TaPHL2 gene

[0118] Using the PCR products obtained in Step 4 of Example 1 as a template, amplification was carried out using primers TaPHL2-110F and TaPHL2-110R to obtain PCR products. The primer sequences are as follows (the ends of the primers are the upstream and downstream sequences of the recognition site of vector BamHI for enzyme digestion respectively, so as to ligate the TaPHL2 fragment into the pWMB110 vector by homologous recombination method, and the recognition site of BamHI for enzyme digestion is underlined):

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

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

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

[0122] The PCR products were recovered and purified using 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 products and the vector backbone were ligated using ClonExpressⅡ One Step Cloning Kit (Vazyme, Code No: C112) to obtain the ligation products.

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

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

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

[0128] (1) Beijing Aoke Dingsheng Biotechnology Co., Ltd. was commissioned to artificially synthesize the fragment shown in Sequence 3, which sequentially includes an SmaI digestion site, a nucleotide sequence shown at positions 180 - 442 of Sequence 1 with a size of 262 bp, a maize alcohol dehydrogenase (Adh) gene sequence with a size of 146 bp (as an intron), a reverse complementary sequence of the nucleotide sequence shown at positions 180 - 442 of Sequence 1 with a size of 262 bp, and a SacI digestion site.

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

[0130] The recombinant vector TaPHL2-pWMB110-RNAi for RNAi interference of TaPHL2 is a vector 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. The structural schematic diagram of the recombinant vector TaPHL2-pWMB110-RNAi for RNAi interference of TaPHL2 is as Figure 1 shown in B.

[0131] 3. Construction of recombinant bacteria

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

[0133] 4. Obtaining and detection of TaPHL2 transgenic wheat

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

[0135] (2) The bacterial liquid obtained in Step (1) was transferred to YEP liquid medium (containing 50 μg / ml rifampicin and kanamycin) and cultured at 28 °C and 200 rpm for about 14 hours (the OD of the bacterial liquid 600 reached 1.5 - 3.0).

[0136] (3) The bacterial cells obtained in Step (2) were collected, centrifuged at 4 °C and 4000 g for 10 min, and diluted with 10% sucrose (containing 0.02% silwet) to an OD 600 of about 1.0.

[0137] (4) In a laminar flow hood, the Agrobacterium colonies were picked with a 1 ml syringe needle and injected into the young embryos of wheat variety Fielder. Then the injected young embryos were cultured under sterile and high humidity conditions until tissue culture wheat seedlings grew out, and the wheat seedlings were continuously cultured until the T0 generation of TaPHL2 overexpressing and RNAi interfering wheat seeds were harvested.

[0138] The T0 generation of TaPHL2 overexpressing and RNAi interfering wheat seeds were respectively sown and self-crossed until the T3 generation of wheat was obtained.

[0139] (5) Detect positive TaPHL2-overexpressing and TaPHL2-RNAi wheat plants using qRT-PCR and PCR.

[0140] The specific steps for PCR detection are as follows: Extract the DNA of different TaPHL2-overexpressing and TaPHL2-RNAi wheat lines and the recipient wheat leaves respectively, and use the extracted DNA as a template for PCR amplification with different primers.

[0141] Among them, the primer sequences 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 for PCR detection of TaPHL2-RNAi 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 Figure 2 and Figure 4 shown. The results indicate that specific bands can be detected in the positive lines, and no specific bands are detected in the recipient Fielder, indicating that the above are all positive lines.

[0148] The specific steps for qRT-PCR detection are as follows: Extract the RNA of different TaPHL2-overexpressing and TaPHL2-RNAi wheat lines and the recipient wheat leaves respectively, reverse transcribe it into cDNA, and perform qRT-PCR detection 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 Figure 3 and Figure 5As shown in the figure, the results showed that the expression levels of TaPHL2 in the TaPHL2 overexpression lines were significantly higher than those in Fielder. Among them, the expression levels of TaPHL2 in lines 2 and 4 were the highest, which were named Ox-TaPHL2-1 and Ox-TaPHL2-2 respectively for subsequent experimental analysis. The expression levels of TaPHL2 in RNAi lines 1 and 5 were significantly lower than those in Fielder, and the expression levels were similar( Figure 5 ), which were named RNAi-TaPHL2-1 and RNAi-TaPHL2-2 respectively for subsequent experimental analysis.

[0152] II. Root structure identification and stress tolerance analysis of TaPHL2 transgenic wheat

[0153] The T3 generation of TaPHL2 overexpression wheat seeds numbered Ox-TaPHL2-1 and Ox-TaPHL2-2, TaPHL2 RNAi transgenic wheat seeds numbered RNAi-TaPHL2-1 and RNAi-TaPHL2-2, and Fielder seeds were grown normally in a hydroponic tray for 7 days and 10 days to observe the root structure, and the main root length and the number of lateral roots were counted respectively; the wheat seeds with consistent germination were planted in soils with different absolute water contents (250%, 350%, 450%) for 20 days and 25 days, and the growth status of the plants was observed after 20 days and 25 days, and the relevant physiological indexes (fresh weight of the above-ground part, CAT content, MDA content, proline content) were measured after 25 days of growth; in a greenhouse environment (16 hours of light, 8 hours of darkness, temperature 25°C), the wheat seeds with consistent germination were planted under normal water conditions with an average absolute water content of 70% and under drought stress conditions with an average absolute water content of 30% during the whole growth period. After growing to complete maturity (100 days), the plant type (plant height, effective tiller number) and yield traits (1000-grain weight, grain weight per plant) of wheat were identified. The specific detection methods for physiological indexes are as follows:

[0154] Determination of CAT content: The CAT content was determined using the Catalase (CAT) kit from Suzhou Comin Biotechnology Co., Ltd. (Comin Company, Code No: EC1.11.1.6). The specific steps are as follows: Weigh about 0.1 g of tissue, add 1 ml of extraction solution, and homogenize in an ice bath. Centrifuge at 8000 g and 4°C for 10 min, take the supernatant, and place it on ice for further measurement. Preheat the spectrophotometer for more than 30 min, adjust the wavelength to 240 nm, and zero with distilled water. Incubate the CAT detection working solution in a water bath at 25°C for 10 min before measurement. Take 1 mL of the CAT detection working solution into a 1 mL quartz cuvette, and then add 35 μL of the sample, mix well for 5 s; immediately measure the initial absorbance value A1 at 240 nm and the absorbance value A2 after 1 min at room temperature, and calculate the CAT content.

[0155] Determination of MDA content: The MDA content was determined using the Malondialdehyde (MDA) test kit from Suzhou Comin Biotechnology Co., Ltd. (Comin Co., Code No: MDA-2-Y). The specific steps are as follows: Weigh approximately 0.1 g of tissue, add 1 ml of extraction solution, and homogenize in an ice bath. Centrifuge at 8000 g for 10 min at 4 °C, take the supernatant, and place it on ice for further measurement. Pipette 0.6 ml of Reagent 1 into a 1.5 ml centrifuge tube, then add 0.2 ml of the sample and mix well. Incubate in a water bath at 95 °C for 30 min, cool in an ice bath, and centrifuge at 10000 g for 10 min at 25 °C. Pipette the supernatant into a 200 μl glass cuvette, measure the absorbance at 532 nm and 600 nm, denoted as A532 and A600, and calculate the MDA content.

[0156] Determination of proline content: The proline (PRO) content was determined using the proline (PRO) content test kit from Suzhou Comin Biotechnology Co., Ltd. (Comin Co., Code No: PRO-2-Y). Weigh approximately 0.1 g of tissue, add 1 mL of extraction solution, and homogenize in an ice bath; then place it in a shaker at 90 °C for extraction for 10 min; centrifuge at 10000 g for 10 min at 25 °C, take the supernatant, and measure after cooling. Warm up the spectrophotometer for more than 30 min, adjust the wavelength to 520 nm, and zero with distilled water. Take 0.5 ml of the sample + 0.5 ml of Reagent 1 + 0.5 ml of Reagent 2 in a capped test tube, incubate in a boiling water bath for 30 min, and shake once every 10 min. After cooling, add 1 ml of Reagent 3 to the test tube, shake for 30 s, let it stand for a while to transfer the pigment to Reagent 3; pipette 0.8 ml - 1 ml of the upper layer solution into a 1 ml glass cuvette, and colorimetric at a wavelength of 520 nm, record the absorbance value A, and calculate the proline content.

[0157] The results of root system structure identification are as Figure 6Shown as follows: Under normal conditions, compared with Fielder plants, the number of lateral roots of TaPHL2-overexpressing wheat plants was significantly reduced, while the number of lateral roots of TaPHL2 RNAi wheat plants was significantly increased. Among them, after 7 days of normal growth, the average number of lateral roots of Fielder plants was 64.7, the average number of lateral roots of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 45.41, the average number of lateral roots of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 42.64, the average number of lateral roots of the TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 99.23, and the average number of lateral roots of the TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 93.7; after 10 days of normal growth, the average number of lateral roots of Fielder plants was 99.52, the average number of lateral roots of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 57.11, the average number of lateral roots of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 61.35, the average number of lateral roots of the TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 123.12, and the average number of lateral roots of the TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 130.12.

[0158] The results of stress tolerance analysis are as Figure 7 Shown as follows: Under water control treatment conditions, the growth of TaPHL2-overexpressing wheat plants was significantly inhibited compared with Fielder plants, with wilting leaves and death of some plants, and the fresh weight of the above-ground part was significantly reduced. In contrast, the leaves of TaPHL2 RNAi wheat plants maintained higher growth vitality compared with Fielder plants, and the fresh weight of the above-ground part was significantly increased. In addition, the activities of catalase (CAT) and the content of proline in TaPHL2-overexpressing wheat lines were significantly lower than those of the Fielder material, while the content of malondialdehyde (MDA) was significantly higher than that of the Fielder material, indicating that the degree of cell damage in TaPHL2-overexpressing wheat lines increased and the antioxidant capacity decreased significantly under water control treatment conditions; while the activities of catalase (CAT) and the content of proline in TaPHL2 RNAi wheat lines were significantly higher than those of the Fielder material, and the content of MDA was significantly lower than that of the Fielder material, indicating that the degree of cell damage in TaPHL2 RNAi wheat lines decreased significantly and the antioxidant capacity increased significantly under water control treatment conditions.

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

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

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

[0162] Under the condition of water content of 250%, the average proline content of Fielder plants was 522.31, the average proline content of the T3 generation of TaPHL2 overexpressing wheat plants numbered Ox-TaPHL2-1 was 485.33, the average proline content of the T3 generation of TaPHL2 overexpressing wheat plants numbered Ox-TaPHL2-2 was 468.42, the average proline content of TaPHL2 RNAi interfering wheat plants numbered RNAi-TaPHL2-1 was 550.57, and the average proline content of TaPHL2 RNAi interfering wheat plants numbered RNAi-TaPHL2-2 was 573.46. Under the condition of water content of 350%, the average proline content of Fielder plants was 386.13, the average proline content of the T3 generation of TaPHL2 overexpressing wheat plants numbered Ox-TaPHL2-1 was 361.25, the average proline content of the T3 generation of TaPHL2 overexpressing wheat plants numbered Ox-TaPHL2-2 was 353.11, the average proline content of TaPHL2 RNAi interfering wheat plants numbered RNAi-TaPHL2-1 was 521.88, and the average proline content of TaPHL2 RNAi interfering wheat plants numbered RNAi-TaPHL2-2 was 512.47. Under the condition of water content of 450%, the average proline content of Fielder plants was 575.43, the average proline content of the T3 generation of TaPHL2 overexpressing wheat plants numbered Ox-TaPHL2-1 was 533.47, the average proline content of the T3 generation of TaPHL2 overexpressing wheat plants numbered Ox-TaPHL2-2 was 543.11, the average proline content of TaPHL2 RNAi interfering wheat plants numbered RNAi-TaPHL2-1 was 684.15, and the average proline content of TaPHL2 RNAi interfering wheat plants numbered 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 Figure 8Shown as follows: Under normal conditions, compared with Fielder plants, the plant height of TaPHL2-overexpressing wheat plants was significantly increased, while that of TaPHL2 RNAi wheat lines decreased; under drought stress conditions, the plant height of TaPHL2-overexpressing wheat plants decreased sharply, while that of TaPHL2 RNAi wheat plants decreased very little. Among them, under normal growth conditions, the average plant height of Fielder plants was 79.03 cm, the average plant height of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 83.68 cm, the average plant height of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 83.12 cm, the average plant height of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 77.26 cm, and the average plant height of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 76.55 cm; under drought stress conditions, the average plant height of Fielder plants was 75.34 cm, the average plant height of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 67.28 cm, the average plant height of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 69.69 cm, the average plant height of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 74.14 cm, and the average plant height of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 77.26 cm.

[0164] Under normal conditions, there was no significant difference in the number of effective tillers among the lines; under drought stress conditions, compared with Fielder plants, the number of effective tillers of TaPHL2-overexpressing wheat plants decreased significantly, while that of TaPHL2 RNAi wheat plants increased significantly. Among them, under drought stress conditions, the average number of effective tillers of Fielder plants was 8.33, the average number of effective tillers of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 6.57, the average number of effective tillers of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 6.29, the average number of effective tillers of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 10.7, and the average number of effective tillers of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 10.7.

[0165] Under normal conditions and drought stress conditions, there was no significant difference in the 1000-grain weight among the lines.

[0166] Under normal conditions, the grain weight per plant of TaPHL2-overexpressing wheat plants decreased significantly, while there was no significant difference in the grain weight per plant between TaPHL2 RNAi wheat plants and Fielder. Among them, the average grain weight per plant of Fielder plants was 9.78 g, the average grain weight per plant of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 9.13 g, the average grain weight per plant of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 8.81 g, the average grain weight per plant of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 10.16 g, and the average grain weight per plant of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 10.1 g. Under drought conditions, compared with Fielder plants, the grain weight per plant of TaPHL2-overexpressing wheat plants decreased sharply, while the grain weight per plant of TaPHL2 RNAi wheat plants increased significantly. Among them, the average grain weight per plant of Fielder plants was 4.85 g, the average grain weight per plant of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-1 was 3.11 g, the average grain weight per plant of the T3 generation TaPHL2-overexpressing wheat plants numbered Ox-TaPHL2-2 was 3.38 g, the average grain weight per plant of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-1 was 8.27 g, and the average grain weight per plant of TaPHL2 RNAi wheat plants numbered RNAi-TaPHL2-2 was 8.3 g.

[0167] Example 3. Subcellular localization analysis of TaPHL2

[0168] I. Construction of TaPHL2-GFP vector

[0169] 1. Using the PCR product obtained in Step 4 of Example 1 as a template, PCR amplification was performed using TaPHL2-GFP-F and TaPHL2-GFP-R to obtain a PCR product, which contained the full-length cDNA of TaPHL2 and carried the GFP vector homologous sequence. The specific primers are as follows:

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

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

[0172] 2. Digest the 16318hGFP vector with BamHI enzyme, and ligate the full-length cDNA of TaPHL2 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 enzyme digestion solution (formula shown in Table 1) and place it in a small beaker.

[0175] 2. Take 10 wheat seedlings with good growth (grown for about 1 week), cut the parts above the stems into thin strips, and place them in the enzyme digestion solution.

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

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

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

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

[0180] 7. Centrifuge at room temperature, 100 g for 1 min with the acceleration set to 2, and discard the supernatant. Suspend the precipitate in each tube gently with 0.5 ml of MMg solution (this step and the following operations are carried out at 23 °C).

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

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

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

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

[0185] The positioning result is as Figure 9 shown, and the result shows that TaPHL2-GFP is located in the nucleus.

[0186] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application is intended to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application and are made with conventional techniques known in the art. The application of some basic features can be made within the scope of the appended claims below.

Claims

1. Use of the TaPHL2 protein or its related biological material in any one of the following 1)-5): 1) Regulating the number of lateral roots of wheat; 2) Regulating the plant height of wheat; 3) Regulating the yield of wheat; 4) Regulating the drought resistance of wheat; 5) Cultivating transgenic wheat with altered number of lateral roots and / or plant height and / or yield and / or drought resistance; The TaPHL2 protein is any one of the following proteins (a1)-(a4): (a1) The protein shown in Sequence 2; (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) A protein related to the number of lateral roots and / or plant height and / or yield and / or drought resistance of wheat obtained by substituting and / or deleting and / or adding one or several amino acid residues to (a1); (a4) A protein having more than 98% identity with (a1) and related to the number of lateral roots and / or plant height and / or yield and / or drought resistance of wheat.

2. The application according to claim 1, wherein: The related 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.

3. The application according to claim 2, wherein: The nucleic acid molecule encoding the TaPHL2 protein is any one of the following DNA molecules (A1) or (A2): (A1) The DNA molecule shown in Sequence 1; (A2) A DNA molecule having more than 75% identity with (A1) and encoding the TaPHL2 protein.

4. The application according to any one of claims 1 to 3, characterized in that: The regulation of wheat yield is the regulation of the grain weight per plant of wheat.

5. Use of a substance that inhibits the TaPHL2 protein described in claim 1 in any one of the following (d1)-(d4): (d1) Regulating the number of lateral roots of wheat; (d2) Regulating the plant height of wheat; (d3) Regulating the drought resistance of wheat; (d4) Cultivating transgenic wheat with altered number of lateral roots and / or plant height and / or drought resistance.

6. A method for cultivating transgenic wheat with altered number of lateral roots and / or plant height and / or yield and / or drought resistance, including the step of increasing the activity and / or content of the TaPHL2 protein described in claim 1 in the recipient wheat to obtain transgenic wheat; the number of lateral roots and / or yield and / or drought resistance of the transgenic wheat is lower than that of the recipient wheat, and the plant height of the transgenic wheat is higher than that of the recipient wheat.

7. The method according to claim 6, wherein: The method for increasing the activity and / or content of the TaPHL2 protein described in claim 1 in the recipient wheat is to overexpress the TaPHL2 protein described in claim 1 in the recipient plant.

8. A method for cultivating transgenic wheat with altered number of lateral roots and / or plant height and / or drought resistance, including the step of reducing the activity and / or content of the TaPHL2 protein described in claim 1 in the recipient plant to obtain transgenic plants; the number of lateral roots and / or drought resistance of the transgenic wheat is higher than that of the recipient wheat, and the plant height of the transgenic wheat is lower than that of the recipient wheat.

9. The method according to claim 8, wherein: The method according to claim 8, characterized in that: the method for reducing the activity and / or content of the TaPHL2 protein described in claim 1 in the recipient plant is to introduce a substance that inhibits the TaPHL2 protein described in claim 1 into the recipient plant. A substance that inhibits the TaPHL2 protein described in claim 1.

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