Application of TaDOF4.7-B gene in improving wheat regeneration ability and lodging resistance

Abstract

The present invention discloses the application of TaDOF4.7-B gene in improving wheat regeneration ability and lodging resistance, and belongs to the technical field of plant genetic engineering. The present invention has discovered a new key regulatory gene capable of regulating wheat regeneration ability. By overexpressing TaDOF4.7-B gene, the regeneration efficiency of wheat immature embryos can be significantly improved; moreover, the plant height of TaDOF4.7-B transgenic plants is significantly reduced compared with Fielder, and can effectively resist lodging; while other agronomic traits such as effective tiller number, spike length, spikelet number, effective spikelet number, grain length, grain width and 100-grain weight have no obvious differences, indicating that TaDOF4.7-B transgenic plants can significantly reduce plant height without affecting other agronomic traits, which is conducive to achieving lodging resistance and stable yield of wheat.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of TaDOF4.7-B gene in improving wheat regeneration ability and lodging resistance. Background Art

[0002] Genetic transformation is a technique that uses recombinant DNA technology, cell tissue culture technology or germplasm system transformation technology to insert exogenous target genes or target DNA fragments into the genome of the recipient plant and obtain new plants through meiosis. Wheat is one of the world's main food crops, but because wheat is a hexaploid plant with many gene copies and poor tissue regeneration ability, there are many obstacles to wheat genetic transformation and in vitro regeneration, and its success rate of transformation and regeneration is lower than that of corn and rice. Therefore, it is necessary to develop a method that is perfectly adapted to wheat genetic transformation.

[0003] In recent years, significant progress has been made in wheat genetic transformation. Wang et al. promoted efficient transformation of wheat by overexpressing the TaWOX5 gene (Wang et al., 2022b). Yu et al. showed that TaLAX1 can promote genetic transformation and genome editing of wheat by activating the TaGRF-TaGIF1 gene, cytokinin biosynthesis and auxin response genes, and promote shoot regeneration (Yu et al., 2024). Although significant progress has been made in wheat transformation, efforts are still needed to develop efficient transformation technologies for cereal crops such as wheat and corn to improve transformation efficiency and regeneration capacity.

[0004] The DOF (DNA-binding with one finger) family is a plant-specific transcription factor that participates in a variety of plant-specific biological processes (Shuichi and Yanagisawa, 2015). The DOF family is very large. In the model plant Arabidopsis, 37 genes encoding DOF protein domains have been identified (Yanagisawa, 2002), and roughly the same number of DOF proteins have been identified in rice (Rojas-Gracia et al., 2019). To date, DOF family proteins have been reported to participate in different plant physiological processes and have functional diversity in plants. For example, in Arabidopsis, DOF protein (OBP1) regulates defense gene expression in response to salicylic acid and oxidative stress signals (Chen et al., 1996). In Arabidopsis, HCA2 (HIGHCAMBIAL ACTIVITY2), TMO6 (TARGET OF MONOPTEROS6), DOF2.1 and DOF6 can be highly expressed at the site of injury, thereby promoting wound healing and tissue regeneration. The DOF protein (NtBBF1) in tobacco induces the activation of the plant oncogene rolB in the apical meristem and vascular tissue to induce auxin expression (Baumann et al., 1999), and participates in the regulation of plant hormones. The DOF protein (PBF) in maize is a transcription factor that controls the endosperm-specific expression of storage proteins (Mena et al., 1998). These results show that DOF proteins play different but very important functions in the growth and development of plants. Therefore, the development of new genes related to wheat regeneration ability from the wheat DOF family is of great significance for wheat genetic breeding and trait improvement. Summary of the invention

[0005] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of TaDOF4.7-B gene in improving wheat regeneration ability and lodging resistance. The present invention has found that overexpression of TaDOF4.7-B gene can improve wheat regeneration ability; moreover, the plant height of transgenic plants overexpressing TaDOF4.7-B is reduced without affecting other ear and grain traits, achieving stable wheat yield while enhancing wheat lodging resistance.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] In a first aspect of the present invention, a use of the TaDOF4.7-B gene in improving the regeneration efficiency of wheat immature embryos is provided; the TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii) or iii):

[0008] i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1;

[0009] ii) a DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i);

[0010] iii) a DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii), and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0011] The term "identity" as used herein refers to sequence similarity to a natural nucleic acid sequence. Homology can be evaluated using computer software, such as the BLAST algorithm (Altschul et al. 1990. Journal of Molecular Biology 215: 403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90: 5873-5877).

[0012] In the above nucleic acid molecules, the 90% or more homology may be at least 90%, 92%, 93%, 95%, 96%, 98% or 99% homology.

[0013] The second aspect of the present invention provides the use of TaDOF4.7-B protein in improving the regeneration efficiency of wheat immature embryos.

[0014] In the above application, the TaDOF4.7-B protein is the protein shown in (A1) or (A2) below:

[0015] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0016] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0017] The third aspect of the present invention provides the use of a recombinant expression vector or genetically engineered bacteria containing the TaDOF4.7-B gene in improving the regeneration efficiency of wheat immature embryos.

[0018] A fourth aspect of the present invention provides a method for improving the regeneration efficiency of wheat immature embryos, comprising the following steps:

[0019] The TaDOF4.7-B gene is connected to an expression vector to construct a recombinant expression vector, and the recombinant expression vector is transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation;

[0020] Agrobacterium strains were used to infect wheat immature embryos to obtain wheat transgenic plants with improved regeneration efficiency.

[0021] In a fifth aspect of the present invention, the use of the TaDOF4.7-B gene described above in any one of the following (1) to (3) is provided:

[0022] (1) Regulating wheat plant height;

[0023] (2) Regulating wheat lodging resistance traits;

[0024] (3) Cultivate dwarf wheat varieties.

[0025] The sixth aspect of the present invention provides the use of the TaDOF4.7-B protein in the following (1) or (2):

[0026] (1) Regulating wheat plant height;

[0027] (2) Regulate wheat lodging resistance.

[0028] The seventh aspect of the present invention provides a recombinant expression vector or genetically engineered bacteria containing the TaDOF4.7-B gene for use in any one of the following (1) to (3):

[0029] (1) Regulating wheat plant height;

[0030] (2) Regulating wheat lodging resistance traits;

[0031] (3) Cultivate dwarf wheat varieties.

[0032] An eighth aspect of the present invention provides a method for improving lodging resistance of wheat, comprising the following steps:

[0033] The TaDOF4.7-B gene is exogenously transferred into the starting wheat plant to overexpress the TaDOF4.7-B gene, thereby obtaining a transgenic wheat plant, wherein the plant height of the transgenic wheat plant is lower than that of the starting wheat plant, and the lodging resistance is improved;

[0034] Alternatively, the expression of the TaDOF4.7-B gene in the wheat genome is upregulated to screen wheat plants with reduced plant height and improved lodging resistance.

[0035] In the above method, the method of exogenously transferring the TaDOF4.7-B gene into wheat includes: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

[0036] Beneficial effects of the present invention:

[0037] The present invention discovered a new key regulatory gene that can regulate the regeneration ability of wheat. By overexpressing the TaDOF4.7-B gene, the regeneration efficiency of wheat immature embryos can be significantly improved. Moreover, the plant height of the TaDOF4.7-B transgenic plants is significantly reduced compared with Fielder, and they can effectively resist lodging. While other agronomic traits such as the number of effective tillers, ear length, number of spikelets, number of effective spikelets, grain length, grain width and 100-grain weight have no obvious differences, indicating that the TaDOF4.7-B transgenic plants can significantly reduce plant height without affecting other agronomic traits, which is beneficial to achieving lodging resistance and stable yield of wheat. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Amino acid sequence alignment of TaDOF4.7A / B / D.

[0039] Figure 2 : Carrier diagram of PC186 no-load control and TaDOF4.7-B-OE.

[0040] Figure 3 :Comparison of the transformation process of Fielder by pUbi::TaDOF4.7-B and the control pC186 empty vector; in the figure, the upper part is the tissue culture process of Fielder wheat immature embryos transformed by pC186 empty vector, and the lower part is the tissue culture process of Fielder wheat immature embryos transformed by pUbi::TaDOF4.7-B. CIM: callus induction culture; SIM: differentiation culture.

[0041] Figure 4 :Identification of T0 generation plants of positive transgenic plants overexpressing TaDOF4.7-B; (A) Bar test strip test results of T0 generation transgenic plants; (B) PCR electrophoresis identification results of vector primers of T0 generation transgenic plants, F: negative control, PC: positive control, M: 2000bp Marker; (C) qRT-PCR expression detection results of T0 generation transgenic plants. Scale bar = 1 cm, *** indicates P < 0.001, ** indicates P < 0.01, * indicates P < 0.05, ns indicates no significant difference.

[0042] Figure 5 : PCR electrophoresis identification results of T1 generation vector primers of transgenic plants; F: negative control; PC: positive control; M: 2000bp Marker.

[0043] Figure 6 : qRT-PCR expression detection results of transgenic plants T1 generation. All are overexpression lines. *** indicates P < 0.001, ns indicates no significant difference.

[0044] Figure 7:TaDOF4.7-B-OE transgenic wheat plant height phenotype; (A) Comparison of wheat plant height between Fielder and TaDOF4.7-B-OE transgenic plants; (B) Comparison of plant height between Fielder and TaDOF4.7-B-OE transgenic plants T1#8 (C) Statistical results of wheat plant height, 10 plants were counted for each line of Fielder and transgenic plants. Error bars indicate standard error, *** indicates P<0.001.

[0045] Figure 8 :Agronomic traits of TaDOF4.7-B-OE transgenic wheat spikelets; (A) Observation of wheat spike length; (B) Statistical results of wheat spike length; (C) Statistical results of wheat spikelet number; (D) Statistical results of wheat effective spikelet number. Five individual plants were counted for each strain of Fielder and TaDOF4.7-B-OE transgenic plants, scale bar = 1 cm. Error bars indicate standard error, ns indicates no significant difference.

[0046] Fig. 9 :Grain traits of TaDOF4.7-B-OE transgenic wheat; (A) Observation of wheat grain morphology and statistical results of 100-grain weight; (B) Observation of wheat grain length and statistical results; (C) Observation of wheat grain width and statistical results. Five individual plants were counted for each strain of Fielder and TaDOF4.7-B-OE transgenic plants, scale bar = 1 cm. Error bars indicate standard error, ns indicates no significant difference. DETAILED DESCRIPTION

[0047] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0048] As mentioned earlier, unlike diploid crops, wheat is a hexaploid plant with a large number of gene copies and poor tissue regeneration ability. The technology of genetically modified organism breeding has developed relatively slowly in the grass crop wheat.

[0049] In view of this, the present invention conducted an in-depth study on the DOF family transcription factor TaDOF4.7, and performed sequence comparison analysis on the amino acid sequences of TaDOF4.7-A, TaDOF4.7-B, and TaDOF4.7-D ( Figure 1), and found that they have extremely high amino acid similarity and all contain conserved DOF protein domains, indicating that the three proteins of DOF family transcription factor TaDOF4.7 may have similar functions. Further, the immature embryos of Fielder wheat were used as explants for wheat genetic transformation, and the expression levels of TaDOF4.7-A / B / D in callus tissue were detected. It was found that the expression level of TaDOF4.7-B was higher than that of TaDOF4.7-A / D. From this analysis, TaDOF4.7-B may play a major role in improving the regeneration ability of wheat. Therefore, the TaDOF4.7-B gene was selected for subsequent experiments.

[0050] The nucleotide sequence of the TaDOF4.7-B gene is shown in SEQ ID NO.1, and is as follows:

[0051] ATGGAAGCTCCTCTGCACCAGCTGGCCCCCATGCAGCAAGCACTGCTCGCCCATCCCCAGAACTGCAAGCACGACGCCGCGGCGACGATGGCCGCGACGGACATGGCCTGCGTCCAGCAGCAGCAGCAGCTGTTGCAGCTGCAGCCGGCGGCGGCGAACCCGAACACGCCCTCGGCCGCGGCGCGGGAGCAGTGCCCGCGGTGCGCGTCGCACGACACCAAGTTCTGCTACTACAACAACTACAACACGTCGCAGCCGCGCCACTTCTGCCGTGCCTGCCGCCGCTACTGGACGCTCGGGGGCTCCCTCCGCAACGTCCCCATCGGGGGCTCCACCCGCAAGCGCCTGCGCCCGGCGCCGCAGCAGGCGATGCGCCGCCCGCCCGTCCACTTCGGCGCGCCTCCGCCACCGATGCCGGCGCAGTCGCACTCCCAGCAGGCTCCGCAAGGCGGACTTCTCAGCTCGCTGTTCGCGCTAGGCGGGGCGCCGCTGTTCGAGGGCCGCGTCGGGTTCGACCTCGGCCTCGGCCTGCCCGGGCTGAGCCAGGTGGGGCTCGGCGGCAGCGCCGGGGAGTTCGGCCTGCACTCCCTCGGGCTCCGGGGCGGCCATGCAGGGACGTCGGCGCCGATGCTCTGGCCAACTGCGTTCTTGGACAACGGCAATGTGGACACGTGGAAGGTGTCCGGCGGCGGGGCGGCTGCCATGTGGGCGCCGGAGTTCTCTTCTGCGCCGACGGTAGCACAGGTCGGCGGCAATGGCATGTTTCATGGCGGGGCCCAGATAATGGGACAACTGTGA。

[0052] The amino acid sequence of TaDOF4.7-B protein is shown in SEQ ID NO.2 as follows:

[0053] MEAPLHQLAPMQQALLAHPQNCKHDAAATMAATDMACVQQQQQLLQLQPAAANPNTPSAAAREQCPRCASHDTKFCYYNNYNTSQPRHFCRACRRYWTLGGSLRNVPIGGSTRKRLRPAPQQAMRRPPVHFGA PPPPMPAQSHSQQAPQGGLLSSLFALGGAPLFEGRVGFDLGLGLPGLSQVGLGGSAGEFGLHSLGLRGGHAGTSAPMLWPTAFLDNGNVDTWKVSGGGAAAMWAPEFSSAPTVAQVGGNGMFHGGAQIMGQL.

[0054] In order to study the function of the TaDOF4.7-B gene, the present invention amplifies the TaDOF4.7-B gene from the cDNA of the stem tip of Chinese spring wheat, constructs an overexpression vector with a DNA sequence containing the full-length CDS fragment of the TaDOF4.7-B gene, and then introduces the overexpression vector into Agrobacterium, and infects wheat embryos by Agrobacterium-mediated method, and then subcultures until a transgenic positive strain is obtained. During the subculture process, the inventors found that overexpression of TaDOF4.7-B can promote the regeneration frequency of wheat and improve the genetic transformation efficiency of wheat; moreover, compared with Fielder, the plant height of the transgenic wheat plant overexpressing TaDOF4.7-B is significantly reduced compared with Fielder, and can effectively resist lodging; while other agronomic traits such as effective tiller number, spike length, spikelet number, effective spikelet number, grain length, grain width and 100-grain weight have no significant differences. These results indicate that TaDOF4.7-B transgenic plants can significantly reduce plant height without affecting other agronomic traits, which is beneficial for achieving lodging resistance and stable yield of wheat.

[0055] In order to enable those skilled in the art to more clearly understand the technical solution of the present application, the technical solution of the present application will be described in detail below in conjunction with specific embodiments.

[0056] The experimental materials used in the examples of the present invention that are not specifically described are all conventional experimental materials in the art and can be purchased through commercial channels. If the specific experimental conditions and methods are not specified in the examples of the present invention, conventional conditions are usually followed, such as J. Sambrook et al., ed., Molecular Cloning Experiment Guide (3rd Edition), Science Press, 2002; DL Spector et al., ed., Cell Experiment Guide, Science Press, 2001; or the conditions recommended by the manufacturer.

[0057] The composition of the culture medium used in the embodiments of the present invention is as follows:

[0058] Co-culture medium: MS powder 2.12 g, sucrose 10 g, 2,4-D 2 mg, glutamine 5 mg, hydrolyzed casein 0.5 g, silver nitrate 4 mg, agar 8 g, distilled water to 1 L; pH = 5.8.

[0059] Recovery medium: MS powder 2.12 g, sucrose 10 g, 2,4-D 2 mg, glycine 2 mg, silver nitrate 4 mg, vitamin B 5 0.5 mg, agar 8 g, distilled water to 1 L; pH = 5.8.

[0060] Callus induction medium A: MS powder 2.12g, sucrose 10g, zeatin 5mg, IAA 0.5mg, glycine 2mg, hygromycin 15mg, silver nitrate 4mg, vitamin B 5 0.5 mg, agar 8 g, distilled water to 1 L; pH = 5.8.

[0061] Callus induction medium B: MS powder 2.12 g, sucrose 10 g, 6-BA 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 30 mg, silver nitrate 4 mg, vitamin B 5 0.5 mg, agar 8 g, distilled water to 1 L; pH = 5.8.

[0062] Differentiation medium: MS powder 2.12 g, sucrose 10 g, zeatin 5 mg, IAA 0.5 mg, glycine 2 mg, hygromycin 15 mg, vitamin B 5 0.5 mg, asparagine 5 mg, glutamine 5 mg, silver nitrate 4 mg, agar 8 g, distilled water to 1 L; pH = 5.8.

[0063] Rooting medium: MS powder 2.12g, sucrose 10g, IAA 0.5mg, paclobutrazol 0.5mg, glycine 1mg, hygromycin 15mg, vitamin B 5 0.5 mg, asparagine 5 mg, glutamine 5 mg, agar 8 g, distilled water to 1 L; pH = 5.8.

[0064] Example 1: Cloning of TaDOF4.7-B gene and construction of plant overexpression vector

[0065] 1. Extraction and purification of total RNA from wheat:

[0066] The TaDOF4.7-B gene was amplified from Chinese spring wheat. The RNA extraction kit used in this experiment was the UltraPure RNA Extraction Kit provided by Beijing Kangwei Reagent Biotechnology Co., Ltd. The specific experimental steps were carried out according to the instructions of the extraction kit.

[0067] To ensure that the RNA quality meets the requirements, the purity and concentration of the purified RNA samples were detected by spectrophotometer and agarose gel electrophoresis, respectively. The purity and concentration standards are as follows: RNA purity is OD 260 / 280 and OD 260 / 230 All were within the range of 1.8-2.0, and the RNA concentration was within the range of 1.0-2.0 μg / μL.

[0068] 2. Synthesis of the first strand of cDNA:

[0069] The first-chain cDNA synthesis kit used in this experiment was completed by FastQuantRT Kit (with gDNase) from Tiangen Biotechnology Co., Ltd.

[0070] 3. Cloning of TaDOF4.7-B gene:

[0071] The reverse transcribed cDNA was used as a template and PCR amplification was performed using the following primer pairs:

[0072] Upstream primer: 5'-ATGGAAGCTCCTCTGCACCAG-3'; (SEQ ID NO.3)

[0073] Downstream primer: 5'-CTAATAAGATCCGTGAGCGTGA-3'. (SEQ ID NO.4)

[0074] The PCR amplification system was 2 μL upstream primer (50 pmol / μL), 2 μL downstream primer (50 pmol / μL), 5 μL 10× PCR buffer, 2 μL dNTP mixed solution (10 mmol / L), 0.5 μL EVO DNA polymerase (5 U), 1 μL cDNA template, and DEPC·H 2 O to make up the total volume to 25 μL;

[0075] The amplification conditions were as follows: pre-denaturation at 94°C for 3 min, denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 1 min, 32 cycles, and extension at 72°C for 7 min.

[0076] 4. Construction of plant expression vector pUbi::TaDOF4.7-B

[0077] (1) Take 4 μL of PCR product and mix with pENTR TM / D-TOPO Vector vector connection, the operation steps are the same as pENTR TM / D-TOPO Vector product manual.

[0078] (2) The ligation product was transformed into Escherichia coli Top10 and cultured overnight on LB solid medium containing kanamycin (100 mg / L).

[0079] (3) Pick a single colony and culture it overnight in LB liquid medium containing kanamycin (100 mg / L). Extract plasmid DNA using the alkaline method and perform sequence determination.

[0080] (4) The amplified product was sequenced and analyzed, and its sequence was shown in SEQ ID NO.1, indicating that the TaDOF4.7-B gene had been connected to pENTR TM / D-TOPO Vector, the cloning vector construction is completed.

[0081] (5) The cloning vector was digested with restriction endonuclease Apa I, and the target fragment was detected and recovered by electrophoresis.

[0082] (6) The cloning vector after restriction digestion was connected to the pC186 vector through LR reaction. The operation steps were carried out according to the LR instructions of the product of Life Technologies Company.

[0083] (7) The ligation product was transformed into Escherichia coli Top10 and cultured overnight on LB solid medium containing kanamycin (50 mg / L).

[0084] (8) Pick a single colony and culture it overnight in LB liquid medium containing kanamycin (50 mg / L). Extract plasmid DNA by alkaline method and perform sequencing analysis to construct an expression vector pUbi::TaDOF4.7-B ( Figure 2 ).

[0085] Example 2: Agrobacterium-mediated transformation of wheat immature embryos and acquisition of positive plants

[0086] 1. Wheat genetic transformation:

[0087] Wheat genetic transformation is carried out by Agrobacterium-mediated transformation. Figure 3 As shown, the specific transformation steps are as follows:

[0088] (1) The expression vector pUbi::TaDOF4.7-B constructed in Example 1 was used to transform Agrobacterium EHA105 to obtain an Agrobacterium infection solution for transformation. The prepared Agrobacterium infection solution was aspirated into a 2 mL centrifuge tube containing Fielder wheat immature embryos, and gently inverted for 45 seconds to immerse the immature embryos in the bacterial solution. If the immature embryos cannot be immersed in the bacterial solution, they can be centrifuged briefly.

[0089] (2) After standing for 5 minutes, pour the bacterial solution and immature embryos into a sterile disposable culture dish and aspirate half of the bacterial solution.

[0090] (3) Use a sterilized and cooled scalpel to pick up the young embryos and place them on the co-culture medium. The scalpel should be sterilized several times to prevent contamination that may affect subsequent experiments.

[0091] (4) Seal the culture dish with sealing film and place it in a dark incubator at 23°C for co-cultivation.

[0092] (5) After 2 days of co-culture, the embryonic axes were cut off and placed on recovery medium at 25°C in the dark.

[0093] (6) After 5 days, the immature embryonic callus began to swell and callus began to form. The swollen callus was transferred to callus induction medium A, and the browned and dead immature embryonic tissue was discarded. The culture was continued at 25°C in the dark.

[0094] (7) After 14 days, a relatively large callus was formed. The callus was cut into two halves and the cut surface in contact with the culture medium was transferred to callus induction medium B and cultured in the dark at 25°C.

[0095] (8) After 21 days, the callus tissue was transferred to the differentiation medium. The callus tissue began to differentiate and sprout. The callus tissue was placed in a light incubator at a temperature of 25°C and a light intensity of 2000 lx for culture.

[0096] (9) After the callus tissue differentiates into green seedlings, it is transferred to the rooting medium until the green seedlings grow 4-5 leaves and then transplanted to a greenhouse or artificial climate chamber for seedling cultivation.

[0097] The pC186 empty vector was transformed as a control, and the transformation conditions remained the same.

[0098] 2. Identification of T0 transgenic plants:

[0099] Cut wheat leaves from 31 T0 transgenic wheat plants and add ddH 2 O Crush the leaves, insert the BAR test strip and let it stand for 10 seconds to observe the test results. When two clear bands appear on the BAR test strip, it proves that the plant has BAR protein and the PC186 vector has been successfully transformed; when the BAR test strip has only one band, the plant does not have BAR protein and the PC186 vector has not been successfully transformed.

[0100] We used BAR test strips to detect 18 resistant plants containing BAR protein ( Figure 4 A).

[0101] To further verify the successful transformation of the target gene TaDOF4.7-B, we designed nested primers for the vector and the target gene based on the Fielder wheat genome as a negative control and PC186 empty vector as a positive control to perform PCR to identify the insertion of TaDOF4.7-B DNA; the sequences of the nested primers are as follows:

[0102] Nested primer F: 5'-AACATTTTGAGGCATTTCGA-3'; (SEQ ID NO.5)

[0103] Nested primer R: 5'-ACAAGGGCATGGCGCCGGAT-3'. (SEQ ID NO.6)

[0104] The electrophoresis results showed that all resistant plants containing BAR protein were positive seedlings ( Figure 4 B) The results showed that TaDOF4.7-B was successfully integrated into the wheat genome.

[0105] In order to further detect whether the positive seedlings are overexpression strains, we cut the leaves of T0 plants to extract RNA for RT-qPCR quantitative detection of the expression of TaDOF4.7-B gene. The results showed that 15 overexpression strains were detected in T0 pUbi::TaDOF4.7-B, and the expression levels were upregulated to varying degrees, among which the expression levels of strains #7, #8, #10 and #11 were relatively high ( Figure 4 C)

[0106] 3. Statistical analysis of conversion data

[0107] In order to ensure the reliability of the experimental data, we repeated the transformation three times and counted the experimental data three times. The results are shown in Tables 1-3.

[0108] Table 1: Fielder's first batch of transformation data

[0109]

[0110] Table 2: Fielder second batch transformation data

[0111]

[0112] Table 3: Fielder third batch transformation data

[0113]

[0114] In Tables 1 to 3:

[0115] Callus proliferation rate = (weight on the last day of callus induction - weight on the first day of callus induction) / weight on the first day of callus induction;

[0116] Regeneration frequency = number of calli with at least one bud / number of inoculated embryos × 100%;

[0117] Regeneration bud rate = number of regenerated buds / number of inoculated young embryos × 100%.

[0118] The results showed that transformation of TaDOF4.7-B could significantly improve the regeneration ability of wheat.

[0119] Example 3: Investigation of agronomic traits of transgenic lines overexpressing TaDOF4.7-B

[0120] Since there are some factors during wheat genetic transformation that cause T0 generation transgenic plants to be stressed and the growth and development process of wheat is affected by external factors, we selected T1 generation stable transgenic plants and wild-type Fielder and observed and measured their agronomic traits.

[0121] According to the expression level of T0 transgenic lines, we selected T0 #8, #11 and #15 lines for planting in the artificial culture room and field. The TaDOF4.7-B-OE T1 transgenic lines were identified, and the results are as follows Figure 5-Figure 6 As shown, the results showed that T1 generation #8-1 / 2 / 3 / 4 / 5 and #11-1 / 2 / 3 / 4 / 5 / 6 / 7 / 8 / 9 / 10 / 11 were all positive transgenic plants, and the expression levels were all high.

[0122] We selected transgenic plants #8, #11, and #15 with higher expression levels of TaDOF4.7-B and planted them in the experimental field with the wild-type Fielder. The experimental conditions were kept consistent, and their plant height, number of tillers, number of effective tillers, panicle length, number of spikelets, number of effective spikelets, number of grains per panicle, grain length, and grain width were statistically analyzed.

[0123] The results showed that TaDOF4.7-B-OE overexpressing plants were fertile. Compared with Fielder wild-type plants, the plant height of TaDOF4.7-B-OE transgenic plants was significantly reduced ( Figure 7 ), while wheat ear traits ( Figure 8 ), wheat grain length, grain width, 100-grain weight and other agronomic traits ( Fig. 9 ) had no significant difference. Therefore, the plant height of transgenic plants overexpressing TaDOF4.7-B was reduced without affecting other ear and grain traits, achieving stable wheat yield while enhancing wheat lodging resistance.

[0124] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Application of TaDOF4.7-B gene in improving the regeneration efficiency of wheat immature embryos; the TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. Application of TaDOF4.7-B protein in improving the regeneration efficiency of wheat immature embryos, wherein the TaDOF4.7-B protein is a protein as shown in (A1) or (A2): (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. Application of recombinant expression vectors or genetically engineered bacteria containing TaDOF4.7-B gene in improving the efficiency of wheat immature embryo regeneration; The TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

4. A method for improving the regeneration efficiency of wheat immature embryos, characterized in that: The following steps are involved: The wheat TaDOF4.7-B gene was linked to an expression vector to construct a recombinant expression vector, and the recombinant expression vector was transferred into Agrobacterium competent cells to obtain an Agrobacterium strain for transformation; Using Agrobacterium strains to infect wheat embryos, transgenic wheat plants with improved regeneration efficiency were obtained; The TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

5. Use of TaDOF4.7-B gene in any of the following (1)-(3): (1) Reduce wheat plant height; (2) Improve wheat's resistance to lodging; (3) Cultivate dwarf wheat varieties; The TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

6. Application of TaDOF4.7-B protein in the following (1) or (2): (1) Reduce wheat plant height; (2) Improve wheat's resistance to lodging; The TaDOF4.7-B protein is the protein shown in (A1) or (A2) below: (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing; (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

7. Use of a recombinant expression vector or genetically engineered bacteria containing the TaDOF4.7-B gene in any of the following (1)-(3): (1) Reduce wheat plant height; (2) Improve wheat's resistance to lodging; (3) Cultivate dwarf wheat varieties; The TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

8. A method for improving lodging resistance of wheat, characterized in that: The following steps are involved: The TaDOF4.7-B gene is exogenously transferred into the starting wheat plant to overexpress the TaDOF4.7-B gene, thereby obtaining a transgenic wheat plant, wherein the plant height of the transgenic wheat plant is lower than that of the starting wheat plant, and the lodging resistance is improved; The TaDOF4.7-B gene is a DNA molecule as shown in the following i) or ii): i) the nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) A DNA molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

9. The method according to claim 8, characterized in that Methods for exogenously transferring the TaDOF4.7-B gene into wheat include: polyethylene glycol method, Agrobacterium-mediated method or gene gun bombardment method.

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