Application of TaDOF5.6 gene in efficient genetic transformation of wheat

Abstract

The present invention discloses the application of TaDOF5.6 gene in efficient genetic transformation of wheat, and belongs to the technical field of plant genetic engineering. The present invention isolates and obtains TaDOF5.6 gene, and conducts genetic transformation test of wheat by Agrobacterium-mediated method, and finds that the gene can greatly improve the transformation efficiency of wheat of different genotypes. The discovery and application of the function of improving wheat transformation efficiency of this gene has very important potential value for broadening the range of wheat genotypes available for transformation, accelerating the functional research and application of genes related to biological stress and abiotic stress such as wheat disease and insect resistance and stress resistance, promoting the use of biotechnology to improve the agronomic traits of main varieties, and promoting wheat biotechnology breeding.

Description

Technical Field

[0001] The invention relates to the technical field of plant genetic engineering, and in particular to the application of TaDOF5.6 gene in efficient genetic transformation of wheat. Background Art

[0002] Continuous population growth, consumption growth and climate change have led to increasing global concerns about food security. Increasing food production and reducing the impact of the environment on food have become urgent requirements. Conventional breeding methods for improving crop agronomic traits are difficult to meet human demand for food. Transgenic technology has advantages that cannot be replaced by traditional breeding methods and has become a powerful tool for improving plant disease resistance, insect resistance, herbicide resistance, drought resistance and other important agronomic traits (Li Yi et al., 2016). Since the first transgenic wheat plant was obtained in the world, wheat transgenic technology has become increasingly mature. However, wheat gene function research and genetic breeding improvement are still seriously lagging behind other staple crops such as rice and corn.

[0003] In recent years, with the rapid development of genome sequencing technology, the genome sequencing of multiple wheat varieties has been completed and published. A large number of studies on the functions of wheat genes need to be carried out quickly, and an efficient, stable and large-scale genetic transformation system needs to be established urgently. However, wheat is an allohexaploid plant, and most genotypes have poor regeneration ability, and there are problems such as low genetic transformation efficiency and poor repeatability. At present, measures to improve the efficiency of wheat genetic transformation mainly include screening wheat genotypes with high regeneration efficiency, optimizing the composition of tissue culture medium and culture conditions (Zhang Wei et al., 2018; Bie et al., 2020), and screening genes that improve wheat transformation efficiency (Debernardi et al., 2020; Wang et al., 2022).

[0004] The DOF family is a type of transcription factor unique to plants. It belongs to the zinc finger protein superfamily and is generally composed of 200-400 amino acids. Because it has a unique single zinc finger conserved DNA binding domain rich in Cys residues, it is named the DOF domain (Yanagisawa and Schmidt, 1999). The transcriptional regulatory domain located at the C-terminus of the DOF protein has a relatively variable amino acid sequence and is not conservative, resulting in the functional diversity of DOF proteins in plants (Yanagisawa, 2002). Studies have shown that DOF family members play an important regulatory role in plant growth and development, light regulation, defense mechanisms, and abiotic stress responses (Lijavetzky et al, 2003; Gupta et al, 2015).

[0005] Hexaploid wheat has undergone natural chromosome doubling during its evolution, and genome expansion has led to a doubling of the number of DOF family genes. Ren et al. (2020) used bioinformatics methods to identify the wheat DOF transcription factor family genome-wide, and isolated a total of 86 genes. These genes are unevenly distributed on wheat chromosomes, and most genes are expressed in different tissues. The DOF5.6 gene, also known as the HCA2 gene, encodes a nuclear-localized transcription factor in Arabidopsis, which can promote the periclinal division of interfascicular parenchyma cells in the apical vascular tissue of the Arabidopsis inflorescence stem, thereby promoting the formation of interfascicular cambium in these vascular tissues (Guo et al., 2009). However, there have been no reports on the functional studies of the TaDOF5.6 gene in wheat. Summary of the invention

[0006] In view of the above-mentioned prior art, the purpose of the present invention is to provide the application of TaDOF5.6 gene in efficient genetic transformation of wheat. The present invention isolates the TaDOF5.6 gene, and conducts a wheat genetic transformation experiment by Agrobacterium-mediated method, and finds that the gene can improve the transformation efficiency of wheat of different genotypes. The discovery and application of the function of improving wheat transformation efficiency of this gene has very important potential value for broadening the range of wheat genotypes available for transformation, accelerating the functional research and application of genes related to biotic stress and abiotic stress such as wheat disease and insect resistance and stress resistance, promoting the use of biotechnology to improve the agronomic traits of the main varieties in production, and promoting wheat biotechnology breeding.

[0007] To achieve the above object, the present invention adopts the following technical solution:

[0008] In a first aspect, the present invention provides the application of TaDOF5.6 gene in efficient genetic transformation of wheat;

[0009] The TaDOF5.6 gene is a nucleic acid molecule as shown in i) or ii) below:

[0010] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0011] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0012] In the above application, improving the genetic transformation efficiency of wheat refers to improving the transformation efficiency of introducing nucleic acid molecules into wheat.

[0013] In the above application, the TaDOF5.6 gene improves the genetic transformation efficiency of wheat by increasing the resistant callus induction rate and the positive seedling transformation rate.

[0014] The second aspect of the present invention provides the use of a protein encoded by the TaDOF5.6 gene in improving the efficiency of wheat genetic transformation;

[0015] The protein encoded by the TaDOF5.6 gene is the protein shown in (A1) or (A2) below:

[0016] (A1) a protein consisting of the amino acid sequence shown in SEQ ID NO.2 in the sequence listing;

[0017] (A2) A fusion protein obtained by linking a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0018] The proteins described in (A1) and (A2) can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0019] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. In order to facilitate the purification of the protein in (A1), a tag can be connected to the amino terminus or carboxyl terminus of the protein in (A1). The tag can be Poly-Arg (usually 6 RRRRR), Poly-His (usually 6 HHHHHH), FLAG (DYKDDDDK), Strep-tagII (WSHPQFEK) or c-Myc (EQKLISEEDL).

[0020] The third aspect of the present invention provides the use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaDOF5.6 gene in promoting the introduction of nucleic acid molecules into wheat.

[0021] A fourth aspect of the present invention provides a method for improving the transformation efficiency of a nucleic acid molecule introduced into a target plant, comprising the following steps:

[0022] The TaDOF5.6 gene and nucleic acid molecule are transferred into the target plant to achieve the purpose of improving the transformation efficiency of the nucleic acid molecule introduced into the target plant;

[0023] The TaDOF5.6 gene is a nucleic acid molecule as shown in i) or ii) below:

[0024] i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1;

[0025] ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

[0026] In the above method, the TaDOF5.6 gene and nucleic acid molecule can be transferred into the target plant via one vector or via different vectors.

[0027] Preferably, the TaDOF5.6 gene and nucleic acid molecule are introduced into the target plant via the pc186 expression vector.

[0028] In the above method, the target plants include but are not limited to monocotyledonous plants such as wheat, corn, rice, and barley.

[0029] Beneficial effects of the present invention:

[0030] The present invention has discovered for the first time that the TaDOF5.6 gene can improve the conversion efficiency of nucleic acid molecules introduced into target plants and / or promote the introduction of nucleic acid molecules into target plants. An overexpression vector is constructed using a DNA sequence containing a full-length fragment of the TaDOF5.6 gene, and then the TaDOF5.6 gene overexpression vector is introduced into an Agrobacterium strain, and the wheat embryo is infected by the Agrobacterium-mediated method. The results were analyzed and found that compared with the control vector, the vector of the TaDOF5.6 gene can promote the entry of nucleic acid molecules into the target plant. The TaDOF5.6 gene can be used to improve the conversion efficiency of the target gene introduced into plants, improve the genetic transformation efficiency of monocotyledonous plants, especially wheat, and has important economic value and social benefits for plant gene function research and improvement of crop agronomic traits. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is a schematic diagram of the partial structure of the plant expression vector pc186-TaDOF5.6;

[0032] Figure 2 It is a schematic diagram of the partial structure of the plant expression vector pc186-GUS vector;

[0033] Figure 3 Schematic diagram of the results of PCR specific amplification of the bar gene in candidate transgenic plants obtained by transforming the plant expression vector pc186-TaDOF5.6. In the figure, 1-14 represent candidate transgenic plants, PC represents positive plasmid, NC represents negative control, CK represents wild-type control, and M represents 2000 bp molecular weight marker. DETAILED DESCRIPTION

[0034] 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.

[0035] As mentioned earlier, wheat is a crop that is difficult to transform genetically, with low efficiency and poor repeatability. There are also large differences in the difficulty of genetic transformation of different genotypes of wheat. Major cultivated varieties such as Jimai 22 are extremely difficult to transform, and it is extremely difficult to use genetic engineering to improve their breeding.

[0036] The DOF5.6 gene is the first transcription factor that regulates the formation of interfascicular cambium discovered by Professor Qu Lijia's research group at Peking University in Arabidopsis, and was selected as one of the important research advances in several fields of Chinese plant science in 2009. However, there is no report on the functional research of the DOF5.6 gene in wheat.

[0037] Based on this, the present invention has conducted an in-depth study on the function of the wheat DOF5.6 gene. The nucleotide sequence of the TaDOF5.6 gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by the TaDOF5.6 gene is shown in SEQ ID NO.2; specifically, as follows:

[0038] TaDOF5.6 Gene:

[0039] ATGATGGCAGGCGCAGCTCATCCGATGCATTTCTGCATGGACTCCGACTGGCTCAAGGGCATCGTTCCCGAGGACCAGGGCGGGATGGGGTCGTCGTCGCCGTCGGAGGAGCTGATCATCGCGTGCCCGGAGCCGATGCAGGCCCAGCAGGCGGCGGACCGGCGGCTGCGCCCGCAGCACGACCAGCCGCTCAAGTGCCCGCGCTGCGACTCCACGCACACCAAGTTCTGCTACTACAACAACTACAGCCTCTCCCAGCCGCGCTACTTCTGCAAGACGTGCCGCCGCTACTGGACCAAGGGCGGCTCCCTCCGCAACGTCCCCGTCGGCGGCGGCTGCCGCAAGAACAAGCGCGCCAGCGCCGCCAAGAAGCCCTCTGCTGCCGCCGTCATCACGCCGCCGATTTCGATGATGCAGCAGCTTCACCACGGGCGACACATGGCAGAGACCGGACTCCACCTGTCCTTCTCCGGGATGCAGCCGCCGGCGGTCTCGGCCGCCGACCCGCTCTGCAGCCTCGGGCTCTTCGACTGGAAGTACGACCACATCCTCTCGGGCTCCGGCGGCTTTGAGAGCGCGAACTCTGAGGCTCACTTCACCGGGCCAGGCATGATGGGCATTGCCAACGGAAGCGGCGGTGGCGGCGCGGAGTACCACGCACTGAACGCGCTCCGGTATGCAGCCGGGCTTGGCGAGCACCTTGCGCTTCCATTTGGTGGCGCGACGTCGCGGGCTGAGCGTGACAGTGTCGTTGCCGAGATGAAGCCGCAGGCGGAGAGGCTGCTGTCGCTGGAGTGGTGCGGCGAGGCGAGCCGCGCGCCGACAGAGACCTCCATCAGCTCCCTGGGCGGGCTCGGCCTGTGGAGCGGCATGATCACCGGCGCCACCCACCATCACCATGGCTCCTCTGCTGCCATCTGA(SEQ ID NO.1)

[0040] Protein encoded by TaDOF5.6 gene:

[0041] MMAGAAHPMHFCMDSDWLKGIVPEDQGGMGSSSPSEELIIACPEPMQAQQAADRRLRPQHDQPLKCPRCDSTHTKFCYYNNYSLSQPRYFCKTCRRYWTKGGSLRNVPVGGGCRKNKRASAAKKPSAAAVITPPISMMQQLHHGRHMAETGLHLS FSGMQPPAVSAADPLCSGLFDWKYDHILSGSGGFESANSEAHFTGPGMMGIANGSGGGGAEYHALNALRYAAGLGEHLALPFGGATSRAERDSVVAEMKPQAERLLSLEWCGEASRAPTETSISSLGGLGLWSGMITGATHHHHGSSAAI(SEQ ID NO.2)

[0042] The present invention conducted genetic transformation experiments on multiple genotypes of wheat through the Agrobacterium-mediated method and found that the TaDOF5.6 gene can improve the transformation efficiency of multiple genotypes of wheat, especially for the extremely difficult to transform wheat variety Jimai 22, it can increase the genetic transformation efficiency to more than 40%, which has great application prospects, and thus the present invention is proposed.

[0043] 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.

[0044] The test materials used in the embodiments of the present invention that are not specifically described are all conventional test materials in the art and can be purchased through commercial channels. The present invention introduces the expression vector into the plant cell, and the introduction methods are all well known to those skilled in the art, including but not limited to: Agrobacterium-mediated method, gene gun bombardment method, electroporation method, ovary injection method, etc. The selection marker gene used in the present invention is the bar gene, which encodes the phosphinothricin acetyltransferase PAT protein, and can further include other selection marker genes and reporter genes such as nptII and hpt. The screening antibiotic selected by the present invention is phosphinothricin, and the selection of bialaphos and other screening agents can also have the same effect; the specific experimental conditions and methods are not indicated in the embodiments of the present invention, usually according to conventional conditions, such as J. Sambrook et al., ed., Science Press, 2002, Molecular Cloning Experiment Guide (3rd Edition); DL Spector et al., ed., Science Press, 2001, Cell Experiment Guide; or according to the conditions recommended by the manufacturer.

[0045] Example 1: Cloning of TaDOF5.6 gene and construction of expression vector

[0046] Total RNA of wheat line Fielder was extracted using Ultrapure RNA Kit (Chemical Industry, Cat. No.: CW0581M).

[0047] cDNA was reverse transcribed according to FastKing RT Kit (With gDNase) (Tiangen Biochemical Technology (Beijing) Co., Ltd., Cat. No.: KR116)).

[0048] Using cDNA as template, PCR amplification was performed using primer pairs (upstream primer: 5'-ATGATGGCAGGCGCAGCTCATC-3', SEQ ID NO.3; downstream primer: 5'-TCAGATGGCAGCAGAGGAGCCAT-3', SEQ ID NO.4). The amplification system was 2 μl upstream primer (10 μmol / μl), 2 μl downstream primer (10 μmol / μl), 12.5 μl 2×Phanta Max MasterMix, 1 μl cDNA template, and ddH2O was added to make the total volume 25 μl. Amplification conditions were: 95°C pre-denaturation for 3 minutes; 95°C denaturation for 15 seconds, 58°C annealing for 15 seconds, 72°C extension for 30 seconds, 35 cycles; 72°C extension for 5 minutes.

[0049] The PCR product obtained by amplification was -Blunt3 Cloning Kit (Beijing Quanshijin Biotechnology Co., Ltd., Catalog No.: CB301-01) Operation steps link -Blunt3 vector to obtain pEASY-Blunt3-TaDOF5.6 vector, and sequenced it.

[0050] Through sequencing analysis, the nucleotide sequence of the PCR amplification product is sequence 1 in the sequence list, and the gene shown in the PCR product is named TaDOF5.6 gene; the protein encoded by the gene is named TaDOF5.6, and the amino acid sequence of the protein is sequence 2 in the sequence list.

[0051] Sequence 1 can also be artificially synthesized and connected to the pEASY-Blunt3 vector to obtain the pEASY-Blunt3-TaDOF5.6 vector.

[0052] Using pEASY-Blunt3-TaDOF5.6 as template, designed primer pair (upstream primer: 5'-CACCATGATGGCAGGCGCAGCT-3', SEQ ID NO.5; downstream primer: 5'-TCAGATGGCAGCAGAGGAGCCAT-3', SEQ ID NO.4) for PCR amplification. The amplification system was 2μl upstream primer (10μmol / μl), 2μl downstream primer (10μmol / μl), 12.5μl 2×PhantaMax Master Mix, 1μl cDNA template, and ddH2O was added to make the total volume 25μl. Amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 30 seconds, 35 cycles; 72℃ extension for 5 minutes.

[0053] The amplified PCR product was referred to pENTR TM Directional Cloning Kit (ThermoScientific TM , Catalog number: K2400-20SP) Operation steps: ligation and sequencing. The correctly sequenced single clone was connected to the pc186 expression vector through LR reaction to obtain the pc186-TaDOF5.6 vector. The schematic diagram of the vector structure is shown in the figure. Figure 1 shown.

[0054] The pc186-TaDOF5.6 was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain that can be used for transformation was obtained and named pc186-TaDOF5.6 / EHA105.

[0055] Example 2: Construction of control vector pc186-GUS

[0056] Refer to NCBI ( https: / / www.ncbi.nlm.nih.gov / ) website Sequence ID: MN266288.1, nucleotides 15108-16919, primer pairs (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3', SEQ ID NO.6; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO.7) were used for PCR amplification. The amplification system was 2μl upstream primer (10μmol / μl), 2μl downstream primer (10μmol / μl), 12.5μl 2×Phanta Max Master Mix, 1μl cDNA template, and ddH2O was added to make the total volume 25μl. Amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 55 seconds, 35 cycles; 72℃ extension for 5 minutes.

[0057] The PCR product obtained by amplification was -Blunt3 Cloning Kit (Cat. No.: CB301-01, Beijing Quanshijin Biotechnology Co., Ltd.) operation steps to obtain pEASY-B3-GUS, and then sequenced.

[0058] After sequencing analysis, the gene indicated by the PCR product was named GUS gene.

[0059] The GUS gene can also be artificially synthesized and connected to the pEASY-Blunt3 vector to obtain pEASY-Blunt3-GUS.

[0060] Using pEASY-Blunt3-GUS as template, designed primer pair (upstream primer: 5'-CACCATGTTACGTCCTGTAGAA-3', SEQ ID NO.8; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID NO.7) for PCR amplification. The amplification system was 2μl upstream primer (10μmol / μl), 2μl downstream primer (10μmol / μl), 12.5μl 2×Phanta MaxMaster Mix, 1μl cDNA template, and ddH2O was added to make the total volume 25μl. Amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 55 seconds, 35 cycles; 72℃ extension for 5 minutes.

[0061] The amplified PCR product was referred to pENTR TM Directional Cloning Kit (Cat. No.: K2400-20SP, Thermo ScientificTM ) Operation steps: ligation and sequencing. The correctly sequenced single clone was ligated to the pc186 expression vector through LR reaction to obtain the pc186-GUS vector. The schematic diagram of the vector structure is shown in the figure. Figure 2 shown.

[0062] The pc186-GUS was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain that can be used for transformation was obtained and named pc186-GUS / EHA105.

[0063] Example 3: Agrobacterium-mediated transformation of wheat immature embryos and identification of resistant plants

[0064] 1. The detailed steps and methods of Agrobacterium-mediated transformation of wheat immature embryos refer to Wheat (Triticum aestivum L.) Transformation Using Immature Embryos (Ishida et al., 2015). The basic steps of genetic transformation are as follows:

[0065] 1. Three days before infection, inoculate EHA105 Agrobacterium of pc186-TaDOF5.6 and pc186-GUS on YEP solid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, respectively, and culture in a dark incubator at 28°C for 2 days. Pick a single colony and inoculate it in YEP liquid medium containing 50 mg / L kanamycin and 50 mg / L rifampicin, and culture it with shaking at 28°C 220rpm overnight; put the above Agrobacterium solution into a 2ml centrifuge tube, centrifuge at 6000rpm for 5 minutes, discard the supernatant, resuspend the precipitate with resuspension solution, and obtain EHA105 Agrobacterium resuspension of pc186-TaDOF5.6 and pc186-GUS, respectively.

[0066] 2. The immature embryos of different wheat genotypes about 14 days after flowering were infected with EHA105 Agrobacterium resuspensions of pc186-TaDOF5.6 and pc186-GUS, respectively, and spread with the scutellum side up on WLS-AS medium (1 / 10MS basic medium, 1 / 10MS vitamins, 10 g / L glucose, 100 μM acetosyringone, and 8 g / L agarose), and cultured in a dark incubator at 23°C for 2 days.

[0067] 3. Transfer the immature embryos after co-cultivation to WLS-Res medium (MS basic medium, MS vitamins, 2,4-D 0.5 mg / L, picloram 2.2 mg / L, glutamine 0.5 g / L, casein 0.1 g / L, MgCl2·6H2O 0.75 g / L, maltose 40 g / L, AgNO3 0.85 mg / L, vitamin C 100 mg / L, carbenicillin 250 mg / L, agarose 5 g / L), and culture in a dark incubator at 25°C for 5 days.

[0068] 4. The callus tissue after recovery culture was transferred to WLS-P5 medium (WLS-Res medium supplemented with PPT 5 mg / L) and cultured in a 25°C incubator in the dark for 14 days.

[0069] 5. The callus was then transferred to WLS-P10 medium (WLS-Res medium supplemented with 10 mg / L PPT) and cultured in a 25°C incubator in the dark for 21 days.

[0070] 6. The callus was transferred to LSZ-P5 medium (MS basic medium, LS vitamins, zeatin 5 mg / L, sucrose 20 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, phytagel 3 g / L), and cultured in a 25°C incubator under light for 2 weeks.

[0071] 7. Transfer the regenerated buds of wheat callus to LSF-P5 medium (MS basic medium, LS vitamins, IBA0.2 mg / L, sucrose 15 g / L, carbenicillin 250 mg / L, PPT 5 mg / L, plant gel 3 g / L), and culture in a 25°C incubator under light until the roots of the regenerated buds are about 1-2 cm long.

[0072] 8. Transplant the strong seedlings with roots into nutrient soil to obtain resistant seedlings of pc186-TaDOF5.6 and pc186-GUS respectively.

[0073] 2. PCR detection of candidate transgenic plants

[0074] The CTAB method (Sambrook and Russell, Molecular Cloning Laboratory Guide, 2001) was used to extract genomic DNA from leaves of T0 generation wheat plants transformed with pc186-TaDOF5.6 and pc186-GUS vectors.

[0075] Primers were designed to detect the bar gene, the primer pair sequence (upstream primer: 5'-GGCGGTCTGCACCATCGTCAACCACTAC-3', SEQ ID NO.9; downstream primer: 5'-AGTCCAGCTGCCAGAAACCCACGTCATG-3', SEQ ID NO.10), and the amplified sequence length was 446bp. The amplification system was 1μl upstream primer (10μmol / μl), 1μl downstream primer (10μmol / μl), 10μl 2×Rapid Taq Master Mix, 1μl cDNA template, and ddH2O was added to make the total volume 20μl. The amplification conditions were: 95℃ pre-denaturation for 3 minutes; 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 15 seconds, 32 cycles; 72℃ extension for 5 minutes.

[0076] PCR identification results Figure 3 As shown, there is no 446 bp bar gene fragment in wild-type wheat.

[0077] 3. Statistics of transformation efficiency of different wheat genotypes

[0078] After wheat immature embryo explants were infected by Agrobacterium-mediated method, the induced resistant callus was transferred to LSF-P5 medium, and after tissue culture until resistant buds were formed, PCR identification was performed. The number of resistant callus and positive seedlings was counted, and the induction rate of resistant callus and transformation efficiency were calculated. The calculation formula is as follows:

[0079] Resistant callus induction rate (%) = (number of resistant callus ÷ total number of young embryos) × 100%;

[0080] Transformation efficiency (%) = (number of positive seedlings / total number of young embryos) × 100%.

[0081] The results are shown in Table 1.

[0082] Table 1: Comparison of transformation efficiency between control vector and pc186-TaDOF5.6 vector

[0083]

[0084] The results showed that compared with the control vector pc186-GUS, the transformation of pc186-TaDOF5.6 vector can improve the transformation efficiency of wheat of the same genotype. In Fielder, Kenong 199 and Jimai 22, the induction rates of resistant callus transformed with pc186-TaDOF5.6 vector were 95.60%, 89.52% and 86.09%, respectively, which were higher than the 91.81%, 84.53% and 77.78% of resistant callus transformed with the control vector pc186-GUS. In terms of transformation efficiency, the transformation efficiency of Fielder transformed with pc186-TaDOF5.6 vector was 180.22%, which was nearly 5 times that of the control vector; the transformation efficiency of Kenong 199 as the recipient genotype was as high as 75.24%, which was much higher than the 0.55% of the control vector pc186-GUS. When Jimai 22 was used as the recipient for genetic transformation, the control vector pc186-GUS failed to obtain positive transgenic plants, while the transformation efficiency of the pc186-TaDOF5.6 vector was 44.35%. From the above results, it can be seen that the TaDOF5.6 gene can greatly improve the transformation efficiency of wheat and partially solve the genotype dependence problem in wheat genetic transformation. In particular, the transformation efficiency is improved more in genotypes that are more difficult to transform.

[0085] 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 overexpression of TaDOF5.6 gene in improving wheat genetic transformation efficiency; The TaDOF5.6 gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

2. The use according to claim 1, characterized in that: The TaDOF5.6 gene improves wheat genetic transformation efficiency by increasing the resistant callus induction rate and transformation efficiency.

3. Application of overexpression of protein encoded by TaDOF5.6 gene in improving wheat genetic transformation efficiency; The protein encoded by the TaDOF5.6 gene 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).

4. Use of an expression cassette, a recombinant expression vector or a recombinant bacterium containing the TaDOF5.6 gene in promoting the introduction of nucleic acid molecules into wheat, characterized in that: The TaDOF5.6 gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) A nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 except i).

5. A method for improving the transformation efficiency of nucleic acid molecules into target plants, characterized in that: The following steps are involved: The TaDOF5.6 gene and nucleic acid molecule are transferred into the target plant to achieve the purpose of improving the transformation efficiency of the nucleic acid molecule introduced into the target plant; The TaDOF5.6 gene is a nucleic acid molecule as shown in i) or ii) below: i) the nucleotide sequence is the nucleic acid molecule shown in SEQ ID NO.1; ii) a nucleic acid molecule encoding the amino acid sequence shown in SEQ ID NO.2 other than i); The target plant is wheat.

6. The method according to claim 5, characterized in that The TaDOF5.6 gene and nucleic acid molecule are transferred into the target plant via the pc186 expression vector.

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