CN116064642A - Application of TaDOF5.6 gene in efficient genetic transformation of wheat - Google Patents

Abstract

The invention discloses an application of TaDOF5.6 gene in efficient genetic transformation of wheat, and belongs to the technical field of plant genetic engineering. According to the invention, the TaDOF5.6 gene is obtained by separation, and a wheat genetic transformation test is carried out by an agrobacterium-mediated method, so that the transformation efficiency of wheat with different genotypes can be greatly improved. The discovery and application of the gene for improving the wheat transformation efficiency function have important potential value for widening the transformation available wheat genotype range, accelerating the functional research and application of biological stress and abiotic stress related genes such as disease and insect resistance and stress resistance of wheat, promoting the improvement of agricultural characters for producing main cultivated varieties by using biotechnology and promoting the biotechnology breeding of wheat.

Description

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

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to application of TaDOF5.6 gene in efficient genetic transformation of wheat.

Background

The growing population, increased consumption and climate change have led to an increasing global concern about grain safety, and increasing grain yield and reducing environmental impact on grain have become urgent demands. Conventional breeding methods for improving agronomic traits of crops are difficult to meet the requirements of human beings on grains. Transgenic technology has the advantage of being irreplaceable 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, etc., 2016). To date, wheat transgenic technology has grown to date from the world in the first instance of transgenic wheat plants. However, research on the gene functions and genetic breeding improvement of wheat still fall behind other staple food crops such as rice, corn and the like.

In recent years, with the rapid development of genome sequencing technology, genome sequencing work of a plurality of wheat varieties has been completed and published, and a large number of studies on wheat gene functions have been required to be rapidly carried out, and a genetic transformation system with high efficiency, stability and scale has been urgently required to be established. However, wheat is a heterologous hexaploid plant, and most genotypes have poor regeneration ability, and have problems of low genetic transformation efficiency and poor reproducibility. Currently, the means for improving the genetic transformation efficiency of wheat mainly include screening for wheat genotypes with high regeneration efficiency, optimizing the medium composition and culture conditions of tissue culture (Zhang Wei et al, 2018; bie et al, 2020), screening for genes that improve the transformation efficiency of wheat, and the like (Debernardi et al,2020;Wang et al,2022).

The DOF family is a plant-specific class of transcription factors belonging to the superfamily of zinc finger proteins, generally consisting of 200-400 amino acids. Because it has a unique single zinc finger conserved DNA binding domain rich in Cys residues, it is named DOF domain (Yanagisawa and Schmidt, 1999). The transcriptional regulatory domain located at the C-terminal end of DOF proteins is variable in amino acid sequence, does not have conservation, and results in functional diversity of DOF proteins in plants (Yanagisawa, 2002). Research has shown that DOF family members have important regulatory roles in plant growth, light regulation, defense mechanisms, abiotic stress response and the like (Lijavetzky et al,2003;Gupta et al,2015).

Hexaploid wheat undergoes natural doubling of chromosomes during evolution, and genome expansion results in doubling of the DOF family genes. The complete genome identification of wheat DOF transcription factor family is carried out by using bioinformatics method in Renzang et al (2020), 86 genes are totally separated, the genes are unevenly distributed on wheat chromosome, and most of the genes are expressed in different tissues. DOF5.6 gene, also known as HCA2 gene, encodes a nuclear-localized transcription factor in arabidopsis that promotes pericycle division of the perivascular parenchymal cells of the apical vascular tissue of the arabidopsis inflorescence stem, and thus promotes formation of the cambium between these vascular bundles (Guo et al, 2009). However, no studies have been reported on the function of the tadof5.6 gene in wheat.

Disclosure of Invention

Aiming at the prior art, the invention aims to provide the application of the TaDOF5.6 gene in the efficient genetic transformation of wheat. According to the invention, the TaDOF5.6 gene is obtained by separation, and a wheat genetic transformation test is carried out by an agrobacterium-mediated method, so that the gene can improve the transformation efficiency of wheat with different genotypes. The functional discovery and application of the gene for improving the wheat transformation efficiency have important potential value for widening the genotype range of the wheat for transformation, accelerating the functional research and application of biological stress and abiotic stress related genes such as disease and insect resistance and stress resistance of the wheat, promoting the improvement of the agronomic characters of the production-promoting varieties by using biotechnology and promoting the biotechnology breeding of the wheat.

In order to achieve the above purpose, the invention adopts the following technical scheme:

in a first aspect of the invention, the application of TaDOF5.6 gene in efficient genetic transformation of wheat is provided;

the TaDOF5.6 gene is a nucleic acid molecule shown in the following i) or ii):

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.

In the above applications, improving the genetic transformation efficiency of wheat means improving the transformation efficiency of a nucleic acid molecule introduced into wheat.

In the application, the TaDOF5.6 gene improves the genetic transformation efficiency of wheat by improving the induction rate of the resistant callus and the transformation rate of positive seedlings.

In a second aspect of the invention, there is provided the use of a protein encoded by the TaDOF5.6 gene for improving the genetic transformation efficiency of wheat;

the TaDOF5.6 gene encodes a protein shown in the following (A1) or (A2):

(A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;

(A2) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.

Wherein, the proteins (A1) and (A2) can be synthesized artificially or can be obtained by synthesizing the encoding genes and then biologically expressing.

Among the above proteins, the protein tag refers to a polypeptide or protein that is fusion expressed together with the target protein by using a DNA in vitro recombination technique, so as to facilitate the expression, detection, tracing and/or purification of the target protein. Wherein, in order to facilitate purification of the protein in (A1), a tag may be attached to the amino-terminal or carboxyl-terminal of the protein of (A1). The tag may be Poly-Arg (typically 6 RRRRRs), poly-His (typically 6 HHHHHH), FLAG (DYKDDDDK), strep-tag II (WSHPQFEK) or c-Myc (EQKLISEEDL).

In a third aspect of the invention, there is provided the use of an expression cassette, recombinant expression vector or recombinant bacterium comprising the TaDOF5.6 gene to facilitate the introduction of a nucleic acid molecule into wheat.

In a fourth aspect of the present invention, there is provided a method for improving the transformation efficiency of a nucleic acid molecule into a plant of interest, comprising the steps of:

transferring TaDOF5.6 gene and nucleic acid molecule into target plant to raise the transformation efficiency of the nucleic acid molecule into target plant;

the TaDOF5.6 gene is a nucleic acid molecule shown in the following i) or ii):

i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.

In the above method, the TaDOF5.6 gene and the nucleic acid molecule may be transferred into the plant of interest by one vector or by a different vector.

Preferably, the TaDOF5.6 gene and nucleic acid molecule are transferred into the plant of interest by the pc186 expression vector.

In the above method, the plant of interest includes, but is not limited to, monocotyledonous plants such as wheat, maize, rice, barley, and the like.

The invention has the beneficial effects that:

the invention discovers that the TaDOF5.6 gene can improve the transformation efficiency of the nucleic acid molecule into the target plant and/or promote the nucleic acid molecule into the target plant for the first time. An over-expression vector is constructed by using a DNA sequence containing a full-length fragment of the TaDOF5.6 gene, and then the TaDOF5.6 gene over-expression vector is introduced into an Agrobacterium strain and infects wheat young embryos by an Agrobacterium-mediated method. As a result, it was found that the vector of TaDOF5.6 gene can promote the entry of a nucleic acid molecule into a plant of interest, as compared with a control vector. The TaDOF5.6 gene can be used for improving the transformation efficiency of a target gene introduced plant, improving the genetic transformation efficiency of monocotyledonous plants, especially wheat, and has important economic value and social benefit for researching plant gene functions and improving crop agronomic traits.

Drawings

FIG. 1 is a schematic diagram of a part of a plant expression vector pc186-TaDOF 5.6;

FIG. 2 is a schematic diagram showing the partial construction of a plant expression vector pc 186-GUS;

FIG. 3 is a schematic diagram showing the result of PCR-specific amplification of bar gene of candidate transgenic plants obtained by transforming plant expression vector pc186-TaDOF 5.6. In the figures, 1-14 represent candidate transgenic plants, PC represents positive plasmid, NC represents negative control, CK represents wild-type control, and M represents 2000bp molecular weight marker.

Detailed Description

It should be noted that the following detailed description is illustrative and is intended to provide further explanation of the present application. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

As described above, wheat belongs to a crop with relatively difficult genetic transformation, the genetic transformation efficiency is low, the repeatability is poor, the genetic transformation difficulties of wheat with different genotypes are also greatly different, and the main cultivars such as Jimai 22 belong to wheat cultivars which are extremely difficult to transform, so that the difficulty of carrying out genetic engineering improvement breeding on the wheat cultivars is great.

DOF5.6 gene is the first transcription factor to regulate the formation of the interbeam forming found in arabidopsis by the professor task group at the university of beijing Qu Lijia, and was selected for important research progress in several fields of the chinese plant science in 2009. However, DOF5.6 gene function research has not been reported in wheat at present.

Based on this, the present invention conducted intensive studies on the function of the wheat DOF5.6 gene. The nucleotide sequence of the TaDOF5.6 gene is shown as SEQ ID NO.1, and the amino acid sequence of the TaDOF5.6 gene encoding protein is shown as SEQ ID NO. 2; the method comprises the following steps:

TaDOF5.6 Gene:

ATGATGGCAGGCGCAGCTCATCCGATGCATTTCTGCATGGACTCCGACTGGCTCAAGGGCATCGTTCCCGAGGACCAGGGCGGGATGGGGTCGTCGTCGCCGTCGGAGGAGCTGATCATCGCGTGCCCGGAGCCGATGCAGGCCCAGCAGGCGGCGGACCGGCGGCTGCGCCCGCAGCACGACCAGCCGCTCAAGTGCCCGCGCTGCGACTCCACGCACACCAAGTTCTGCTACTACAACAACTACAGCCTCTCCCAGCCGCGCTACTTCTGCAAGACGTGCCGCCGCTACTGGACCAAGGGCGGCTCCCTCCGCAACGTCCCCGTCGGCGGCGGCTGCCGCAAGAACAAGCGCGCCAGCGCCGCCAAGAAGCCCTCTGCTGCCGCCGTCATCACGCCGCCGATTTCGATGATGCAGCAGCTTCACCACGGGCGACACATGGCAGAGACCGGACTCCACCTGTCCTTCTCCGGGATGCAGCCGCCGGCGGTCTCGGCCGCCGACCCGCTCTGCAGCCTCGGGCTCTTCGACTGGAAGTACGACCACATCCTCTCGGGCTCCGGCGGCTTTGAGAGCGCGAACTCTGAGGCTCACTTCACCGGGCCAGGCATGATGGGCATTGCCAACGGAAGCGGCGGTGGCGGCGCGGAGTACCACGCACTGAACGCGCTCCGGTATGCAGCCGGGCTTGGCGAGCACCTTGCGCTTCCATTTGGTGGCGCGACGTCGCGGGCTGAGCGTGACAGTGTCGTTGCCGAGATGAAGCCGCAGGCGGAGAGGCTGCTGTCGCTGGAGTGGTGCGGCGAGGCGAGCCGCGCGCCGACAGAGACCTCCATCAGCTCCCTGGGCGGGCTCGGCCTGTGGAGCGGCATGATCACCGGCGCCACCCACCATCACCATGGCTCCTCTGCTGCCATCTGA(SEQ ID NO.1)

TaDOF5.6 gene encoded protein:

MMAGAAHPMHFCMDSDWLKGIVPEDQGGMGSSSPSEELIIACPEPMQAQQAADRRLRPQHDQPLKCPRCDSTHTKFCYYNNYSLSQPRYFCKTCRRYWTKGGSLRNVPVGGGCRKNKRASAAKKPSAAAVITPPISMMQQLHHGRHMAETGLHLSFSGMQPPAVSAADPLCSLGLFDWKYDHILSGSGGFESANSEAHFTGPGMMGIANGSGGGGAEYHALNALRYAAGLGEHLALPFGGATSRAERDSVVAEMKPQAERLLSLEWCGEASRAPTETSISSLGGLGLWSGMITGATHHHHGSSAAI(SEQ ID NO.2)

according to the invention, a genetic transformation test of wheat polygenotypes is carried out by an agrobacterium-mediated method, and the TaDOF5.6 gene is found to improve the transformation efficiency of wheat with a plurality of genotypes, especially for wheat variety Jimai 22 which is extremely difficult to transform, the genetic transformation efficiency can be improved to more than 40%, and the genetic transformation test has a great application prospect, so that the genetic transformation test method is provided.

In order to enable those skilled in the art to more clearly understand the technical solutions of the present application, the technical solutions of the present application will be described in detail below with reference to specific embodiments.

The test materials used in the examples of the present invention, which are not specifically described, are all conventional in the art and are commercially available. The present invention introduces expression vectors into plant cells, all of which are well known to those skilled in the art, and include, but are not limited to: agrobacterium mediating method, gene gun bombardment method, electric excitation method, ovary injection method, etc. The selectable marker gene used in the invention is the bar gene, codes for the PAT protein of the glufosinate acetyltransferase, and can further comprise other selectable marker genes such as nptII, hpt and the like and a reporter gene. The selected screening antibiotic is glufosinate, and the screening agents such as biamap and the like can also have the same effect; specific experimental conditions and methods are not noted in the examples of the present invention, and generally conventional conditions, such as j. Sambrook et al, scientific press, 2002, guidelines for molecular cloning experiments (third edition); D.L. speket et al, scientific press, 2001, guidance of cell experiments; or according to manufacturer recommended conditions.

Example 1: cloning of TaDOF5.6 gene and construction of expression vector

Total RNA from wheat strain Fielder was extracted using Ultrapure RNA Kit (well known as century, cat# CW 0581M).

The cDNA was reverse transcribed With reference to the FastKing RT Kit (With gDNase) Kit (Tiangen Biochemical technology (Beijing), inc.: KR 116).

PCR amplification was performed using cDNA as a template and a primer pair (upstream primer: 5'-ATGATGGCAGGCGCAGCTCATC-3', SEQ ID No.3; downstream primer: 5'-TCAGATGGCAGCAGAGGAGCCAT-3', SEQ ID No. 4). The amplification system was 2. Mu.l of the upstream primer (10. Mu. Mol/. Mu.l), 2. Mu.l of the downstream primer (10. Mu. Mol/. Mu.l), 12.5. Mu.l of 2X Phanta Max Master Mix, 1. Mu.l of cDNA template, plus ddH ₂ O the total volume was made up to 25. Mu.l. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 30 seconds, and cycling for 35 times; extension was carried out at 72℃for 5 minutes.

PCR products obtained by amplification are referred to

-Blunt3 Cloning Kit (Beijing full gold Biotechnology Co., ltd.; cat# CB 301-01) operating procedure connection +.>

-Blunt3 vector, pEASY-Blunt3-TaDOF5.6 vector was obtained and sequenced.

Through sequencing analysis, the nucleotide sequence of the PCR amplification product is sequence 1 in a sequence table, and the gene shown by the PCR product is named as TaDOF5.6 gene; the protein coded by the gene is named as TaDOF5.6, and the amino acid sequence of the protein is sequence 2 in a sequence table.

Or artificially synthesizing the sequence 1, and connecting the sequence to a pEASY-Blunt3 vector to obtain the pEASY-Blunt3-TaDOF5.6 vector.

Design with pEASY-Blunt3-TaDOF5.6 as templateThe primer pair (upstream primer: 5'-CACCATGATGGCAGGCGCAGCT-3', SEQ ID No.5; downstream primer: 5'-TCAGATGGCAGCAGAGGAGCCAT-3', SEQ ID No. 4) was PCR amplified. The amplification system was 2. Mu.l of the upstream primer (10. Mu. Mol/. Mu.l), 2. Mu.l of the downstream primer (10. Mu. Mol/. Mu.l), 12.5. Mu.l of 2X Phanta Max Master Mix, 1. Mu.l of cDNA template, plus ddH ₂ O the total volume was made up to 25. Mu.l. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 30 seconds, and cycling for 35 times; extension was carried out at 72℃for 5 minutes.

PCR products obtained by amplification were referred to pENTR ^TM Directional

Cloning Kit(Thermo Scientific ^TM Cargo number: k2400-20 SP) ligation sequencing. The monoclonal with correct sequencing is connected to a pc186 expression vector through an LR reaction to obtain a pc186-TaDOF5.6 vector, and the partial structure of the vector is shown in figure 1.

The competent cells of Agrobacterium EHA105 were transformed with pc186-TaDOF5.6 and the Agrobacterium strain available for transformation was obtained and designated pc186-TaDOF5.6/EHA105.

Example 2: construction of control vector pc186-GUS

Reference NCBI%https://www.ncbi.nlm.nih.gov/) Website Sequence ID: the 15108-16919 nucleotide of MN266288.1 was PCR amplified using a primer pair (upstream primer: 5'-ATGTTACGTCCTGTAGAA-3', SEQ ID No.6; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID No. 7). The amplification system was 2. Mu.l of the upstream primer (10. Mu. Mol/. Mu.l), 2. Mu.l of the downstream primer (10. Mu. Mol/. Mu.l), 12.5. Mu.l of 2X Phanta Max Master Mix, 1. Mu.l of cDNA template, plus ddH ₂ O the total volume was made up to 25. Mu.l. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 55 seconds, and cycling for 35 times; extension was carried out at 72℃for 5 minutes.

PCR products obtained by amplification are referred to

Blunt3 Cloning Kit (cat# CB301-01, beijing full gold biotechnology)Available from the company of the surgical Co., ltd.) to obtain pEASY-B3-GUS, and sequencing.

The gene shown in the PCR product was designated GUS gene by sequencing analysis.

Or artificially synthesizing GUS gene, and connecting the GUS gene with pEASY-Blunt3 vector to obtain pEASY-Blunt3-GUS.

PCR amplification was performed using pEASY-Blunt3-GUS as a template, and a primer set (upstream primer: 5'-CACCATGTTACGTCCTGTAGAA-3', SEQ ID No.8; downstream primer: 5'-TCATTGTTTGCCTCCCTG-3', SEQ ID No. 7) was designed. The amplification system was 2. Mu.l of the upstream primer (10. Mu. Mol/. Mu.l), 2. Mu.l of the downstream primer (10. Mu. Mol/. Mu.l), 12.5. Mu.l of 2X Phanta Max Master Mix, 1. Mu.l of cDNA template, plus ddH ₂ O the total volume was made up to 25. Mu.l. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 55 seconds, and cycling for 35 times; extension was carried out at 72℃for 5 minutes.

PCR products obtained by amplification were referred to pENTR ^TM Directional

Cloning Kit (cat# K2400-20SP,Thermo Scientific) ^TM ) The operating procedure is connected with sequencing. The monoclonal with correct sequencing is connected to a pc186 expression vector through an LR reaction to obtain a pc186-GUS vector, and the partial structure of the vector is shown in figure 2.

The pc186-GUS was transformed into Agrobacterium EHA105 competent cells, and an Agrobacterium strain was obtained for transformation, designated pc186-GUS/EHA105.

Example 3 Agrobacterium-mediated transformation of wheat young embryo and identification of resistant plants

1. Agrobacterium-mediated method for Wheat young embryos detailed procedures and methods reference wheats (Triticum aestivum l.) Transformation Using Immature Embryos (Ishida et al 2015). The basic steps of genetic transformation are as follows:

1. 3 days before infection, EHA105 Agrobacterium of pc186-TaDOF5.6 and pc186-GUS were inoculated on YEP solid medium containing 50mg/L kanamycin and 50mg/L rifampicin, respectively, and the incubator was dark-cultured at 28℃for 2 days. Single colonies were picked and inoculated into YEP liquid medium containing 50mg/L kanamycin and 50mg/L rifampicin, and shake-cultured overnight at 28℃at 220 rpm; the above Agrobacterium solution was placed in a 2ml centrifuge tube, centrifuged at 6000rpm for 5 minutes, the supernatant was discarded, and the pellet was resuspended with a resuspension to obtain EHA105 Agrobacterium resuspension of pc186-TaDOF5.6 and pc186-GUS, respectively.

2. Young embryos of different wheat genotypes about 14 days after flowering were infested with EHA105 Agrobacterium heavy suspensions of pc186-TaDOF5.6 and pc186-GUS, respectively, scutellum facing upwards was plated on WLS-AS medium (1/10 MS minimal medium, 1/10MS vitamins, glucose 10g/L, acetosyringone 100. Mu.M, agarose 8 g/L) and incubated in darkness for 2 days at 23 ℃.

3. Transferring the co-cultured young embryo to WLS-Res culture medium (MS minimal medium, MS vitamins, 2, 4-D0.5 mg/L, picloram 2.2mg/L, glutamine 0.5g/L, casein 0.1g/L, mgCl) ₂ ·6H ₂ O0.75 g/L, maltose 40g/L, agNO ₃ 0.85mg/L, 100mg/L vitamin C, 250mg/L carbenicillin, 5g/L agarose), and the incubator was dark-cultured at 25℃for 5 days.

4. The callus after recovery culture was transferred to WLS-P5 medium (PPT 5mg/L was added to WLS-Res medium) and cultured in the dark at 25℃for 14 days.

5. The callus was transferred to WLS-P10 medium (10 mg/L of PPT was added to WLS-Res medium) and cultured in the dark at 25℃for 21 days.

6. The callus was transferred to LSZ-P5 medium (MS minimal medium, LS vitamins, zeatin 5mg/L, sucrose 20g/L, carbenicillin 250mg/L, PPT 5mg/L, plant gel 3 g/L) and incubated with light at 25℃for 2 weeks.

7. The regenerated buds of the wheat callus are transferred to LSF-P5 culture medium (MS basic culture medium, LS vitamins, IBA 0.2mg/L, sucrose 15g/L, carbenicillin 250mg/L, PPT mg/L, plant gel 3 g/L) and cultured in an incubator at 25 ℃ until the roots of the regenerated buds grow about 1-2 cm.

8. Transplanting the strong seedlings with long roots into nutrient soil to obtain resistant seedlings of pc186-TaDOF5.6 and pc186-GUS respectively.

2. PCR detection of candidate transgenic plants

The genomic DNA of the leaves of the T0 generation transformed pc186-TaDOF5.6 and pc186-GUS vectors was extracted by CTAB (Sambrook and Russell, molecular cloning Experimental guidelines, 2001).

The primers were designed to detect the bar gene and the primer pair sequences (upstream primer: 5'-GGCGGTCTGCACCATCGTCAACCACTAC-3', SEQ ID No.9; downstream primer: 5'-AGTCCAGCTGCCAGAAACCCACGTCATG-3', SEQ ID No. 10) were 446bp in length. The amplification system was 1. Mu.l of the upstream primer (10. Mu. Mol/. Mu.l), 1. Mu.l of the downstream primer (10. Mu. Mol/. Mu.l), 10. Mu.l of 2X Rapid Taq Master Mix, 1. Mu.l of cDNA template, and ddH ₂ O the total volume was made up to 20. Mu.l. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95℃for 15 seconds, annealing at 58℃for 15 seconds, extension at 72℃for 15 seconds, and circulation 32 times; extension was carried out at 72℃for 5 minutes.

The PCR identification result is shown in FIG. 3, and the bar gene fragment of 446bp is not found in the wild type wheat.

3. Transformation efficiency statistics of wheat of different genotypes

After wheat young embryo explants are infected by an agrobacterium-mediated method, induced and formed resistant callus is transferred to an LSF-P5 culture medium, and after tissue culture is carried out until resistant buds are formed, PCR identification is carried out. Counting the number of the resistant calli and the number of positive seedlings, and calculating the induction rate and the conversion efficiency of the resistant calli, wherein the calculation formula is as follows:

induction rate of resistant calli (%) = (number of resistant calli/(total number of young embryos) ×100%;

conversion efficiency (%) = (number of positive seedlings/(total number of young embryos) ×100%.

The results are shown in Table 1.

Table 1: comparison of transformation efficiency of control vector and pc186-TaDOF5.6 vector

The results show that: compared with a control vector pc186-GUS, the transformation efficiency of the same-genotype wheat can be improved by transforming the pc186-TaDOF5.6 vector. In Fielder, kenong 199 and Jimai 22, the induction rates of the resistant calli of the transformed pc186-TaDOF5.6 vector were 95.60%, 89.52% and 86.09%, respectively, which were higher than the induction rates of the resistant calli of the transformed control vector pc186-GUS by 91.81%, 84.53% and 77.78%, respectively. In terms of transformation efficiency, the transformation efficiency of the Fielder transformed pc186-tadof5.6 vector was 180.22%, approaching 5-fold that of the control vector; the transformation efficiency of the Kenong 199 receptor genotype is up to 75.24%, which is far higher than that of the control vector pc186-GUS by 0.55%. When the Jimai 22 is used as a receptor for genetic transformation, a positive transgenic plant cannot be obtained by the control vector pc186-GUS, and the transformation efficiency of the transformation pc186-TaDOF5.6 vector is 44.35%. From the above results, it is clear that the TaDOF5.6 gene can greatly improve the transformation efficiency of wheat and partially solve the genotype dependence problem in the genetic transformation of wheat. Particularly, in genotypes with more difficult transformation, the transformation efficiency is improved to a larger extent.

The foregoing description is only of the preferred embodiments of the present application and is not intended to limit the same, but rather, various modifications and variations may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims (8)

1. Application of TaDOF5.6 gene in improving genetic transformation efficiency of wheat;

   the TaDOF5.6 gene is a nucleic acid molecule shown in the following i) or ii):

   i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

   ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
2. 2. The use according to claim 1, wherein the tadof5.6 gene increases wheat genetic transformation efficiency by increasing the induction rate and transformation efficiency of resistant calli.
3. Application of TaDOF5.6 gene encoded protein in improving genetic transformation efficiency of wheat.
4. 4. The use according to claim 3, wherein the protein encoded by the tadof5.6 gene is a protein represented by the following (A1) or (A2):

   (A1) A protein consisting of an amino acid sequence shown as SEQ ID NO.2 in a sequence table;

   (A2) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in (A1) with a protein tag.
5. 5. Use of an expression cassette, recombinant expression vector or recombinant bacterium comprising the tadof5.6 gene to facilitate the introduction of a nucleic acid molecule into wheat.
6. 6. A method for improving the transformation efficiency of a nucleic acid molecule introduced into a plant of interest, comprising the steps of:

   transferring TaDOF5.6 gene and nucleic acid molecule into target plant to raise the transformation efficiency of the nucleic acid molecule into target plant;

   the TaDOF5.6 gene is a nucleic acid molecule shown in the following i) or ii):

   i) The nucleotide sequence is a nucleic acid molecule shown as SEQ ID NO. 1;

   ii) a nucleic acid molecule other than i) encoding the amino acid sequence shown in SEQ ID NO. 2.
7. 7. The method of claim 6, wherein the tadof5.6 gene and nucleic acid molecule are transferred into the plant of interest by a pc186 expression vector.
8. 8. The method according to claim 6 or 7, wherein the plant of interest is wheat.

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