CN118005757A - Wheat cold-resistant gene TaERF-like and application thereof - Google Patents

Abstract

The present invention relates to the technical field of plant genetic engineering, and in particular to a wheat cold-resistant gene TaERF7-like and an application thereof. The present invention provides an application of a wheat ethylene response element binding protein family AP2/EREBP transcription factor gene TaERF7-like in plant cold resistance and advancing the flowering time of plants. Plants transformed with the gene can tolerate low temperature stress and advance the flowering time. The nucleotide sequence of the gene and the amino acid sequence of the encoded protein are shown in SEQ ID NO.1 and SEQ ID NO.2, respectively. By overexpressing the gene in Arabidopsis thaliana, the cold resistance of transgenic plants can be significantly improved, and the flowering time of plants can be advanced. The TaERF7-like gene of the present invention can be used as a gene resource for genetic improvement of crops, and has broad application prospects.

Description

Wheat cold-tolerant gene TaERF7-like and its application

Technical Field

The present invention relates to the technical field of plant genetic engineering, and in particular to a wheat gene TaERF7-like and an application thereof.

Background technique

Low temperatures can cause wheat seedlings to freeze and die, and in severe cases, it can cause crop failure, posing a huge threat to wheat production. Therefore, breeding cold-resistant varieties is an important technical approach to deal with wheat frost damage. Using Agrobacterium-mediated genetic transformation technology to overexpress stress-resistant genes in target plants and develop new transgenic plant varieties with better stress resistance is a technology with broad application prospects. At present, a large number of stress-resistant genes have been cloned and transferred into a variety of plants for use in molecular stress-resistant breeding and research on stress response mechanisms.

ERF (ethylene responsive factor) is a subgroup of AP2/ERFBP (Ethylene responsive element-binding proteins) transcription factors that exist only in plants. ERF transcription factors contain a conserved 60-amino acid AP2/ERF domain that can recognize the GCC-box element present in the promoter of ethylene-responsive genes. More and more studies have shown that ERF transcription factors are involved in plant resistance to various adversities such as pathogens, drought, salinity, and heavy metals, but there are no reports on the role of ERF transcription factors in plant cold tolerance. The functions of most ERF transcription factors are still unclear and need to be further explored.

Summary of the invention

The present invention provides a wheat cold-resistant gene TaERF7-like and its application, and provides an application of the wheat ERF transcription factor gene TaERF7-like in cold-resistant aspects, and the gene can be used to breed cold-resistant crop varieties. The present invention also finds that the gene TaERF7-like can advance the flowering time of plants and promote early maturity of plants.

The present invention provides a protein TaERF7-like, wherein the protein TaERF7-like has any of the following amino acid sequences:

(1) the amino acid sequence shown in SEQ ID NO.2;

(2) an amino acid sequence having the same functional protein obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO.2;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 2; preferably, the homology is at least 90%; more preferably, 95%; and even more preferably, 99%.

The present invention also provides a gene TaERF7-like, wherein the gene TaERF7-like is used to encode the protein TaERF7-like;

Preferably, the gene TaERF7-like has any of the following nucleotide sequences:

(1) the nucleotide sequence shown in SEQ ID NO.1;

(2) A nucleotide sequence that is complementary to, homologous to, or encoding a protein with the same function as the sequence shown in SEQ ID NO. 1, or obtained by substitution, insertion or deletion of one or more nucleotides.

Preferably, the cDNA sequence of the wheat cold-resistant gene TaERF7-like is shown as SEQ ID NO.1.

The present invention also provides a primer for amplifying the gene TaERF7-like.

Preferably, the nucleotide sequence included in the primer includes the nucleotide sequence shown in SEQ ID NO.5-6.

The present invention also provides a biological material, comprising the gene TaERF7-like.

According to the biological material, the biological material is any one of a recombinant vector, an expression cassette, a recombinant bacterium or a host cell.

Preferably, the host cell includes a host cell that can develop into a plant and/or a host cell that cannot develop into a plant.

The present invention also provides an overexpression vector PBI121::TaERF7-like containing the above-mentioned wheat cold-resistant gene, which can be used in any species that can be genetically transformed.

The present invention also provides the use of the protein TaERF7-like, the gene TaERF7-like or the biomaterial in any of the following:

1) Regulate plant cold tolerance;

Preferably, the regulation is positive regulation;

2) Application in plant breeding;

3) preparing cold-resistant plants;

4) Regulate the flowering time of plants;

Preferably, the plant is caused to bloom early;

5) Preparing early flowering plants.

The present invention also provides a method for regulating plant cold resistance and/or regulating plant flowering time, comprising: regulating the expression level of the gene encoding the TaERF7-like protein in the plant.

According to the method for regulating plant cold resistance and/or regulating plant flowering time, the cold resistance and/or flowering time of the plant are affected by increasing the expression level of the gene encoding the TaERF7-like protein in the plant.

The present invention also provides a method for constructing a transgenic plant, wherein the gene TaERF7-like is introduced into a target plant to obtain a transgenic plant with improved cold resistance and/or an early flowering time.

According to the application, the method for regulating plant cold resistance and/or regulating plant flowering time, and the method for constructing a transgenic plant, the plant is Arabidopsis, wheat, barley, rice or corn.

The present invention provides the application of wheat ERF transcription factor gene TaERF7-like in cultivating cold-resistant crops. The present invention clones the protein coding sequence of wheat cold-resistant transcription factor gene TaERF7-like from wheat variety Zhongmai 8444, and overexpresses the gene in plant cells by adding an enhanced promoter to the vector. In order to facilitate the screening of transgenic plants or cell lines, a reporter gene (GUS gene or GFP fluorescent reporter gene) or a biotin marker gene with resistance (hygromycin, kanamycin, gentamicin, etc.) can be added. The expression vector containing the TaERF7-like gene of the present invention can be used to transform plant cells or tissues by conventional biological methods such as gene guns and Agrobacterium-mediated, and continue to be cultivated into a complete plant. The transformed plant can be a dicotyledonous plant or a monocotyledonous plant such as Arabidopsis, wheat, rice, corn and soybean.

The present invention cloned the wheat cold-resistant gene TaERF7-like for the first time using plant genetic engineering technology, and transferred the gene into Arabidopsis thaliana by Agrobacterium-mediated genetic transformation. By identifying the cold-resistant phenotype of wild-type and transgenic plants, it was confirmed that the overexpression of the gene of the present invention can significantly improve the cold resistance of plants. The present invention will provide new gene resources for the genetic improvement of crop cold resistance and has broad application prospects.

The present invention also found that the gene TaERF7-like can advance the flowering time of plants and promote early maturity of plants.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to more clearly illustrate the technical solutions in the present invention or the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art.

Figure 1 is a graph showing the expression level of the TaERF7-like gene in leaves of wheat at the three-leaf and one-heart stage provided in Example 1 of the present invention, measured by qRT-PCR after being treated with -5°C low temperature stress for 0h, 1h, 3h, 6h, and 12h.

FIG. 2 is a graph showing the expression levels of the TaERF7-like gene detected by semi-quantitative PCR in the wild-type and transgenic Arabidopsis plants overexpressing the TaERF7-like gene provided in Example 3 of the present invention.

FIG3 shows wild-type and transgenic Arabidopsis seedlings before freezing treatment and after 7-day recovery according to Example 4 of the present invention; the transgenic seedlings exhibit stronger cold tolerance.

FIG4 is a statistical table of survival rates of wild-type and transgenic Arabidopsis seedlings after low temperature stress treatment provided in Example 4 of the present invention.

FIG5 shows the wild-type and transgenic Arabidopsis plants before freezing treatment and after 7-day recovery according to Example 4 of the present invention; the transgenic plants exhibit stronger cold tolerance.

Figure 6 shows the phenotypes of wheat transfected with BSMV:γ0 and BSMV:TaERF7 before and after low temperature stress treatment as provided in Example 5 of the present invention; barley mosaic virus successfully induced the down-regulation of TaER7-like gene expression in wheat, and the cold resistance of the plant was significantly reduced.

FIG. 7 shows that transgenic Arabidopsis thaliana blooms earlier than wild-type Arabidopsis thaliana under a long photoperiod provided in Example 6 of the present invention.

Detailed ways

In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Example 1 Cloning and expression analysis of wheat genes

1.1 Extraction of total RNA from wheat

Total RNA was extracted from plant tissues using Biozol total RNA extraction reagent (Biozol Biotechnology Co., Ltd., Beijing).

a. Plant tissue homogenate. Add steel beads to a 2 ml centrifuge tube in advance, take an appropriate amount of tissue leaves and put them into the tube. After fully cooling in liquid nitrogen, use a high-throughput tissue grinder with a frequency of 45 Hz to grind the leaves into powder for 60 seconds. Add 1 ml of Biozol total RNA extraction reagent, mix up and down, and vortex for 1 minute. Let stand at room temperature for 5 minutes.

b. Separation: Add 200 μl of chloroform to the centrifuge tube, vortex for 30 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12,000×g and 4°C for 10 minutes. Pipette the upper aqueous phase containing total RNA into a new centrifuge tube, add 500 μl of isopropanol, invert several times to mix, and let stand at room temperature for 10 minutes.

c. Precipitation: Centrifuge at 12000×g, 4℃ for 10min. At this time, white RNA precipitate can be seen at the bottom of the tube. Discard the supernatant. Add 1ml 75% ethanol and gently invert to mix so that the white RNA precipitate floats. Centrifuge at 12000×g, 4℃ for 2min and discard the supernatant. Leave to dry at room temperature for 10min.

e. Dissolution and concentration determination: Add appropriate amount of DEPC water to dissolve RNA precipitate. Use ultra-micro-volume UV spectrophotometer (Thermo Fisher NanoDrop ^TM One/OneC) to determine the concentration and dilute to the same concentration. Store at -80℃.

1.2 Synthesis of first-strand cDNA

use All-in-One First-Strand cDNA Synthesis SuperMix forqPCR (One-Step gDNA Removal) Kit synthesizes the first-strand cDNA. Reaction components: Total RNA 2μl, 5× All-in-One SuperMix for qPCR 4μl, gDNA Remover 1μl, RNase-free Water 13μl, gently pipette to mix. The reaction procedure is: incubate at 42℃ for 15min, heat at 85℃ for 5s to inactivate RT/RI and gDNARemover.

1.3 Cloning of the TaERF7-like gene CDS segment

Based on the TaERF7-like gene sequence published in the wheat Chinese Spring reference genome (RefSeq v.1.1) as the reference sequence, specific primers were designed for PCR amplification. The primers used for the full-length sequence were: Forward: 5'-ATGAGGAACGGCAGCACCG-3' (SEQ ID No.7); Reverse: 5'-CTAGAAGAGCCGCAGCTCCGT-3' (SEQID No.8), and the primers for the 292bp fragment amplification were Forward: 5'-GCTCTTCTAGGACGGATCCA-3' (SEQ ID No.9); Reverse: 5'-GAATGCTCTCCGGTTACACT-3' (SEQ ID No.10). The cDNA of wheat (Triticum aestivum L.) variety Zhongmai 8444 was used as the amplification template, and the PCR reaction system was 40 μl: 1 μl of amplification template, 2× MaxMaster Mix 20μl, upstream primer (10μM) 1μl, downstream primer (10μM) 1μl, RNase-free Water 17μl. Reaction procedure: 95℃ pre-denaturation for 30s, 1 cycle; 95℃ denaturation for 5s, 60℃ annealing for 30s, 72℃ extension for 1min, amplification for 35 cycles; 72℃ full extension for 10min. Detect PCR results by 1.5% agarose gel electrophoresis.

1.4 Analysis of the expression pattern of wheat TaERF7-like gene after low temperature induction

Wheat seedlings at the three-leaf and one-heart stage were subjected to low temperature stress at -5°C. Wheat leaves at 0h, 1h, 3h, 6h and 12h were collected and total RNA was extracted and the first-strand cDNA was synthesized. qRT-PCR was used to analyze the changes in the expression of TaERF7-like genes in the samples. Three biological replicates were performed for each sample, and three technical replicates were performed for each biological replicate. qRT-PCR amplification system and procedure:

The first-strand cDNA synthesized by reverse transcription was diluted 4 times and used as a qRT-PCR template. The expression of the target gene in the leaf tissue of Zhongmai 8444 was analyzed using a CFX96 TouchReal-Time PCR Detection System real-time fluorescence quantitative RT-PCR instrument (BIORAD, USA), with wheat actin ACTIN as the internal reference gene and specific primers (see Table 1).

Table 1 Primers used for real-time fluorescence quantitative PCR

Primer name Primer sequences Actin-RT-F 5'-GGTAACATTGTGCTCAGTGGTGG-3' (SEQ ID No. 3) Actin-RT-R 5'-AACGACCTTAATCTTCATGCTGC-3' (SEQ ID No. 4) ERF7-RT-F 5'-CAGGATGCCAGAGCAACTG-3' (SEQ ID No. 5) ERF7-RT-R 5'-GGTTGAGGTCGAACACGAAG-3' (SEQ ID No. 6)

Reaction system: cDNA template 1 μl, 2× Green qPCR SuperMix 5μl, upstream primer (10μM) 0.3μl, downstream primer (10μM) 0.3μl, RNase-free Water 3.4μl. The reaction procedure was: 94℃ pre-denaturation for 30 seconds, 1 cycle; 94℃ denaturation for 5 seconds, 60℃ annealing for 30 seconds, amplification for 40 cycles; collect fluorescence serial number during melting curve stage. The relative expression of genes was calculated by 2 ^-ΔΔCt method, and the results are shown in Figure 1.

Example 2 Recombinant plasmid vector construction

2.1 Construction of overexpression vector

The gene overexpression vector PBI121 was linearized with restriction endonucleases SacⅠ and XbaⅠ. The enzyme digestion system was: PBI121 vector 40μl, restriction endonucleases SacⅠ1μl, XbaⅠ1μl, 10×Cutsmart Buffer 10μl, ddH ₂ O48μl. After 37℃ enzyme digestion reaction for 3h, 500μl of anhydrous ethanol was added and mixed by inversion, centrifuged at 12000×g for 10min, dried and dissolved in water.

The TaERF7-like gene CDS sequence recovered from the gel was connected with the linearized PBI121 vector sequence using a seamless cloning kit. The connection system was: 2×One Step Cloning Mix 5μl, linearized vector 1μl (50ng/μl), insert fragment 2μl (150ng/μl), ddH ₂ O 2μl. The connection procedure was: 50℃ reaction for 30min.

After the ligation reaction, the ligation system was transformed into E. coli DH5α competent cells, and positive clones were identified by PCR amplification. After PCR amplification and identification, Sanger sequencing was performed to verify that the recombinant plasmid was named PBI121::TaERF7-like.

2.2 Construction of expression silencing vector

The pCa-γbLIC vector was linearized with restriction endonuclease ApaⅠ. The enzyme digestion system was: 40μL pCa-γbLIC vector, 1μL restriction endonuclease ApaⅠ, 10μL 10×Cutsmart Buffer, 49μL ddH ₂ O. The digestion reaction procedure was 37℃, 3h. 500μL of anhydrous ethanol was added and mixed by inversion, centrifuged at 12000rpm for 10min, dried and dissolved in water. The 292bp TaERF7-like gene CDS sequence recovered from the gel was connected with the linearized pCa-γbLIC vector sequence using a seamless cloning kit. After the connection reaction was completed, the connection system was transformed into Escherichia coli DH5α competent cells, and positive clones were identified by PCR amplification. After the PCR amplification was correctly identified, Sanger sequencing was performed for verification, and the recombinant plasmid was named BSMV::TaERF7-like.

2.3 Transform the recombinant plasmid into Agrobacterium competent cells

Take GV3101 Agrobacterium competent cells and place them on ice to melt. Pipette 1μg plasmid and 100μl Agrobacterium GV3101 competent cells and mix them evenly, freeze them in liquid nitrogen for 5min, quickly transfer to 37℃ to melt for 3min, and finally place them on ice for 3min; add 800μl liquid LB medium without antibiotics, shake and recover at 28℃ and 200rpm for 3h; centrifuge for 2min to collect bacteria; leave 100μl supernatant in the tube, blow and resuspend the bacteria with a pipette tip, and apply the resuspended liquid to an LB plate containing 25μg/ml Kan and 25μg/ml Rif. Incubate at 28℃ inverted for 48h until monoclonal antibodies grow. Use a pipette tip to pick up monoclonal antibodies to 3ml LB liquid medium (containing 25μg/ml kanamycin and 25μg/ml rifampicin), shake and culture at 28℃ and 200rpm overnight. After the bacterial solution PCR amplification is correctly identified, add 50% glycerol at 1 times the volume of the bacterial solution and store at -80℃.

Example 3 Creation of transgenic Arabidopsis

3.1 Arabidopsis thaliana cultivation

Take an appropriate amount of Arabidopsis and place it in clean water. After 48 hours of imbibition at 4°C, sterilize it with 75% ethanol for 5 minutes, and then sterilize it with 10% pasteurized solution for 10 minutes. After disinfection, rinse the seeds with sterile water 5 times, and finally sow the seeds evenly in a culture dish containing 1/2MS culture medium. After the seeds grow true leaves, transplant them into a 7cm×7cm×8cm flower pot and culture them in an artificial culture box with a long light period until flowering.

3.2 Agrobacterium-mediated stable genetic transformation of Arabidopsis

After activating the Agrobacterium containing the PBI121::TaERF7-like recombinant vector, shake the culture in large quantities. When the bacterial solution OD ₆₀₀ is about 0.8, centrifuge at 4500×g for 10 minutes to collect the cells, add Arabidopsis transformation resuspension to resuspend the cells, adjust to OD ₆₀₀ = 0.6, and then add silwet77 transformation adjuvant at 1/1000 of the resuspension volume. Soak the Arabidopsis inflorescence in the Agrobacterium resuspension for 1 minute, gently shake off the excess bacterial solution on the surface, lay the plant flat on a tray to culture overnight in the dark, and then transfer it to normal conditions for culture until it is strong, and collect T ₁ generation seeds.

3.3 Screening of Arabidopsis transgenic positive lines

The _T1 generation seeds were sown on 1/2MS medium plates containing 40 mg/L kanamycin, and transgenic positive plants were screened and transplanted into soil for culture. Leaves were collected to extract DNA, and PCR amplification was performed to further identify positive plants. After they matured, individual plants were harvested to obtain _T2 generation seeds.

3.4 Expression level determination of transgenic Arabidopsis lines

Total RNA was extracted from leaves of transgenic strains with overexpressed TaERF7-like gene, and the expression of TaERF7-like gene was identified by semi-quantitative PCR with ACTIN as internal reference gene. The same volume of PCR product was used for agarose gel electrophoresis detection, and the expression of target gene was determined according to the brightness of bands between different samples. The results showed that the expression of gene in overexpressed strains was significantly higher than that in wild type, and these three strains were selected for the next test.

Example 4 Identification of cold tolerance of transgenic Arabidopsis

4.1 Method for identifying cold tolerance of Arabidopsis thaliana at the seedling stage

After disinfection, Arabidopsis seeds were sown in 1/2MS medium and cultured under normal conditions until the two-leaf stage for low temperature stress. The conditions of low temperature stress were: cold induction at -2°C in a dark environment for 48 hours, then the temperature was lowered to -8°C for 10 hours, and finally returned to normal temperature for 7 days. The seedlings were photographed and the survival rate was calculated. The results showed that TaERF7-like overexpression enhanced the cold resistance of Arabidopsis, and the survival rate of TaERF7-like overexpression strains after low temperature stress was significantly higher than that of the wild type (Figures 3 and 4).

4.2 Methods for identifying cold tolerance of adult Arabidopsis

Arabidopsis plants were treated with low temperature stress when they reached the rosette stage: cold induction was carried out at 4°C in a dark environment for 24 hours, and then the temperature was lowered to -5°C for 5-7 hours, and then the temperature was raised to 10°C for 6 hours to slow down the seedlings, and finally adjusted to normal environmental conditions for cultivation, and the frostbite of the plants was observed by taking pictures. The results showed that the growth status of the TaERF7-like overexpression line and the wild-type plants was similar before treatment, and the frostbite rate after low temperature stress was lower than that of the wild-type (Figure 5).

Example 5 Identification of cold tolerance after gene expression silencing in wheat

5.1 Wheat Planting

Take wheat seeds with full grains and place them in a culture dish containing moist filter paper. Let them swell at room temperature until the seeds turn white. Then select seeds with consistent growth and sow them in flower pots. Sow 4 seeds in each flower pot and culture them in an artificial incubator until they reach the two-leaf and one-heart stage.

5.2 Tobacco cultivation and injection

Take an appropriate amount of Nicotiana benthamiana seeds and place them in clean water. Place them at room temperature to absorb for 48 hours. Use filter paper to absorb the moisture on the surface of the seeds, and then evenly spread them on the surface of the fully moistened nutrient soil. Cover the flowerpot with plastic wrap or transparent plastic cloth to keep the soil moist. When the seedlings grow to 2-4 leaves, use tweezers to dig out the seedlings and transplant them into 7cm×7cm×8cm flowerpots. Transplant one seedling to each flowerpot and cultivate them in an artificial incubator until the 6-8 leaf stage.

After the Agrobacterium containing the target vector is activated, shake the bacteria in large quantities. When the bacterial solution OD600 is about 0.8, centrifuge at 4500×g for 10 minutes to collect the bacteria, add tobacco transformation resuspension to resuspend the bacteria twice, and finally add an appropriate amount of resuspension to adjust to OD600=0.6. Mix equal volumes (1:1:1) of the resuspended Agrobacterium containing α, β, and γ vectors, stand at 25℃ in the dark for 3 hours, and then use a 5ml needleless syringe to penetrate and inject the unfolded leaves. After 10 days, take tobacco leaves with virus symptoms, add an appropriate amount of 20μM phosphate buffer (pH 7.2) and a small amount of diatomaceous earth in a mortar, grind the leaves into a homogenate, and rub and inoculate the second leaf of two-leaf one-heart stage wheat.

5.3 Identification of wheat cold resistance

The virus-inoculated wheat was cultured at 22°C for 10-14 days, and a small amount of leaves were taken to extract RNA. The silencing effect of the target gene was detected by qRT-PCR. The plants inoculated with BSMV:γ0 virus were used as controls to identify the cold tolerance of plants with downregulated expression of TaERF7-like gene in a low-temperature incubator. The treatment conditions were: cold induction at 4°C for 12 hours in a dark environment, then the temperature was lowered to -6°C for 5 hours, and then the temperature was raised to 10°C for 2 hours. The results showed that the expression of TaERF7 gene in wheat transfected with BSMV:TaERF7 was significantly lower than that in the control (BSMV:γ0 plants) (Figure 6B); and when the third or fourth leaves of the plant began to show wilting and water-soaked frostbite phenotype, the BSMV:γ0 virus-inoculated plants still performed normally (Figure 6A and C).

Example 6 Identification of Plant Flowering Period

Transgenic and wild-type Arabidopsis seeds were placed in clean water, imbibed at 4°C for 48 hours, and then sterilized with 75% ethanol for 5 minutes, and then sterilized with 10% pasteurized solution for 10 minutes. After disinfection, the seeds were rinsed with sterile water 5 times, and finally the seeds were evenly sown in a culture dish containing 1/2MS culture medium. One week later, the seedlings were transplanted into a 7cm×7cm×8cm flower pot and cultured in an artificial incubator for 3-4 weeks using a long photoperiod. The growth environment was 16h of light, 22°C, 8h of darkness, 20°C, 50% relative humidity, and a photosynthetic photon flux density of 450μmol·m ^-2 ·s ^-1 of light intensity. The flowering of wild-type and transgenic plants was observed and counted. The results showed that Arabidopsis overexpressing TaERF7-like bloomed earlier than wild-type plants (Figure 7).

The SEQ ID NO.1-2 sequences involved in the present invention are as follows:

Information of SEQ ID NO.1

Sequence characteristics:cDNA

Length: 582 bp

Type: Nucleotide

Chain: Single chain

SEQ ID NO.1

Information about SEQ ID NO.2

Length: 193aa

Type: Amino Acid

SEQ ID NO.2

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A protein TaERF-like, wherein the protein TaERF-like has any one of the following amino acid sequences:

(1) An amino acid sequence as shown in SEQ ID NO. 2;

(2) Amino acid sequence with the same functional protein obtained by substituting, inserting or deleting one or more amino acids in the amino acid sequence shown as SEQ ID NO. 2;

(3) An amino acid sequence having at least 80% homology with the amino acid sequence shown in SEQ ID NO. 2; preferably, the homology is at least 90%; more preferably 95%; further preferably 99%.

2. A gene TaERF-like, wherein said gene TaERF-like is used to encode a protein TaERF7-like of claim 1;

preferably, the gene TaERF, 7-like, has any one of the following nucleotide sequences:

(1) A nucleotide sequence shown as SEQ ID NO. 1;

(2) A nucleotide sequence encoding the same functional protein that is complementary, homologous, or obtained by substitution, insertion or deletion of one or more nucleotides to the sequence shown in SEQ ID NO. 1.

3. A primer for amplifying the gene TaERF-like of claim 2;

Preferably, the primer comprises a nucleotide sequence comprising the nucleotide sequence shown in SEQ ID NO. 5-6.

4. A biological material comprising the gene TaERF-like of claim 2.

5. The biological material according to claim 4, wherein the biological material is any one of a recombinant vector, an expression cassette, a recombinant bacterium, or a host cell;

Preferably, the host cell comprises a host cell that can develop into a plant and/or a host cell that cannot develop into a plant.

6. Use of a protein TaERF, a 7-like according to claim 1, a gene TaERF, 7-like according to claim 2 or a biological material according to claim 4 in any of the following:

1) Regulating and controlling cold resistance of plants;

Preferably, the modulation is positive modulation;

2) Application in plant breeding;

3) Preparing cold-resistant plants;

4) Regulating and controlling the flowering time of plants;

Preferably, the plant is flowering in advance;

5) Preparing plants flowering in advance.

7. A method of regulating cold tolerance and/or regulating flowering time in a plant comprising: regulating the expression level of the gene encoding the TaERF-like protein according to claim 1 in a plant.

8. The method for controlling plant cold tolerance and/or flowering-time according to claim 7, wherein the plant cold tolerance and/or flowering-time is affected by increasing the expression level of the gene encoding the TaERF-like protein in the plant.

9. A method for constructing transgenic plant features that the gene TaERF-like is introduced into target plant to obtain transgenic plant with improved cold resistance and/or early flowering time.

10. The use according to claim 6, the method for regulating cold tolerance and/or flowering time in plants according to any one of claims 7 to 8, the method for constructing transgenic plants according to claim 9, characterized in that the plants are dicotyledonous plants, also monocotyledonous plants such as: arabidopsis, wheat, barley, rice, maize and soybean;

More preferably Arabidopsis thaliana or wheat.