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

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to a wheat cold-resistant gene TaERF-like and application thereof. The invention provides an application of a wheat ethylene response element binding protein family AP2/EREBP transcription factor gene TaERF-like in plant cold resistance and plant flowering time advance. The plant transformed with the gene can endure low temperature stress and lead flowering time to be advanced, and the nucleotide sequence of the gene and the amino acid sequence of the coded protein are respectively shown as SEQ ID NO.1 and SEQ ID NO. 2. Through over-expression of the gene in arabidopsis, the cold resistance of transgenic plants can be obviously improved, and the flowering time of plants is advanced.

Description

Wheat cold-resistant gene TaERF-like and application thereof

Technical Field

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

Background

The wheat seedlings can be frozen and dead at low temperature, and can be dead in severe cases, so that the wheat production is threatened greatly. Therefore, the cultivation of cold-resistant varieties is an important technical approach for coping with wheat freezing injury. The agrobacterium-mediated genetic transformation technology is utilized to over-express the stress-resistant gene in the target plant, so that a new transgenic plant variety with better stress resistance is developed, and the technology has a wide application prospect. At present, a large number of stress-resistant genes have been cloned and transferred into various plants for molecular stress-resistant breeding and research of stress response mechanisms.

ERF (ethylene responsive factor) is a subset of the class of AP2/ERFBP (Ethylene responsive element-binding proteins) transcription factors that are present only in plants. The ERF transcription factor comprises a conserved 60 amino acid AP2/ERF domain that recognizes the GCC-box element present in the ethylene response gene promoter. More and more researches show that ERF transcription factors are involved in the resistance reaction of plants to various adverse conditions such as bacteria, drought, salt, heavy metals and the like, but the effect of the ERF transcription factors in the cold-resistant process of plants is not reported. The function of most ERF transcription factors remains unclear and remains to be explored further.

Disclosure of Invention

The invention provides a wheat cold-resistant gene TaERF-like and application thereof, and provides application of a wheat ERF transcription factor gene TaERF-like in cold resistance, wherein the gene can be used for cultivating cold-resistant crop varieties. The invention also discovers that the gene TaERF-like can lead the flowering time of plants to be advanced and promote the plants to be early matured.

The invention provides 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%.

The invention also provides a gene TaERF-like, wherein the gene TaERF-like is used for encoding the protein TaERF7-like;

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.

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

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

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

The invention also provides a biological material comprising the gene TaERF-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 comprises a host cell that can develop into a plant and/or a host cell that cannot develop into a plant.

The invention also provides an over-expression vector PBI121: taERF7-like containing the wheat cold-resistant gene, which can be applied to any species capable of carrying out genetic transformation.

The invention also provides the use of said protein TaERF7-like, said gene TaERF7-like or said biological material 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.

The invention also provides a method for regulating cold resistance and/or flowering time of plants, comprising the following steps: regulating the expression level of the coding gene of TaERF-like protein in the plant.

According to the method for regulating the cold resistance and/or the flowering time of the plant, the cold resistance and/or the flowering time of the plant is influenced by improving the expression quantity of the coding gene of the TaERF-like protein in the plant.

The invention also provides a construction method of the transgenic plant, which is to introduce the gene TaERF-like into a target plant to obtain the transgenic plant with improved cold resistance and/or advanced flowering time.

According to the application, the method for regulating plant cold resistance and/or regulating plant flowering time and the construction method of the transgenic plant, the plant is arabidopsis thaliana, wheat, barley, rice or corn.

The invention provides an application of wheat ERF transcription factor gene TaERF-like in cultivating cold-resistant crops. The invention clones the protein coding sequence of wheat cold-resistant transcription factor gene TaERF-like from wheat 8444 in wheat variety, and the gene is over-expressed in plant cells by adding enhanced promoter into the vector. To facilitate the screening of transgenic plants or cell lines, a reporter gene (GUS gene or GFP fluorescein reporter gene) or a resistant antibiotic marker gene (hygromycin, kanamycin, gentamicin, etc.) or the like may be added. The expression vector containing TaERF-like gene of the present invention can be transformed into cells or tissues of plants by using conventional biological methods such as gene gun, agrobacterium mediation, etc., and further cultured into complete plants. The transformed plant may be either a dicotyledonous plant or a monocotyledonous plant such as: arabidopsis thaliana, wheat, rice, maize, soybean, and the like.

The invention uses plant genetic engineering technology to clone wheat cold-resistant gene TaERF-like for the first time, and transfers the gene into Arabidopsis through agrobacterium-mediated genetic transformation method. The cold-resistant phenotype identification of wild type and transgenic plants proves that the cold resistance of the plants can be obviously improved after the gene is over-expressed. The invention provides new gene resources for cold-resistant genetic improvement of crops and has wide application prospect.

The invention also discovers that the gene TaERF-like can lead the flowering time of plants to be advanced and promote the plants to be early matured.

Drawings

In order to more clearly illustrate the technical solutions of the present invention or of the prior art, the following description will make a brief introduction to the drawings used as required in the description of the embodiments or of the prior art.

FIG. 1 shows the expression level of TaERF-like gene in leaf after 0h, 1h, 3h, 6h, 12h of low temperature stress treatment of wheat in the trefoil-heart stage provided in example 1 of the present invention.

FIG. 2 shows the detection of TaERF-like gene expression level by semi-quantitative PCR in transgenic Arabidopsis plants over-expressing TaERF-like gene and wild-type provided in example 3 of the present invention.

FIG. 3 is a graph of wild type and transgenic Arabidopsis seedlings recovered for 7 days before and after freezing treatment provided in example 4 of the present invention; transgenic seedlings exhibited greater cold tolerance.

FIG. 4 is a table showing the survival rate statistics of wild type and transgenic Arabidopsis seedlings after low temperature stress treatment as provided in example 4 of the present invention.

FIG. 5 is a wild-type and transgenic Arabidopsis plant recovered for 7 days before and after freezing treatment as provided in example 4 of the present invention; transgenic plants exhibit greater cold tolerance.

FIG. 6 shows the phenotype of wheat transfected with BSMV: gamma 0 and BSMV: taERF, before and after low temperature stress treatment, provided in example 5 of the present invention; the barley mosaic virus successfully induces TaER-like genes to be down-regulated and expressed in wheat, and the cold resistance of plants is obviously reduced.

FIG. 7 shows that the transgenic Arabidopsis thaliana flowers earlier than the wild Arabidopsis thaliana in long photoperiod provided in example 6 of the present invention.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings, and it is apparent that the described embodiments are some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1 cloning and expression analysis of wheat Gene

1.1 Extraction of wheat Total RNA

Extraction of total RNA from plant tissues was performed with Biozol total RNA extraction reagent (Bai Rui Biotech Co., ltd., beijing).

A. homogenizing plant tissue. Steel balls are added into a 2ml centrifuge tube in advance, a proper amount of tissue leaves are taken and put into the tube, and after the tissue leaves are fully cooled in liquid nitrogen, the tissue leaves are ground into powder by using a high-flux tissue grinder with the frequency of 45Hz for 60 seconds. Adding 1ml Biozol total RNA extraction reagent, mixing up and down, and vortex shaking for 1min. Standing at room temperature for 5min.

B. separating: 200 μl chloroform was added to the centrifuge tube, vortexed for 30s, and allowed to stand at room temperature for 5min. Centrifuge at 12000 Xg and 4℃for 10min. The upper aqueous phase containing total RNA was aspirated into a new centrifuge tube, 500. Mu.l of isopropanol was added, mixed several times upside down, and allowed to stand at room temperature for 10min.

C. precipitation: centrifugation was performed at 12000 Xg for 10min at 4℃at which time white RNA precipitate was seen at the bottom of the tube and the supernatant was discarded. 1ml of 75% ethanol was added and the mixture was gently inverted and mixed to float the white RNA pellet. Centrifuge at 12000 Xg for 2min at 4deg.C, discard supernatant. Air-drying at room temperature for 10min.

E. dissolution and concentration measurement: an appropriate amount of DEPC water was added to dissolve the RNA precipitate. The concentration was determined using an ultra-micro ultraviolet spectrophotometer (NanoDrop ^TM One/OneC) and diluted to the same concentration. Stored at-80 ℃.

1.2 Synthesis of first Strand cDNA

UsingThe All-in-One First-STRAND CDNA SYNTHESIS SuperMix for qPCR (One-Step gDNA Removal) kit synthesizes First strand cDNA. The reaction components are as follows: total RNA 2. Mu.l, 5×All-in-One SuperMix for qPCR. Mu.l, gDNA remote 1. Mu.l, rnase-FREE WATER. Mu.l were gently mixed by pipetting. The reaction procedure is: incubation at 42℃for 15min and heating at 85℃for 5s for deactivationRT/RI and gDNARemover.

1.3 Cloning of TaERF-like Gene CDS segments

According to the TaERF-like gene sequence published in the wheat China spring reference genome (RefSeq v.1.1) as a reference sequence, designing a specific primer for PCR amplification, wherein a primer ：Forward:5'-ATGAGGAACGGCAGCACCG-3'(SEQ ID No.7);Reverse:5'-CTAGAAGAGCCGCAGCTCCGT-3'(SEQ ID No.8),292bp fragment amplification primer for the full-length sequence is Forward:5'-GCTCTTCTAGGACGGATCCA-3' (SEQ ID No. 9); reverse:5'-GAATGCTCTCCGGTTACACT-3' (SEQ ID No. 10). Wheat (Triticum aestivum l.) variety middle wheat 8444cDNA was used as amplification template, and the PCR reaction system was 40 μl: amplification template 1. Mu.l, 2XMax Master Mix 20. Mu.l, upstream primer (10. Mu.M) 1. Mu.l, downstream primer (10. Mu.M) 1. Mu.l, rnase-FREE WATER. Mu.l. The reaction procedure: pre-denaturation at 95 ℃ for 30s,1 cycle; denaturation at 95℃for 5s, annealing at 60℃for 30s, extension at 72℃for 1min, amplification for 35 cycles; extending at 72 deg.C for 10min. The PCR results were detected by electrophoresis on a 1.5% agarose gel.

1.4 Analysis of expression Pattern of wheat TaERF-like Gene after Low temperature Induction

Wheat seedlings of the three leaves in one heart stage are subjected to low temperature stress treatment at the temperature of minus 5 ℃, wheat leaves treated for 0h, 1h, 3h, 6h and 12h are collected and extracted with total RNA and first strand cDNA is synthesized. The qRT-PCR analysis samples were analyzed for changes in TaERF-like gene expression levels. Each sample was subjected to 3 biological replicates and each biological replicate was subjected to 3 technical replicates. qRT-PCR amplification system and procedure:

The first strand cDNA synthesized by reverse transcription was diluted 4-fold in concentration to be used as qRT-PCR template. The expression of the target gene in leaf tissue of the middle wheat 8444 was analyzed using a CFX96 Touch Real-Time PCR Detection System Real-time fluorescent quantitative RT-PCR apparatus (BIORAD, USA) with wheat ACTIN as an internal reference gene and specific primers (see Table 1).

TABLE 1 primers for real-time fluorescent 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)

The reaction system: cDNA template 1 μl,2×GREEN QPCR Supermix 5. Mu.l, upstream primer (10. Mu.M) 0.3. Mu.l, downstream primer (10. Mu.M) 0.3. Mu.l, RNase-FREE WATER 3.4.4. Mu.l. The reaction procedure is: pre-denaturation at 94 ℃ for 30 seconds, 1 cycle; denaturation at 94℃for 5 seconds, annealing at 60℃for 30 seconds, amplification for 40 cycles; fluorescent serial numbers were collected during the dissolution profile phase. The relative expression level of the gene was calculated by the 2 ^-ΔΔCt method, and the result is shown in FIG. 1.

EXAMPLE 2 construction of recombinant plasmid vector

2.1 Construction of the super-expression vector

The gene overexpression vector PBI121 was linearized with the restriction enzymes SacI and XbaI. The enzyme digestion system is as follows: PBI121 vector 40. Mu.l, restriction enzyme SacI 1. Mu.l, xbaI 1. Mu.l, 10X Cutsmart Buffer. Mu.l, ddH ₂ O48. Mu.l. After enzyme digestion reaction for 3 hours at 37 ℃,500 μl of absolute ethanol is added for inversion and mixing, 12000 Xg is centrifugated for 10 minutes, and water is added for dissolution after airing.

The gel recovered TaERF-like gene CDS sequence was ligated with linearized PBI121 vector sequence using a seamless cloning kit. The connection system is as follows: 2X One Step Cloning Mix. Mu.l, linearized vector 1. Mu.l (50 ng/. Mu.l), insert 2. Mu.l (150 ng/. Mu.l), ddH ₂ O2. Mu.l. The connection procedure is as follows: the reaction was carried out at 50℃for 30min.

After the ligation reaction is completed, the ligation system is transformed into E.coli DH5 alpha competent cells, and positive clones are identified by PCR amplification. After the PCR amplification is correctly identified, sanger sequencing verification is carried out, and the recombinant plasmid is named as PBI121: taERF7-like.

2.2 Expression silencing vector construction

The pCa-gamma bLIC vector was linearized with the restriction enzyme ApaI. The enzyme digestion system is as follows: 40. Mu.L of pCa-gamma bLIC vector, 1. Mu.L of restriction enzyme ApaI, 10X Cutsmart Buffer. Mu.L, and 49. Mu.L of ddH ₂ O. Enzyme digestion reaction procedure: 37℃for 3h. Adding 500 μl of absolute ethanol, mixing, centrifuging at 12000rpm for 10min, air drying, and dissolving in water. The gel recovered CDS sequence of the 29p TaERF7-like gene was ligated with the linearized pCa-gamma bLIC vector sequence using a seamless cloning kit. After the ligation reaction is completed, the ligation system is transformed into E.coli DH5 alpha competent cells, and positive clones are identified by PCR amplification. After the PCR amplification is correctly identified, sanger sequencing verification is carried out, and the recombinant plasmid is named BSMV:: taERF-like.

2.3 Transformation of recombinant plasmids into Agrobacterium competent cells

GV3101 agrobacterium competent cells were thawed on ice. Sucking 1 μg of plasmid, mixing with 100 μl of Agrobacterium GV3101 competent cells, quick freezing with liquid nitrogen for 5min, rapidly transferring to 37deg.C, thawing for 3min, and placing on ice for 3min; adding 800 μl of liquid LB medium without antibiotics, and shaking at 28deg.C and 200rpm for resuscitation for 3 hr; centrifuging for 2min to collect thalli; 100. Mu.l of the supernatant was left in the tube, the resuspended cells were blown with a gun head and the resuspension was plated onto LB plates containing 25. Mu.g/ml Kan and 25. Mu.g/ml Rif. Culturing at 28 deg.c for 48 hr until monoclonal antibody grows out. Monoclonal antibodies were picked up with a gun head to 3ml of LB liquid medium (containing 25. Mu.g/ml kanamycin and 25. Mu.g/ml rifampicin), and incubated overnight at 28℃with shaking at 200 rpm. After the correct identification of the bacterial liquid by PCR amplification, 50% glycerol of 1 time of the bacterial liquid volume is added and stored at-80 ℃.

EXAMPLE 3 creation of transgenic Arabidopsis thaliana

3.1 Arabidopsis thaliana planting

Placing appropriate amount of Arabidopsis thaliana in clear water, swelling at 4deg.C for 48 hr, sterilizing with 75% ethanol for 5min, and sterilizing with 10% pasteurization solution for 10min. After sterilization, the seeds were rinsed 5 times with sterile water and finally the seeds were evenly spot-sown into a petri dish containing 1/2MS medium. Transplanting the seeds into a flowerpot with the length of 7cm multiplied by 8cm after the true leaves of the seeds grow, and culturing the seeds in an artificial incubator with a long photoperiod until the seeds bloom.

3.2 Agrobacterium-mediated stable genetic transformation of Arabidopsis thaliana

Agrobacterium comprising the PBI121: taERF7-like recombinant vector is activated and subjected to a large amount of shaking, when the bacterial liquid OD ₆₀₀ is about 0.8, 4500 Xg is centrifuged for 10min to collect bacterial cells, the Arabidopsis thaliana is added to transform heavy suspension to resuspend the bacterial cells, the OD ₆₀₀ =0.6 is adjusted, and then silwet77 transformation auxiliary agent with the volume of 1/1000 of the heavy suspension is added. The arabidopsis inflorescence is soaked in agrobacterium resuspension for 1min, superfluous bacterial liquid on the surface is gently thrown away, the plant is horizontally placed on a tray for light-proof culture overnight, and then is transferred to normal conditions for culture until the plant is firm, and T ₁ generation seeds are collected.

3.3 Screening of Arabidopsis transgenic Positive lines

Sowing the T ₁ generation seeds on a 1/2MS culture medium flat plate containing 40mg/L kanamycin, screening transgenic positive plants, transplanting the transgenic positive plants into soil for culture, collecting leaves for extracting DNA, carrying out PCR amplification to further determine positive plants, and collecting the single plants after the positive plants are mature to obtain the T ₂ generation seeds.

3.4 Determination of expression level of transgenic Arabidopsis lines

And extracting total RNA from transgenic plant leaves over-expressed by TaERF-like genes, taking ACTIN as an internal reference gene, and identifying the expression condition of TaERF-like genes by utilizing semi-quantitative PCR. And taking the PCR products with the same volume to carry out agarose gel electrophoresis detection, and judging the expression quantity of the target gene according to the strip brightness among different samples. The results show that the expression level of the gene of the over-expressed strain is obviously higher than that of the gene in the wild type, and the three strains are selected for the next test.

Example 4 identification of Cold resistance of transgenic Arabidopsis thaliana

4.1 Method for identifying cold resistance of arabidopsis thaliana in seedling stage

The arabidopsis seeds are sown in a 1/2MS culture medium after being disinfected, and are cultured to a two-leaf stage under normal conditions for low-temperature stress. The conditions of low temperature stress are: cold inducing at-2deg.C for 48 hr in dark environment, freezing at-8deg.C for 10 hr, recovering at normal temperature for 7 days, photographing the seedlings, and counting survival rate. The results show that TaERF-like overexpression enhances cold resistance of Arabidopsis, and that the survival rate of TaERF-like overexpressed lines after low temperature stress is significantly increased compared to wild-type (FIGS. 3 and 4).

4.2 Method for identifying cold resistance of arabidopsis thaliana in adult stage

The arabidopsis plants grow to a rosette stage and are subjected to low-temperature stress treatment: cold induction is carried out for 24 hours at the temperature of 4 ℃ in a dark environment, then the temperature is reduced to-5 ℃ for 5-7 hours, then the temperature is raised to 10 ℃ for 6 hours, finally, the culture is carried out under the normal environment condition, and the frostbite condition of the plants is observed through photographing. The results show that the pre-treatment TaERF-like over-expressed strain has a similar growth status to the wild-type strain and a lower frostbite rate after low temperature stress than the wild-type strain (FIG. 5).

Example 5 identification of Cold resistance of Gene after expression silencing in wheat

5.1 Wheat planting

Placing wheat seeds with full seeds in a culture dish containing moist filter paper, sucking and expanding at room temperature until the seeds are exposed to white, selecting seeds with consistent growth vigor, dibbling the seeds into flowerpots, dibbling 4 seeds from each flowerpot, and culturing the seeds in an artificial incubator until two leaves are in a one-heart period.

5.2 Tobacco planting and injection

And (3) placing a proper amount of the raw tobacco seeds in clean water, standing at room temperature for swelling for 48 hours, then sucking the water on the surfaces of the seeds by using filter paper, and uniformly broadcasting the water on the surfaces of the fully-wetted nutrient soil. The flowerpot is covered by a preservative film or transparent plastic cloth to keep the soil moist. When the seedlings grow to 2-4 leaves, the seedlings are dug out by forceps and transplanted into flowerpots with the length of 7cm multiplied by 8cm, one seedling is transplanted into each flowerpot, and the seedlings are cultivated in an artificial incubator until the period of 6-8 leaves.

And (3) activating agrobacterium containing the target carrier, performing a large amount of shaking, centrifuging 4500 Xg for 10 minutes when the OD600 of the bacterial liquid is about 0.8, collecting the bacterial body, adding the tobacco transformation heavy suspension to resuspend the bacterial body for 2 times, and finally adding a proper amount of heavy suspension to adjust the OD600 to be 0.6. The re-suspended agrobacteria with alpha, beta and gamma carriers respectively are mixed in equal volumes (1:1:1), and after standing for 3 hours at 25 ℃ in the dark, the unfolded leaves are infiltrated and injected by a 5ml needleless injector. Tobacco leaves with viral symptoms were taken after 10 days, and appropriate amount of 20 μm phosphate buffer (pH 7.2) and a small amount of diatomaceous earth were added to a mortar, and the leaves were ground to homogenate, and the second leaf of the two-leaf one-heart wheat was inoculated by friction.

5.3 Identification of Cold resistance of wheat

Culturing wheat after virus inoculation at 22 ℃ for 10-14 days, taking a small number of leaves to extract RNA, and detecting the silencing effect of the target gene by qRT-PCR. And (3) taking plants inoculated with BSMV: gamma 0 virus as a control, and identifying the cold resistance of TaERF-like gene down-regulating expression plants in a low-temperature incubator, wherein the treatment conditions are as follows: cold inducing at 4deg.C for 12 hr in dark environment, freezing at-6deg.C for 5 hr, and raising the temperature to 10deg.C for 2 hr. The results show that TaERF gene expression level in wheat transfected with BSMV TaERF was significantly reduced compared to control (BSMV: gamma 0 plants) (B in FIG. 6); and the third or fourth leaf of the plant starts to show the frostbite phenotype of wilting water immersion, the BSMV: gamma 0 virus inoculated plant still shows normal (A and C in FIG. 6).

Example 6 identification of flowering period in plants

Placing transgenic and wild Arabidopsis seeds in clear water, swelling at 4deg.C for 48 hr, sterilizing with 75% ethanol for 5min, and sterilizing with 10% pasteurization solution for 10min. After sterilization, the seeds were rinsed 5 times with sterile water and finally the seeds were evenly spot-sown into a petri dish containing 1/2MS medium. After one week, transplanting the seedlings into a flowerpot with the length of 7cm multiplied by 8cm, and culturing for 3-4 weeks in an artificial incubator by using a long photoperiod, wherein the growth environment is 16h under illumination at 22 ℃ and 8h under darkness at 20 ℃, the relative humidity is 50%, and the photosynthetic photon flux density of light intensity is 450 mu mol.m ^-2·s^-1. And observing and counting flowering conditions of wild type and transgenic plants. The results showed that TaERF-like overexpressed Arabidopsis were bloomed earlier than the wild-type plants (FIG. 7).

The sequence of SEQ ID NO.1-2 related to the invention is as follows:

information of SEQ ID NO.1

Sequence characterization cDNA

Length: 582bp

Type (2): nucleotide(s)

Chain-based properties: single strand

SEQ ID NO.1

Information of SEQ ID NO.2

Length: 193aa

Type (2): amino acids

SEQ ID NO.2

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart 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.