Wheat cold-tolerant gene TaERF7-like and its application
By cloning and overexpressing the wheat ERF transcription factor gene TaERF7-like, the problem of insufficient plant cold resistance was solved, and the effects of improved cold resistance and advanced flowering time were achieved, providing a new genetic improvement path for crops such as wheat.
Patent Information
- Application Number
- CN202311838098.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-12-28
- Publication Date
- 2025-09-26
- Estimated Expiration
- 2043-12-28
AI Technical Summary
In the prior art, the role of ERF transcription factors in plant cold tolerance has not been reported, and methods for breeding cold-tolerant wheat varieties are limited, resulting in wheat being susceptible to freezing damage at low temperatures, resulting in crop failure.
The wheat ERF transcription factor gene TaERF7-like was cloned and overexpressed in plants through Agrobacterium-mediated genetic transformation technology to improve the cold tolerance of plants. At the same time, the flowering time of plants was affected by regulating the expression level of the gene encoding the TaERF7-like protein.
It significantly improves the cold resistance of plants, advances their flowering time, promotes early maturity, and provides new genetic resources for genetic improvement of crop cold resistance.
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Figure CN118005757B_ABST
Abstract
Description
Technical Field
[0001] 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 Art
[0002] Low temperatures can cause wheat seedlings to freeze and die, and in severe cases, even total crop failure, posing a significant threat to wheat production. Therefore, breeding cold-tolerant varieties is an important approach to combating winter damage in wheat. Using Agrobacterium-mediated genetic transformation to overexpress stress-resistant genes in target plants, thereby developing new transgenic plant varieties with enhanced stress tolerance, is a promising technology. Currently, a large number of stress-resistant genes have been cloned and introduced into various plant species for molecular stress-resistance breeding and research into stress response mechanisms.
[0003] ERFs (ethylene responsive factors) are a subgroup of AP2 / ERFBP (ethylene responsive element-binding proteins) transcription factors found only in plants. ERFs contain a conserved 60-amino acid AP2 / ERF domain that recognizes the GCC-box element present in the promoters of ethylene-responsive genes. A growing number of studies have shown that ERFs are involved in plant resistance to various stresses, including pathogens, drought, salinity, and heavy metals. However, no studies have examined the role of ERFs in plant cold tolerance. The functions of most ERFs remain unclear and require further investigation. Summary of the Invention
[0004] The present invention provides a wheat cold-tolerance gene, TaERF7-like, and its applications, as well as applications of the wheat ERF transcription factor gene TaERF7-like in cold-tolerance research. This gene can be used to cultivate cold-tolerant crop varieties. The present invention also discovered that the TaERF7-like gene can advance plant flowering and promote early maturity.
[0005] The present invention provides a protein TaERF7-like, wherein the protein TaERF7-like has any of the following amino acid sequences:
[0006] (1) the amino acid sequence shown in SEQ ID NO. 2;
[0007] (2) an amino acid sequence of a protein having the same function obtained by replacing, inserting or deleting one or more amino acids in the amino acid sequence shown in SEQ ID NO. 2;
[0008] (3) An amino acid sequence having at least 80% homology to 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%.
[0009] The present invention also provides a gene TaERF7-like, wherein the gene TaERF7-like is used to encode the protein TaERF7-like;
[0010] Preferably, the gene TaERF7-like has any one of the following nucleotide sequences:
[0011] (1) the nucleotide sequence shown in SEQ ID NO. 1;
[0012] (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.
[0013] Preferably, the cDNA sequence of the wheat cold-resistant gene TaERF7-like is shown as SEQ ID NO.1.
[0014] The present invention also provides a primer for amplifying the gene TaERF7-like.
[0015] Preferably, the nucleotide sequence included in the primer includes the nucleotide sequence shown in SEQ ID NO.5-6.
[0016] The present invention also provides a biological material comprising the gene TaERF7-like.
[0017] 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.
[0018] 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.
[0019] 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.
[0020] The present invention also provides use of the protein TaERF7-like, the gene TaERF7-like or the biomaterial in any of the following:
[0021] 1) Regulate plant cold tolerance;
[0022] Preferably, the regulation is positive regulation;
[0023] 2) Application in plant breeding;
[0024] 3) preparing cold-resistant plants;
[0025] 4) Regulate the flowering time of plants;
[0026] Preferably, the plant is caused to bloom early;
[0027] 5) Preparing early flowering plants.
[0028] 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.
[0029] 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.
[0030] 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 early flowering time.
[0031] 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 thaliana, wheat, barley, rice or corn.
[0032] The present invention provides the application of wheat ERF transcription factor gene TaERF7-like in cultivating cold-resistant crops. The present invention cloned the protein coding sequence of wheat cold-resistant transcription factor gene TaERF7-like from wheat variety Zhongmai 8444, and overexpressed 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 then continue to cultivate into complete plants. The transformed plants can be dicotyledons or monocotyledons such as Arabidopsis thaliana, wheat, rice, corn and soybean.
[0033] This study, using plant genetic engineering techniques, cloned the wheat cold-tolerance gene TaERF7-like for the first time and introduced it into Arabidopsis thaliana via Agrobacterium-mediated genetic transformation. Phenotypic characterization of cold-tolerance in wild-type and transgenic plants confirmed that overexpression of the gene significantly improved plant cold tolerance. This study will provide a new gene resource for genetically improving cold-tolerance in crops and has broad application prospects.
[0034] The present invention also discovered that the gene TaERF7-like can advance the flowering time of plants and promote early maturity of plants. BRIEF DESCRIPTION OF THE DRAWINGS
[0035] In order to more clearly illustrate the technical solutions of 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.
[0036] Figure 1 The wheat at the three-leaf and one-heart stage provided in Example 1 of the present invention was subjected to -5°C low temperature stress for 0 h, 1 h, 3 h, 6 h, and 12 h, and the expression level of the TaERF7-like gene in the leaves was determined by qRT-PCR.
[0037] Figure 2 The expression level of the TaERF7-like gene was 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.
[0038] Figure 3 Wild-type and transgenic Arabidopsis seedlings before and after 7-day recovery from freezing treatment provided in Example 4 of the present invention; the transgenic seedlings showed stronger cold tolerance.
[0039] Figure 4 This 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.
[0040] Figure 5 Wild-type and transgenic Arabidopsis plants before and after freezing treatment and 7 days of recovery provided in Example 4 of the present invention; the transgenic plants showed stronger cold tolerance.
[0041] Figure 6 This is the phenotype 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.
[0042] Figure 7 The transgenic Arabidopsis thaliana provided in Example 6 of the present invention blooms earlier than the wild-type Arabidopsis thaliana under the long photoperiod. DETAILED DESCRIPTION
[0043] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.
[0044] Example 1 Cloning and expression analysis of wheat genes
[0045] 1.1 Extraction of total RNA from wheat
[0046] Total RNA was extracted from plant tissues using Biozol total RNA extraction reagent (Biozol Biotechnology Co., Ltd., Beijing).
[0047] a. Homogenize the plant tissue. Pre-fill a 2 ml centrifuge tube with steel balls. Place an appropriate amount of tissue leaves into the tube. After sufficient cooling in liquid nitrogen, grind the leaves using a high-throughput tissue grinder at 45 Hz for 60 seconds until they are powdered. Add 1 ml of Biozol Total RNA Extraction Reagent, mix thoroughly, and vortex for 1 minute. Let stand at room temperature for 5 minutes.
[0048] b. Separation: Add 200 μl of chloroform to a centrifuge tube, vortex for 30 seconds, and let stand at room temperature for 5 minutes. Centrifuge at 12,000 × g at 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.
[0049] c. Precipitation: Centrifuge at 12,000 × g, 4°C for 10 minutes. A white RNA precipitate will be visible at the bottom of the tube. Discard the supernatant. Add 1 ml of 75% ethanol and gently invert to mix until the white RNA precipitate floats. Centrifuge at 12,000 × g, 4°C for 2 minutes. Discard the supernatant. Allow to air dry at room temperature for 10 minutes.
[0050] e. Dissolution and concentration determination: Add appropriate amount of DEPC water to dissolve the RNA precipitate. Use ultraviolet spectrophotometer (Thermo Fisher NanoDrop TM One / OneC) to determine the concentration and dilute to the same concentration. Store at -80°C.
[0051] 1.2 Synthesis of first-strand cDNA
[0052] use All-in-One First-Strand cDNA Synthesis SuperMix for qPCR (One-Step gDNA Removal) Kit synthesizes 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 gDNA Remover.
[0053] 1.3 Cloning of the TaERF7-like gene CDS segment
[0054] Based on the TaERF7-like gene sequence published in the wheat Chinese Spring reference genome (RefSeq v.1.1), specific primers were designed for PCR amplification. The full-length primers used were: Forward: 5'-ATGAGGAACGGCAGCACCG-3' (SEQ ID No. 7); Reverse: 5'-CTAGAAGAGCCGCAGCTCCGT-3' (SEQ ID No. 8). The 292 bp fragment amplification primers were: Forward: 5'-GCTCTTCTAGGACGGATCCA-3' (SEQ ID No. 9); Reverse: 5'-GAATGCTCTCCGGTTACACT-3' (SEQ ID No. 10). Wheat (Triticum aestivum L.) cultivar Zhongmai 8444 cDNA was used as the amplification template. The PCR reaction system was 40 μl: 1 μl amplification template, 2× MaxMaster Mix 20 μl, upstream primer (10 μM) 1 μl, downstream primer (10 μM) 1 μl, RNase-free water 17 μl. Reaction procedure: 1 cycle of initial denaturation at 95°C for 30 s; 35 cycles of denaturation at 95°C for 5 s, annealing at 60°C for 30 s, and extension at 72°C for 1 min; and full extension at 72°C for 10 min. PCR results were verified by 1.5% agarose gel electrophoresis.
[0055] 1.4 Analysis of the expression pattern of wheat TaERF7-like gene after low temperature induction
[0056] Wheat seedlings at the three-leaf, one-heart stage were subjected to -5°C low-temperature stress. Wheat leaves were collected at 0, 1, 3, 6, and 12 hours of treatment, and total RNA was extracted and first-strand cDNA synthesized. qRT-PCR was used to analyze the expression of the TaERF7-like gene 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:
[0057] The first-strand cDNA synthesized by reverse transcription was diluted 4-fold and used as a template for qRT-PCR. The expression of target genes in Zhongmai 8444 leaf tissue was analyzed using a CFX96 TouchReal-Time PCR Detection System (BIORAD, USA) with wheat actin ACTIN as the internal reference gene and specific primers (see Table 1).
[0058] Table 1 Primers used for real-time fluorescence quantitative PCR
[0059] 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)
[0060] 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; fluorescence serial number was collected during the melting curve. -ΔΔCt The relative gene expression was calculated by Figure 1 .
[0061] Example 2 Construction of recombinant plasmid vector
[0062] 2.1 Overexpression vector construction
[0063] Linearize the gene overexpression vector PBI121 with restriction endonucleases SacⅠ and XbaⅠ. The digestion system is as follows: 40 μl of PBI121 vector, 1 μl of restriction endonucleases SacⅠ and XbaⅠ, 10 μl of 10× Cutsmart Buffer, and 48 μl of ddH2O. Incubate the digestion at 37°C for 3 hours, then add 500 μl of anhydrous ethanol and mix by inversion. Centrifuge at 12,000 × g for 10 minutes, air dry, and dissolve in water.
[0064] Use a seamless cloning kit to ligate the TaERF7-like gene CDS sequence recovered from the gel with the linearized PBI121 vector sequence. The ligation system consists of 5 μl of 2× One Step Cloning Mix, 1 μl of linearized vector (50 ng / μl), 2 μl of insert (150 ng / μl), and 2 μl of ddH2O. Ligation procedure: 50°C for 30 min.
[0065] 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 the recombinant plasmid, and the recombinant plasmid was named PBI121::TaERF7-like.
[0066] 2.2 Construction of expression silencing vector
[0067] The pCa-γbLIC vector was linearized with the restriction endonuclease Apa I. The digestion system consisted of 40 μL of the pCa-γbLIC vector, 1 μL of the restriction endonuclease Apa I, 10 μL of 10× Cutsmart Buffer, and 49 μL of ddH2O. The digestion reaction was performed at 37°C for 3 h. The mixture was mixed by inversion with 500 μL of anhydrous ethanol, centrifuged at 12,000 rpm for 10 min, air-dried, and then dissolved in water. The 292-bp TaERF7-like gene CDS sequence, recovered from the gel, was ligated with the linearized pCa-γbLIC vector sequence using a seamless cloning kit. Following the ligation reaction, the ligation system was transformed into competent Escherichia coli DH5α cells, and positive clones were identified by PCR amplification. After positive PCR amplification, the recombinant plasmid was verified by Sanger sequencing. The recombinant plasmid was named BSMV::TaERF7-like.
[0068] 2.3 Transform the recombinant plasmid into Agrobacterium competent cells
[0069] Thaw GV3101 Agrobacterium competent cells on ice. Pipette 1μg of plasmid and 100μl of Agrobacterium GV3101 competent cells, mix well, quick-freeze in liquid nitrogen for 5 minutes, quickly transfer to 37°C to thaw for 3 minutes, and finally place on ice for 3 minutes; add 800μl of liquid LB medium without antibiotics, and shake at 28°C, 200rpm for 3 hours to recover; centrifuge for 2 minutes to collect the cells; leave 100μl of supernatant in the tube, use a pipette tip to resuspend the cells, and spread the resuspension onto an LB plate containing 25μg / ml Kan and 25μg / ml Rif. Incubate at 28°C upside down for 48 hours until monoclonal antibodies grow. Use a pipette tip to pick up the monoclonal antibodies and add them to 3ml of LB liquid medium (containing 25μg / ml kanamycin and 25μg / ml rifampicin) and shake and culture at 28°C, 200rpm overnight. After the bacterial solution was correctly amplified and identified by PCR, 50% glycerol was added at 1 times the volume of the bacterial solution and stored at -80°C.
[0070] Example 3 Creation of transgenic Arabidopsis
[0071] 3.1 Arabidopsis thaliana cultivation
[0072] Place an appropriate amount of Arabidopsis thaliana in clean water and allow to imbibe for 48 hours at 4°C. Disinfect with 75% ethanol for 5 minutes, followed by 10% pasteurization for 10 minutes. After disinfection, rinse the seeds five times with sterile water and evenly sow them in a Petri dish containing 1 / 2 MS medium. Once true leaves develop, transplant the seeds into a 7 cm × 7 cm × 8 cm pot and incubate them in a long-light-period incubator until flowering.
[0073] 3.2 Agrobacterium-mediated stable genetic transformation of Arabidopsis
[0074] After activating the Agrobacterium containing the PBI121::TaERF7-like recombinant vector, shake the bacteria in large quantities. 600 When the OD value is about 0.8, centrifuge at 4500×g for 10 min to collect the cells, add Arabidopsis transformation resuspension solution to resuspend the cells, and adjust to OD 600 =0.6, then add Silwet77 transformation aid at 1 / 1000 the volume of the resuspension. Soak the Arabidopsis inflorescences in the Agrobacterium resuspension for 1 minute, gently shake off any excess bacterial solution, and incubate the plants flat on a tray in the dark overnight. Then, transfer the plants to normal conditions and culture until they set, and collect T1 seeds.
[0075] 3.3 Screening of Arabidopsis transgenic positive lines
[0076] The T1 generation seeds were sown on 1 / 2MS culture medium plates containing 40 mg / L kanamycin, and transgenic positive plants were screened and transplanted into soil for culture. The leaves were collected to extract DNA, and PCR amplification was used to further identify positive plants. After they matured, individual plants were harvested to obtain T2 generation seeds.
[0077] 3.4 Expression level determination of transgenic Arabidopsis lines
[0078] Total RNA was extracted from leaves of transgenic lines overexpressing the TaERF7-like gene. Semi-quantitative PCR was used to characterize TaERF7-like gene expression, using ACTIN as a reference gene. Equal volumes of PCR product were subjected to agarose gel electrophoresis, and the expression level of the target gene was determined based on the brightness of the bands in the different samples. The results showed that the expression levels of the gene in the overexpressing lines were significantly higher than those in the wild-type lines. These three lines were selected for further experiments.
[0079] Example 4 Identification of Cold Tolerance of Transgenic Arabidopsis
[0080] 4.1 Method for identifying cold tolerance of Arabidopsis thaliana at the seedling stage
[0081] After disinfection, Arabidopsis seeds were sown in 1 / 2MS medium and cultured under normal conditions until the two-leaf stage, where they were subjected to low-temperature stress. The low-temperature stress conditions were: cold induction at -2°C in a dark environment for 48 hours, followed by freezing at -8°C for 10 hours, and finally returning to room temperature for 7 days. The seedlings were photographed and their survival rates were calculated. The results showed that TaERF7-like overexpression enhanced the cold resistance of Arabidopsis, and the survival rate of TaERF7-like overexpressing strains after low-temperature stress was significantly higher than that of the wild type ( Figure 3 and Figure 4 ).
[0082] 4.2 Identification of cold tolerance in adult Arabidopsis
[0083] Arabidopsis plants were subjected to low temperature stress treatment when they reached the rosette stage: they were kept in a dark environment at 4°C for cold induction for 24 hours, then the temperature was lowered to -5°C for freezing treatment for 5-7 hours, and then the temperature was raised to 10°C for seedling retrieval for 6 hours. Finally, the plants were adjusted to normal environmental conditions and photographed to observe the frostbite of the plants. The results showed that the growth status of the TaERF7-like overexpressing line and the wild-type plants before treatment was similar, and the frostbite rate after low temperature stress was lower than that of the wild-type ( Figure 5 ).
[0084] Example 5 Identification of cold tolerance after gene expression silencing in wheat
[0085] 5.1 Wheat Planting
[0086] 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 cultivate them in an artificial incubator until they reach the two-leaf and one-heart stage.
[0087] 5.2 Tobacco cultivation and injection
[0088] Place an appropriate amount of Nicotiana benthamiana seeds in clean water and allow them to swell at room temperature for 48 hours. Use filter paper to blot the seeds dry and then evenly spread them over the surface of moist soil. Cover the pot with plastic wrap or transparent plastic sheeting to keep the soil moist. When the seedlings have 2-4 leaves, remove them with tweezers and transplant them into 7cm x 7cm x 8cm pots, one seedling per pot. Cultivate in an incubator until they reach the 6-8 leaf stage.
[0089] After activating the Agrobacterium containing the target vector, shake the culture extensively. When the OD600 of the culture solution is approximately 0.8, centrifuge at 4500 × g for 10 minutes to collect the cells. Resuspend the cells twice in tobacco transformation resuspension solution, and finally adjust the OD600 to 0.6 by adding an appropriate amount of resuspension solution. Equal volumes of resuspended Agrobacterium containing the α, β, and γ vectors are mixed (1:1:1) and allowed to stand at 25°C in the dark for 3 hours before inoculating the expanded leaves with a 5 ml needleless syringe. Ten days later, tobacco leaves showing viral symptoms are removed and ground into a homogenous slurry in a mortar with an appropriate amount of 20 μM phosphate buffer (pH 7.2) and a small amount of diatomaceous earth. The leaves are then inoculated with the second leaf of two-leaf, one-heart stage wheat by friction.
[0090] 5.3 Identification of wheat cold resistance
[0091] The wheat inoculated with the virus was cultured at 22°C for 10-14 days, and a small amount of leaves was 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 the control, and the cold resistance of the plants with downregulated expression of TaERF7-like gene was identified 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 level of TaERF7 gene in wheat transfected with BSMV:TaERF7 was significantly lower than that in the control (BSMV:γ0 plants) ( Figure 6 Middle B); and when the third or fourth leaves of the plant began to wilt and water-soaked like frostbite phenotype, the BSMV:γ0 virus-inoculated plants still appeared normal ( Figure 6 A and C).
[0092] Example 6 Identification of Plant Flowering Period
[0093] Take transgenic and wild-type Arabidopsis seeds and place them in clean water. After imbibition at 4°C for 48 hours, disinfect them with 75% ethanol for 5 minutes, and then disinfect them 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. One week later, transplant the seedlings into a 7cm×7cm×8cm pot and culture them in an artificial incubator with a long photoperiod for 3-4 weeks. The growth environment is 16 hours of light, 22°C, 8 hours of darkness, 20°C, 50% relative humidity, and a photosynthetic photon flux density of 450μmol·m -2 ·s -1 The flowering of wild-type and transgenic plants was observed and counted. The results showed that TaERF7-like overexpressed Arabidopsis flowers earlier than wild-type plants ( Figure 7 ).
[0094] The SEQ ID NO.1-2 sequences involved in the present invention are as follows:
[0095] Information about SEQ ID NO.1
[0096] Sequence characteristics: cDNA
[0097] Length: 582 bp
[0098] Type: Nucleotide
[0099] Chain: Single chain
[0100] SEQ ID NO.1
[0101]
[0102] Information about SEQ ID NO.2
[0103] Length: 193aa
[0104] Type: Amino Acid
[0105] SEQ ID NO.2
[0106]
[0107] 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 various embodiments of the present invention.
Claims
1. Use of the protein TaERF7-like, its encoding gene, and a biomaterial containing the encoding gene in any of the following: 1) Regulating plant cold tolerance; 2) Preparation of cold-resistant plants; 3) Make the plants bloom earlier; 4) Preparation of early flowering plants; The amino acid sequence of the protein TaERF7-like is shown in SEQ ID NO.2; The plant is Arabidopsis thaliana or wheat.
2. The application according to claim 1, characterized in that The regulation is positive regulation.
3. A method for improving plant cold resistance and / or advancing plant flowering time, characterized in that: include: Increase the expression of genes encoding TaERF7-like proteins in plants; The amino acid sequence of the protein TaERF7-like is shown in SEQ ID NO.2; The plant is Arabidopsis thaliana or wheat.
4. A method for constructing a transgenic plant, characterized in that: The gene encoding the TaERF7-like protein is introduced into the target plant to obtain a transgenic plant with improved cold tolerance and / or early flowering time; The amino acid sequence of the protein TaERF7-like is shown in SEQ ID NO.2; The plant is Arabidopsis thaliana or wheat.
5. The use according to claim 1 or 2, the method for improving plant cold resistance and / or advancing plant flowering time according to claim 3, and the method for constructing a transgenic plant according to claim 4, characterized in that: The nucleotide sequence of the gene encoding the TaERF7-like protein is shown in SEQ ID NO.
1.
6. The use according to claim 1 or 2, characterized in that: The biological material is any one of a recombinant vector, an expression cassette, a recombinant bacterium or a host cell; The host cell is a host cell that cannot develop into a plant.