Application of wheat transcription factor TaERF112 in response to low phosphorus stress

Abstract

The invention relates to a wheat low-phosphorus response transcription factor TaERF112 and application thereof, and belongs to the technical field of plant bioengineering. The low-phosphorus response transcription factor TaERF112 gene is cloned from wheat, the CDS region sequence of the gene is 882bp, 294 amino acids are encoded, the molecular weight is 31 and 572.92 Da, and the gene is hydrophilic protein. By analyzing the TaERF112 gene structure, protein physicochemical properties, subcellular localization and species evolution characteristics, the characteristics and function prediction of the gene are known. Furthermore, a wheat taerf112 mutant is created by using a CRISPR / Cas9 technology, phenotypic characters, biomass and phosphorus utilization characteristics of a transformed strain under different phosphorus conditions are evaluated, and the important effect of the wheat taerf112 mutant in regulation and control of a wheat root system and phosphorus homeostasis is preliminarily revealed. Theoretical basis and gene resources are provided for efficient genetic improvement of wheat phosphorus.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of plant bioengineering and transgenic technology, and particularly relates to a wheat low-phosphorus response transcription factor TaERF112 coding gene and application thereof. BACKGROUND

[0002] Wheat is one of the main food crops in China, and its yield is directly related to the national food security. Phosphorus plays an important role in the growth and development of wheat, but the utilization rate of phosphorus fertilizer in wheat in China is only maintained at 19%. About 70% of the global arable land is facing phosphorus limitation due to the lack of available phosphorus in soil. This is mainly due to the fact that phosphorus in soil is easily combined with metal ions to form insoluble phosphate or exists in the form of organic phosphorus, thereby resulting in low content of available phosphorus in soil and restricting the growth and development of crops. At the same time, the excessive application of phosphorus fertilizer in farmland ecosystem leads to the rapid accumulation of phosphorus and accelerates the loss of phosphorus to water, causing environmental problems such as water eutrophication. Therefore, it is of great significance to identify and develop excellent germplasm resources with high phosphorus utilization efficiency, determine genetic loci related to phosphorus efficiency, and analyze the genetic mechanism of phosphorus efficiency for cultivating phosphorus-efficient varieties, reducing phosphorus fertilizer application, and promoting the development of green agriculture.

[0003] AP2 / ERF is a large family of plant-specific transcription factors. The gene family has an AP2 / ERF type DNA binding domain of about 60-70 amino acids, which was first discovered in the homologous gene APETALA2 (AP2) of Arabidopsis thaliana. AP2, ERF, RAV and DREB are four subfamilies of the AP2 / ERF gene family. The AP2 subfamily is different from the other three subfamilies because it has two AP2 / ERF domains, while the ERF, DREB and RAV three subfamilies all have one AP2 / ERF domain. The difference between RAV and ERF and DREB subfamilies is that it has an additional B3 DNA binding domain, while the difference between DREB and ERF subfamilies is that the 14th and 19th amino acids of the AP2 / ERF domain are different, where DREB is valine (V) and glutamic acid (E), and ERF is alanine (A) and aspartic acid (D). Ethylene response factor (ERF) was first isolated from Nicotiana tabacum, and members of the ERF subfamily regulate the expression of PR genes by binding to the GCC box (AGCCGCC) element. In addition, ERF is also involved in the signal pathways of salicylic acid, jasmonic acid and ethylene, which are essential for plant stress response and development. Because of its ability to coordinate with multiple signal and hormone pathways, ERF is considered an ideal candidate gene for improving plant tolerance to biotic and abiotic stress. The present application analyzes the gene structure, protein physicochemical properties, subcellular localization and species evolution characteristics of TaERF112 to understand the characteristics and function prediction of the gene. Further, using CRISPR / Cas9 technology, wheat taerf112 mutants are created, and the phenotypic traits, biomass and phosphorus utilization characteristics of the transformed lines under different phosphorus conditions are evaluated. This provides material basis and theoretical basis for further research on the function and mechanism of TaERF112 gene, and also provides gene resources for genetic improvement and utilization of phosphorus-efficient wheat varieties. There is no report on wheat TaERF112 gene and its application related to low phosphorus stress in the prior art. SUMMARY

[0004] The key technical problem to be solved by the present application is to provide a wheat low phosphorus response transcription factor TaERF112 coding gene and its application. To solve the above problems, the technical scheme adopted by the present application is as follows:

[0005] 1. A wheat low phosphorus response transcription factor TaERF112, wherein the transcript sequence of the TaERF112 gene is shown as SEQ ID NO: 1, and the CDS coding region is 147-1,028 bp.

[0006] 2. A vector comprising a nucleotide fragment as shown in SEQ No. 1.

[0007] 3. A method for cloning and vector construction of a wheat low-phosphorus response transcription factor TaERF112, comprising the following steps:

[0008] (1) Selection of plant materials and reagents;

[0009] (2) RNA extraction and reverse transcription;

[0010] (3) Primer design and gene cloning;

[0011] (4) Bioinformatics analysis of TaERF112 gene;

[0012] (5) Construction of subcellular localization vector.

[0013] 4. A method for creating a wheat taerf112 mutant, comprising the following steps:

[0014] (1) Designing a wheat phosphorus response transcription factor TaERF112 target, the target is selected according to the conserved sequence in the three subgenomes of wheat, designing gRNA for TaERF112-4A, TaERF112-4B and TaERF112-4D respectively, connecting the purified fragment with the pCambia3300 / Cas9-bar linearized vector digested by BsaI enzyme through overlap extension PCR.

[0015] (2) Transforming wheat with the gene coding vector pCambia3300 / Cas9-TaERF112 using Agrobacterium-mediated wheat immature embryo transformation method, selecting T0 positive plants through Bar gene (herbicide resistance marker) screening, using Hi-TOM second-generation sequencing platform to perform high-throughput sequencing on the edited target site of T0 and T1 plants, and screening to obtain gene editing mutant lines taerf112-1 and taerf112-2.

[0016] The nucleotide sequence of the wheat phosphorus response transcription factor TaERF112 coding gene is shown in SEQ ID NO. 1.

[0017] 5. A method for verifying the function of a wheat low-phosphorus response transcription factor TaERF112 gene, comprising: (1) subcellular localization of TaERF112; and (2) analysis of the low-phosphorus tolerance of a wheat taerf112 mutant.

[0018] 6. Overexpression of a TaERF112 gene enhances the resistance of wheat to low-phosphorus stress, wherein the transcript sequence of the TaERF112 gene is shown in SEQ ID NO: 1, and the CDS coding region is 147-1,028 bp.

[0019] 7. The TaERF112 gene overexpression enhances the absorption capacity of wheat to phosphorus elements under low phosphorus environment, wherein the transcript sequence of the TaERF112 gene is shown as SEQ ID NO: 1, and the CDS coding region is 147-1,028 bp.

[0020] The present application has at least the following beneficial effects: the TaERF112 gene is successfully cloned in wheat by the method of gene cloning, the characteristics and function prediction of the gene are understood by using bioinformatics, and the subcellular localization vector is constructed. The subcellular localization of Arabidopsis protoplast shows that TaERF112 is mainly located in the nucleus; the gene editing vector is further constructed, and the phenotype traits, biomass and phosphorus utilization characteristics of the T2 generation taerf112 mutant under low phosphorus stress are analyzed after the wheat is successfully transformed, and the results show that the wheat TaERF112 is a positive regulatory factor responding to low phosphorus stress. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 The figure is the transcript sequence of the TaERF112 gene. In the figure, the bold part is the CDS sequence, and the underlined part is the UTR sequence.

[0022] Figure 2 The figure is the PCR amplification of the TaERF112 gene. In the figure, M: D2,000 Marker; 1 is the PCR product of the TaERF112 gene.

[0023] Figure 3 The figure is the basic physicochemical property analysis diagram of the TaERF112 protein. Figure 3 A represents the composition of amino acids, Figure 3 B represents the hydrophilic-hydrophobic analysis, Figure 3 C represents the transmembrane domain prediction, Figure 3 D represents the nuclear localization signal prediction.

[0024] Figure 4 The figure is the functional structure analysis diagram of the TaERF112 protein. Figure 4 A represents the primary structure functional domain of the protein, Figure 4 B represents the secondary structure, Figure 4 C represents the tertiary structure, Figure 4 D represents the signal peptide, Figure 4 E represents the phosphorylation site analysis.

[0025] Figure 5 The figure is the TaERF112 homologous protein system evolution tree analysis. In the figure, the numbers represent the support rate of each branch, and the Bar represents the evolution tree branch length, which is used to reflect the difference between different sequences.

[0026] Figure 6Structure diagram of subcellular localization vector PC1300S-GFP-TaERF112. In the figure, the TaERF112 gene connection position is between the two enzyme digestion sites of KpnI and BamHI.

[0027] Figure 7 PCR identification of transgenic wheat T0 generation positive plants. In the figure, M represents DL, 2000 DNA Marker; 1-73 represents the T0 generation line number of transgenic wheat; WT represents the wild type plant control; - represents the pure water blank control.

[0028] Figure 8 Homozygous mutant line target site design site and gene editing type diagram. The underlined part in the figure represents the PAM sequence.

[0029] Figure 9 TaERF112 gene subcellular localization result diagram in Arabidopsis protoplast. In the figure, the scale bar is 10 μm.

[0030] Figure 10 Phenotypic traits, biomass and phosphorus content results of taerf-112 mutant under low phosphorus stress. Figure 10 A is the phenotype of taerf112-1, taerf112-2 mutant and KN199 wild type (WT) grown under normal phosphorus (normal-Pi) and low phosphorus (low-Pi) conditions for 20 days (scale bar = 5 cm). (B-E) The root dry weight (RDW) (B), stem dry weight (SDW) (C), root phosphorus concentration (RPC) (D) and stem phosphorus concentration (SPC) (E) of the mutant and wild type plants were measured after growing for 20 days under two phosphorus conditions. Data represent the mean ± standard deviation (SD) of three biological replicates. The asterisk (*) indicates statistically significant differences (P < 0.05) determined by Student's t test. Figure 10 Figure 10 Figure 10 Figure 10 Specific implementation method

[0031] ​​​​The methods and devices used in the following examples of the present application are conventional unless otherwise specified, and the apparatuses and reagents used are conventional apparatuses and reagents purchased from reagent companies. In order to make the purpose, technical scheme and advantages of the present application clearer, the specific embodiments of the present application will be described in detail below with reference to the drawings. The examples are used only to explain the present application and are not intended to limit the scope of the present application. Examples of these preferred embodiments are illustrated in the accompanying drawings. The embodiments of the present application shown in the drawings and described according to the drawings are merely exemplary, and the present application is not limited to these embodiments. Here, it should be noted that, in order to avoid obscuring the technical scheme of the present application due to unnecessary details, only the processing steps closely related to the scheme according to the present application are shown in the drawings, and other details that are not closely related are omitted.

[0032] Example 1

[0033] The present embodiment provides a wheat phosphorus response transcription factor TaERF112, the full-length transcript sequence of which is shown as SEQ ID NO: 1, wherein the CDS coding region is 147-1,028 bp.

[0034] Example 2

[0035] The present embodiment provides a cloning and vector construction method of a wheat low-phosphorus response transcription factor TaERF112, including gene cloning, bioinformatics analysis and subcellular localization vector construction.

[0036] 1. Gene cloning

[0037] The wheat material A076 used in the present application is preserved in the laboratory and planted in an artificial climate incubator. The root tissues of the wheat material A076 used are collected under different phosphorus levels, and the specific extraction steps are performed according to the instructions of the plant total RNA extraction kit (DP432). The RNA concentration is detected using an ultramicro spectrophotometer (Pultton P100+), and the RNA quality is detected using 1% agarose gel electrophoresis. The first strand cDNA synthesis kit is used to reverse transcribe the RNA into cDNA, and the reverse transcription product is stored at -80℃.

[0038] According to the gene sequence open reading frame sequence corresponding to the wheat TaERF112 gene in the Chinese Spring v2.0 reference sequence of the wheat Omics genome database, the amplification primers TaERF112-CDS-F: 5'-GAGGGTAGCGACCGACAAG-3' and TaERF112-CDS-R: 5'-CTACTTGCACGCGCGCG-3' were designed in the 5'-UTR and 3'-UTR regions of the TaERF112 gene using SnapGene v6.0.2 software. The CDS of the TaERF112 gene was amplified using cDNA as a template, and FastPfu DNA Polymerase (AP221-01) high-fidelity DNA polymerase was used for PCR amplification. The PCR product was 1,107 bp, and the PCR amplification system is shown in Table 1. The PCR amplification program was set as 95°C pre-denaturation for 4 min; 95°C denaturation for 20 s, 56°C annealing for 20 s, 72°C extension for 30 s, 36 cycles; and 72°C final extension for 5 min. FastPfu DNA Polymerase (AP221-01) high-fidelity DNA polymerase was used for PCR amplification, and the PCR product was 1,107 bp. The PCR amplification system is shown in Table 1. The PCR amplification program was set as 95°C pre-denaturation for 4 min; 95°C denaturation for 20 s, 56°C annealing for 20 s, 72°C extension for 30 s, 36 cycles; and 72°C final extension for 5 min.

[0039] Table 1 PCR reaction system for amplifying the CDS of the TaERF112 gene

[0040]

[0041] The PCR product was verified by 1% agarose gel electrophoresis (containing 0.5x TAE buffer and GelView nucleic acid dye). The target band was quickly cut under ultraviolet light and then recovered according to the OMEGA Gel Extraction Kit D2500 kit operation steps. The gel recovery product was mixed with Zero Cloning Vector, and after the reaction, the Trans1-T1 E. coli competent cells were heat-shocked and transformed. The cells were plated on LB plates containing ampicillin (Amp) resistance, and incubated at 37°C for 12-16 h. Single colonies were inoculated in LB liquid medium (containing Amp resistance) and incubated at 37°C for 10 h. After positive clones were verified by bacterial liquid PCR, they were sent to Shengong Biotechnology (Shanghai) Co., Ltd. for sequencing. The single clones with correct sequencing were further cultured, and the plasmid was extracted and stored at -20°C for later use.

[0042] 2. Bioinformatics analysis

[0043] The CDS sequence of TaERF112 gene was translated into protein sequence using SnapGene software. The physicochemical properties of TaERF112 were analyzed through Expasy online platform, including molecular weight, theoretical isoelectric point, fatty index, instability index and hydrophilic-hydrophobic index. The transmembrane region of the protein was predicted using the TMHMM website, and the subcellular localization and nuclear localization signal of the protein were predicted using the WoLF PSORT II and NLS Mapper websites, respectively. The primary structure domain of the protein was analyzed using the online software SMART. The secondary structure and tertiary structure of the protein were predicted using the PSIPRED and SWISS-MODEL websites, respectively, and the PSIPRED prediction was beautified and displayed using the Neopybio online tool. The signal peptide of TaERF112 protein was analyzed using the Signal P website, and the phosphorylation site of the protein was predicted using the NetPhos website.

[0044] Based on the WheatOmics omics big data platform developed by the Wheat Alliance, the gDNA, cDNA and protein sequence information of TaERF112 in wheat A, B and D subgenomes were identified by homologous gene alignment. The gene structure analysis was completed using the GSDS online tool developed by Gao-Lab. Multiple alignment of protein sequences was achieved by Clustal Omega, and the results after alignment were visualized using Jalview software. Evolutionary analysis of TaERF112 protein was performed by BlastP alignment of homologous protein sequences in UniProt, NCBI and wheat genome databases, and the phylogenetic tree was constructed using Mega12 software.

[0045] 3. Construction of subcellular localization vector

[0046] According to the experimental requirements, a primer containing a KpnI and BamHI homologous arm linker (Table 2) was designed, and a suitable TaERF112 gene cloning product plasmid was used as a template for PCR amplification. The amplified product was recovered by agarose gel to obtain the target fragment; the PC1300S-GFP vector was treated with restriction endonucleases KpnI and BamHI, and the PCR gel was recovered and linearized. The homologous recombination connection reaction was performed using the ClonExpress II One Step Cloning Kit (Novagen) kit, and the reaction system is shown in the table. Use a pipette to mix gently (do not shake to mix), and immediately ice bath after 37°C reaction for 30 min. The ligation product was transformed into E. coli DH5α competent cells, plated on LB plates, and incubated at 37°C for 16 h. Single colonies were picked for colony PCR verification, and positive clones were sent for sequencing. The sequencing primers are shown in Table 2. The appropriate expression vector was named PC1300S-TaERF112-GFP.

[0047] Table 2 Primers used for constructing the TaERF112 subcellular localization vector.

[0048]

[0049]

[0050] Note: Underlined lines indicate restriction endonuclease KpnI and BamHI cleavage sites.

[0051] The results showed that PCR amplification using cDNA as a template contained the coding region sequence of the target gene. The PCR products were detected by 1% agarose gel electrophoresis. Figure 2 A single, clear band appeared at approximately 1,100 bp. After gel extraction and purification of the target fragment, it was ligated into a T-cloning vector. Positive clones were selected and tested, successfully obtaining the complete CDS sequence of the target gene, which was 882 bp in length. Expasy-ProtParam predicted the TaERF112 protein to have a molecular weight of 31,572.92 Da, with alanine (Ala) being the most abundant amino acid at 46, accounting for 15.70% of the total amino acid composition. Figure 3 A). The theoretical isoelectric point (pI) of TaERF112 protein is 4.73, containing 48 negatively charged amino acid residues (Asp+Glu) and 32 positively charged amino acid residues (Arg+Lys). The protein instability index is 57.10 (an instability coefficient less than 40 indicates a stable protein), and the protein lipid index is 67.13. The overall average hydrophilicity index (GRAVY) is -0.400 (positive values ​​indicate hydrophobic proteins, negative values ​​indicate hydrophilic proteins). Leucine (L) at position 151 exhibits the highest hydrophobicity with an index of 1.911, while glycine (G) at position 78 exhibits the highest hydrophilicity with an index of -2.289. Based on these findings, TaERF112 protein is classified as a hydrophilic protein (Figure). Figure 3 B). Using the online tool TMHMM, TaERF112 protein was predicted to have no obvious transmembrane region, suggesting it is a non-transmembrane protein. Figure 3 C). WoLF PSORTII was used to predict subcellular localization of the TaERF112 protein in the nucleus, and NLS Mapper predicted the presence of two nuclear localization signal sequences: VHLLPKRPRVDDF and the LASPKKRPRIE motif. Figure 3 D).

[0052] The primary and spatial structures of proteins are fundamental to their biological functions. The online tool SMART was used to predict the functional domains of the primary structure of the TaERF112 protein. Figure 4A), the results showed that the protein only contains one AP2 domain (located at 75-138) (SMART ACC: SM000380), and the 14th and 19th amino acids of the AP2 domain are alanine (A) and aspartic acid (D), respectively, which is consistent with previous studies, further confirming that the protein belongs to the ERF family members. Then, the secondary structure of TaERF112 protein was predicted by PSIPRED 4.0, as shown in Figure 4 B, there are 204 amino acids forming random coil in the TaERF112 protein, accounting for 69.62%; there are 74 amino acids forming α-helix, accounting for 25.26%; there are 15 amino acids forming β-extended strand, accounting for 5.12%. The tertiary structure of the protein was predicted by online software SWISS-MODEL ( Figure 4 C), the results showed that the AP2 domain of TaERF112 protein has a basic (the pI of 75-102 amino acids is 11) hydrophilic region composed of three anti-parallel β-folds at the N terminal, which is essential for its recognition of cis-acting elements. In addition, the C terminal contains an amphipathic α-helix structure, which may mediate the interaction of the protein with other transcription factors or DNA. The Signal P 4.1 website analysis did not predict the presence of signal peptide in TaERF112 protein ( Figure 4 D). Net Phos 3.1 prediction showed that the TaERF112 protein contains 39 sites with phosphorylation potential greater than 0.5 ( Figure 4 E), among which, there are 31, 2 and 6 phosphorylation sites of serine (Serine), tyrosine (Tyrosine) and threonine (Threonine), respectively. It shows that the protein may have complex regulatory functions in cells.

[0053] Further analysis of the evolutionary relationship between TaERF112 protein and other species was carried out to infer its potential function. Based on the homologous alignment results of UniProt, NCBI and wheat genome website protein database, the phylogenetic tree of wheat TaERF112 protein and 14 species was constructed, including Triticum urartu, Aegilops tauschii subsp. strangulata, Triticum dicoccoides, Triticum turgidum subsp. durum, Hordeum vulgare subsp. vulgare, Sorghum bicolor, Zea mays, Oryza sativa, Panicum miliaceum, Brachypodium distachyon, Lolium multiflorum, Glycine max and Arabidopsis thaliana. First, the optimal model for constructing the phylogenetic tree was calculated by MEGA 12 as JTT+G+I+F, and then the phylogenetic tree of 21 gene protein sequences was constructed based on this model using the maximum likelihood method. Figure 5 It is shown that the wheat (AABBDD) TaERF112 protein is most closely related to the homologous proteins of T. turgidum subsp. durum (AABB) and T. dicoccoides (AABB), and TaERF112 is most closely related to the homologous proteins of TraesCS4A03G0008700 in 4A subgenome, T. urartu (AA), T. turgidum subsp. durum (AABB) and T. dicoccoides (AABB), indicating that the TaERF112 gene evolution is highly conserved among wheat and its ancestral species, and suggesting that subgenome-specific retention may occur during the evolution from tetraploid ancestor (AABB) to hexaploid (AABBDD). In addition, TaERF112 and its homologous proteins of rice OsERF64, maize Zm00001d027928 and barley HORVU.MOREX.r2.4HG0337730 form a specific evolution branch of monocotyledonous plants, while they are obviously differentiated from dicotyledonous model plant Arabidopsis thaliana AtERF71 / 73 and soybean GmERF1 / 5 proteins, revealing the functional specificity evolution of ERF gene family after the differentiation of monocotyledonous and dicotyledonous plants.

[0054] To determine the subcellular localization of TaERF112 protein, its coding region was fused with the GFP gene of PC1300S vector (Fig. 1) and expressed driven by the strong CaMV-35S promoter. Figure 6

[0055] Example 3

[0056] This example provides a method for creating a wheat taerf112 mutant, comprising the following steps:

[0057] 1. Design of gene editing target

[0058] For the TaERF112 gene identified in this study, CRISPR / Cas9 was used to create wheat gene editing mutant materials. The complete coding sequence of the three orthologous genes (TraesCS4A02G078700, TraesCS4B03G0787300 and TraesCS4D02G078700) in the three subgenomes of TaERF112-A / B / D was subjected to multiple sequence alignment using DNAMAN software, and the conserved sequence of the exon region in the three subgenomes was screened. The conserved sequence was submitted to the CRISPR-P online design platform (http: / / crispr.hzau.edu.cn), and the parameters were set as SpCas9 recognition PAM sequence "NGG". The system scoring criteria include target sequence specificity (60%), three subgenome targeting efficiency consistency (30%), and GC content (40-60%). After whole genome off-target analysis using WheatCRISPR database, the target sgRNA (5'-GCCGCCCAGGAGCGTCACGGCGG-3') located in the exon and front of the ERF functional domain was finally selected, which is completely conserved in A, B and D genomes.

[0059] 2. Construction of gene editing vector

[0060] (1) Connection of sgRNA and vector: The designed specific sgRNA sequence (5'-GCCGCCCAGGAGCGTCACGGCGG-3') was connected with the wheat U6 promoter by overlap extension PCR, and the PCR product was separated by 1% agarose gel electrophoresis and the gel was recovered. The purified fragment was recombined with the BsaI enzyme-digested pCambia3300 / Cas9-bar linearized vector, and after 37°C reaction for 30 min, it was placed on ice or 4°C for storage.

[0061] ​(2) E. coli transformation: Take 10 μL ligation product into 100 μL DH5a competent cells, the competent cells are taken out from -80 °C refrigerator immediately and placed on ice, 5 min later, the ligation product is added after the bacterial block is dissolved, and the ice is placed for 25 min. After 42 °C heat shock for 45 s, immediately ice bath for 2 min (avoid shaking), add 100 μL antibiotic-free LB liquid medium, 37 °C, 200 rpm shaking culture for 1 h. The bacterial solution is coated on the LB plate containing the corresponding bacterial antibiotic, and cultured at 37 °C for 16-24 h.

[0062] (3) Positive clone screening: 12 single colony bacteria are randomly selected from the transformation plate, and are respectively inoculated in 2 ml EP tubes containing 500 μL LB medium, and are cultured at 37 °C for 6 h. Take 5 μL bacterial solution for colony PCR verification, and select the correct PCR verification colony for sequencing confirmation.

[0063] (4) Transformation of Agrobacterium: Take 20 μL EHA105 Agrobacterium competent cells, add 1 μL sequencing correct plasmid DNA, ice bath for 5 min, liquid nitrogen freeze for 5 min, 37 °C water bath for 5 min, ice bath for 5 min again. Add 100 μL antibiotic-free LB liquid medium, 28 °C, 200 rpm shaking culture for 2 h, then coat on the plate containing the corresponding bacterial antibiotic and 50 mg / L rifampicin, and culture at 28 °C for 48 h. Pick a single colony and inoculate in 2 mL antibiotic-containing LB medium, and culture at 28 °C overnight. Take 400 μL bacterial solution and mix with 100 μL 75% sterile glycerol, and store at -80 °C for standby.

[0064] 3. Agrobacterium-mediated wheat genetic transformation

[0065] Wuhan Nami Biotechnology Co., Ltd. is entrusted to complete the wheat genetic transformation. The gene coding vector is transformed into the recipient material KN199 by using the Agrobacterium-mediated wheat embryo transformation method.

[0066] 4. Identification of wheat taerf112 positive mutant

[0067] (1) Transgenic plants bar gene detection: To screen transgenic positive plants, bar gene (herbicide resistance marker) PCR detection was used. Young leaves of each generation of plants were taken, and wheat genomic DNA was extracted by CTAB method. Primer design reference vector sequence: BAR-F (5'-ATGAGCCCAGAACGACGCC-3') and BAR-R (5'-TCAGATCTCGGTGACGGGC-3'), expected amplification fragment 447 bp. The PCR reaction system (20 μL) contained 2 × Taq Master Mix 10 μL, DNA template 1 μL (about 50 ng), upstream and downstream primers 0.5 μL (10 μM) each, and ddH2O to supplement. The reaction program was as follows: 94°C pre-denaturation for 5 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 35 cycles; 72°C extension for 5 min. The amplification product was detected by 1% agarose gel electrophoresis ( Figure 7 ), and positive plants were retained for subsequent analysis.

[0068] (2) Target site mutation detection: The HiTOM second-generation sequencing platform was used to perform high-throughput sequencing verification on the edited target sites of T0 and T1 plants. First, TaERF112-A / B / D target site-specific primers (SSP) were designed, and the primer design followed the principle of single-end effective sequencing length of 123 bp, ensuring that the primer distance from the target site was not more than 100 bp. The SSP primers were located in the conserved region of the homologous genes, and the amplification product was a mixture of homologous genes, which was distinguished by sequencing after sequence difference. The primer sequences are shown in Table 3. The first round of PCR reaction system was 20 μL, containing total genomic DNA (25 ng / μL) 2 μL, forward and reverse primers (10 μM) 0.4 μL each, 2 × Phanta Max Master Mix 10 μL, and ddH2O 7.2 μL. The PCR reaction program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 30 sec, 68°C annealing for 30 sec, 72°C extension for 20 sec, 35 cycles; 72°C extension for 3 min, 4°C storage. The first round of PCR product was sent to the HiTOM platform for second round of barcode PCR amplification in a 96-well plate kit. The HiTOM platform used the Illumina HiSeq platform to sequence the library, and the data volume of each sample was selected as 5000 reads, and the filter threshold was set as 1%. After sequencing, the sequencing data was uploaded to the Hi-TOM online analysis platform, and the system automatically decoded and analyzed the mutation, determined the editing type of all homologous copy target points of each sample, and distinguished the homologous genes according to the sequence difference. The system could automatically identify and distinguish the editing types of each homologous gene, including insertion, deletion and other mutation forms, and calculate the ratio of each mutation type. Finally, taerf112 homozygous mutant lines ( Figure 8 ) were obtained.

[0069] ​Table 3 Primers used for the first round of PCR amplification for Hi-Tom sequencing

[0070]

[0071] Note: underlined indicates bridging sequence.

[0072] Example 4

[0073] The embodiment provides a wheat low-phosphorus response transcription factor TaERF112 gene function verification method, and comprises the following steps:

[0074] 1. Subcellular localization analysis of TaERF112

[0075] Select 4-5 weeks old healthy Arabidopsis thaliana leaves, avoid the main vein and cut about 0.5g leaf tissue, and separate the protoplasts by using the enzymatic method. Take 200μL protoplast suspension, respectively add 20μg PC1300S-TaERF112-GFP recombinant plasmid and PC1300S-GFP empty plasmid and 210μL PEG solution (40% PEG4000, 0.2M mannitol, 100mM CaCl2) for transformation, and the successfully transformed cells are observed by using a laser confocal microscope to observe the localization.

[0076] 2. Analysis of low-phosphorus tolerance of wheat taerf112 mutants

[0077] Wild type KN199 (WT) and gene editing homozygous mutant lines (taerf112-1 and taerf112-2) are planted in an artificial climate room of Gansu Agricultural University. Two phosphorus concentration treatments are set: normal phosphorus (normal-Pi, 500μM KH2PO4) and low phosphorus (low-Pi, 6.5μM KH2PO4). In order to obtain uniform germination effect, the wheat seeds with consistent fullness are selected, disinfected with 3% NaClO for 10min, then washed clean with distilled water, and placed in a germination box lined with two layers of filter paper. After 7 days of germination, the seedlings with consistent growth are selected, removed from the seeds and transplanted into 10L hydroponic boxes. After 20 days of normal phosphorus and low phosphorus treatment, phenotype analysis and index determination are performed. The nutrient solution is replaced every 3 days during the experiment.

[0078] The results show that: pC1300S-GFP empty plasmid is used as a control. The plasmid with the target gene and the empty pC1300S-GFP are respectively transformed into Arabidopsis thaliana protoplasts, and the fluorescence signal distribution is observed by using a confocal laser microscope after 8-10h of culture under weak light. As shown in FIG. 2, the fluorescence signal of the protoplasts transformed with the empty pC1300S-GFP plasmid is mainly distributed in the cytoplasm, and the fluorescence signal of the protoplasts transformed with the pC1300S-TaERF112-GFP plasmid is mainly distributed in the nucleus. Figure 9The fluorescence signal of PC1300S-GFP control group was mainly distributed in the cytoplasm, while the fluorescence signal of PC1300S-GFP-TaERF112 was significantly enriched in the nuclear region. It is shown that TaERF112 acts as a nuclear localization protein, which is consistent with the prediction results of previous studies and WoLF PSORT II, NLS Mapper( Figure 3 D).

[0079] In order to further confirm the role of TaERF112 in low-phosphorus tolerance of wheat, we created CRISPR-Cas9-based TaERF112 knockout transgenic lines (taerf112-1 and taerf112-2) with KN199 (low-phosphorus-tolerant material) as the background. Figure 8 After low-phosphorus treatment, we found that the stem and root dry weights of the transgenic lines with loss-of-function of TaERF112 gene were significantly less than those of the control group after 20 days of low-phosphorus treatment. Figure 10 B, 10C). Under low-phosphorus conditions, the P concentrations in the stems and roots of the two mutants were significantly lower than those of the control group. Figure 10 D, 10E), which indicates that the mutation of TaERF112 may reduce the phosphorus uptake of the root system under low-phosphorus conditions. It is preliminarily confirmed that TaERF112 gene plays a positive regulatory role in the response of wheat to low-phosphorus stress.

[0080] In summary, the present application preliminarily reveals the important role of TaERF112 in regulating the root system and phosphorus homeostasis of wheat under low-phosphorus stress. It provides a theoretical basis and gene resources for the genetic improvement of phosphorus-efficient wheat.

[0081] The above description is merely a specific implementation of the present application. It should be noted that, for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, which should also be considered as falling within the scope of protection of the present application.

Claims

1. A wheat low phosphorus response transcription factor TaERF112, characterized in that The transcript sequence of the TaERF112 gene is shown as SEQ ID NO: 1, wherein the CDS coding region is 147-1,028 bp.

2. A vector, characterized in that The vector comprises a nucleotide fragment shown as SEQ ID NO.

1.

3. A method for cloning and vector construction of a wheat low phosphorus response transcription factor TaERF112, characterized in that The method comprises the following steps: (1) plant material and reagent selection; (2) RNA extraction and reverse transcription; (3) primer design and gene cloning; (4) bioinformatics analysis of the TaERF112 gene; (5) construction of a subcellular localization vector.

4. A method of creating a wheat taerfl 12 mutant, characterized by The method comprises the following steps: (1) design of a wheat phosphorus response transcription factor TaERF112 target, the target is selected according to the conserved sequence in three subgenomic copies of wheat, gRNA is designed for TaERF112-4A, TaERF112-4B and TaERF112-4D respectively, and the purified fragment is recombined and connected with a linearized pCambia3300 / Cas9-bar vector subjected to BsaI enzyme digestion; (2) the gene coding vector pCambia3300 / Cas9-TaERF-112 is transformed into wheat by using an Agrobacterium-mediated wheat young embryo transformation method, T0 generation positive plants are selected by Bar gene screening, high-throughput sequencing is performed on the edited target sites of T0 and T1 generation plants by using a Hi-TOM second-generation sequencing platform, and gene editing mutant lines taerf112-1 and taerf112-2 are obtained by screening.

5. A method for verifying the function of a wheat low phosphorus response transcription factor TaERF112 gene, characterized in that The method comprises: (1) subcellular localization of TaERF112; (2) analysis of the low-phosphorus tolerance of a wheat taerf112 mutant.

6. Overexpression of a TaERF112 gene enhances the resistance of wheat to low-phosphorus stress, the transcript sequence of the TaERF112 gene is shown as SEQ ID NO: 1, wherein the CDS coding region is 147-1,028 bp.

7. Overexpression of a TaERF112 gene enhances the phosphorus element absorption capacity of wheat under a low-phosphorus environment, the transcript sequence of the TaERF112 gene is shown as SEQ ID NO: 1, wherein the CDS coding region is 147-1,028 bp.

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