Wheat phospho-hunger response factor taPHR1-like 7, coding gene and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a wheat phosphorus starvation response factor TaPHR1-like7, a coding gene thereof and application. The wheat phosphorus starvation response factor TaPHR1-like7, and an amino acid sequence of the wheat phosphorus starvation response factor TaPHR1-like7 is shown as SEQ ID NO. 1. The application analyzes the action mechanism of the wheat phosphorus starvation response factor TaPHR1-like7 in nitrogen and other nutrient metabolisms, and simultaneously finds that the TaPHR1-like7 can make the wheat kernel length larger, and can provide important gene resources and related germplasm resources for cultivating wheat varieties with efficient nitrogen utilization and longer kernel length.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a wheat phosphorus starvation response factor TaPHR1-like7 and its encoding gene and applications. Background Technology

[0002] Wheat is one of the world's most important cereal crops, accounting for one-third of global food production. Wheat yield is determined by three factors: the number of spikes per unit area, the number of grains per spike, and the grain weight. Therefore, the spike traits of wheat directly determine its yield, and the number of spikelets per spike is closely related to the number of grains per spike.

[0003] MYB transcription factors are one of the largest transcription factor families in plants, possessing diverse biological functions and participating in the regulation of plant growth and development, primary metabolism, and secondary metabolism. Currently, 155, 197, and 393 MYB family transcription factors have been identified in rice, Arabidopsis thaliana, and wheat, respectively. The MYB-CC subfamily of PHR1 proteins are important central regulators in the phosphorus signaling pathway, containing both a MYB domain and a CC domain at their C-terminus. The MYB domain is a DNA-binding domain, while the CC domain facilitates the accurate binding of the MYB domain to cis-acting elements. PHR1 contains a DNA activation domain at its N-terminus. Current research has found that PHR1 binds to the P1BS cis-acting elements of target genes in a dimer form, thereby regulating the expression of downstream genes. It has been reported that overexpression of TaPHR1 increases the number of grains per spike in wheat, thus increasing yield; ectopic expression of TaPHR3 in rice leads to increased biomass, number of grains per spike, and spike branching.

[0004] TaPHR1-like7 is a transcription factor containing a MYB domain and a core regulator of the phosphorus starvation signaling pathway. It specifically binds to conserved DNA sequences to activate the expression of phosphorus starvation-induced genes. Furthermore, TaPHR1-like7 plays an important regulatory role in the growth, development, and nitrogen metabolism pathways of plant organs such as roots and leaves; however, whether it responds to phosphorus starvation induction and, if so, by which signal. The inventors have elucidated the mechanism of action of TaPHR1-like7 in nitrogen and other nutrient metabolism, and discovered that TaPHR1-like7 can increase wheat grain length, providing important genetic resources and related germplasm resources for breeding wheat varieties with high nitrogen utilization and longer grain size. Currently, there are no reports on the effects of TaPHR1-like7 on increasing nitrogen utilization and wheat grain length.

[0005] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0006] The purpose of this invention is to provide a wheat phosphorus starvation response factor TaPHR1-like7, its encoding gene, and its applications.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] The first objective of this invention is to provide a wheat phosphorus starvation response factor TaPHR1-like7, the amino acid sequence of which is shown in SEQ ID NO.1.

[0009] A second objective of this invention is to provide a coding gene that encodes the wheat phosphorus starvation response factor TaPHR1-like7, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0010] A third objective of this invention is to provide a gene-encoding vector containing the encoded gene.

[0011] A fourth objective of this invention is to provide the application of the wheat phosphorus starvation response factor TaPHR1-like7, or the encoding gene, or the gene encoding vector in wheat breeding.

[0012] More specifically, the wheat breeding includes responding to phosphorus hunger, improving wheat nitrogen utilization, increasing wheat grain length, increasing wheat yield, and improving effective tillering and thousand-grain weight.

[0013] The fifth objective of this invention is to provide a method for obtaining wheat grain length-increasing breeding materials, comprising the following steps:

[0014] S1. The target of the wheat phosphorus starvation response factor TaPHR1-like7 was designed. The target was selected based on the conserved sequence in three wheat copies. Two gRNAs were designed for TaPHRL7-1A, TaPHRL7-1B and TaPHRL7-1D, respectively, and annealed to form double strands. TaPHR1-like7 was recombined into the two-element expression vector PLH3 containing the UBI promoter by homologous recombination to construct the gene coding vector PLH3-TaPHR1-like7.

[0015] S2. The gene-coding vector PLH3-TaPHR1-like7 was transformed into wheat using Agrobacterium-mediated in situ transformation of mature wheat embryos. T0 generation positive plants were selected by Bar tag rapid test paper, and T1 generation edited heterozygous mutants were obtained by sequencing. After further generations, T2 generation homozygous transgenic lines aabbdd and their editing types were obtained by Hi-TOM next-generation sequencing. Homozygous mutant lines phl7-1, phl7-2 and phl7-3 were obtained, which are the breeding materials for increased wheat grain length.

[0016] More specifically, in S1, the amino acid sequence of the wheat phosphorus starvation response factor TaPHR1-like7 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding the wheat phosphorus starvation response factor TaPHR1-like7 is shown in SEQ ID NO.2.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention elucidates the mechanism of action of the wheat phosphorus starvation response factor TaPHR1-like7 in nitrogen and other nutrient metabolism. It also discovers that TaPHR1-like7 can increase wheat grain length, providing important gene resources and related germplasm resources for breeding wheat varieties with high nitrogen utilization and longer grain diameter. Attached Figure Description

[0019] Figure 1 Diagram showing target sites and gene editing types for homozygous mutant lines;

[0020] Figure 2 The graph shows the response of TaPHR1-like7 to phosphorus starvation in wheat seedlings.

[0021] Figure 3 Figure showing the total nitrogen accumulation and GS enzyme activity assay results in gene-edited homozygous mutant materials;

[0022] Figure 4 Phenotypic diagram of seed size-related traits in gene-edited homozygous mutant materials;

[0023] Figure 5 The graph shows the impact of gene-edited homozygous mutant materials on yield.

[0024] Explanation of key figure labels:

[0025] exist Figure 1-5 In this context, WT represents the wheat receptor Zhengmai 7698 (ZM7698), also known as the wild type; the gene knockout / editing homozygous mutant lines used for subsequent research include three types, namely phl7-1, phl7-2, and phl7-3. Detailed Implementation

[0026] The technical solution of this invention patent will be clearly and completely described below. Obviously, the described embodiments are some embodiments of this invention, but not all embodiments.

[0027] Example 1: Wheat phosphorus starvation response factor TaPHR1-like7

[0028] A wheat phosphorus starvation response factor TaPHR1-like7, the amino acid sequence of which is shown in SEQ ID NO.1; and the nucleotide sequence encoding which is shown in SEQ ID NO.2.

[0029] Example 2: Method for obtaining breeding materials with increased wheat grain length

[0030] A method for obtaining wheat grain length enhancement breeding materials includes the following steps:

[0031] S1. Target sites for the wheat phosphorus starvation response factor TaPHR1-like7 were designed. Target selection was based on conserved sequences in three wheat copies. First, the CDS sequence of the wheat TaPHR1-like7 gene was input into a CRISPR 2.0 online database to design two gRNAs for TaPHRL7-1A, TaPHRL7-1B, and TaPHRL7-1D, respectively, for subsequent gene editing vector construction. The primer pairs corresponding to the two gRNAs are shown in Table 1 below.

[0032] Table 1. Primer sequences for two gRNAs corresponding to two pairs of target sites.

[0033]

[0034] The two single-stranded gRNAs synthesized above were annealed to form double-stranded DNA;

[0035] Table 2 Annealing PCR Reaction System

[0036]

[0037] The PCR-obtained fragments were sequentially ligated into the linearized intermediate vector TaU3 using homologous recombination. The restriction enzymes used in the linearized intermediate vector TaU3 were BsmBI and BbsI, respectively. The linearization system is shown in Table 3 below.

[0038] Table 3. Linearization system of TaU3 carrier

[0039]

[0040] Enzyme ligation system reference The IIOne Step Cloning Kit (Vazyme, C112-02) was used, and then the intermediate vector TaU3 containing gRNA was ligated to the two-element expression vector PLH3 containing the UBI promoter via homologous recombination. The ligation reaction system was the same as the kit above (Vazyme, C112-02).

[0041] The above ligation product was transformed into Escherichia coli DH5α (transformation procedure as per Qingke Biotechnology competent cells), positive clones were identified, sequenced, and plasmids were extracted (Beijing Quanshijin Plasmid Mini-Prep Kit, EM101-02).

[0042] The gene-encoding vector PLH3-TaPHR1-like7 was constructed and transformed into Agrobacterium, following the Agrobacterium C58C1 instruction manual (Shanghai Weidi Biotechnology). The identified positive clones were activated by overnight incubation at 28°C and 200 rpm in liquid LB medium containing the corresponding antibiotics and rifampin. The bacterial culture was then mixed with 50% glycerol at a 1:1 volume ratio and stored at -80°C. The culture was then removed from the refrigerator for activation when infecting callus tissue.

[0043] The amino acid sequence of wheat phosphorus starvation response factor TaPHR1-like7 is shown in SEQ ID NO.1, and the nucleotide sequence of the gene encoding wheat phosphorus starvation response factor TaPHR1-like7 is shown in SEQ ID NO.2.

[0044] S2. The gene-coding vector PLH3-TaPHR1-like7 was transformed into wheat (wild-type Zhengmai 7698) using Agrobacterium-mediated in situ transformation of mature wheat embryos. The experiment was completed by the Crop Genomics and Molecular Breeding Center of Henan Agricultural University and T0 generation seedlings were delivered.

[0045] T0 generation positive plants were selected by screening with Bar / PAT rapid test strips (Shanghai Youlong Biotechnology Co., Ltd., AA1032-LS). The seeds of the obtained T0 generation positive plants were then used to generate T1 seedlings. T1 seedling DNA was crudely extracted. Using this DNA as a template, the TaPHR1-Like7 gene A, B, and D chromosome-specific primers (AF / R, BF / R, DF / R) were designed according to the Toyobo KOD One Mix instructions for amplification to detect the gene editing / knockout type.

[0046] Table 4 A-F / R, BF / R and DF / R sequences

[0047]

[0048] Heterozygous mutants of the T1 generation were obtained through sequencing and screening. T1 generation seeds were harvested and, after further generations, T2 generation seedlings were obtained. Crude DNA was extracted from these seedlings. Using this DNA as a template, the TaPHR1-Like7 gene was amplified using universal ABD chromosome primers (F1 / R1) designed according to the Hi-TOM next-generation sequencing guide (http: / / 121.40.237.174 / Hi-TOM / Sample_acceptance_sanyang.php). Homozygous transgenic lines and their editing types were obtained using Hi-TOM next-generation sequencing. Homozygous mutant lines phl7-1, phl7-2, and phl7-3 were selected, which are the breeding materials for increased wheat grain length. The target sites and gene editing types for the homozygous mutant lines are detailed below. Figure 1 .

[0049] Table 5. Universal primer F1 / R1 sequences

[0050]

[0051] Example 3: Identification of TaPHR1-like7, a phosphorus starvation response factor in wheat.

[0052] Wild-type Zhengmai 7698 (WT) and gene-edited homozygous mutant lines (phl7-1, phl7-2, and phl7-3) were germinated in deionized water for one week to deplete the nutrients stored in the seeds. They were then transferred to Hoagland nutrient solutions (containing 0.2 mM and 0.01 mM KH2PO4, respectively) and cultured for 12 days, with the nutrient solution changed every 3 days. When the seedlings reached the three-leaf stage, total RNA was extracted from the wheat roots using a plant RNA rapid extraction kit (Beijing Nobel Biotechnology Co., Ltd., RNE05). III. The cDNA was reverse transcribed using the RT SuperMix for qPCR (+gDNAwiper) kit (Vazyme, R323-01). This cDNA was then used as a template for qRT-PCR analysis of root-related phosphorus starvation response genes. The primers used for qRT-PCR are shown in Table 6 below. The qRT reaction system and procedure followed the ChamQ Blue Universal SYBR qPCR MasterMix quantitative kit (Vazyme, Q312-02), with an annealing temperature of 60℃. Results are shown below. Figure 2 .in, Figure 2 HP was cultured in Hoagland nutrient solution containing 0.2 mM; LP was cultured in Hoagland nutrient solution containing 0.01 mM.

[0053] Table 6 Primer names and sequences

[0054]

[0055] Depend on Figure 2 It can be seen that TaPHR1-like7 significantly positively regulates the expression of phosphorus starvation-related genes, thereby responding to phosphorus starvation.

[0056] Example 4: Effects of wheat phosphorus starvation response factor TaPHR1-like7 on nitrogen accumulation and GS enzyme activity

[0057] Wheat gene-edited homozygous mutant lines phl7-1, phl7-2, phl7-3 and wild-type (WT) seeds were selected and germinated in deionized water for about one week until the nutrients in the seeds were exhausted and the seedlings grew to the one-leaf-one-heart stage. They were then transferred to Hoagland nutrient solution (containing HP: 0.2mM KH2PO4 and LP: 0.01mM KH2PO4, respectively). The seedlings were fixed with sponges. The nutrient solution was changed every 3 days. After 12 days of cultivation, the surface moisture of the aboveground and underground parts was absorbed with absorbent paper. Samples were taken and placed in kraft paper bags and dried at 80℃ for 3 days until constant weight. The dry weight was recorded.

[0058] The dried sample was ground into powder using a grinder. 0.1 g of the dried sample was weighed into a 50 ml digestion tube, sulfuric acid was added, and the sample was carbonized overnight. After digestion with sulfuric acid and hydrogen peroxide, the nitrogen accumulation in the plant was determined using an AA3 flow analyzer. Separately, grains at the peak grain-filling stage were analyzed for glutamine synthetase activity. Specific methods were based on a glutamine synthetase kit (Nanjing Jiancheng). Results are shown below. Figure 3 .

[0059] Depend on Figure 3 It was found that after the taphr1-like7 mutation, the total nitrogen accumulation under high phosphorus conditions was downregulated compared to the wild-type WT, while the total nitrogen accumulation increased under low phosphorus conditions. This indicates that TaPHR1-like7 inhibits total nitrogen accumulation under low phosphorus conditions. In addition, the results of GS enzyme activity assay in grains during the peak grain-filling stage showed that the GS enzyme activity of the taphr1-like7 mutant was increased compared to the wild-type WT.

[0060] Example 5: Effects of TaPHR1-like7 mutant plants on wheat grain size and yield

[0061] Seeds from the obtained homozygous gene-edited mutant lines (phl7-1, phl7-2, and phl7-3) were conventionally cultivated in the field. Grain size characteristics were investigated at maturity, and yield per plant, effective tillers, and thousand-grain weight were statistically analyzed. Results are shown below. Figure 4 and Figure 5 .

[0062] Depend on Figure 4It can be seen that, compared with wild-type WT, the taphr1-like7 mutant has a larger grain length, but the grain width is not significantly different. This indicates that knocking out TaPHR1-like7 can change the grain length, meaning that TaPHR1-like7 can be used to breed wheat varieties with increased grain length.

[0063] Depend on Figure 5 It was found that, compared with wild-type WT, the taphr1-like7 mutant had increased effective tillers, thousand-grain weight, and yield. This indicates that knocking out TaPHR1-like7 can increase the number of effective tillers, thousand-grain weight, and yield of wheat.

[0064] In summary, this invention elucidates the mechanism of action of the wheat phosphorus starvation response factor TaPHR1-like7 in nitrogen and other nutrient metabolism. It also discovers that TaPHR1-like7 can increase wheat grain length, providing important gene resources and related germplasm resources for breeding wheat varieties with efficient nitrogen utilization and longer grain diameter.

[0065] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. The application of a wheat phosphorus starvation response factor TaPHR1-like7 in wheat breeding, characterized in that, In the process of wheat breeding, the wheat phosphorus starvation response factor TaPHR1-like7 is knocked out to increase wheat grain length, increase wheat yield, improve effective tillering and thousand-grain weight, and improve wheat nitrogen utilization under low phosphorus conditions; wherein, the amino acid sequence of the wheat phosphorus starvation response factor TaPHR1-like7 is shown in SEQ ID NO.

1.

2. A method for obtaining breeding materials with increased wheat grain length, characterized in that, Includes the following steps: S1. Design the target of wheat phosphorus starvation response factor TaPHR1-like7. The target was selected based on the conserved sequence in three wheat copies. Two gRNAs were designed for TaPHRL7-1A, TaPHRL7-1B and TaPHRL7-1D, respectively, and annealed to form double strands. TaPHR1-like7 was recombined into the two-element expression vector PLH3 containing the UBI promoter by homologous recombination to construct the gene coding vector PLH3-TaPHR1-like7. S2. The gene-coding vector PLH3-TaPHR1-like7 was transformed into wheat using Agrobacterium-mediated in situ transformation of mature wheat embryos. T0 positive plants were selected by Bar tag screening, and T1 generation edited heterozygous mutants were obtained by sequencing screening. T2 generation transgenic lines were obtained by adding generations, and T2 generation homozygous transgenic lines were obtained by Hi-TOM next-generation sequencing. Gene-edited homozygous mutant lines were screened to obtain breeding materials with increased wheat grain length. Among them, in S1, the amino acid sequence of wheat phosphorus starvation response factor TaPHR1-like7 is shown in SEQ ID NO.1.

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