Application of corn ZmMYB80 gene in regulation and control of corn root development and low phosphorus stress tolerance
By overexpressing the maize ZmMYB80 gene, the root development of maize was regulated, which solved the problem of root development inhibition under low phosphorus stress, achieved stronger tolerance to low phosphorus stress, and promoted the breeding of low phosphorus tolerant maize varieties.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INSTITUTE OF CROP SCIENCE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-19
AI Technical Summary
In existing technologies, root development in maize is hindered under low phosphorus stress, resulting in stunted plants and a significant reduction in aboveground biomass. Therefore, it is difficult to cultivate maize varieties that are tolerant to low phosphorus stress.
By overexpressing the maize ZmMYB80 gene, its expression level in maize was enhanced, and root development was regulated to improve the ability to withstand low phosphorus stress. The recombinant vector CUB-ZmMYB80 was used for gene transformation to obtain overexpressing maize materials OE#1 and OE#2.
Under low phosphorus conditions, the total root length of the ZmMYB80 overexpression lines decreased less than that of the wild type, and the aboveground biomass loss was reduced, showing a stronger ability to tolerate low phosphorus stress, which laid the foundation for breeding new maize varieties tolerant to low phosphorus stress.
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Figure CN122060752A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to maize. ZmMYB80 Application of genes in regulating maize root development and tolerance to low phosphorus stress. Background Technology
[0002] Phosphorus plays a crucial role in plant growth, being the second most essential nutrient and often referred to as the "element of life." It is a component of various intermediate metabolites and participates in the metabolism of carbohydrates, lipids, and proteins. Phosphorus is closely related to the biochemical reactions of plant energy, is a component of nucleic acids (the genetic material of organisms), and influences cell division and meristematic development. Phosphorus utilization and metabolism accompany the entire life cycle of a plant. During evolution, plants have developed a series of mechanisms for the absorption, transport, and recycling of inorganic phosphates to maintain the effective phosphorus concentration within the plant and ensure its growth and development. The root system is not only the main organ for plants to obtain nutrients and water from the soil but also the earliest and most important organ for sensing various environmental stimuli. When the aboveground parts receive phosphorus stress signals transmitted from the roots, the entire plant responds rapidly to nutrient stress, taking corresponding regulatory measures to adapt to the low-phosphorus environment. Studies in plants such as Arabidopsis thaliana, maize, and rice have found that the effective phosphorus concentration in the soil can alter root morphology, including regulating the formation of lateral root primordia, the growth of the taproot and lateral roots, changes in the angle of lateral root growth, and increasing root hair density. When plants are under low phosphorus stress, the synthesis of proteins in their bodies is inhibited, and the plants become stunted; at the same time, the root system usually becomes more developed, which is reflected in the increased root-to-shoot ratio under low phosphorus conditions.
[0003] As a globally important food, feed, and industrial raw material crop, maize has a huge demand for phosphorus during its production process. However, approximately 60% of the soil in my country's main maize-producing areas suffers from varying degrees of phosphorus deficiency. Therefore, developing maize varieties tolerant to low phosphorus stress has become an urgent need to ensure the sustainable development of the maize industry. Thus, identifying key genes for maize tolerance to low phosphorus stress is of great significance for breeding such varieties.
[0004] The MYB transcription factor family, one of the largest transcriptional regulatory families in plants, is characterized by its conserved MYB domain and plays a crucial regulatory role in plant growth, development, secondary metabolism, and stress responses. Numerous studies have confirmed that MYB family members mediate plant adaptive responses to stresses such as drought, salinity, and nutrient deficiency by recognizing specific cis-regulatory elements in the promoter regions of downstream target genes, thereby activating or inhibiting gene expression. Whether the ZmMYB80 transcription factor in maize participates in plant responses to low phosphorus stress remains unstudied. Summary of the Invention
[0005] The purpose of this invention is to provide corn ZmMYB80Application of genes in regulating maize root development and tolerance to low phosphorus stress.
[0006] corn ZmMYB80 Genes, the corn ZmMYB80 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1 of the sequence listing; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating maize root development and tolerance to low phosphorus stress. (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating maize root development and tolerance to low phosphorus stress.
[0007] The corn ZmMYB80 protein, wherein the amino acid sequence of the corn ZmMYB80 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating maize root development and tolerance to low phosphorus stress.
[0008] Containing the corn ZmMYB80 The carrier of genes.
[0009] Containing the corn ZmMYB80 Engineered bacteria with gene vectors.
[0010] Amplify the corn ZmMYB80 Primers for any segment of a gene.
[0011] The corn ZmMYB80 Application of genes in regulating maize root development and tolerance to low phosphorus stress.
[0012] A method to regulate maize root development and tolerance to low phosphorus stress by increasing the expression level of ZmMYB80 protein in maize.
[0013] Beneficial effects of the present invention: The present invention has found that, under normal phosphorus supply and phosphorus deficiency conditions, compared with the wild type, ZmMYB80 Overexpression significantly increased the total root length of maize, and the decrease in total root length in wild-type maize under low phosphorus conditions was significantly greater than that in overexpressing lines. Under low phosphorus stress, the aboveground biomass of wild-type maize decreased by up to 30%; in contrast, ZmMYB80 The reduction rates of the overexpression lines OE#1 and OE#2 were only 12% and 22%, respectively, indicating that... ZmMYB80 The overexpression material exhibits greater tolerance to low phosphorus stress. This discovery lays the foundation for breeding new maize varieties that regulate root development and are tolerant to low phosphorus stress. Attached Figure Description
[0014] Figure 1 This is a schematic diagram of the CUB-ZmMYB80 carrier.
[0015] Figure 2 for ZmMYB80 Overexpression maize plants ZmMYB80 Gene expression level detection.
[0016] Figure 3 for ZmMYB80 Total root length of maize after 7 days of hydroponic treatment with different phosphorus concentrations.
[0017] Figure 4 The relative fresh weight of the aboveground parts of ZmMYB80-overexpressing maize under low and normal phosphorus conditions. Detailed Implementation
[0018] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.
[0019] The maize inbred lines used in the following examples were: B73 and CAL50; strains: Escherichia coli strain DH5α and Agrobacterium strain GV3101; and the overexpression vector was CUB.
[0020] Example 1: Construction of the recombinant vector CUB-ZmMYB80 Total RNA was extracted from the aboveground parts of B73 seedlings and reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Using this cDNA as a template, the upstream primer 5'-CG was used... GGATCC ATGCAGGCAGATGACAAACTCG-3' (underscore sequence is...) BamH I site) and downstream primer 5'-CG GGATCC CTAGTGATTTTCTGCTTCGCC-3' (underlined sequence is...) BamH PCR amplification was performed at site I, and a DNA fragment of approximately 1320 bp was purified and recovered to obtain the full-length sequence of the ZmMYB80 gene coding region. BamH The CUB plasmid vector was digested with NEB restriction enzyme, and the digestion products were recovered. T4 DNA ligase was then used to ligate... ZmMYB80 The PCR product was ligated with a linearized CUB plasmid vector to obtain the recombinant vector CUB-ZmMYB80 ( Figure 1 The recombinant vector was transformed into *E. coli* competent cells DH5α using the heat shock method, plated on LB agar plates containing kanamycin resistance, and positive clones were selected for sequencing. The correctly sequenced plasmid was then transformed into *Agrobacterium tumefaciens* competent cells GV3101 using electroporation.
[0021] Example 2 ZmMYB80 Obtaining maize materials through overexpression The embryos of the maize inbred line CAL50 were transformed using Agrobacterium-mediated transformation. Following infection, co-culture, callus induction, selection, differentiation, and seedling formation, T0 generation seeds were harvested in a greenhouse. After further propagation in Hainan and Beijing, two homozygous transgenic maize lines were obtained. OE #1 and OE #2 Total RNA was extracted from the aboveground parts of OE #1, OE #2, and wild-type CAL50 seedlings, respectively. cDNA was obtained by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). This cDNA was then used as a template for quantitative real-time PCR detection. For quantitative real-time PCR, the primers for ZmMYB80 detection were 5'-TGGAACAACGCTACCGGAAG-3' and 5'-CACACATTCCATCTAGAAGTT-3'; ZmTub was used as an internal control for the maize gene, with primer sequences of 5'-ACACCACCATTGGGAGTCTA-3' and 5'-TTGTGGGGACCACTACTTTC-3'. Real-time quantitative PCR was performed using the Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific), with each experiment set to three replicates. -ΔΔCT The relative expression level was calculated using this method. The results are as follows: Figure 2 As shown, OE#1 and OE#2 Transgenic pure lines ZmMYB80 Gene expression levels were significantly increased compared to the wild type (CAL50).
[0022] Example 3 ZmMYB80Analysis of the response of maize overexpression materials to low phosphorus stress The experimental plants were: OE#1 , OE#2 T2 generation plants of the strain, wild-type CAL50 material.
[0023] Hydroponic conditions for corn: Select plump corn seeds of uniform size, soak them in 3% NaClO for 20 min, and then rinse them three times with distilled water. Soak the seeds in distilled water at room temperature for 6 h, then place them on moist filter paper; incubate them in the dark at 28℃ for 2 days. When the radicle grows to 1.5 cm, roll the seedlings in filter paper and place them in distilled water for further cultivation, and cover them with a black plastic bag to block out light. When the corn seedlings have one leaf and one bud, select seedlings of uniform growth, remove the endosperm, and transfer them to a 3 L hydroponic container. First, treat them with a semi-nutrient solution for 2 days, then treat them with a full nutrient solution (250 μM PO4). 3- ) and low phosphorus nutrient solution (5 μM PO4) 3- Treatment: Change the nutrient solution every 2 days during hydroponics. The plant culture conditions are 14 h light / 10 h darkness, 28℃ / 22℃.
[0024] right ZmMYB80 Overexpression lines OE#1 , OE#2 After 7 days of hydroponic treatment with normal and low phosphorus levels, root images were acquired using a root scanner, and the total root length was quantitatively analyzed using WinRHIZO Pro 2019 software. The results are shown in Figure 3. Under normal phosphorus supply and phosphorus deficiency conditions, compared with the wild type, ZmMYB80 Overexpression significantly increased the total root length of maize; furthermore, compared with normal phosphorus, wild-type maize under low phosphorus conditions and... ZmMYB80 The total root length of all overexpression lines showed a decreasing trend, but the decrease was significantly greater in the wild type: compared to normal phosphorus supply conditions, the total root length of the wild type decreased by 25.86%, while the total root length of the OE#1 and OE#2 lines decreased by only 7.6% and 8.08%, respectively. Simultaneously, the aboveground biomass accumulation characteristics were measured, and the results are as follows: Figure 4 As shown, under low phosphorus stress, the aboveground biomass of wild-type plants decreased by up to 30%; in contrast, ZmMYB80 The reduction rates of the overexpression lines OE#1 and OE#2 were only 12% and 22%, respectively, indicating that... ZmMYB80 Overexpression materials are more tolerant to low phosphorus stress.
[0025] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.
Claims
1. Corn ZmMYB80 Genes, characterized by, The corn ZmMYB80 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1 of the sequence listing; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of regulating maize root development and tolerance to low phosphorus stress. (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the function of regulating maize root development and tolerance to low phosphorus stress.
2. Corn ZmMYB80 protein, characterized in that, The amino acid sequence of the corn ZmMYB80 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the function of regulating maize root development and tolerance to low phosphorus stress.
3. Containing the corn as described in claim 1 ZmMYB80 The carrier of genes.
4. Containing the corn as described in claim 3 ZmMYB80 Engineered bacteria with gene vectors.
5. The maize according to claim 1 ZmMYB80 Primers for any segment of a gene.
6. The corn according to claim 1 ZmMYB80 Application of genes in regulating maize root development and tolerance to low phosphorus stress.
7. A method for regulating maize root development and tolerance to low phosphorus stress, characterized in that, Increase the expression level of ZmMYB80 protein in maize.