Zmbzip68 protein and its encoding gene in regulating corn heat resistance

Abstract

The application relates to application of a ZmbZIP68 protein and an encoding gene thereof in regulating corn heat resistance. It is found that the corn ZmbZIP68 gene can positively regulate plant heat resistance, and through overexpression of the expression amount of the ZmbZIP68 gene, the heat resistance of the plant can be effectively increased. Discovery of the heat resistance function of the ZmbZIP68 gene provides a new gene target and resource for breeding heat-resistant plant varieties, has important significance for research on the heat-resistant molecular mechanism of corn, and lays a certain theoretical foundation for research on a mechanism of plant response to high-temperature stress and a molecular mechanism of resisting adverse environment.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically the application of the ZmbZIP68 protein and its encoding gene in regulating heat resistance in maize. More particularly, it relates to the application of the ZmbZIP68 protein and its encoding gene in regulating maize's tolerance to high-temperature stress. Background Technology

[0002] Maize (Zea mays), belonging to the genus Zea in the family Poaceae, is an important food and feed crop, and also the world's highest-yielding crop. However, since the Industrial Revolution, global warming caused by human activities has increased the risk of plants suffering from heat stress. Literature reports that rising temperatures cause a significant decrease in crop yields: for every 1°C increase in global average temperature, staple crops will reduce yields by 19.7% [maize (Zea mays): 7.4%; wheat (Triticumaestivum): 6.0%; rice (Oryza sativa): 3.2%; soybean (Glycine max): 3.1%]. Taking maize as an example, it often suffers frequent and severe heat stress during its growth period, manifested as reduced photosynthetic rate, increased transpiration rate, decreased pollen viability, accelerated cell senescence, and shortened grain-filling time, resulting in reduced ear count and grain weight, ultimately causing a serious negative impact on yield.

[0003] bZIP transcription factors are among the most numerous transcription factors in plants, playing a crucial role in plant responses to biotic and abiotic stresses. Plant bZIP domains contain 60-80 amino acids, characterized by: an N-terminal basic domain containing a nuclear localization signal (NLS) and an N-x7-R / K motif that binds to a specific DNA sequence; and a C-terminal leucine zipper region, which often forms α-helical homodimers or heterodimers through hydrophobic interactions.

[0004] bZIP transcription factors play a crucial role in regulating plant responses to abiotic stress. Taking drought stress as an example, some bZIP family members can enhance plant drought tolerance by mediating the ABA pathway. Some bZIP family members can regulate the expression of drought-related genes by binding to promoter cis-acting elements. Transgenic plants overexpressing bZIP show significantly higher activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) than those overexpressing WT, and these substances are particularly important for improving plant drought resistance. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide an application of the ZmbZIP68 protein and its encoding gene in regulating heat resistance in maize. This application involves increasing the concentration of ZmbZIP68 protein in the plant. ZmbZIP68 The expression level of the gene and / or the activity of the ZmbZIP68 protein can effectively improve the heat resistance of plants.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: Applications of the ZmbZIP68 protein, or its encoding gene, in any of the following aspects: (1) Regulating plant heat resistance; Specifically, the heat resistance can be expressed as the survival rate after recovering at room temperature after being exposed to a high temperature of 45 degrees Celsius for 2-3 days; (2) Regulating the survival rate and / or growth of plants under high temperature conditions; (3) Select and breed transgenic plants with heat resistance and / or increased yield; (4) Improvement of heat-resistant plant germplasm resources; The amino acid sequence of the ZmbZIP68 protein is as follows: (1) The amino acid sequence shown in SEQ ID NO.1, encoded by the nucleotide sequence shown in SEQ ID NO.2; or, (2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids as shown in SEQ ID NO.1; The nucleotide sequence of the gene encoding the ZmbZIP68 protein is as follows: (1) The nucleotide sequence as shown in SEQ ID NO.2; or, (2) A nucleotide sequence encoding a protein with the same function obtained by substituting, deleting, or inserting one or more nucleotides into the nucleotide sequence shown in SEQ ID NO.2; or, (3) A nucleotide sequence that hybridizes with a nucleotide sequence as shown in SEQ ID NO.2.

[0007] The nucleotide sequence shown in SEQ ID NO.2 is the cDNA sequence of the ZmbZIP68 protein in maize, consisting of 1867 bases. The gene reading frame consists of 3 exons. Considering codon degeneracy, all nucleotide sequences encoding the ZmbZIP68 protein are within the scope of protection of this invention.

[0008] Based on the above plan, The application enhances the heat resistance of the plant by increasing the expression level of the ZmbZIP68 protein encoding gene and / or the activity of the ZmbZIP68 protein, thereby increasing the survival rate of the plant under high temperature conditions, breeding transgenic plants with heat resistance and / or increased yield, and improving heat-resistant plant germplasm resources.

[0009] Preferably, the increase in the expression level of the ZmbZIP68 protein in the plant is achieved by introducing an overexpression vector containing the encoding gene of the ZmbZIP68 protein into the plant.

[0010] Transgenic maize lines were obtained by introducing an overexpression vector containing the coding gene of the ZmbZIP68 protein into maize using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, or Agrobacterium-mediated transformation.

[0011] The primer pair used for amplifying the ZmbZIP68 protein-coding gene as described in claim 1 is characterized by: The nucleotide sequences of the primer pairs are shown in SEQ ID NO.4-5.

[0012] The specific amplification steps are as follows: (1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use cDNA as template to amplify the CDS sequence (SEQ ID NO.3) of the ZmbZIP68 gene, and ligate the amplification product into a plant expression vector to obtain a recombinant expression vector; (2) Agrobacterium was transformed using the recombinant expression vector obtained in step (1) to obtain recombinant Agrobacterium; (3) The recombinant Agrobacterium obtained in step (2) was used to infect maize callus tissue, and positive transgenic plants were screened to obtain heat-resistant transgenic maize.

[0013] Should ZmbZIP68 Transgenic maize plants overexpressing this gene showed significantly increased heat resistance compared to wild-type maize.

[0014] A biomaterial, characterized in that the biomaterial contains the gene encoding the ZmbZIP68 protein as described in claim 1.

[0015] Based on the above plan, The biomaterials include expression cassettes, vectors, host cells, or recombinant bacteria.

[0016] This invention provides cloning vectors or various expression vectors containing the coding gene of the ZmbZIP68 protein. This invention also provides host cells containing the vector, transformed plant cells containing the coding gene of the ZmbZIP68 protein, or transgenic plants.

[0017] The above-mentioned biomaterial can be used in any of the following applications: (1) Regulating plant heat resistance; (2) Regulating the survival rate and / or growth of plants under high temperature conditions; (3) Select and breed transgenic plants with heat resistance and / or increased yield; (4) Improvement of heat-resistant plant germplasm resources.

[0018] In this invention, the plant is a monocotyledonous plant. The monocotyledonous plant is a member of the Poaceae family. More preferably, it is maize.

[0019] The beneficial effects of the ZmbZIP68 protein and its encoding gene described in this invention in regulating heat resistance in maize are as follows: This invention discovered corn ZmbZIP68 Genes can positively regulate plant heat resistance by increasing... ZmbZIP68 The expression level of genes can effectively enhance the heat resistance of plants. This invention constructs... ZmbZIP68 Transgenic maize plants overexpressing this gene exhibit significantly enhanced heat resistance compared to wild-type maize. ZmbZIP68 The discovery of the heat-resistant function of genes provides new gene targets and resources for breeding heat-resistant plant varieties. It is of great significance for the study of the molecular mechanism of heat resistance in maize and lays a certain theoretical foundation for studying the mechanism of plant response to high temperature stress and the molecular mechanism of resisting adverse environments. Attached Figure Description

[0020] The present invention includes the following figures: Figure 1 This image shows the growth of the overexpression line bZIP68-OE in Example 2 of this invention after recovery from high-temperature treatment; where WT represents wild-type maize plants and bZIP68-OE represents the overexpression line.

[0021] Figure 2 In the overexpression line bZIP68-OE of Example 2 of this invention ZmbZIP68 Gene expression levels were detected, where WT represents wild-type maize plants and bZIP68-OE represents overexpression lines.

[0022] Figure 3 This is a statistical chart showing the survival rate of the overexpression line bZIP68-OE in Example 2 of the present invention; where WT represents wild-type maize plants and bZIP68-OE represents the overexpression line.

[0023] Figure 4 In the wild type of embodiment 3 of the present invention ZmbZIP68 Gene induction under heat stress. Detailed Implementation

[0024] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.

[0025] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 21), or as recommended by the manufacturer's instructions.

[0026] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Agrobacterium EHA105 strain was provided by the Crop Functional Genomics Platform of the College of Biological Sciences, China Agricultural University, and is commercially available (Ma et al., 2009, Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic riceis mediated by alteration in cell cycle and ectopic expression of stress genes. Plant Physiol. 150, 244–256). In the following examples, various restriction endonucleases, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, and KOD were purchased from NEB, Toyobo, and other biotechnology companies; dNTPs were purchased from Genestar; plasmid miniprep kits and agarose gel extraction kits were purchased from Shanghai Jierui Biotechnology Co., Ltd.; agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampin (Rif), and other antibiotics, as well as glucose, BSA, LB medium, etc., were purchased from Sigma, Bio-Rad, and other companies; reagents used for real-time quantitative PCR were purchased from TaKaRa; all other chemical reagents used in the following examples were imported or domestically produced analytical grade reagents. Primers used in the following examples were synthesized by Huada Biotechnology Co., Ltd., and related sequencing was performed. Example 1 ZmbZIP68 Construction and identification of gene overexpression maize The maize functional genomics platform of China Agricultural University was used to construct a genome containing... ZmbZIP68The gene vector was transformed into Agrobacterium strain EHA105, which was then used to infect maize callus tissue to obtain transgenic seedlings. The specific method was as follows: Agrobacterium containing the target vector was inoculated into 100 mL of LB triple-antibiotic liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), and cultured overnight at 28°C with shaking until OD was reached. 600 The bacterial cells were collected by centrifugation at 50×g for 15 min at room temperature, with a value of 1.0-2.0. The cells were then resuspended in 2 mL of transformation buffer (1 / 2 MS, 5% sucrose, 40 μL Silwet L-77). Corn callus tissue was immersed in the Agrobacterium transformation buffer and sealed. The tissue was then returned to a light-treated culture rack and allowed to grow normally until plants emerged. The resulting seeds were then screened and subjected to high-temperature stress treatment experiments.

[0027] In this embodiment, the overexpression line bZIP68-OE was isolated, and the expression level in the obtained overexpression line was detected by real-time quantitative PCR. ZmbZIP68 Gene expression, specifically through the following methods: (1) Extract total RNA from maize and reverse transcribe it to obtain cDNA.

[0028] (2) After diluting the cDNA obtained by reverse transcription by 3 times, real-time quantitative PCR was performed using the Takara kit. The reaction system used was: 2×SYBR Premix ExTaq buffer, 0.2 μL DyII, 0.4 μL Primer (F / R), 2 μL cDNA template, and finally ddH2O to make up to 20 μL. After thorough mixing, it was placed in an ABI PRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions were: 95℃ for 30 s; 95℃ for 5 s; 60℃ for 40 s; 40 cycles.

[0029] The amplification primer pairs are shown in SEQ ID NO.4-5.

[0030] Simultaneously with the amplification of the identified genes, each sample was amplified using UBI as an internal control. After the PCR reaction was completed, according to 2... -Δ（ΔCt） The relative expression levels between wild-type and overexpression lines were calculated based on the principle of [previous method] and plotted for analysis. The results are as follows: Figure 2 As shown, the results indicate that in bZIP68-OE ZmbZIP68 Gene expression was upregulated by about 1.5 times.

[0031] Example 2 Overexpression ZmbZIP68 Tests on the heat resistance of genetically modified corn First, seeds of wild-type maize (control group WT) and the overexpression line bZIP68-OE were sown in small pots containing black soil, imported soil, and vermiculite (1:1:1), with 12 seeds in each pot. Each pot was then covered with 2 cm of soil and placed on a tray. The soil was watered until completely moist, and the pots were placed in a 23 ℃ incubator with 16 hours of light and 8 hours of darkness. After 12 days of growth, the plants were treated at 45 ℃ until the second leaf withered and shriveled. They were then removed and placed in a 23 ℃ incubator for one week to recover before the survival rate was recorded.

[0032] Phenotypic characteristics of wild-type plants and overexpressing line bZIP68-OE after high-temperature treatment recovery are as follows: Figure 1 As shown, compared with the control, the survival rate of the overexpression line bZIP68-OE was significantly increased, indicating that the overexpression line bZIP68-OE exhibits a heat-resistant phenotype. In the above experiments, 12-16 seedlings were used in each experiment, and the experiments were independently repeated 3 times.

[0033] Example 3 Detection ZmbZIP68 Gene induction at high temperatures Wild-type plants that had grown for approximately 12 days were placed at 42 ℃ for 6 h and 12 h. Root samples were then collected to extract RNA, which was reverse transcribed into cDNA and detected by qPCR. Specific detection methods are described in Example 1. Experimental results are as follows: Figure 3 As shown Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

[0034] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

Claims

1. Applications of ZmbZIP68 protein or its encoding gene in any of the following aspects: (1) Enhance the heat resistance of plants; (2) Improve the survival rate of plants under high temperature conditions, wherein the high temperature condition is 45 degrees Celsius; (3) Selecting and breeding transgenic plants with improved heat resistance; (4) Improvement of heat-resistant plant germplasm resources; The amino acid sequence of the ZmbZIP68 protein is shown in SEQ ID NO.1; the encoding gene of the ZmbZIP68 protein is shown in SEQ ID NO.3; the plant is maize.

2. The application as described in claim 1, characterized in that: The application enhances the heat resistance of the plant by increasing the expression level of the ZmbZIP68 encoding gene, thereby increasing the survival rate of the plant under high temperature conditions, breeding transgenic plants with improved heat resistance, and improving heat-resistant plant germplasm resources.

3. An application of a biomaterial in any of the following aspects: (1) Enhance the heat resistance of plants; (2) Improve the survival rate of plants under high temperature conditions, wherein the high temperature condition is 45 degrees Celsius; (3) Selecting and breeding transgenic plants with improved heat resistance; (4) Improvement of heat-resistant plant germplasm resources; The plant in question is corn; The biological material is an expression cassette, vector, or host cell containing the gene encoding the ZmbZIP68 protein as described in claim 1.

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