Application of ZmbZIP68 protein and coding gene thereof in regulation and control of heat resistance of corn

Abstract

The invention relates to application of a ZmbZIP68 protein and a coding gene thereof in regulation and control of heat resistance of corn. It is found that the corn ZmbZIP68 gene can positively regulate the heat resistance of plants, and the heat resistance of the plants can be effectively improved by overexpressing the expression quantity of the ZmbZIP68 gene. The discovery of the heat-resistant function of the ZmbZIP68 gene provides a new gene target and resource for cultivating heat-resistant plant varieties, has important significance on research on a heat-resistant molecular mechanism of corn, and lays a certain theoretical foundation for research on a high-temperature stress response mechanism and a molecular mechanism for resisting adverse environments of plants.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and specifically relates to the application of a ZmbZIP68 protein and its encoding gene in regulating heat resistance in maize. In particular, it refers to the application of the ZmbZIP68 protein and its encoding gene in regulating the tolerance of maize to high temperature stress. Background Art

[0002] Maize (Zea mays) belongs to the genus Zea of the Gramineae family, and is an important food crop and feed crop, and also the crop with the highest total global output. After the Industrial Revolution, global warming caused by human activities has increased the risk of plants suffering from high temperature stress. There are literature reports that an increase in temperature will cause a significant decrease in crop yields: for every 1°C increase in the global average temperature, the main food crops will reduce production by 19.7% [maize (Zea mays): 7.4%; wheat (Triticum aestivum): 6.0%; rice (Oryza sativa): 3.2%; soybean (Glycine max): 3.1%]. Taking maize as an example, it often suffers from frequent and severe high temperature stress during its growth period, manifested as reduced photosynthetic rate, increased transpiration rate, decreased pollen viability, accelerated cell senescence, shortened filling time, etc., resulting in a decrease in the number of grains per ear and grain weight, and ultimately having a serious negative impact on the yield.

[0003] The bZIP transcription factor is one of the transcription factors with the largest number of members in plants, and plays an important role in the process of plants responding to biotic and abiotic stresses. The plant bZIP domain contains 60 - 80 amino acids, and its structural characteristics are: the N-terminal is a basic domain, containing a nuclear localization signal (NLS) and an N-x7-R / K motif that binds to a specific DNA sequence; the C-terminal is a leucine zipper region, and this region often forms homo- or hetero-dimers in the form of α-helices through the interaction of hydrophobic surfaces.

[0004] The bZIP transcription factor plays an important role in regulating the response of plants to abiotic stresses. Taking drought stress as an example, some members of the bZIP family can enhance the drought tolerance of plants by mediating the ABA pathway. Some members of the bZIP family can regulate the expression of drought-related genes by binding to the cis-acting elements of the promoters of drought-resistant genes. The activities of antioxidant enzymes such as superoxide dismutase, catalase (CAT), and peroxidase (POD) in transgenic plants overexpressing bZIP are significantly higher than those of the WT, and these substances are particularly important for improving the drought resistance of plants. Summary of the Invention

[0005] Aiming at the defects existing in the prior art, the purpose of the present invention is to provide an application of ZmbZIP68 protein and its encoding gene in regulating heat resistance of maize. This application effectively improves the heat resistance of plants by increasing the expression level of the ZmbZIP68 gene and / or the activity of the ZmbZIP68 protein in plants.

[0006] To achieve the above object, the technical solution adopted by the present invention is:

[0007] Application of ZmbZIP68 protein, or its encoding gene, in any of the following aspects:

[0008] (1) Regulating plant heat resistance;

[0009] Specifically, the heat resistance can be manifested as the survival rate after recovery at normal temperature after 2 - 3 days at a high temperature of 45 degrees Celsius.

[0010] (2) Regulating the survival rate and / or growth of plants under high temperature conditions;

[0011] (3) Breeding transgenic plants with improved heat resistance and / or yield;

[0012] (4) Improving plant heat-resistant germplasm resources;

[0013] The amino acid sequence of the ZmbZIP68 protein is:

[0014] (1) The amino acid sequence shown in SEQ ID NO.1, which is encoded by the nucleotide sequence shown in SEQ ID NO.2; or,

[0015] (2) An amino acid sequence of a protein with the same function obtained by substitution, insertion or deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO.1;

[0016] The nucleotide sequence of the encoding gene of the ZmbZIP68 protein is:

[0017] (1) The nucleotide sequence shown in SEQ ID NO.2; or,

[0018] (2) A coding nucleotide sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.2; or,

[0019] (3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO.2.

[0020] The nucleotide sequence shown in SEQ ID NO.2 is the cDNA sequence of the ZmbZIP68 protein in maize, consisting of 1,867 bases, and the open reading frame of this gene consists of 3 exons. Considering the degeneracy of codons, all nucleotide sequences encoding the ZmbZIP68 protein are within the protection scope of the present invention.

[0021] On the basis of the above solution,

[0022] The application enhances the heat resistance of the plant, enhances the survival rate of the plant under high-temperature conditions, breeds transgenic plants with improved heat resistance and / or increased yield, and improves the heat-resistant germplasm resources of the plant by increasing the expression level of the gene encoding the ZmbZIP68 protein and / or the activity of the ZmbZIP68 protein in the plant.

[0023] Preferably, the increase in the expression level of the ZmbZIP68 protein in the plant is achieved by introducing an overexpression vector containing the coding gene of the ZmbZIP68 protein into the plant.

[0024] An overexpression vector containing the coding gene of the ZmbZIP68 protein is introduced into maize by using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to obtain transgenic maize lines.

[0025] The primer pair for amplifying the gene encoding the ZmbZIP68 protein in claim 1 is characterized in that:

[0026] The nucleotide sequences of the primer pair are as shown in SEQ ID NO.4-5.

[0027] The specific amplification steps are as follows:

[0028] (1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use the cDNA as a template to amplify the CDS sequence (SEQ ID NO.3) of the ZmbZIP68 gene, and ligate the amplification product to a plant expression vector to obtain a recombinant expression vector;

[0029] (2) Transform Agrobacterium with the recombinant expression vector obtained in step (1) to obtain recombinant Agrobacterium;

[0030] (3) Infect maize callus with the recombinant Agrobacterium obtained in step (2), screen positive transgenic plants, and obtain heat-resistant transgenic maize.

[0031] Compared with wild-type maize, the heat resistance of this ZmbZIP68 overexpression transgenic maize plant is significantly increased.

[0032] A biological material, characterized in that the biological material contains the coding gene of the ZmbZIP68 protein described in claim 1.

[0033] On the basis of the above solution,

[0034] The biological material includes an expression cassette, a vector, a host cell or a recombinant bacterium.

[0035] The present invention provides a cloning vector or various expression vectors containing the coding gene of the ZmbZIP68 protein. The present invention also provides a host cell containing the vector, a transformed plant cell containing the coding gene of the ZmbZIP68 protein, or a transgenic plant.

[0036] The application of the above biological material in any of the following aspects:

[0037] (1) Regulating plant heat resistance;

[0038] (2) Regulating the survival rate and / or growth of plants under high-temperature conditions;

[0039] (3) Breeding transgenic plants with improved heat resistance and / or yield;

[0040] (4) Improving plant heat-resistant germplasm resources.

[0041] In the present invention, the plant is a monocotyledonous plant. The monocotyledonous plant is a gramineous plant. More preferably, it is maize.

[0042] The application of the ZmbZIP68 protein and its coding gene described in the present invention in regulating maize heat resistance has the following beneficial effects:

[0043] The present invention discovers that the maize ZmbZIP68 gene can positively regulate plant heat resistance. By increasing the expression level of the ZmbZIP68 gene, the heat resistance of plants can be effectively enhanced. The present invention constructs an overexpressing transgenic maize plant of ZmbZIP68. Compared with wild-type maize, the heat resistance of this plant is significantly enhanced. The discovery of the heat resistance function of the ZmbZIP68 gene provides a new gene target and resource for cultivating heat-tolerant plant varieties, has important significance for the research on the heat tolerance molecular mechanism of maize, and lays a certain theoretical foundation for the research on the mechanism of plant response to high-temperature stress and the molecular mechanism of resisting adverse environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The present invention has the following drawings:

[0045] Figure 1 It is the plant growth situation of the overexpressing line bZIP68-OE after high-temperature treatment and recovery in Example 2 of the present invention; wherein, WT represents the wild-type maize plant, and bZIP68-OE represents the overexpressing line.

[0046] Figure 2 It is for the detection of the expression level of the ZmbZIP68 gene in 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.

[0047] Figure 3 It is a statistical chart of the survival rate results 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.

[0048] Figure 4 It is the induction situation of the ZmbZIP68 gene in wild-type under heat stress in Example 3 of the present invention. Detailed implementation manners

[0049] The preferred implementation manners of the present invention will be described in detail below in conjunction with the embodiments. It should be understood that the following embodiments are given only for the purpose of illustration and are not used to limit the scope of the present invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0050] The experimental methods used in the following embodiments are all conventional methods unless otherwise specified. If not specifically indicated, the embodiments are carried out under conventional experimental conditions, such as those in the Molecular Cloning Experiment Manual by Sambrook et al. (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 21), or according to the conditions recommended by the manufacturer's instructions.

[0051] The materials, reagents, etc. used in the following examples can be obtained from commercial sources without special instructions. The Agrobacterium EHA105 strain was kindly provided by the Crop Functional Genomics Platform of the College of Biological Sciences, China Agricultural University and can be purchased on the market (Ma et al., 2009, Enhanced tolerance to chilling stress in OsMYB3R-2 transgenic rice is 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, KOD were purchased from biological companies such as NEB and Toyobo; dNTPs were purchased from Genestar Company; plasmid mini-prep kits and agarose gel recovery kits were purchased from Shanghai Jierui Biotechnology Company; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampicin (Rif), and Glucose, BSA, LB Medium, etc. were purchased from companies such as Sigma and Bio-Rad; the reagents used for real-time quantitative PCR were purchased from TaKaRa; all other chemical reagents used in the following examples were imported or domestic analytical pure reagents. The primers used in the following examples were synthesized by Liuhe Huada Company and subjected to relevant sequencing

[0052] Example 1 Construction and Identification of Maize Overexpressing ZmbZIP68 Gene

[0053] The vector containing the ZmbZIP68 gene constructed by the Maize Functional Genomics Platform of China Agricultural University was transformed into the Agrobacterium EHA105 strain, and then maize calli were infected to obtain transgenic seedlings. The specific method was as follows: The 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. When the OD 600 value was 1.0 - 2.0, it was centrifuged at 50 × g for 15 min at room temperature to collect the cells; the cells were suspended with 2 mL of transformation solution (1 / 2 MS, 5% sucrose, 40 μL of Silwet L-77); the maize calli were soaked in the Agrobacterium transformation solution and sealed. It was placed back on the light culture rack and allowed to grow normally until plants emerged. Then, the seeds obtained by screening were subjected to high-temperature stress treatment experiments.

[0054] In this example, the overexpression strain bZIP68-OE was isolated, and real-time quantitative PCR was used to detect the gene expression of ZmbZIP68 in the obtained overexpression strain. The specific method is as follows:

[0055] (1) Extract total RNA from maize and reverse transcribe to obtain cDNA.

[0056] (2) After diluting the reverse-transcribed cDNA 3-fold, use the Takara kit for real-time quantitative PCR. The reaction system used is: 2×SYBR Premix ExTaq buffer, 0.2 μL DyII, 0.4 μL Primer (F / R), 2 μL cDNA template, and finally make up to 20 μL with ddH2O. After mixing well, put it into the ABIPRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions are: 95°C for 30 s; 95°C for 5 s; 60°C for 40 s; 40 cycles.

[0057] The sequences of the amplification primer pairs are shown in SEQ ID NO.4-5.

[0058] While amplifying the identified gene, UBI was used as an internal reference for simultaneous amplification in each sample. After the PCR reaction was completed, the relative expression levels between the wild type and the overexpression strain were calculated according to the 2 -Δ(ΔCt) principle and plotted for analysis. The results are as Figure 1 shown. The results show that the ZmbZIP68 gene in bZIP68-OE was up-regulated by about 1.5 times.

[0059] Example 2 Detection of the high-temperature resistance of maize overexpressing the ZmbZIP68 gene

[0060] First, sow the seeds of wild-type maize (control group WT) and the overexpression strain bZIP68-OE in small pots filled with black soil, imported soil, and vermiculite (1:1:1). Put 12 seeds in each pot, cover with 2 cm of soil, place in a tray, water until the soil is completely wet, and place in a 23°C incubator with 16 h of light and 8 h of darkness. After growing for 12 days, perform a high-temperature treatment at 45°C until the second leaf shrinks and wilts, then take it out and place it in a 23°C incubator to recover for one week, and then count the survival rate.

[0061] The phenotypes of the wild-type plants and the overexpression strain bZIP68-OE after the high-temperature treatment and recovery are as Figure 1 shown. Compared with the control, the survival rate of the overexpression strain bZIP68-OE was significantly increased, indicating that the overexpression strain bZIP68-OE showed a heat-resistant phenotype. In the above experiments, 12-16 seedlings were used in each experiment, and the experiments were independently repeated 3 times.

[0062] Example 3: Detecting the induction of the ZmbZIP68 gene under high temperature

[0063] Wild-type plants grown for about 12 days were placed at 42 °C for 6 h and 12 h, and the roots were sampled for RNA extraction. After reverse transcription into cDNA, qPCR was performed for detection. The specific detection method is shown in Example 1. The experimental results are as Figure 3 shown

[0064] 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 based on the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of the present invention claimed.

[0065] The content not described in detail in this specification belongs to the prior art well known to those skilled in the art.

Claims

1. Use of the ZmbZIP68 protein, or its coding gene, in any of the following aspects: (1) Regulating plant heat tolerance; (2) Regulating the survival rate and / or growth of plants under high-temperature conditions; (3) Breeding transgenic plants with improved heat tolerance and / or yield; (4) Improving plant heat-resistant germplasm resources; The amino acid sequence of the ZmbZIP68 protein is: (1) The amino acid sequence shown in SEQ ID NO.1, which is 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 substitution, insertion or deletion of one or more amino acids in the amino acid sequence shown in SEQ ID NO.1; The nucleotide sequence of the coding gene of the ZmbZIP68 protein is: (1) The nucleotide sequence shown in SEQ ID NO.2; or, (2) A coding nucleotide sequence of a protein with the same function obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.2; or, (3) A nucleotide sequence that hybridizes with the nucleotide sequence shown in SEQ ID NO.

2.

2. The use according to claim 1, characterized in that: The use enhances the heat tolerance of the plant, enhances the survival rate of the plant under high-temperature conditions, breeds transgenic plants with improved heat tolerance and / or yield, and improves plant heat-resistant germplasm resources by increasing the expression level of the ZmbZIP68 protein coding gene and / or the activity of the ZmbZIP68 protein in the plant.

3. A primer pair for amplifying the ZmbZIP68 protein coding gene according to claim 1, characterized in that: The nucleotide sequence of the primer pair is as shown in SEQ ID NO.4-5.

4. A biological material, characterized in that, The biological material contains the coding gene of the ZmbZIP68 protein according to claim 1.

5. The biomaterial according to claim 4, wherein The biological material includes an expression cassette, a vector, a host cell or a recombinant bacterium.

6. Use of a biological material according to claim 4 or 5 in any of the following aspects: (1) Regulating plant heat tolerance; (2) Regulating the survival rate and / or growth of plants under high-temperature conditions; (3) Breeding transgenic plants with improved heat tolerance and / or yield; (4) Improving plant heat-resistant germplasm resources.

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