Application of ZmAHA1 protein and coding gene thereof in regulation and control of heat stress resistance of corn

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of ZmAHA1 protein and a coding gene thereof in regulation and control of heat stress resistance of corn, through overexpression of ZmAha1 gene in corn, it is determined that an overexpressed plant has obvious high-temperature-resistant phenotype, it is verified that the corn ZmAha1 gene can positively regulate and control plant heat resistance, and through the expression quantity of the overexpressed ZmAha1 gene, the corn ZmAha1 gene can positively regulate and control plant heat stress resistance. The heat resistance of plants can be effectively improved. The discovery of the heat-resistant function of the ZmAha1 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 particularly relates to the application of ZmAHA1 protein and its encoding gene in regulating heat stress tolerance in maize. 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. Global warming has increased the risk of plants suffering from heat 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 heat 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] Heat shock proteins (Hsp), also known as heat shock proteins, are a class of highly conserved stress proteins widely present in various organisms. Most of the currently known heat shock proteins belong to molecular chaperones, and their main functions are to assist in the correct folding, assembly, transportation, and degradation of proteins; under stress conditions, they can stabilize the structures of proteins and membranes and prevent the aggregation of denatured proteins. Hsp90 is an ATP-regulated dimeric molecular chaperone that contains three highly conserved domains, namely an N-terminal domain of approximately 25 kDa, a 35 kDa middle domain (M), and a 12 kDa C-terminal domain. Its N-terminal domain has an ATP binding site and endogenous ATPase activity. Although its ATPase activity is weak, the results of mutation studies show that ATPase activity is necessary for Hsp90 to perform its biological functions. The realization of the function of Hsp90 in the cytoplasm of eukaryotes requires a series of co-chaperones and cofactors to regulate its activity and its interaction with target proteins and endow it with various physiological activities. Among them, AHA1 (Activator of 90 kDa heat shock protein ATPase) plays an important role. Studies have shown that its N-terminal domain can bind to the middle domain of Hsp, and the C-terminal domain of AHA1 can bind to the N-terminal of Hsp to activate its ATPase activity. Heterologous expression of DgAHA in yeast (Saccharomyces cerevisiae) cells and Arabidopsis thaliana plants enhanced their heat tolerance. However, so far, there has been little research on AHA1 protein in maize, and the roles of these members in maize's resistance to stress are still unclear. Summary of the Invention

[0004] Aiming at the deficiencies in the prior art, the object of the present invention is to provide the application of ZmAHA1 protein and its encoding gene in regulating heat stress tolerance in maize.

[0005] In the present invention, in a multi-gene overexpressing maize population, using the relative leaf injury area as an index, a preliminary screening of high-temperature phenotypes was carried out. The overexpressing lines with high-temperature tolerance phenotypes obtained from the preliminary screening were re-screened to determine their high-temperature related phenotypes, and the overexpressed genes of the lines with significantly high-temperature tolerance phenotypes obtained from the screening were determined. Through the above screening, the target gene GRMZM2G021816 (Activator of 90 kDa heat shock protein ATPase) was targeted to be related to heat stress tolerance in maize, and it was predicted that GRMZM2G021816 is an activator enzyme of HSP90 ATPase. By overexpressing the ZmAha1 gene, it was found that the overexpressing plants had obvious high-temperature tolerance phenotypes. The present invention further proves that the ZmAha1 gene positively regulates the heat resistance of plants, and the heat resistance of plants can be improved by increasing the expression level of the ZmAha1 gene in plants.

[0006] To solve the above technical problems, the technical solution provided by the present invention is:

[0007] Use of the ZmAHA1 protein or its coding gene or a biological material containing its coding gene in any of the following aspects:

[0008] A1) Use in improving the heat tolerance of maize;

[0009] A2) Use in improving the survival rate of maize under high-temperature environments;

[0010] A3) Use in breeding maize with improved heat tolerance and / or increased yield;

[0011] A4) Use in improving the heat-tolerant germplasm resources of maize.

[0012] Preferably, the temperature of the high-temperature environment is 42 - 45 °C.

[0013] Preferably, the ZmAHA1 protein has any of the following amino acid sequences:

[0014] B1) The amino acid sequence shown in SEQ ID NO.1;

[0015] B2) 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] Preferably, the coding sequence of the ZmAha1 gene has any of the following nucleotide sequences:

[0017] C1) The nucleotide sequence shown in SEQ ID NO.2;

[0018] C2) A coding nucleotide sequence that encodes a protein with the same function and is obtained by substitution, deletion or insertion of one or more nucleotides in the nucleotide sequence shown in SEQ ID NO.2;

[0019] C3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.2 under stringent conditions.

[0020] Preferably, the biological material is any one of the following D1) to D9):

[0021] D1) A nucleic acid molecule encoding the protein described in claim 3;

[0022] D2) An expression cassette containing the nucleic acid molecule described in D1);

[0023] D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2);

[0024] D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3);

[0025] D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3);

[0026] D6) A transgenic plant tissue containing the nucleic acid molecule described in D1), or a transgenic plant tissue containing the expression cassette described in D2), or a transgenic plant tissue containing the recombinant vector described in D3);

[0027] D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2), or a transgenic plant organ containing the recombinant vector described in D3).

[0028] Preferably, it is achieved by increasing the expression level or activity of the ZmAHA1 protein, or increasing the expression level of the ZmAha1 gene.

[0029] The primer sequences for amplifying the ZmAha1 gene are shown in SEQ ID No.3 and SEQ ID No.4.

[0030] A method for improving the heat tolerance of maize, which improves the heat tolerance of maize in a high-temperature environment by increasing the expression level or activity of the ZmAHA1 protein in maize, or increasing the expression level of the ZmAha1 gene; the ZmAHA1 protein has any one of the following amino acid sequences:

[0031] A1) The amino acid sequence shown in SEQ ID NO.1;

[0032] A2) An amino acid sequence of a protein with the same function obtained by substitution, insertion or deletion of one or more amino acids from the amino acid sequence shown in SEQ ID NO.1.

[0033] A method for increasing the yield of maize in a high-temperature environment, by increasing the expression level or activity of ZmAHA1 protein in maize, or increasing the expression level of ZmAha1 gene, to improve the heat tolerance of maize in a high-temperature environment; the ZmAHA1 protein has any one of the following amino acid sequences:

[0034] A1) The amino acid sequence shown in SEQ ID NO.1;

[0035] A2) An amino acid sequence of a protein with the same function obtained by substitution, insertion or deletion of one or more amino acids from the amino acid sequence shown in SEQ ID NO.1.

[0036] Preferably, increasing the expression level of the ZmAha1 gene in maize is achieved by introducing an overexpression vector containing the coding gene of the ZmAHA1 protein into the maize.

[0037] Preferably, it includes using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroconductivity or Agrobacterium-mediated method to introduce the overexpression vector containing the coding gene of the ZmAHA1 protein into maize to obtain transgenic maize lines.

[0038] The beneficial effects of the present invention are:

[0039] The present invention verifies that the maize ZmAha1 gene can positively regulate plant heat tolerance. By increasing the expression level of the ZmAha1 gene, the heat tolerance of plants can be effectively enhanced. The present invention constructs ZmAha1 overexpression transgenic maize plants. Compared with wild-type maize, the heat tolerance of these plants is significantly enhanced, providing new gene targets and resources for cultivating heat-tolerant plant varieties, having important significance for the research on the heat tolerance molecular mechanism of maize, and laying a certain theoretical foundation for studying the mechanism of plant response to high-temperature stress and the molecular mechanism of resisting adverse environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The drawings are used to provide further understanding of the present invention and constitute a part of the specification. Together with the embodiments of the present invention, they are used to explain the present invention and do not constitute a limitation to the present invention. In the drawings:

[0041] Figure 1 A shows the plant growth situation of the overexpression line OE#5100 after high-temperature treatment recovery in Example 2 of the present invention; wherein, WT represents wild-type maize plants, and OE#5100 represents the overexpression line.

[0042] Figure 1 B shows the detection of the expression level of the ZmAha1 gene in the overexpression line OE#5100 in Example 2 of the present invention, where WT represents wild-type maize plants and OE#5100 represents the overexpression line.

[0043] Figure 1 C is a statistical chart of the survival rate of the overexpression line OE#5100 in Example 2 of the present invention; where WT represents wild-type maize plants and OE#5100 represents the overexpression line.

[0044] Figure 1 D shows the induction of the ZmAah1 gene in wild-type under heat stress in Example 3 of the present invention. Detailed implementation manners

[0045] The following is a description of the preferred examples of the present invention with reference to the accompanying drawings. It should be understood that the following examples are given only for the purpose of illustration and are not intended 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.

[0046] The experimental methods used in the following examples are all conventional methods unless otherwise specified. If not specifically indicated, the examples are carried out under conventional experimental conditions, such as those described in the Molecular Cloning Laboratory 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.

[0047] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. 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, and KOD were purchased from biological companies such as NEB and Toyobo; dNTPs were purchased from Genestar; the plasmid miniprep kit and agarose gel recovery kit were purchased from Shanghai Jierui Bio-Engineering Co., Ltd.; antibiotics such as agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), and rifampicin (Rif), as well as Glucose, BSA, and LB Medium 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 BGI and subjected to relevant sequencing.

[0048] The gene ID of the ZmAha1 gene is GRMZM2G021816 (Activator of 90 kDa heat shock protein ATPase). The amino acid sequence of the ZmAHA1 protein is shown in SEQ ID No. 1 and consists of 348 amino acid residues. The nucleotide sequence of its CDS is shown in SEQ ID No. 2 and consists of 1047 bases.

[0049] Example 1 Construction and identification of maize ZmAha1 gene overexpression plants

[0050] The vector containing the ZmAha1 gene constructed by the maize functional genomics platform of China Agricultural University was transformed into the Agrobacterium EHA105 strain, specifically: (1) extracting maize total RNA, reverse transcribing to obtain cDNA, using the cDNA as a template, amplifying the CDS sequence of the ZmAha1 gene, and connecting the amplified product to the maize expression vector to obtain a recombinant expression vector; (2) using the recombinant expression vector obtained in step (1) to transform the Agrobacterium EHA105 strain to obtain a recombinant Agrobacterium; (3) using the recombinant Agrobacterium obtained in step (2) to infect maize callus tissue, screen positive transgenic plants, and obtain ZmAha1 gene overexpression seedlings.

[0051] The specific method is as follows: inoculate the Agrobacterium containing the target vector into 100 mL LB triple-antibody liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), culture at 28°C overnight with shaking, and wait until OD 600 The value was 1.0-2.0, centrifuged at 50×g for 15 minutes at room temperature, and the cells were collected; the cells were suspended in 2mL of transformation solution (1 / 2MS, 5% sucrose, 40μL Silwet L-77); the corn callus was immersed in the transformation solution of Agrobacterium and sealed. It was placed back on the light culture rack and grown normally until the plants grew out. Then the seeds of the ZmAha1 gene overexpression material obtained by screening were subjected to high temperature stress treatment experiments.

[0052] In this example, an overexpression strain OE#5100 was isolated and the gene expression of ZmAha1 in the obtained overexpression strain OE#5100 was detected by real-time quantitative PCR. The specific method is as follows:

[0053] (1) Extract total RNA from corn and obtain cDNA by reverse transcription.

[0054] (2) After the cDNA obtained by reverse transcription was diluted 3 times, real-time quantitative PCR was performed using the Takara kit. The reaction system used was: 2×SYBR Premix ExTaqbuffer, 0.2μL DyII, 0.4μL Primer (F / R), the primer sequences were as shown in SEQ ID No. 3 and SEQ ID No. 4, 2μL cDNA template, and finally ddH2O was used to fill it to 20μL. After thorough mixing, it was placed in the ABIPRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions were: 95℃30s; 95℃5s; 60℃40s; 40cycles. While amplifying the identified gene, each sample was amplified simultaneously using UBI as an internal reference. After the PCR reaction was completed, according to 2 -Δ(ΔCt) The relative expression levels between the wild type and overexpression strains were calculated and analyzed graphically. Figure 1As shown in Figure B, the results indicate that the expression of the ZmAha1 gene in OE#5100 was upregulated by approximately 2- to 3-fold (P value < 0.01).

[0055] Example 2 Detection of the High Temperature Tolerance of Maize Plants Overexpressing the ZmAha1 Gene

[0056] First, seeds of wild-type maize (control group WT) and the overexpression line OE#5100 were sown in small pots filled with black soil, imported soil, and vermiculite (1:1:1), with 12 seeds placed in each pot. Then, 2 cm of soil was covered and the pots were placed in a tray. The soil was watered until it was completely wet and then placed in an incubator at 23°C with a 16-h light and 8-h dark cycle. After growing for 12 days, the plants were subjected to a high temperature treatment at 45°C for 2-3 days until the second leaf shrank and wilted. Then, the plants were taken out and placed in an incubator at 23°C for one week to recover, and their survival rates were counted.

[0057] The phenotypes of the wild-type plants and the overexpression line OE#5100 after the high temperature treatment and recovery are shown in Figure 1 Figure A. Compared with the control group, the survival rate of the ZmAha1 gene overexpression line OE#5100 was significantly increased. As shown in Figure 1 Figure C, after performing a T-test analysis on the survival rates of wild-type plants and overexpressing plants, it was found that the survival rate of overexpressing plants was significantly higher than that of wild-type plants (P value < 0.01), indicating that the ZmAha1 gene overexpression line OE#5100 exhibited a heat-tolerant phenotype. In the above experiments, 12-16 seedlings were used in each experiment, and the experiments were independently repeated 3 times.

[0058] Example 3 Detection of the Induction of the ZmAha1 Gene under High Temperature

[0059] Wild-type maize plants grown for about 12 days were placed at 42°C for 1 h and 6 h, and then the roots were collected for RNA extraction. After reverse transcription into cDNA, qPCR was performed for detection. The specific detection method is described in Example 1.

[0060] The results are shown in Figure 1 Figure D. This gene can be induced by heat stress, and its expression level increased by approximately 7- to 8-fold after 6 h of heat treatment.

[0061] The above results indicate that overexpression of the ZmAha1 gene can improve the heat tolerance of maize.

[0062] The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0063] Finally, it should be noted that the above are only preferred examples of the present invention and are not used to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or perform equivalent replacements on some of the technical features. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of the ZmAHA1 protein, or its coding gene, or a biological material containing its coding gene, in any of the following aspects: A1) Use in improving the heat tolerance of maize; A2) Use in increasing the survival rate of maize under high-temperature environments; A3) Use in breeding maize with improved heat tolerance and / or increased yield; A4) Use in improving the heat-tolerant germplasm resources of maize.

2. The use according to claim 1, wherein the temperature of the high-temperature environment is 42 to 45 °C.

3. The application according to claim 1, wherein The ZmAHA1 protein has any of the following amino acid sequences: B1) The amino acid sequence shown in SEQ ID NO.1; B2) 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.

4. The application according to claim 1, wherein The coding sequence of the ZmAha1 gene has any of the following nucleotide sequences: C1) The nucleotide sequence shown in SEQ ID NO.2; C2) 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; C3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.2 under stringent conditions.

5. The application according to claim 1, characterized in that, The biological material is any one of the following D1) to D9): D1) A nucleic acid molecule encoding the protein described in claim 3; D2) An expression cassette containing the nucleic acid molecule described in D1); D3) A recombinant vector containing the nucleic acid molecule described in D1), or a recombinant vector containing the expression cassette described in D2); D4) A recombinant microorganism containing the nucleic acid molecule described in D1), or a recombinant microorganism containing the expression cassette described in D2), or a recombinant microorganism containing the recombinant vector described in D3); D5) A transgenic plant cell line containing the nucleic acid molecule described in D1), or a transgenic plant cell line containing the expression cassette described in D2), or a transgenic plant cell line containing the recombinant vector described in D3); D6) A transgenic plant tissue containing the nucleic acid molecule described in D1), or a transgenic plant tissue containing the expression cassette described in D2), or a transgenic plant tissue containing the recombinant vector described in D3); D7) A transgenic plant organ containing the nucleic acid molecule described in D1), or a transgenic plant organ containing the expression cassette described in D2), or a transgenic plant organ containing the recombinant vector described in D3).

6. The use according to claim 1, wherein it is achieved by increasing the expression level or activity of the ZmAHA1 protein, or increasing the expression level of the ZmAha1 gene.

7. The primer sequences for amplifying the ZmAha1 gene are shown in SEQ ID No.3 and SEQ ID No.

4.

8. A method for improving the heat tolerance of maize, wherein by increasing the expression level or activity of the ZmAHA1 protein in maize, or increasing the expression level of the ZmAha1 gene, the heat tolerance of maize under high-temperature environments is improved; the ZmAHA1 protein has any of the following amino acid sequences: A1) The amino acid sequence shown in SEQ ID NO.1; An amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids from the amino acid sequence shown in SEQ ID NO.1, which has the same functional protein.

9. A method for increasing the yield of maize in a high-temperature environment, characterized in that by increasing the expression level or activity of ZmAha1 protein in maize, or increasing the expression level of ZmAha1 gene, the heat tolerance of maize in a high-temperature environment is improved; the ZmAHA1 protein has any of the following amino acid sequences: A1) The amino acid sequence shown in SEQ ID NO.1; A2) An amino acid sequence obtained by substitution, insertion or deletion of one or more amino acids from the amino acid sequence shown in SEQ ID NO.1, which has the same functional protein.

10. The method according to claim 8 or 9, characterized in that increasing the expression level of the ZmAha1 gene in maize is achieved by introducing an overexpression vector containing the coding gene of the ZmAHA1 protein into the maize.

Images