Application of ZmAAAP59 protein and its coding gene in regulating corn tolerance to high temperature stress

Abstract

The present application relates to the field of plant genetic engineering technology, in particular to the application of ZmAAAP59 protein and its coding gene in regulating corn tolerance to high temperature stress, by knocking out ZmAAAP59 gene in corn, it is determined that the mutant has obvious high temperature tolerance phenotype, it is verified that corn ZmAAAP59 gene can negatively regulate plant heat resistance, by reducing the expression amount of ZmAAAP59 gene in plants, the heat resistance of plants can be effectively increased.The discovery of the heat resistance function of ZmAAAP59 gene provides a new gene target and resource for breeding heat-resistant plant varieties, has important significance for the study of the heat tolerance molecular mechanism of corn, and lays a certain theoretical foundation for the study of the mechanism of plant response to high temperature stress and the molecular mechanism of resisting adverse environment.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the ZmAAAP59 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. Global warming increases 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 (Triticum aestivum): 6.0%; Rice (Oryza sativa): 3.2%; Soybean (Glycine max): 3.1%].

[0003] Corn requires many nutrients during its growth, among which nitrogen is crucial for its development. Corn primarily utilizes inorganic nitrogen (nitrates and ammonium) and organic nitrogen (amino acids, peptides, and proteins) from the soil or growing medium. Amino acids are key molecules in protein synthesis, energy metabolism, and the biosynthesis of neurotransmitters and peptide hormones. Generally, amino acid transport is mediated by proton-coupled transporters. In plants, amino acid transporters are classified into two main families based on their absorption characteristics and sequence similarity: the AAAP (amino acid / auxin permease) family and the APC (amino acid-polyamine organic cation) family.

[0004] In maize, 71 potential AAAP family members were identified using bioinformatics methods. These genes have high repetition rates and are unevenly distributed across the 10 maize chromosomes. They play an important role in the absorption and utilization of amino nitrogen under conditions of limited inorganic nitrogen supply or excessive amino nitrogen supply in the soil. AtAAP1, ZmAAP4, and ZmVAAT3 are amino acid transporters identified for the first time in Arabidopsis and maize, respectively. Although the important roles of many amino acid transporters have been extensively studied in Arabidopsis and rice, research on AAAP family amino acid transporters in maize has been limited to date, and the roles of these members in maize's resistance to abiotic stress remain unclear. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of the ZmAAAP59 protein and its encoding gene in regulating maize's tolerance to high-temperature stress.

[0006] This invention employs a preliminary screening of high-temperature phenotypes in a maize CRISPR knockout mutant library, using relative leaf injury area as an indicator. Lines exhibiting high-temperature sensitivity / tolerance phenotypes from the initial screening are then re-screened to determine their high-temperature-related phenotypes. The knockout gene for the selected high-temperature phenotype lines is then identified. Through this screening, the target gene GRMZM2G145989 (amino acid / auxin permease59) was identified as being associated with maize's tolerance to high-temperature stress, and GRMZM2G145989 was predicted to be an amino acid / auxin permease. Knocking out the ZmAAAP59 gene revealed that the knockout lines exhibited a significant high-temperature tolerance phenotype. This invention further demonstrates that the ZmAAAP59 gene negatively regulates plant heat resistance, and that reducing the expression level of the ZmAAAP59 gene in plants can improve plant heat tolerance.

[0007] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0008] The use of the ZmAAAP59 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects:

[0009] A1) Application in improving the heat resistance of corn;

[0010] A2) Application in improving the survival rate of corn under high temperature conditions;

[0011] A3) Application in the breeding of heat-resistant and / or yield-enhancing maize;

[0012] A4) Application in improving heat-resistant maize germplasm resources.

[0013] Furthermore, the temperature of the high-temperature environment is 45°C.

[0014] Furthermore, the ZmAAAP59 protein has any of the following amino acid sequences:

[0015] B1) The amino acid sequence as shown in SEQ ID NO.1;

[0016] B2) 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.

[0017] Furthermore, the gene coding sequence of the ZmAAAP59 protein has any of the following nucleotide sequences:

[0018] C1) The nucleotide sequence shown in SEQ ID NO.2;

[0019] C2) A nucleotide sequence encoding a protein with the same function obtained by substituting, deleting or inserting one or more nucleotides as shown in SEQ ID NO.2;

[0020] C3) A nucleotide sequence that can hybridize with a nucleotide sequence such as SEQ ID NO.2 under strict conditions.

[0021] Furthermore, the biomaterial is any one of the following D1) to D9):

[0022] D1) A nucleic acid molecule encoding the protein described in claim 3;

[0023] D2) An expression cassette containing the nucleic acid molecules described in D1);

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

[0025] D4) Recombinant microorganisms containing the nucleic acid molecules described in D1), or recombinant microorganisms containing the expression cassette described in D2), or recombinant microorganisms containing the recombinant vector described in D3);

[0026] 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);

[0027] D6) Transgenic plant tissue containing the nucleic acid molecules described in D1), or transgenic plant tissue containing the expression cassette described in D2), or transgenic plant tissue containing the recombinant vector described in D3);

[0028] 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).

[0029] Furthermore, this can be achieved by inhibiting or reducing the expression level or activity of the ZmAAP59 protein, or by silencing or reducing the expression level of the ZmAAP59 gene.

[0030] The primer sequences for amplifying the ZmAAP59 gene are shown in SEQ ID No. 4 and SEQ ID No. 5.

[0031] A method for improving the heat resistance of maize involves inhibiting or reducing the expression level or activity of the ZmAAP59 protein in maize to obtain mutant plants, thereby improving their heat resistance under high-temperature environments; wherein the ZmAAP59 protein has any of the following amino acid sequences:

[0032] A1) The amino acid sequence as shown in SEQ ID NO.1;

[0033] A2) An amino acid sequence of a protein with the same function obtained by substituting, inserting or deleting one or more amino acids from the amino acid sequence shown in SEQ ID NO.1.

[0034] Furthermore, the gene encoding the ZmAAP59 protein in maize was edited using CRISPR / Cas9 technology to inhibit the expression or activity of the ZmAAP59 protein; the gRNA target sequence is shown in SEQ ID NO.3.

[0035] The method for constructing heat-resistant transgenic maize of the present invention is achieved through transgenic, hybridization, backcrossing, self-pollination or asexual reproduction.

[0036] Furthermore, the transgenic process involves introducing a repressor gene containing the ZmAAAP59 protein into maize using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electrical conductivity, or Agrobacterium-mediated transformation to obtain transgenic maize lines.

[0037] The inhibitory factors include gRNAs or interfering RNAs capable of inhibiting ZmAAAP59 expression.

[0038] A ZmAAP59 gene mutant material, wherein the mutant material has the following nucleotide sequence of the ZmAAP59 gene: the sequence shown in SEQ ID NO.2 has 17 bases deleted from position 323 to position 339.

[0039] The beneficial effects of this invention are:

[0040] This invention verifies that the maize ZmAAAP59 gene can negatively regulate plant heat tolerance, and that mutating the ZmAAAP59 gene can effectively enhance plant heat resistance. This invention constructed a CRISPR knockout plant of the ZmAAAP59 gene, which showed significantly enhanced heat resistance compared to wild-type maize. The discovery of the heat-resistant function of the ZmAAAP59 gene provides a new gene target and resource for breeding heat-resistant plant varieties, and is of great significance for the study of the molecular mechanism of heat tolerance in maize. It lays a theoretical foundation for studying the mechanisms by which plants respond to high-temperature stress and resist adverse environments. Attached Figure Description

[0041] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used in conjunction with embodiments of the invention to explain the invention and do not constitute a limitation thereof. In the drawings:

[0042] Figure 1A represents the plant growth of the mutant line CR#39900 in Example 3 of this invention after recovery from high-temperature treatment; where WT represents wild-type maize plants and CR#39900 represents mutant lines.

[0043] Figure 1 B is a schematic diagram showing the identification results of the CRISPR / Cas9 knockout mutant of the ZmAAAP59 gene in the mutant line CR#39900 in Example 2 of this invention.

[0044] Figure 1 C is a statistical chart showing the survival rate of the mutant line CR#39900 in Example 3 of this invention; where WT represents wild-type maize plants and CR#39900 represents mutant lines. Detailed Implementation

[0045] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the following embodiments are given for illustrative purposes only 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 its spirit and essence.

[0046] 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 Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 21), or as recommended by the manufacturer's instructions.

[0047] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Agrobacterium EHA105 strain was kindly 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 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 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.

[0048] The gene number of ZmAAAP59 is GRMZM2G145989 (amino acid / auxin permease59). The amino acid sequence of the ZmAAAP59 protein is shown in SEQ ID No.1, consisting of 471 amino acid residues; the CDS nucleotide sequence of its encoding gene is shown in SEQ ID No.2, consisting of 1416 bases.

[0049] Example 1: Construction and identification of maize ZmAAAP59 gene knockout mutant plants

[0050] The CRISPR / Cas9 vector containing ZmAAAP59-gRNA, constructed from the maize functional genomics platform of China Agricultural University, was transformed into Agrobacterium EHA105 strain. The gRNA sequence is: TCAAGAGCAACCTGGGCGG (SEQ ID No. 3). The vector was then used to infect maize callus tissue to obtain transgenic seedlings.

[0051] The specific method is as follows: Agrobacterium containing the target vector is 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 is 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 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 callus was then placed back on a light-controlled culture rack and allowed to grow normally until plants emerged. A homozygous stable knockout line was obtained in the T0 generation. Then, through self-pollination in the F2 generation, a homozygous knockout line ZmAAAP59-CR#39900 with CRISPR / Cas9 background removed was obtained. The resulting seeds were then subjected to high-temperature stress treatment experiments.

[0052] In this embodiment, the mutant line CR#39900 was isolated. PCR was used to detect the DNA sequence differences of the ZmAAAP59 gene in the mutant material. The PCR amplification method is as follows:

[0053] The primer sequences for amplifying the ZmAAAP59 gene are as follows:

[0054] ZmAAAP59-CRISPR-F: 5'-CTGTCGCTGGCGTGGGCGAT-3' (SEQ ID No. 4)

[0055] ZmAAAP59-CRISPR-R: 5'-GCCGTGCCAGTGGAAGCAGTT-3' (SEQ IDNo.5)

[0056] The PCR reaction system is shown in Table 1 below:

[0057] Table 1

[0058]

[0059] Reaction procedure setup:

[0060] The PCR reaction procedure is shown in Table 2 below:

[0061] Table 2

[0062]

[0063] The band size was detected by electrophoresis at 220V using a 1% agarose gel, and the sample was sent to a sequencing company for sequencing.

[0064] like Figure 1As shown in B, the mutant CR#39900, compared with wild-type maize B73-329, has a deletion of 17 bases CCGTCAAGAGCAACCTG in the coding sequence of the ZmAAAP59 gene in the maize genome on both homologous chromosomes. This results in a premature stop codon in the ZmAAAP59 coding sequence, thus knocking out the ZmAAAP59 gene.

[0065] Example 2: Detection of high-temperature resistance in ZmAAAP59 gene knockout maize

[0066] First, seeds of wild-type maize (control group WT) and the gene knockout line CR#39900 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 2cm 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 maize was treated at 45℃ for 2-3 days until the second leaf wrinkled and wilted. Afterward, the maize was removed and placed back in a 23℃ incubator for a week to recover before the survival rate was recorded.

[0067] Phenotypic characteristics of wild-type plants and gene knockout line CR#39900 after high-temperature treatment recovery are as follows: Figure 1 As shown in Figure A, compared to the control group, the gene knockout line CR#39900 exhibited a heat-resistant phenotype. Figure 1 C represents the statistical test results of the survival rate of the mutant line CR#39900 and the control group. The survival rate of the ZmAAAP59 gene knockout line CR#39900 was significantly increased. In the above experiments, 12-16 seedlings were used in each experiment, and the experiments were independently repeated 3 times.

[0068] The above results indicate that knocking out the ZmAAAP59 gene can improve the heat resistance of maize.

[0069] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0070] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended 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 make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Knockout ZmAAAP59 The application of genes in any of the following aspects is characterized by, The applications include: A1) Application in improving the heat resistance of corn; A2) Application in improving the survival rate of corn under high temperature conditions; A3) Application in improving heat-resistant maize germplasm resources; The application involves knocking out corn. ZmAAP59 Genetic realization; The ZmAAP59 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1; The temperature of the high-temperature environment is 45°C.

2. The application according to claim 1, characterized in that, The ZmAAAP59 The coding sequence of the gene is shown in SEQ ID NO.

2.

3. A method for improving the heat resistance of corn, characterized in that, By knocking out corn ZmAAP59 Genes were used to obtain mutant plants, which improved their heat resistance in high-temperature environments; The ZmAAP59 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.1; The temperature of the high-temperature environment is 45°C.

4. The method according to claim 3, characterized in that, The gene encoding the ZmAAP59 protein in maize was knocked out using CRISPR / Cas9 technology; the gRNA target sequence is shown in SEQ ID NO.3.

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