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

Abstract

The invention relates to the technical field of plant genetic engineering, in particular to application of ZmAAAP59 protein and a coding gene thereof in regulation and control of high temperature stress tolerance of corn, the ZmAAAP59 gene is knocked out in corn, it is determined that a mutant of the ZmAAAP59 gene has an obvious high temperature resistance phenotype, it is verified that the corn ZmAAAP59 gene can negatively regulate and control plant heat resistance, and by reducing the expression quantity of the ZmAAAP59 gene in the plant, the corn ZmAAAP59 gene can negatively regulate and control plant heat resistance. The heat resistance of plants can be effectively improved. The discovery of the heat-resistant function of the ZmAAAP59 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 ZmAAAP59 protein and its coding gene in regulating the high-temperature stress tolerance of maize. Background Art

[0002] Maize (Zea mays) belongs to the genus Zea in the family Poaceae, 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 high-temperature stress. There are literature reports that rising temperatures will cause a significant decline 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%].

[0003] Maize requires many nutrient elements during its growth process, and among these, nitrogen is crucial for the growth and development of maize. Maize mainly utilizes inorganic nitrogen (nitrate and ammonium) and organic nitrogen (amino acids, peptides, and proteins) in the soil or medium, and among these, amino acids are key molecules for 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 divided into two main families according to their absorption characteristics and sequence similarities, namely the AAAP (amino acid / auxin permease) and APC (amino acid-polyamine organic cation) families.

[0004] In maize, 71 possible members of the AAAP family have been identified by bioinformatics methods. These genes have a high repetition rate and are unevenly distributed on 10 maize chromosomes. They play an important role in the absorption and utilization of amino nitrogen when the supply of soil inorganic nitrogen is limited or the supply of soil amino nitrogen is excessive. AtAAP1, ZmAAP4, and ZmVAAT3 are the first identified amino acid transporters in Arabidopsis and maize, respectively. Although the important roles of many amino acid transporters have been deeply studied in Arabidopsis and rice, so far, the research on amino acid transporters of the AAAP family in maize has been relatively less, and the role of these members in maize's resistance to stress is still unclear. Summary of the Invention

[0005] Aiming at the deficiencies in the prior art, the purpose of the present invention is to provide the application of ZmAAAP59 protein and its coding gene in regulating the high-temperature stress tolerance of maize.

[0006] In the present invention, in a maize CRISPR knockout mutant library, with the relative leaf injury area as an index, a preliminary screening of high-temperature phenotypes was carried out. Lines with high-temperature sensitive / tolerant phenotypes obtained from the preliminary screening were re-screened to determine their high-temperature related phenotypes, and the knockout genes of the lines with high-temperature phenotypes obtained from the screening were determined. Through the above screening, it was targeted that the target gene GRMZM2G145989 (amino acid / auxin permease 59) is related to maize tolerance to high-temperature stress, and it was predicted that GRMZM2G145989 is an amino acid / auxin permease. By knocking out the ZmAAAP59 gene, it was found that its knockout lines had obvious high-temperature tolerance phenotypes. The present invention further proves that the ZmAAAP59 gene negatively regulates the heat resistance of plants, and the heat resistance of plants can be improved by reducing the expression level of the ZmAAAP59 gene in plants.

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

[0008] Use of the ZmAAAP59 protein or its coding gene or biological material containing its coding gene in any of the following aspects:

[0009] A1) Use in improving the heat resistance of maize;

[0010] A2) Use in improving the survival rate of maize under high-temperature environments;

[0011] A3) Use in breeding maize with improved heat resistance and / or increased yield;

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

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

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

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

[0016] 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.

[0017] Further, 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 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;

[0020] C3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO.2 under stringent conditions.

[0021] Furthermore, the biological material 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 molecule 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) 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);

[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) 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);

[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, it is achieved by inhibiting or reducing the expression level or activity of the ZmAAP59 protein, or silencing the expression 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 tolerance of maize, by inhibiting or reducing the expression level or activity of the ZmAAP59 protein in maize to obtain mutant plants, thereby improving the heat tolerance in high-temperature environments; the ZmAAAP59 protein has any one of the following amino acid sequences:

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

[0033] A2) 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.

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

[0035] The method for constructing the heat-resistant transgenic maize of the present invention is achieved by methods such as transgenic, hybridization, backcross, self-cross or asexual reproduction.

[0036] Furthermore, the transgenic includes introducing an inhibitor containing the gene encoding the ZmAAAP59 protein into maize by using a Ti plasmid, a plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation or Agrobacterium-mediated method to obtain a transgenic maize line.

[0037] The inhibitor includes gRNA or interfering RNA that can inhibit the expression of ZmAAAP59.

[0038] A ZmAAP59 gene mutant material, wherein the nucleotide sequence of the ZmAAP59 gene of the mutant material is that 17 bases are deleted from positions 323 to 339 of the sequence shown in SEQ ID NO.2.

[0039] The beneficial effects of the present invention are:

[0040] The present invention verifies that the maize ZmAAAP59 gene can negatively regulate plant heat tolerance. By mutating the ZmAAAP59 gene, the heat resistance of plants can be effectively enhanced. The present invention constructs a CRISPR knockout plant of the ZmAAAP59 gene. Compared with wild-type maize, the heat resistance of this plant is significantly enhanced. The discovery of the heat-resistant function of the ZmAAAP59 gene provides a new gene target and resource for cultivating heat-resistant plant varieties, which is of great significance for the study of the heat-resistant molecular mechanism of 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The drawings are used to provide a 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:

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

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

[0044] Figure 1 C is a statistical chart of the survival rate of mutant line CR#39900 in Example 3 of the present invention; wherein, WT represents wild-type maize plants, and CR#39900 represents the mutant line. 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 illustrative purposes 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] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods. Unless otherwise specified, 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] Materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The Agrobacterium tumefaciens strain EHA105 was kindly provided by the Crop Functional Genomics Platform of the College of Biology, China Agricultural University and can be purchased commercially (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; plasmid mini-prep kits and agarose gel recovery kits were purchased from Shanghai Jierui Biotechnology Co., Ltd.; 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; reagents used for real-time quantitative PCR were purchased from TaKaRa; various other chemical reagents used in the following examples were all imported or domestic analytical pure reagents. Primers used in the following examples were synthesized by Liuhe BGI and subjected to relevant sequencing.

[0048] The gene ID of the ZmAAAP59 gene is GRMZM2G145989 (amino acid / auxin permease 59). The amino acid sequence of the ZmAAAP59 protein is shown in SEQ ID No. 1 and consists of 471 amino acid residues; the CDS nucleotide sequence of its encoding gene is shown in SEQ ID No. 2 and consists of 1416 bases.

[0049] Example 1 Construction and Identification of Mutant Plants with Knockout of the Maize ZmAAAP59 Gene

[0050] The CRISPR / Cas9 vector containing ZmAAAP59-gRNA constructed by the Maize Functional Genomics Platform of China Agricultural University was transformed into the Agrobacterium tumefaciens strain EHA105. The gRNA sequence: TCAAGAGCAACCTGGGCGG (SEQ ID No. 3), and then used to infect maize callus to obtain transgenic seedlings.

[0051] The specific method is as follows: Inoculate the Agrobacterium containing the target vector into 100 mL of LB triple-antibiotic liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), and culture it overnight with shaking at 28°C. Wait until the OD 600 value is 1.0 - 2.0, centrifuge at 50×g for 15 min at room temperature to collect the bacterial cells; suspend the bacterial cells with 2 mL of transformation solution (1 / 2MS, 5% sucrose, 40 μL of Silwet L-77); soak the maize callus in the Agrobacterium transformation solution and seal it. Place it back on the light culture rack and grow normally until plants grow out. Obtain a homozygous and stable knockout line in the T0 generation, and then obtain a homozygous knockout line ZmAAAP59-CR#39900 without the CRISPR / Cas9 background through self-crossing in the F2 generation. Conduct a high-temperature stress treatment experiment on the obtained seeds.

[0052] In this example, the mutant line CR#39900 was isolated, and the DNA sequence differences of the ZmAAAP59 gene in the mutant material were detected by PCR. The PCR amplification method is as follows:

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

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

[0055] ZmAAAP59-CRISPR-R: 5’-GCCGTGCCAGTGGAAGCAGTT-3’ (SEQ ID No.5)

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

[0057] Table 1

[0058]

[0059] Setting of the reaction program:

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

[0061] Table 2

[0062]

[0063] Use 1% agarose gel electrophoresis at 220 V voltage to detect that the band size is about 300 bp, and send it to a sequencing company for sequencing.

[0064] As Figure 1As shown in Figure B, in mutant CR#39900, compared with wild-type maize B73-329, in two homologous chromosomes, 17 bases CCGTCAAGAGCAACCTG were deleted at positions 323 to 339 of the coding sequence of the ZmAAAP59 gene in the maize genome, resulting in a premature termination codon in the ZmAAAP59 coding sequence, thus knocking out the ZmAAAP59 gene.

[0065] Example 2 Detection of High Temperature Resistance of Maize with ZmAAAP59 Gene Knocked Out

[0066] First, seeds of wild-type maize (control group WT) and gene knockout line CR#39900 were sown in small pots filled with black soil, imported soil and vermiculite (1:1:1). 12 seeds were placed in each pot, and then covered with 2 cm of soil and placed in a tray. Water was added until the soil was completely wet, and then placed in an incubator at 23 °C with 16 h of light and 8 h of darkness. After growing for 12 days, they were subjected to a high temperature treatment at 45 °C for 2 - 3 days until the second leaf shrank and wilted, and then taken out and placed in an incubator at 23 °C to recover for one week, and then the survival rate was counted.

[0067] The phenotypes of wild-type plants and gene knockout line CR#39900 after recovery from high temperature treatment are as Figure 1 shown in Figure A. Compared with the control group, gene knockout line CR#39900 showed a heat-tolerant phenotype. Figure 1 Figure C shows the statistical test results of the survival rates of mutant line CR#39900 and the control group. The survival rate of the ZmAAAP59 gene knockout line CR#39900 increased significantly. In the above experiments, 12 - 16 seedlings were used each time, 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 content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0070] 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 for 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 ZmAAAP59 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, characterized in that the temperature of the high-temperature environment is 45 °C.

3. The application according to claim 1, characterized in that The ZmAAAP59 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 gene coding sequence of the ZmAAAP59 protein 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, characterized in that it is achieved by inhibiting or reducing the expression level or activity of the ZmAAP59 protein, or by silencing the expression or reducing the expression level of the ZmAAP59 gene.

7. The primer sequences for amplifying the ZmAAP59 gene are shown in SEQ ID No.4 and SEQ ID No.

5.

8. A method for improving the heat tolerance of maize, characterized in that by inhibiting or reducing the expression level or activity of the ZmAAP59 protein in maize, a mutant plant is obtained, and the heat tolerance under high-temperature environments is improved; the ZmAAAP59 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 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.

9. The method according to claim 8, wherein the coding gene of the ZmAAP59 protein is edited in maize by the CRISPR / Cas9 technology to inhibit the expression level or activity of the ZmAAP59 protein; the gRNA target sequence is shown in SEQ ID NO.

3.

10. A ZmAAP59 gene mutant material, wherein the mutant material is: the nucleotide sequence of its ZmAAP59 gene has a 17-base deletion from position 323 to position 339 of the sequence shown in SEQ ID NO.2.

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