Application of ZmbHLH103 protein and coding gene thereof in regulation and control of heat resistance of corn

Abstract

The invention relates to application of a ZmbHLH103 protein and a coding gene thereof in regulation and control of heat resistance of corn. It is found that the corn ZmbHLH103 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 ZmbHLH103 gene. The discovery of the heat-resistant function of the ZmbHLH103 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 to the application of ZmbHLH103 protein and its coding gene in regulating heat resistance of maize. More particularly, it refers to the application of ZmbHLH103 protein and its coding gene in regulating maize tolerance 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 rising temperatures will cause a significant decrease in crop yields: for every 1°C increase in the global average temperature, the staple 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 bHLH transcription factor is the second largest class of transcription factors in plants and plays an important role in the growth, development and stress response processes of plants. It is predicted that there are about 200 or more bHLH members in maize according to the sequence. Its bHLH domain contains about 50 - 60 amino acids and is composed of a basic amino acid region and a helix-loop-helix region, and the N-terminal basic domain binds to the DNA cis-element G-box (CACGTG). Although bHLH family members have been reported to be involved in plant responses to biotic or abiotic stresses, there are few reports on bHLH family members in maize responses to high temperature stress. Summary of the Invention

[0004] Aiming at the defects existing in the prior art, the purpose of the present invention is to provide the application of ZmbHLH103 protein and its coding gene in regulating heat resistance of maize. This application effectively improves the heat resistance of plants by increasing the expression level of the ZmbHLH103 gene and / or the activity of the ZmbHLH103 protein in the plants.

[0005] In the present invention, in a maize population with CRISPR mutations, using the relative leaf injury area as an index, a preliminary screening of high-temperature phenotypes was carried out. Mutant lines with high-temperature phenotypes obtained from the preliminary screening were re-screened to determine their high-temperature-related phenotypes, and the mutant genes of the lines with significant high-temperature phenotypes obtained from the screening were determined. Through the above screening, the target gene GRMZM2G301089 (bHLH-transcription factor 103) was found to be possibly related to maize tolerance to high-temperature stress, and GRMZM2G301089 was predicted to be a member of the bHLH transcription factor family. Through the CRISPR lines of the ZmbHLH103 gene, it was found that they had obvious high-temperature sensitive phenotypes. The present invention further proved through experiments that ZmbHLH103 positively regulates the heat resistance of plants, and the heat resistance of plants can be improved by increasing the expression level of the ZmbHLH103 gene in plants.

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

[0007] The application of the ZmbHLH103 protein, or its coding gene, in any of the following aspects:

[0008] (1) Regulating the heat resistance of plants;

[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 ZmbHLH103 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 coding gene of the ZmbHLH103 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 ZmbHLH103 protein in maize, consisting of 5136 bases, and the open reading frame of this gene consists of 7 exons. Considering the degeneracy of codons, all nucleotide sequences encoding the ZmbHLH103 protein are within the protection scope of the present invention.

[0021] On the basis of the above scheme,

[0022] The application enhances the heat resistance of the plant, enhances the survival rate of the plant under high temperature conditions, selects and breeds transgenic plants with improved heat resistance and / or increased yield, and improves the heat-resistant germplasm resources of plants by increasing the expression level of the gene encoding the ZmbHLH103 protein and / or the activity of the ZmbHLH103 protein in the plant.

[0023] Preferably, the increase in the expression level of the ZmbHLH103 protein in the plant is achieved by introducing an overexpression vector containing the gene encoding the ZmbHLH103 protein into the plant.

[0024] A suppressor for inhibiting the expression of the above-mentioned gene encoding the ZmbHLH103 protein, characterized in that: the suppressor is:

[0025] (1) The gRNA sequence shown in SEQ ID NO.3;

[0026] (2) An interfering RNA that inhibits the gene encoding the ZmbHLH103 protein.

[0027] By using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroconductivity or Agrobacterium-mediated method to introduce a suppressor containing the gene encoding the ZmbHLH103 protein into maize, a Zmbhlh103 transgenic maize mutant line is obtained, and its heat resistance is significantly reduced compared with wild-type maize.

[0028] Similarly, by using methods such as Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroconductivity or Agrobacterium-mediated method, an overexpression vector containing the gene encoding the ZmbHLH103 protein can be introduced into maize to obtain a transgenic maize line.

[0029] The primer pair for amplifying the above-mentioned ZmbHLH103 protein-coding gene is characterized in that:

[0030] The nucleotide sequences of the primer pair are shown in SEQ ID NO.4-5.

[0031] The specific amplification steps are as follows:

[0032] (1) Extract total RNA from maize, reverse transcribe to obtain cDNA, use the cDNA as a template to amplify the CDS sequence of the ZmbHLH103 gene, and ligate the amplification product to a plant expression vector to obtain a recombinant expression vector;

[0033] (2) Transform Agrobacterium with the recombinant expression vector obtained in step (1) to obtain recombinant Agrobacterium;

[0034] (3) Infect maize callus with the recombinant Agrobacterium obtained in step (2), screen positive transgenic plants, and obtain heat-resistant transgenic maize.

[0035] Compared with wild-type maize, the heat resistance of the ZmbHLH103 overexpression transgenic maize plants is significantly increased.

[0036] A biological material, characterized in that the biological material contains the coding gene of the ZmbHLH103 protein described in claim 1.

[0037] On the basis of the above scheme,

[0038] The biological material includes an expression cassette, a vector, a host cell or a recombinant bacterium.

[0039] The present invention provides a cloning vector or various expression vectors containing the coding gene of the ZmbHLH103 protein. The present invention also provides a host cell containing the vector, a transformed plant cell containing the coding gene of the ZmbHLH103 protein or a transgenic plant.

[0040] The application of the above biological material in any of the following aspects:

[0041] (1) Regulating plant heat resistance;

[0042] (2) Regulating the survival rate and / or growth of plants under high-temperature conditions;

[0043] (3) Breeding transgenic plants with improved heat resistance and / or yield;

[0044] (4) Improving plant heat-resistant germplasm resources.

[0045] In the present invention, the plant is a monocotyledonous plant. The monocotyledonous plant is a gramineous plant. More preferably, it is maize.

[0046] The application of the ZmbHLH103 protein and its coding gene according to the present invention in regulating heat resistance of maize has the beneficial effects as follows:

[0047] The present invention discovers that the maize ZmbHLH103 gene can positively regulate plant heat resistance. By overexpressing the expression level of the ZmbHLH103 gene, the heat resistance of plants can be effectively increased. The discovery of the heat resistance function of the ZmbHLH103 gene provides new gene targets and resources 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 studying the mechanism of plant response to high temperature stress and the molecular mechanism of resisting adverse environments. Brief Description of the Drawings

[0048] The present invention has the following drawings:

[0049] Figure 1 It shows the plant growth situation of the overexpression line BHLH103-MYC after high temperature treatment recovery in Example 2 of the present invention; wherein, WT represents wild-type maize plants, and BHLH103-MYC represents the overexpression line.

[0050] Figure 2 It shows the detection of the expression level of ZmbHLH103 protein in the overexpression line BHLH103-MYC in Example 2 of the present invention; wherein, WT represents wild-type maize plants, and BHLH103-MYC represents the overexpression line.

[0051] Figure 3 It is a statistical chart of the survival rate of the overexpression line BHLH103-MYC in Example 2 of the present invention; wherein, WT represents wild-type maize plants, and BHLH103-MYC represents the overexpression line.

[0052] Figure 4 It shows the plant growth situation of the mutant lines #12300 and #12500 after high temperature treatment recovery in Example 4 of the present invention; wherein, WT represents wild-type maize plants, and #12300 and #12500 represent the mutant lines.

[0053] Figure 5 It is a schematic diagram of the identification results of the CRISPR / Cas9 knockout mutants of the ZmbHLH103 gene in the mutant lines #12300 and #12500 in Example 4 of the present invention.

[0054] Figure 6 It is a statistical chart of the survival rate of the mutant lines #12300 and #12500 in Example 2 of the present invention; wherein, WT represents wild-type maize plants, and #12300 and #12500 represent the mutant lines. Detailed Embodiments

[0055] The preferred embodiments 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.

[0056] 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: A 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.

[0057] The content not described in detail in this specification belongs to the prior art well-known to those skilled in the art.

[0058] The materials, reagents, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The Agrobacterium tumefaciens EHA105 strain was kindly provided by the Crop Functional Genomics Platform of the College of Biology, 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; plasmid mini-prep kits and agarose gel recovery kits were purchased from Shanghai Jierui Biotechnology Co., Ltd.; agar powder, agarose, antibiotics such as 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 BGI and subjected to relevant sequencing.

[0059] Example 1 Construction and Identification of Maize Overexpressing ZmbHLH103 Gene

[0060] The vector containing the ZmbHLH103 gene constructed by the maize functional genomics platform of China Agricultural University was transformed into the Agrobacterium tumefaciens strain EHA105, and then the maize callus was infected to obtain transgenic seedlings. The specific method is as follows: The Agrobacterium tumefaciens 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 OD600 value reached 1.0 - 2.0, it was centrifuged at 50×g for 15 min at room temperature to collect the bacteria; the bacteria were suspended with 2 mL of transformation solution (1 / 2MS, 5% sucrose, 40 μL of Silwet L-77); the maize callus was soaked in the Agrobacterium tumefaciens 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 a high-temperature stress treatment experiment.

[0061] In this example, the overexpression line BHLH103-MYC was isolated, and the expression of the ZmbHLH103 gene in the obtained overexpression line BHLH103-MYC was detected by Western Blot method.

[0062] Example 2 Detection of the high-temperature resistance of maize overexpressing the ZmbHLH103 gene

[0063] First, the seeds of wild-type maize (control group WT) and the overexpression line BHLH103-MYC were sown in small pots filled with black soil, imported soil, and vermiculite (1:1:1), with 12 seeds in each pot. Then, 2 cm of soil was covered and placed in a tray, watered until the soil was completely wet, and 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 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.

[0064] After the high-temperature treatment and recovery, the phenotypes of the wild-type plants and the overexpression line BHLH103-MYC were as Figure 1 shown. Compared with the control, the survival rate of the overexpression line BHLH103-MYC was significantly increased, indicating that the overexpression line BHLH103-MYC exhibited a heat-resistant phenotype. In the above experiments, 12 - 16 seedlings were used in each experiment, and the experiments were independently repeated 3 times.

[0065] Example 3 Construction and identification of maize with the ZmbHLH103 gene knocked out

[0066] The CRISPR / Cas9 vector containing Zmbhlh103-gRNA constructed by the maize functional genomics platform of China Agricultural University was transformed into Agrobacterium tumefaciens strain EHA105. The gRNA sequence: GCCTCGTTCCAGGACTACA (SEQ ID NO.3), and then maize callus was infected to obtain transgenic seedlings. The specific method is 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 bacteria; the bacteria were suspended with 2 mL of transformation solution (1 / 2MS, 5% sucrose, 40 μL Silwet L-77); the maize callus was 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. Homozygous and stable knockout lines were obtained in the T0 generation, and then homozygous knockout lines without the CRISPR / Cas9 background were obtained by self-crossing the F2 generation. The obtained seeds were subjected to high-temperature stress treatment experiments.

[0067] In this example, mutant lines #12300 and #12500 were isolated. The DNA sequence differences of the ZmbHLH103 gene in the materials were detected by PCR. The PCR amplification method is as follows:

[0068] The primer sequences for amplifying the ZmbHLH103 gene are:

[0069] ZmbHLH103-CRISPR-F: 5’-TGCAGATGGACTCCTACAACT-3’ (SEQ ID NO.4)

[0070] Zm bHLH103-CRISPR-R: 5’-GTAGCTCTGGCTCAGCTTCT-3’ (SEQ ID NO.5)

[0071] The PCR reaction system is shown in Table 1 below:

[0072] Table 1

[0073]

[0074] The setting of the reaction program:

[0075] The PCR reaction program is shown in Table 2 below:

[0076] Table 2

[0077]

[0078] The size of the bands was detected by electrophoresis using 1% agarose gel at 220 V, and the bands were about 400 - 500 bp in size. Then they were sent to a sequencing company for sequencing.

[0079] Example 4 Detection of the high - temperature resistance of ZmbHLH103 gene - knockout maize

[0080] First, the seeds of wild - type maize (control group WT) and mutant lines #12300 and #12500 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. 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, they were subjected to a high - temperature treatment at 45°C until the second leaf shrank and wilted, and then taken out and placed in an incubator at 23°C to recover for one week. Then the survival rate was counted.

[0081] After the recovery from the high - temperature treatment, the phenotypes of the wild - type plants and mutant lines #12300 and #12500 were as Figure 4 shown. Compared with the control, the survival rates of mutant lines #12300 and #12500 were significantly reduced, indicating that the mutant lines showed a high - temperature - sensitive phenotype. In the above experiments, 12 - 16 seedlings were used each time, and the experiments were independently repeated 3 times.

[0082] 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 ZmbHLH103 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 ZmbHLH103 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 ZmbHLH103 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, wherein: 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 ZmbHLH103 protein coding gene and / or the activity of the ZmbHLH103 protein in the plant.

3. An inhibitor for suppressing the expression of the ZmbHLH103 protein-coding gene described in claim 1, characterized in that: The inhibitor is: The gRNA sequence shown in SEQ ID NO.

3.

4. A primer pair for amplifying the ZmbHLH103 protein coding gene described in claim 1, wherein: The nucleotide sequence of the primer pair is as shown in SEQ ID NO.4-5.

5. A biological material, characterized in that, The biological material contains the coding gene of the ZmbHLH103 protein described in claim 1.

6. The biomaterial according to claim 5, characterized in that, The biological material includes an expression cassette, a vector, a host cell or a recombinant bacterium.

7. Use of a biological material according to claim 5 or 6 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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