Application of PBS1 protein and coding gene thereof in regulation and control of heat resistance of plants

Abstract

The invention relates to the technical field of genetic engineering, in particular to application of PBS1 protein and a coding gene thereof in regulation and control of plant heat resistance. The application comprises the steps of regulating and controlling the heat resistance of plants, cultivating heat-resistant plants and improving planting resources of the plants. According to the PBS1 protein related to plant heat resistance and the coding gene thereof, gene editing is carried out on plants based on the PBS1 protein, the heat resistance of the plants can be effectively improved, and the PBS1 protein has important value in the field of plant heat resistance improvement.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, and in particular to application of a PBS1 protein and a gene encoding the same in regulating plant heat resistance. Background Art

[0002] In plant breeding, stress tolerance is a crucial factor in plant growth and development. For example, the yield of several major crops (wheat, rice, corn, and soybeans) is often closely linked to their stress tolerance. In particular, studies have shown that with rising global temperatures, the yields of various crops have declined to varying degrees. Therefore, existing crop cultivation has placed higher demands on plant heat tolerance, and improving crop heat tolerance and adaptability is urgent. Breeding high-temperature-tolerant food crop varieties has important scientific significance and production value.

[0003] Arabidopsis thaliana PBS1 The gene is a gene encoding Arabidopsis cytoplasmic receptor kinase. Studies have found that this gene is mainly related to the immune response of plants. No existing technology has found that Arabidopsis PBS1 Associations between genes and heat tolerance in plants. Summary of the Invention

[0004] In order to solve the problems existing in the prior art, the present invention provides a PBS1 protein and an encoding gene thereof for use in regulating plant heat tolerance.

[0005] The present invention is directed to the gene of Arabidopsis thaliana cytoplasmic receptor kinase PBS1 PBS1 A study on AvrPphB susceptible protein 1 (AvrPphB susceptible protein 1) was conducted and it was found that transgenic plants overexpressing this gene showed obvious basal heat tolerance and acquired heat tolerance phenotypes.

[0006] In a first aspect, the present invention provides the use of PBS1 protein, or a gene encoding the protein, or a biological material comprising the gene encoding the protein, in regulating the heat tolerance of plants.

[0007] The present invention further provides the use of PBS1 protein, or a gene encoding the protein, or a biological material containing the gene encoding the protein, in cultivating heat-resistant plants.

[0008] The present invention further provides the use of PBS1 protein, or its encoding gene, or biological materials containing its encoding gene in improving plant germplasm resources.

[0009] Furthermore, by increasing the expression level of the PBS1 protein in the plant, the heat tolerance of the plant is improved.

[0010] Furthermore, the PBS1 protein comprises any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO. 1; (2) The amino acid sequence of a protein with the same function as the amino acid sequence shown in SEQ ID NO. 1 obtained by replacing, inserting or deleting one or more amino acids.

[0011] Furthermore, the gene encoding the PBS1 protein includes any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO. 2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO. 2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

[0012] PBS1 The gene originates from Arabidopsis thaliana of the Columbia ecotype and is accessioned as At5g13160 in the Arabidopsis thaliana genome database. The PBS1 gene has a nucleotide sequence shown in SEQ ID No. 3, consisting of 2,829 bases. The gene contains four introns, and the nucleotide sequence of its CDS region is shown in SEQ ID No. 2, consisting of 1,371 bases.

[0013] Furthermore, the plant is a monocotyledonous plant or a dicotyledonous plant, preferably Arabidopsis thaliana.

[0014] Furthermore, the biological material is an expression cassette, a vector or a transgenic cell.

[0015] The transgenic cells described in the present invention do not include transgenic cells that have the ability to independently develop into complete individuals, that is, they do not include plant varieties.

[0016] In a second aspect, the present invention provides a method for regulating heat tolerance of a transgenic gene, comprising: The expression level of PBS1 protein in the plant is regulated.

[0017] The PBS1 protein includes any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO. 1; (2) The amino acid sequence of a protein with the same function as the amino acid sequence shown in SEQ ID NO. 1 obtained by replacing, inserting or deleting one or more amino acids.

[0018] Furthermore, the expression level of PBS1 protein in plants is regulated by gene editing; or heat-resistant strains are cultivated by hybridizing plants overexpressing the PBS1 protein with wild-type plants.

[0019] Furthermore, the gene editing method includes: one or more of the CRISPR / Cas9 system, gene gun, Agrobacterium-mediated or viral vector system.

[0020] After overexpressing the gene encoding the PBS1 protein of the present invention, the plant exhibits a heat-resistant phenotype. To facilitate identification and screening of transgenic plant cells or plants, the vector used can be processed, such as by adding a plant selectable marker or an antibiotic resistance marker.

[0021] In a third aspect, the present invention provides a primer pair for amplifying a gene encoding PBS1 protein.

[0022] The distance between the upstream primer and the downstream primer is between 1 and 1371 bases; the length of each primer in the primer pair is 20 bases, and the sequence is as follows: Upstream primer: 5'-ATGGGTTGTTTCTCGTGTTT -3' (nucleotide sequence shown in SEQ ID No. 4), Downstream primer: 5'-CTACCCGGTACTGTTGCTCT-3' (nucleotide sequence shown in SEQ ID No. 5).

[0023] The present invention has the following beneficial effects: This study discovered the relationship between the PBS1 protein and plant heat-tolerant phenotypes. Experimental results confirm that overexpressing the PBS1 protein gene in plants can effectively improve their heat tolerance. The application of the PBS1 protein provided by this invention provides a theoretical basis for breeding heat-tolerant food crop varieties and provides genetic resources for breeding new heat-tolerant plant varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the present invention or the prior art, a brief introduction will be given below to the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0025] Figure 1 This is a diagram of PBS1 protein detection in the overexpression strain PBS1-GFP provided in Example 1 of the present invention; wherein #4 and #6 represent overexpression strains.

[0026] Figure 2This is a photograph of the plant recovery after testing the basic heat resistance and acquired heat resistance of the overexpression strain PBS1-GFP provided in Example 2 of the present invention; wherein Col is the wild type, and PBS1-GFP / Col#4 and PBS1-GFP / Col#6 represent overexpression strains.

[0027] Figure 3 This is a statistical diagram of plant survival rates after testing the basic heat resistance and acquired heat resistance of the overexpression strain PBS1-GFP provided in Example 2 of the present invention; wherein PBS1-GFP#4 and PBS1-GFP#6 represent overexpression strains. DETAILED DESCRIPTION

[0028] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods involved in the following examples are all conventional methods in the art. For example, reference can be made to experimental manuals in the art, or the conditions recommended by the manufacturer's instructions.

[0030] Unless otherwise specified, the experimental materials and reagents involved in the following examples can be obtained from commercial sources.

[0031] In the following examples, the pSuper-1300 vector was obtained from the laboratory; pCAMBIA1300 is a commonly used cloning vector and is commercially available; the Arabidopsis thaliana variety is the Columbia ecotype; and the Agrobacterium tumefaciens GV3101 strain is a commonly used cloning vector and is available in most molecular biology laboratories.

[0032] The main reagents used in the following examples are as follows: various restriction endonucleases, Taq enzyme, KOD, etc. were purchased from biological companies such as TAKARA (Dalian), NEB, and Toyobo; homologous recombination enzymes were purchased from Novozymes; dNTPs were purchased from Genestar; plasmid miniprep kit and agarose gel recovery kit were purchased from Shanghai Jierui Bioengineering Co., Ltd.; MS medium, agar powder, agarose, antibiotics such as ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampicin (Rif), and LB medium were purchased from Sigma; nitrocellulose membranes were purchased from Bio-Rad and other companies; various other chemical reagents used in the examples were imported or domestically produced analytical grade reagents.

[0033] The primers used in the examples were synthesized by Liuhe BGI and the related sequencing was performed.

[0034] Example 1 Construction and screening of PBS1 gene overexpressing plants of the present invention 1. In order to fully understand the effect of cytoplasmic receptor kinase on plant stress resistance, the present invention is to use Arabidopsis thaliana ( Arabidopsis thaliana A gene encoding a plant serine / threonine protein kinase was cloned from L. PBS1 According to the sequence analysis of the coding region, the present invention designed primers to amplify the coding region of the gene and connect it to the overexpression vector with pSuper promoter. pSuper-1300 The primers used were: Upstream primer: 5'-ATGGGTTGTTTCTCGTGTTT-3' (nucleotide sequence shown in SEQ ID No. 4), Downstream primer: 5'-CCCGGTACTGTTGCTCTCTG-3' (nucleotide sequence shown in SEQ ID No. 6).

[0035] 2. The present invention will PBS1 The gene was then linked to a vector with a Super promoter pSuper-1300-GFP The specific method is as follows: the first step is to use Arabidopsis cDNA as a template (total RNA is extracted from plants and cDNA is obtained by reverse transcription), and to use upstream primers and downstream primers to PBS1 The gene was amplified (the total length of the PCR product was 1368 bp, excluding the stop codon); the second step was to use Kpn I endonuclease, enzyme digestion pSuper-1300-GFP The vector is linearized; in the third step, the PCR product is ligated with the homologous recombination enzyme pSuper-1300-GFP The vectors are connected and the ligation product is named PBS1-pSuper-1300-GFP .

[0036] The plasmid obtained in the previous step was digested and tested. Kpn I and will PBS1-pSuper- 1300-GFP After enzyme digestion, run the plasmid on a 1% agarose gel at 120V and 50mA using a UVP Gel Documentation system. After successful enzyme digestion, sequence the plasmid and proceed to the next step.

[0037] 3. The correctly constructed PBS1-pSuper-1300-GFPThe vector was transformed into Agrobacterium tumefaciens GV3101 strain by electroporation, and then the positive monoclonal bacteria containing the above vector were transformed into Arabidopsis wild-type plants to obtain transgenic Arabidopsis seedlings. The specific method is as follows: Agrobacterium containing the above vector was inoculated into 100 mL LB triple-antibody liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), and cultured with shaking at 28°C overnight to an OD of 0. 600 to 1.0-2.0; centrifuge at 4000 rpm for 15 min at room temperature to collect the cells; suspend the cells in 100 mL of transformation solution (1 / 2MS, 5% sucrose, 40 μL Silwet L-77) and adjust the cell concentration to OD 600 to 1.0; soak the Arabidopsis inflorescence in the Agrobacterium transformation solution for about 90 seconds, then take it out, cover it with a black garbage bag to keep it moist, and place the plant in a dark place away from light overnight. After 12 hours, take out the plant and place it on a light culture rack to grow normally until harvest.

[0038] 4. The above pSuper-1300-GFP The vector carries the hygromycin resistance gene, and transgenic Arabidopsis seedlings are screened for hygromycin resistance to obtain homozygous transgenic plants. The specific method is as follows: Hygromycin-resistant positive seedlings from the T1 generation obtained above are harvested individually to obtain the T2 generation; these T2 seeds are tested for hygromycin resistance, and strains are selected where 3 / 4 are resistant and 1 / 4 are not, indicating that the overexpression vector containing the target gene has been inserted as a single copy in these strains; hygromycin-resistant plants from these strains are removed and harvested individually to obtain the T3 generation; these T3 seeds are screened for hygromycin resistance, and homozygous transgenic strains that do not segregate are selected. These homozygous plants can be used for seed propagation and low-temperature stress treatment experiments.

[0039] In this experiment, the overexpression homozygous strain PBS1-GFP was obtained through transformation and screening.

[0040] 5. Western blotting was used to detect PBS1-GFP protein expression in the resulting overexpression lines. The specific method was as follows: total protein was extracted from wild-type (WT) and PBS1-GFP transgenic plants; the protein concentration of the extracted samples was determined and adjusted to a consistent total protein amount (30 μg); and PBS1 protein content in the overexpression plants was detected using a protein immunoblotting technique. Anti-GFP antibodies were used to detect PBS1 protein, and anti-actin antibodies were used to detect the internal control protein, actin.

[0041] Test results such as Figure 1 As shown, it can be seen that PBS1 protein can be detected in the overexpression line.

[0042] The method of overexpressing PBS1 protein in other plants can refer to this example.

[0043] Example 2 Detection of high temperature resistance of plants overexpressing PBS1 protein In the present invention, wild-type Arabidopsis seedlings and the PBS1 protein overexpressing strain obtained in Example 1 were grown on MS solid medium, totaling 18 materials. After the seedlings grew for 9 days, the seedlings were divided into two groups, with 9 materials in each group.

[0044] In the first group, seedlings with basic heat tolerance were subjected to high temperature treatment in a dark water bath at 40°C and were taken out after 25 minutes of treatment.

[0045] In the second group, the seedlings were first treated in a 37°C dark-proof water bath for 30 min, then placed in a 22°C incubator to recover for 2 h, and then subjected to high temperature treatment again in a 40°C dark-proof water bath for 100 min.

[0046] The cells were then cultured under normal light conditions for 7 days. During this period, tissues injured by high temperature would turn yellow and die during the culture process, while tissues that could resist low temperature damage would gradually turn green and resume growth. Photos were taken and the survival rate was calculated.

[0047] PBS1 The phenotypes of the overexpression strains and the wild-type control strains after high-temperature treatment of basic heat resistance or acquired heat resistance are as follows: Figure 2 As shown in the figure, it can be seen that both basic heat resistance and acquired heat resistance treatments b and c (overexpression lines) are better than a (wild type) in heat resistance.

[0048] The survival rate statistics are as follows Figure 3 As shown in the figure, it can be seen that the survival rates of PBS1-GFP#4 and PBS1-GFP#6 (57.63% and 62.33% under basic heat resistance treatment, 68.92% and 70.80% under acquired heat resistance treatment) are significantly higher than those of the wild type (32.06% under basic heat resistance treatment, 27.10% under acquired heat resistance treatment).

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of PBS1 protein, or its encoding gene, or biological materials containing its encoding gene in regulating plant heat tolerance.

2. Use of PBS1 protein, or its encoding gene, or biological materials containing its encoding gene in cultivating heat-resistant plants.

3. Application of PBS1 protein, or its encoding gene, or biological materials containing its encoding gene in plant germplasm resource improvement.

4. The use according to any one of claims 1 to 3, characterized in that By increasing the expression level of the PBS1 protein in the plant, the heat tolerance of the plant is improved.

5. The use according to any one of claims 1 to 4, characterized in that The PBS1 protein includes any one of the following amino acid sequences: (1) the amino acid sequence shown in SEQ ID NO. 1; (2) The amino acid sequence of a protein with the same function as the amino acid sequence shown in SEQ ID NO. 1 obtained by replacing, inserting or deleting one or more amino acids.

6. The use according to any one of claims 1 to 5, characterized in that The coding gene of the PBS1 protein includes any one of the following nucleotide sequences: (1) the nucleotide sequence shown in SEQ ID NO. 2; (2) A nucleotide sequence encoding a protein with the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO. 2; (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

7. The use according to any one of claims 1 to 6, characterized in that The plant is a monocotyledonous plant or a dicotyledonous plant, preferably Arabidopsis thaliana.

8. The use according to any one of claims 1 to 7, characterized in that The biological material is an expression cassette, a vector or a transgenic cell.

9. A method for regulating heat tolerance of transgenic plants, characterized in that: include: The expression level of PBS1 protein in the plant is regulated.

10. The method according to claim 9, characterized in that The expression level of PBS1 protein in plants is regulated by gene editing; or heat-resistant strains are cultivated by hybridizing plants overexpressing the PBS1 protein with wild-type plants.

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