Application of SNRK2.1 protein and its encoding gene in improving cold tolerance of maize

Abstract

This invention relates to the field of biotechnology, specifically to the application of the SNRK2.1 protein and its encoding gene in improving the cold tolerance of maize. This invention demonstrates that overexpression of the SNRK2.1 gene in maize can enhance the plant's cold tolerance, and further proposes the application of the maize SNRK2.1 protein or its encoding gene, or biological materials containing its encoding gene, in any of the following aspects: (1) altering the cold tolerance of plants; (2) improving the survival rate of plants under low-temperature conditions; (3) breeding transgenic plants with improved cold tolerance; (4) improving cold-tolerant plant germplasm resources. This invention provides new gene resources for breeding new cold-tolerant plant varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the application of the SNRK2.1 protein and its encoding gene in improving the cold tolerance of maize. Background Technology

[0002] Maize (Zeamays L.) is an economic crop originating in tropical low-latitude regions. Although it has gradually spread to high-latitude and high-altitude temperate regions during human domestication and cultivation, maize remains highly sensitive to low-temperature damage. Therefore, it is still necessary to cultivate cold-resistant maize varieties when expanding maize cultivation in temperate regions. Maize's sensitivity to low temperatures is mainly due to reduced photosynthetic capacity and metabolic disorders. Short-term exposure of maize seedlings to low temperatures leads to decreased photosynthetic activity, subsequently affecting the transport of assimilates through dissipation mechanisms and the antioxidant system.

[0003] The maize SNRK2.1 homolog in Arabidopsis, SNRK2.8, ​​encodes a member of the SNF1-related protein kinase family, involved in salt and osmotic stress, but its role in plant cold tolerance has not been reported. Existing transgenic technologies can introduce stress-resistance genes into maize genetic material requiring improvement, resulting in offspring exhibiting stably inherited stress resistance and providing superior varietal resources for agricultural production. Discovering new genes regulating maize cold tolerance is of great significance for maize breeding and later production stages. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of SNRK2.1 protein and its encoding gene in improving the cold tolerance of maize. By screening a maize library of transgenic overexpression lines and observing their phenotypes, it was found that different overexpression gene lines exhibited different phenotypes, and several lines overexpressing the SNRK2.1 gene all showed obvious cold tolerance phenotypes.

[0005] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0006] The use of the SNRK2.1 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects:

[0007] (1) Improve the cold resistance of corn;

[0008] (2) Breeding transgenic corn with improved cold resistance;

[0009] (3) Improve cold-resistant maize germplasm resources.

[0010] Preferred,

[0011] The cold tolerance of maize can be improved by increasing the expression level and / or activity of SNRK2.1 protein in maize.

[0012] Preferred,

[0013] The amino acid sequence of the corn SNRK2.1 protein is any one of the following:

[0014] (A1) The amino acid sequence shown in SEQ ID NO.2;

[0015] (A2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0016] Preferred,

[0017] The cDNA nucleotide sequence of the corn SNRK2.1 protein is any one of the following:

[0018] (B1) The nucleotide sequence shown in SEQ ID NO.1;

[0019] (B2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.1 that has been substituted, deleted and / or added with one or more nucleotides and expresses a protein with the same function;

[0020] (B3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO.1.

[0021] Preferred,

[0022] The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

[0023] This invention also provides cloning vectors or various expression vectors containing the SNRK2.1 gene sequence or fragment thereof for low-temperature tolerance in maize, host cells containing the vectors, transformed plant cells containing the gene sequence or specific fragment thereof, and transgenic plants. The overexpression vector containing the SNRK2.1 gene is a pBCXUN vector containing the Ubi promoter.

[0024] Primers for amplifying the cDNA of the SNRK2.1 gene, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0025] This invention also provides a method for breeding cold-resistant maize.

[0026] By increasing the expression level and / or activity of SNRK2.1 protein, plants with enhanced cold resistance were obtained; the amino acid sequence of the SNRK2.1 protein is any of the following:

[0027] (A1) The amino acid sequence shown in SEQ ID NO.2;

[0028] (A2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0029] Preferred methods for breeding cold-resistant maize include the following steps:

[0030] (1) Amplify the full-length cDNA sequence of the SNRK2.1 gene (as shown in SEQ ID NO.1);

[0031] (2) Construct an overexpression vector for the SNRK2.1 gene;

[0032] (3) Construct recombinant Agrobacterium containing an overexpression vector of the SNRK2.1 gene;

[0033] (4) Using Agrobacterium infection method, transgenic plants overexpressing the SNRK2.1 gene were constructed.

[0034] Specifically, the gene encoding the SNRK2.1 protein was ligated into the pBCXUN vector to obtain the recombinant vector pBCXUN-SNRK2.1 containing the SNRK2.1 coding sequence. This recombinant vector was then transformed into Agrobacterium EHA105 to obtain recombinant Agrobacterium EHA105 / pBCXUN-SNRK2.1 containing the recombinant vector pBCXUN-SNRK2.1. The recombinant Agrobacterium EHA105 / pBCXUN-SNRK2.1 was then used to infect maize and introduced into recipient plants to obtain transgenic plants with increased drought resistance.

[0035] The amino acid sequence of the SNRK2.1 protein is any of the following:

[0036] (A1) The amino acid sequence shown in SEQ ID NO.2;

[0037] (A2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID NO.2.

[0038] Preferred,

[0039] The primers for detecting the ZmMYB121 gene are shown in SEQ ID No. 5-SEQ ID No. 6.

[0040] Preferred,

[0041] Methods include introducing a recombinant expression vector containing the maize SNRK2.1 gene into maize using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, gene gun, electroporation, and Agrobacterium-mediated transformation to obtain transgenic maize lines.

[0042] The beneficial effects of this invention are:

[0043] This invention cloned the SNRK2.1 gene, constructed transgenic plants and Crispr / Cas9 mutant materials with SNRK2.1 gene overexpression, and verified that the SNRK2.1 gene participates in regulating maize's cold tolerance; overexpression of the SNRK2.1 gene enables maize to acquire stronger low-temperature tolerance. This invention provides new gene resources for breeding new cold-tolerant plant varieties and lays a theoretical foundation for studying the mechanism of maize's response to low-temperature stress. Attached Figure Description

[0044] 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:

[0045] Figure 1 The figure shows the results of the SNRK2.1 gene overexpression test in the WT group and the maize overexpression line in Example 2 of the present invention; in the figure, *** represents P<0.001.

[0046] Figure 2 These are photographs of the plant growth of the WT group and the maize overexpression lines after low-temperature treatment and recovery in Example 3 of this invention.

[0047] Figure 3 This is a statistical chart of ion leakage rates in the WT group and maize overexpression lines in Example 3 of the present invention; in the chart, * represents P<0.05. Detailed Implementation

[0048] 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.

[0049] This invention provides the application of the SNRK2.1 protein and its encoding gene in improving the cold tolerance of maize. Overexpressing maize populations were screened, using the relative leaf injury area as an indicator for preliminary screening of low-temperature phenotypes. Overexpressing lines showing the initial phenotype were further screened to determine their low-temperature-related phenotypes. By consulting the overexpression information table, the gene number of the overexpressed gene in this line was found to be GRMZM2G056270, which was further identified as maize serine-threonine protein kinase SNRK2.1 based on gene annotations on the MaizeGDB website. This invention identifies the SNRK2.1 gene as a potential key gene for cold tolerance in maize, and by overexpressing the SNRK2.1 gene in maize, cold-resistant transgenic plants were obtained.

[0050] The cDNA sequence of the maize SNRK2.1 protein involved in this invention is as follows:

[0051] 1) The nucleotide sequence shown in SEQ ID No. 1;

[0052] 2) A nucleotide sequence that expresses a protein with the same function but with one or more nucleotides substituted, deleted, and / or added to the nucleotide sequence shown in SEQ ID No. 1;

[0053] 3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID No. 1.

[0054] The cDNA of the maize SNRK2.1 gene consists of 1367 bases, and its sequence is shown in SEQ ID No. 1. The reading frame of this gene consists of 9 exons. The amino acid sequence of the protein encoded by the maize SNRK2.1 gene is shown in SEQ ID No. 2.

[0055] The corn SNRK2.1 protein of this invention has any one of the following amino acid sequences:

[0056] 1) The amino acid sequence shown in SEQ ID No. 2;

[0057] 2) The amino acid sequence of a protein with the same function obtained by substituting, deleting or inserting one or more amino acid residues of the amino acid sequence shown in SEQ ID No. 2.

[0058] It should be understood that those skilled in the art can, based on the amino acid sequence disclosed in this invention, substitute, delete, and / or add one or more amino acids to obtain the mutant sequence of the protein without affecting its activity. The following examples are used to illustrate the invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al.'s Molecular Cloning Laboratory Manual (Sambrook J & Russell DW, Molecular cloning: alaboratory manual, 21), or according to the conditions recommended in the manufacturer's instructions. The main reagents used in the following examples were: 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; and all other chemical reagents used in the examples were imported or domestically produced analytical grade reagents. Primers used in the examples were synthesized by Huada Biotechnology Co., Ltd., and related sequencing was performed.

[0059] Example 1: Construction and detection of SNRK2.1 gene overexpression vector

[0060] Total RNA was extracted from B73 maize (Zea mays L.), and cDNA was obtained by reverse transcription. Using cDNA as a template and F and R as primers, the SNRK2.1 gene was amplified. The primers contained restriction enzyme sites, and after restriction enzyme digestion, the gene was ligated into an overexpression vector.

[0061] The method for constructing the SNRK2.1 gene overexpression vector is as follows:

[0062] (1) Total RNA was extracted from B73 maize using the Magen RNA Extraction Kit. The specific steps were described in the kit instructions.

[0063] (2) Use the Thermo Reverse Transcription Kit to reverse transcribe RNA into cDNA. Refer to the kit instructions for specific steps.

[0064] (3) Using maize cDNA as a template and F and R as primers, amplify the cDNA of SNRK2.1 (as shown in SEQ ID No.1, whose encoded amino acid sequence is shown in SEQ ID No.2). The amplified product is then electrophoresed and gel-cleaved for recovery. The recovery method is as described in the kit instructions of Tiangen Company.

[0065] The primers used to amplify the SNRK2.1 gene cDNA are:

[0066] Upstream primer F: 5'-ATGGAGCGGTACGAGGT-3' (SEQ ID No. 3);

[0067] Downstream primer R: 5'-CACTGCACAAACGAAGTC-3' (SEQ ID No. 4).

[0068] (4) The recovered SNRK2.1 gene cDNA and pBCXUN vector were double-digested with Xba I and Cla I. The digestion products were then recovered by electrophoresis and gel extraction. The recovered products were ligated with T4 ligase to obtain the digestion-ligation system product. The SNRK2.1 gene was ligated into the pBCXUN vector to prepare the SNRK2.1 gene overexpression vector, which was then used to drive the expression of the SNRK2.1 gene using the Ubi promoter. The digestion and ligation system was based on the Takara kit instructions.

[0069] The pBCXUN vector is obtained by linking a hygromycin resistance gene into the commercial vector pCAMBIA1300 as the backbone (Guo et al., 2018 Stepwise cis-regulatory changes in ZCN8 contribute to maize flowering-time adaptation. Current Bio. 28, 3005–3015); at the same time, the promoter of the maize ubiquitin gene Ubi is cloned into the vector through enzyme digestion and ligation to drive the transcription of downstream overexpressed genes.

[0070] (5) Take 5 μL of the product from the enzyme digestion-ligation system and transform it into competent E. coli cells. Screen on LB plates containing 50 μg / mL kanamycin. Identify single clones by colony PCR and select positive clones for sequencing. The recombinant expression vector with correct sequencing results is named SNRK2.1-pBCXUN.

[0071] The obtained plasmid vector SNRK2.1-pBCXUN was digested with enzymes and then detected by electrophoresis. The specific method was as follows: SNRK2.1-pBCXUN was digested with XbaI and ClaI, electrophoresed on a 1% agarose gel at 120V and 50mA, and then scanned and imaged using a UVP GelDocumentation gel analysis system.

[0072] Example 2: Construction and detection of plants overexpressing the SNRK2.1 gene

[0073] The SNRK2.1-pBCXUN vector prepared in Example 1 was transformed into Agrobacterium EHA105 strain, and then infected with maize LH244 callus tissue to obtain transgenic seedlings.

[0074] The specific method is as follows: Agrobacterium EHA105 containing the SNRK2.1-pBCXUN 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 until OD was reached. 600 The bacterial cells were collected by centrifugation at 50g for 15 min at room temperature, with a value of 1.0-2.0. The cells were then 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, sealed, and returned to a light-treated culture rack for normal growth until plants emerged. The resulting seeds were then screened and subjected to low-temperature stress treatment experiments.

[0075] In this embodiment, overexpression lines OE-1 and OE-2 with high SNRK2.1 gene expression levels were isolated. Specifically, real-time quantitative PCR was used to detect SNRK2.1 gene expression in the obtained overexpression lines OE-1 and OE-2. The specific method is as follows:

[0076] 1) Extract total RNA from plants and reverse transcribe it to obtain cDNA.

[0077] 2) After diluting the cDNA obtained from reverse transcription 5 times, perform real-time quantitative PCR using a Takara kit. The reaction system included: 2×SYBR Premix ExTaq buffer, 0.2 μL DyII, 0.4 μL Primer (F1 / R1), 2 μL cDNA template, and finally add ddH2O to a final volume of 20 μL. After thorough mixing, the mixture was placed in an ABI PRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions were: 95℃ for 30 s; 95℃ for 5 s; 60℃ for 40 s; 40 cycles.

[0078] The sequences of primers F1 and R1 (primers for qRT-PCR) are as follows:

[0079] F1: 5'-ACTGTTGGCACAACCTGCATA-3' (SEQ ID No. 5);

[0080] R1: 5'-TCTCTCGGGTCATCAGGGTCT-3' (SEQ ID No. 6).

[0081] After the PCR reaction is completed, according to 2 -Δ(ΔCt)The principle was used to calculate and plot the relative expression levels between the wild-type (WT group) and overexpression lines (OE). Three biological replicates were performed, and the trends were similar across the three replicates. Simultaneously with the amplification of the identified genes, the UBI gene was amplified as an internal control for each sample. The test results are shown below. Figure 1 ,from Figure 1 The results showed that the expression level of the overexpression strain was significantly higher than that of the WT control group.

[0082] Example 3: Detection of low-temperature resistance in plants overexpressing the SNRK2.1 gene

[0083] First, seeds from the WT group (wild-type maize) and OE-1 and OE-2 obtained in Example 2 were sown in small pots (10cm long, 10cm wide, and 10cm high) containing black soil, imported soil, and vermiculite (mass ratio 1:1:1). Five seeds were placed in each pot, covered with 2cm of soil, and placed on a tray. The pots were watered until the soil was completely moist and placed in a 23°C incubation room with 16 hours of light and 8 hours of darkness. After 14 days of growth, the plants were treated at 4°C for 4 days until the second leaf withered and shriveled. They were then removed and placed in a 23°C incubation room for two days to recover before photographing and collecting samples for ion leakage rate analysis. Three seedlings from each overexpression line (OE) and wild type (WT) were taken for testing, and three biological replicates were performed.

[0084] The plant growth of the WT group and maize overexpression lines after low-temperature treatment recovery is as follows: Figure 2 As shown (the left image is the control group without low-temperature treatment, and the right image is the experimental group after low-temperature treatment and recovery). The results showed that the wild-type WT leaves were severely wilted, dried out, and even unable to stand upright, while the overexpression lines OE-1 and OE-2 only had slight damage to the leaf tips and remained upright, showing a low-temperature resistant phenotype.

[0085] In this embodiment, the ion leakage rate was statistically analyzed by measuring the relative conductivity of the leaves, L = (S1-S0) / (S2-S0)*100%. After low-temperature treatment, an entire corn plant was placed in a 15ml centrifuge tube containing 10ml of distilled water. The tube was evacuated using a vacuum pump for 30 minutes, then placed on a shaker at room temperature for 1 hour. The initial conductivity value, S1, was then measured using a conductivity meter. The sample was then placed in a boiling water bath for 15 minutes, removed, and placed on a shaker for 2 hours. The conductivity was measured again and recorded as S2. S0 represents the conductivity of the blank control distilled water.

[0086] The results are as follows Figure 3 As shown in Table 1, compared with wild-type WT plants, the ion leakage rates of overexpression lines OE-1 and OE-2 were reduced by 66.10% and 67.34%, respectively (the average difference between the ion leakage rates of wild-type WT plants and overexpression lines in three trials), reaching a significant difference (P<0.05), indicating that overexpression of the SNRK2.1 gene can enhance the cold resistance of maize.

[0087] Table 1. Ion leakage rate values ​​(%) from three independent experiments.

[0088] WT OE-1 OE-2 93.75697 25.72782 21.97902 90.02732 27.36804 24.46877 87.94857 20.32647 23.25247

[0089] The contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0090] 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. The use of the SNRK2.1 protein or its encoding gene, or biological material containing its encoding gene, in any of the following aspects: (1) Improve the cold resistance of corn; (2) Breeding transgenic maize with improved cold tolerance; (3) Improve cold-resistant maize germplasm resources; The application is achieved by increasing the expression level of SNRK2.1 protein in maize; The amino acid sequence of the corn SNRK2.1 protein is as follows: (A1) The amino acid sequence shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The cDNA nucleotide sequence of the corn SNRK2.1 protein is any one of the following: (B1) The nucleotide sequence shown in SEQ ID NO.1; (B2) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO.

1.

3. The application according to claim 1 or 2, characterized in that, The biological material is an expression cassette, vector, host cell, or recombinant bacteria.

4. A method for breeding cold-resistant maize, characterized in that, By increasing the expression level of SNRK2.1 protein, maize with improved cold tolerance was obtained; the amino acid sequence of the SNRK2.1 protein is as follows: (A1) The amino acid sequence shown in SEQ ID NO.

2.

5. The breeding method for cold-resistant maize according to claim 4, comprising the following steps: The gene encoding the SNRK2.1 protein was linked to pBCXUN Carrier, to obtain containing SNRK2.1 Recombination vector of coding sequence pBCXUN-SNRK2.1 Transform the recombinant vector into Agrobacterium EHA105 Obtaining a substance containing a recombinant vector pBCXUN-SNRK2.1 Recombinant Agrobacterium EHA105 / pBCXUN-SNRK2.1 Recombinant Agrobacterium EHA105 / pBCXUN-SNRK2.1 Infecting corn and introducing it into recipient plants yields transgenic plants with increased drought resistance; The amino acid sequence of the SNRK2.1 protein is as follows: (A1) The amino acid sequence shown in SEQ ID NO.

2.

6. The method for breeding cold-resistant maize according to claim 5, characterized in that, Detection ZmMYB121 The primers for the gene are shown in SEQ ID NO.5-SEQ ID NO.

6.

7. The method for breeding cold-resistant plants according to claim 4, characterized in that, This includes methods such as using Ti plasmids, plant virus vectors, direct DNA transformation, microinjection, gene guns, electrical conductivity, and Agrobacterium-mediated transformation to generate maize containing the aforementioned... SNRK2.1 Gene recombinant expression vectors are introduced into maize to obtain transgenic maize lines.

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