Application of MIEL1 protein and coding gene thereof in improving cold resistance of corn

Abstract

The invention relates to the technical field of biology, in particular to application of MIEL1 protein and a coding gene thereof in improving cold resistance of corn. The invention shows that overexpression of the MIEL1 gene in corn can enhance the low temperature resistance of the plant, and further provides application of the corn MIEL1 protein or the coding gene thereof or a biological material containing the coding gene in any one of the following aspects: (1) changing the cold resistance of the plant; (2) the survival rate of the plants in a low-temperature environment is improved; (3) breeding a transgenic plant with improved cold resistance; and (4) improving plant cold-resistant germplasm resources. The invention provides a new gene resource for cultivating new varieties of low-temperature-resistant plants.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to application of MIEL1 protein and its encoding gene in improving the cold tolerance of corn. Background Art

[0002] Maize (Zeamays L.) is an economic crop that originated in tropical low-latitude areas. Although it has gradually entered high-latitude and high-altitude temperate regions during human domestication and cultivation, maize is still very sensitive to low temperature damage. Therefore, it is still necessary to cultivate cold-resistant maize when expanding maize cultivation in temperate regions. Maize's sensitivity to low temperatures is mainly caused by weakened photosynthesis capacity and its own metabolic disorders. Short-term exposure of maize seedlings to low temperatures will lead to reduced photosynthesis activity, followed by the involvement of dissipation mechanisms and antioxidant systems, affecting the transport of assimilates.

[0003] The MIEL1 gene, the homolog of maize MIEL1 in Arabidopsis, encodes the RING-type E3 ubiquitin ligase MYB83 protein that interacts with MYB30 and ubiquitinates it, leading to proteasomal degradation of MYB30 and downregulating its transcriptional activity. Since MYB30 is a positive regulator of HR and defense responses in Arabidopsis, MIEL1 is involved in the negative regulation of these processes, but its role in plant cold tolerance has not been reported. Existing transgenic technology can introduce plant stress resistance genes into the genetic material of maize that needs to be improved, and make its offspring show stable inherited stress resistance, providing excellent variety resources for agricultural production. The study of new genes regulating maize cold tolerance is of great significance to maize breeding and later production. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention aims to provide an application of MIEL1 protein and its encoding gene in improving the cold tolerance of corn. By screening a corn library of transgenic overexpression strains and observing the phenotypes, it was found that the phenotypes of different overexpression gene strains were different. Several strains overexpressing the MIEL1 gene showed obvious cold tolerance phenotypes compared with wild-type plants.

[0005] In order to solve the above technical problems, the technical solution provided by the present invention is:

[0006] Use of MIEL1 protein or its encoding gene, or biological material containing its encoding gene in any of the following aspects:

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

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

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

[0010] Preferably,

[0011] By increasing the expression level and / or activity of MIEL1 protein in corn, the cold tolerance of corn is improved.

[0012] Preferably,

[0013] The amino acid sequence of the corn MIEL1 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 the amino acid sequence shown in SEQ ID NO.2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0016] Preferably,

[0017] The cDNA nucleotide sequence of the corn MIEL1 protein is any one of the following:

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

[0019] (B2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID NO.1 and the nucleotide sequence 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] Preferably,

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

[0023] The present invention also provides cloning vectors or various expression vectors containing the low-temperature-resistant MIEL1 gene sequence of corn or its fragments, host cells containing the vectors, transformed plant cells and transgenic plants containing the gene sequence or its specific fragments. Among them, the overexpression vector containing the MIEL1 gene is a pBCXUN vector containing an Ubi promoter.

[0024] The nucleotide sequences of the primers for amplifying the cDNA of the MIEL1 gene are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0025] The present invention also provides a breeding method for cold-tolerant corn.

[0026] By increasing the expression level and / or activity of MIEL1 protein, a plant with improved cold resistance is obtained; the amino acid sequence of the MIEL1 protein is any one of the following:

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

[0028] (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0029] Preferably, the breeding method of cold-tolerant corn comprises the following steps:

[0030] (1) amplifying the full-length gene cDNA sequence of MIEL1 gene (as shown in SEQ ID NO.1);

[0031] (2) constructing an overexpression vector of the MIEL1 gene;

[0032] (3) constructing a recombinant Agrobacterium containing an overexpression vector of the MIEL1 gene;

[0033] (4) The transgenic plants overexpressing MIEL1 gene were constructed by Agrobacterium infection.

[0034] Specifically, the gene encoding MIEL1 protein is connected to the pBCXUN vector to obtain a recombinant vector pBCXUN-MIEL1 containing the MIEL1 coding sequence, the recombinant vector is transformed into Agrobacterium EHA105 to obtain a recombinant Agrobacterium EHA105 / pBCXUN-MIEL1 containing the recombinant vector pBCXUN-MIEL1, the recombinant Agrobacterium EHA105 / pBCXUN-MIEL1 is infected with corn, and introduced into a recipient plant to obtain a transgenic plant with increased drought resistance;

[0035] The amino acid sequence of the MIEL1 protein is any one of the following:

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

[0037] (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0038] Preferably,

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

[0040] Preferably,

[0041] The method comprises the steps of using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation and Agrobacterium-mediated method to introduce a recombinant expression vector containing the corn MIEL1 gene into corn to obtain a transgenic corn strain.

[0042] The beneficial effects of the present invention are:

[0043] The present invention cloned the MIEL1 gene, constructed a transgenic plant with overexpression of the MIEL1 gene, and verified that the MIEL1 gene is involved in regulating the cold tolerance of corn. Overexpression of the MIEL1 gene can enable corn to obtain stronger low temperature tolerance. The present invention provides a new gene resource for breeding new varieties of low temperature tolerant plants, and at the same time lays a certain theoretical foundation for studying the mechanism of corn response to low temperature stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The accompanying 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 of the present invention. In the accompanying drawings:

[0045] Figure 1 This is a graph showing the test results of MIEL1 gene overexpression in the WT group and corn overexpression strains in Example 2 of the present invention; in the graph, *** represents P<0.001.

[0046] Figure 2 The photos are of the growth of plants of the WT group and the corn overexpression strain after recovery from low temperature treatment in Example 3 of the present invention;

[0047] Figure 3 This is a statistical graph of ion leakage rates of the WT group and corn overexpression strains in Example 3 of the present invention; in the graph, * represents P<0.05. DETAILED DESCRIPTION

[0048] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided only for the purpose of illustration and are not intended to limit the scope of the present invention. Those skilled in the art may make various modifications and substitutions to the present invention without departing from the purpose and spirit of the present invention.

[0049] The present invention provides the application of MIEL1 protein and its encoding gene in improving the cold tolerance of corn, screens the overexpression corn population, uses the relative injured area of ​​leaves as an index, performs preliminary screening of low temperature phenotypes, rescreens the overexpression strains with phenotypes in the initial screening, determines the low temperature related phenotypes, and finds that the gene number of the overexpressed gene of the strain is GRMZM2G056270 by consulting the overexpression information table, and further determines that it is corn E3 ubiquitin ligase MIEL1 according to the gene annotation on the MaizeGDB website. The present invention studies and determines that the MIEL1 gene may be a key gene for cold tolerance and cold resistance of corn, and obtains cold resistant transgenic plants by overexpressing the MIEL1 gene in corn.

[0050] The cDNA sequence of the maize MIEL1 protein involved in the present invention is:

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

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

[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 MIEL1 gene consists of 1498 bases, and the sequence is shown in SEQ ID No. 1. The gene reading frame consists of 12 exons. The amino acid sequence of the protein encoded by the maize MIEL1 gene is shown in SEQ ID No. 2.

[0055] The corn MIEL1 protein of the present 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 the amino acid sequence shown in SEQ ID No. 2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

[0058] It should be understood that those skilled in the art can replace, delete and / or add one or more amino acids based on the amino acid sequence disclosed in the present invention without affecting its activity to obtain a mutant sequence of the protein.

[0059] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the examples are all based on conventional experimental conditions, such as Sambrook et al. Molecular cloning laboratory manual (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 21), or the conditions recommended by the manufacturer's instructions.

[0060] The main reagents in the following examples are: various restriction endonucleases, Taq DNA polymerase, T4 ligase, Pyrobest Taq enzyme, KOD purchased from NEB, Toyobo and other biological companies; dNTPs purchased from Genestar; plasmid mini-extraction kit and agarose gel recovery kit purchased from Shanghai Jierui Bioengineering Company; agar powder, agarose, ampicillin (Amp), kanamycin (Kan), gentamicin sulfate (Gen), rifampicin (Rif) and other antibiotics and Glucose, BSA, LBMedium etc. purchased from Sigma, Bio-Rad and other companies; the reagents used for real-time quantitative PCR were purchased from TaKaRa, and various other chemical reagents used in the examples were imported or domestic analytical reagents. The primers used in the examples were synthesized by Liuhe Huada Company and related sequencing was performed.

[0061] Example 1 Construction and detection of MIEL1 gene overexpression vector

[0062] Total RNA was extracted from B73 corn (Zea mays L.), and cDNA was obtained by reverse transcription. The MIEL1 gene was amplified using cDNA as a template and F and R as primers. The primers had restriction sites and were connected to the overexpression vector after restriction digestion.

[0063] The specific method for constructing the MIEL1 gene overexpression vector is as follows:

[0064] (1) Total RNA of B73 maize was extracted using the RNA extraction kit of Magen Company. The specific steps were referred to the instructions of the kit.

[0065] (2) Use the reverse transcription kit of Thermo Company to reverse transcribe RNA into cDNA. For specific steps, refer to the kit instructions.

[0066] (3) Using maize cDNA as a template and primers F and R, the cDNA of the MIEL1 gene (as shown in SEQ ID No. 1, and its encoded amino acid sequence is shown in SEQ ID No. 2) was amplified, and the amplified product was recovered by electrophoresis and gel cutting. The recovery method was referred to the instructions of the kit of Tiangen Company.

[0067] The primer sequences used to amplify MIEL1 gene cDNA are:

[0068] Upstream primer F: 5'-ATGACGTCGCTACTCTTCC-3' (SEQ ID No. 3);

[0069] Downstream primer R: 5'-TATGTTGTTGTCGGATGAGTC-3' (SEQ ID No. 4).

[0070] (4) The recovered MIEL1 gene cDNA and pBCXUN vector were double-digested with Xba I and Cla I, and the digestion products were recovered by electrophoresis and gel cutting. The recovered products were ligated with T4 ligase to obtain the product of the digestion-ligation system, and the MIEL1 gene was ligated to the pBCXUN vector to prepare the MIEL1 gene overexpression vector, and the expression of the MIEL1 gene was driven by the Ubi promoter.

[0071] The pBCXUN vector is based on the commercial vector pCAMBIA1300, into which the hygromycin resistance gene is linked (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 by enzyme ligation to drive the transcription of downstream overexpressed genes.

[0072] (5) Take 5 μL of the product of the restriction-ligation system, transform it into competent E. coli, and screen it on an LB plate containing 50 μg / mL kanamycin. Use colony PCR to identify single clones, and select positive clones for sequencing. The recombinant expression vector with correct sequencing is named pBCXUN-MIEL1.

[0073] The obtained plasmid vector pBCXUN-MIEL1 was digested with enzymes and then subjected to electrophoresis detection. The specific method was as follows: pBCXUN-MIEL1 was digested with Xba I and Cla I, electrophoresed with 1% agarose gel, 120V, 50mA, and scanned with UVP Gel Documentation gel analysis system for imaging.

[0074] Example 2 Construction and detection of MIEL1 gene overexpressing plants

[0075] The pBCXUN-MIEL1 vector prepared in Example 1 was transformed into Agrobacterium EHA105 strain, and then infected corn LH244 callus to obtain transgenic seedlings.

[0076] The specific method is as follows: inoculate Agrobacterium EHA105 containing the pBCXUN-MIEL1 vector into 100 mL LB triple-antibody liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), culture at 28°C overnight with shaking, and wait until OD 600 The value was 1.0-2.0, centrifuged at 50g for 15 minutes at room temperature, and the cells were collected; the cells were suspended in 2mL of transformation solution (1 / 2MS, 5% sucrose, 40μL Silwet L-77); the corn callus was immersed in the transformation solution of Agrobacterium, sealed, and placed back on the light culture rack for normal growth until the plants grew out. Then, the selection was carried out, and the seeds obtained were subjected to the low temperature stress treatment experiment.

[0077] In this example, overexpression strains OE-1 and OE-2 with high MIEL1 gene expression levels were isolated. Real-time quantitative PCR was used to detect the MIEL1 gene expression in the obtained overexpression strains OE-1 and OE-2. The specific method is as follows:

[0078] 1) Extract total RNA from plants and reverse transcribe it to obtain cDNA.

[0079] 2) After the cDNA obtained by reverse transcription was diluted 5 times, real-time quantitative PCR was performed using the Takara kit. The reaction system used included: 2×SYBR Premix ExTaq buffer, 0.2μL DyII, 0.4μL Primer (F1 / R1), 2μL cDNA template, and finally filled to 20μL with ddH2O. After thorough mixing, the cDNA was placed in an ABIPRISM 75 real-time quantitative PCR instrument for two-step PCR amplification. The reaction conditions were: 95℃30s; 95℃5s; 60℃40s; 40 cycles.

[0080] Among them, the sequences of primers F1 and R1 (primers for qRT-PCR) are as follows:

[0081] F1: 5'-ACTGTGGACACACAATGCAC-3' (SEQ ID No. 5);

[0082] R1: 5'-CGTTGCAAAGCACCCAAATCT-3' (SEQ ID No. 6).

[0083] After the PCR reaction is completed, -Δ(ΔCt) The relative expression between the wild type (WT group) and the overexpression strain (OE) was calculated and analyzed by the principle of the method. Three biological replicates were performed and the trends were similar. While amplifying the identified genes, each sample was amplified simultaneously with the UBI gene as an internal reference. The test results are shown in Figure 1 ,from Figure 1It can be seen that the expression level of the overexpression strain was significantly higher than that of the WT control group.

[0084] Example 3 Detection of low temperature resistance of plants overexpressing MIEL1 gene

[0085] First, the seeds of the WT group (wild-type corn) and OE-1 and OE-2 obtained in Example 2 were sown in small pots with a length of 10 cm, a width of 10 cm, and a height of 10 cm containing black soil, imported soil, and vermiculite (mass ratio 1:1:1). Five seeds were placed in each pot, and then covered with 2 cm of soil and placed in a tray. Water was poured until the soil was completely moistened, and placed in a culture room at 23°C, with 16 hours of light and 8 hours of darkness. After 14 days of growth, the seeds were treated with a low temperature of 4°C for 4 days until the second leaf shrank and wilted. After being taken out and placed in a 23°C culture room for recovery for two days, photos were taken and samples were collected for statistics of ion leakage rate. Three seedlings were taken from each overexpression line (OE) and wild type (WT) for measurement, and three biological replicates were performed.

[0086] The growth of the WT group and the maize overexpression lines after recovery from low temperature treatment is shown in Figure 2. Figure 2 As shown (the left picture is the control group without low temperature treatment, and the right picture is the experimental group after low temperature treatment recovery). The results showed that the leaves of the wild-type WT were severely wilted, dried up, and even unable to stand upright, while the overexpression lines OE-1 and OE-2 only had slight injuries on the leaf tips, and still remained upright, showing a phenotype of low temperature resistance.

[0087] In this embodiment, the statistics of ion leakage rate are performed by measuring the relative conductivity of the leaves L = (S1-S0) / (S2-S0)*100%. A whole corn plant after low temperature treatment is placed in a 15ml centrifuge tube filled with 10ml distilled water, evacuated with a vacuum pump for 30 minutes, and then placed on a shaker at room temperature for 1 hour, and then the initial conductivity is measured by a conductivity meter as S1, and then the sample is placed in a boiling water bath for 15 minutes, taken out and placed on a shaker for 2 hours, and then the conductivity is measured and recorded as S2. S0 is the conductivity of the blank control distilled water.

[0088] The results are as follows Figure 3 As shown in Table 1, compared with the wild-type WT plants, the ion leakage rates of the overexpression lines OE-1 and OE-2 were reduced by 51.5% and 48.0%, respectively (the average of the three differences in ion leakage rates between the wild-type WT plants and the overexpression lines), reaching a significant difference, P < 0.05, indicating that overexpression of the MIEL1 gene can enhance the cold resistance of corn.

[0089] Table 1 Ion leakage rate values ​​of three independent experiments (%)

[0090]

[0091]

[0092] The contents not described in detail in this specification belong to the prior art known to professional and technical personnel in this field.

[0093] Finally, it should be noted that the above description is only a preferred example of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. Application of MIEL1 protein or its encoding gene, or biological materials containing its encoding gene in any of the following aspects: (1) Improve the cold tolerance of corn; (2) Breeding transgenic corn with improved cold tolerance; (3) Improve corn cold-resistant germplasm resources.

2. The use according to claim 1, characterized in that: By increasing the expression level and / or activity of MIEL1 protein in corn, the cold tolerance of corn is improved.

3. The use according to claim 1 or 2, characterized in that: The amino acid sequence of the corn MIEL1 protein is any one of the following: (A1) the amino acid sequence shown in SEQ ID NO.2; (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

4. The use according to claim 1 or 2, characterized in that: The cDNA nucleotide sequence of the corn MIEL1 protein is any one of the following: (B1) the nucleotide sequence shown in SEQ ID NO.1; (B2) a nucleotide sequence in which one or more nucleotides are substituted, deleted and / or added to the nucleotide sequence shown in SEQ ID No. 1 and the nucleotide sequence expresses a protein with the same function; (B3) A nucleotide sequence that is completely complementary to the nucleotide sequence shown in SEQ ID NO.

1.

5. The use according to claim 1 or 2, characterized in that: The biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

6. Primers for amplifying cDNA of MIEL1 gene, the nucleotide sequences of which are shown in SEQ ID NO.3 and SEQ ID NO.

4.

7. A method for breeding cold-tolerant corn, characterized in that: A plant with improved cold tolerance is obtained by increasing the expression level and / or activity of the MIEL1 protein; the amino acid sequence of the MIEL1 protein is any one of the following: (A1) the amino acid sequence shown in SEQ ID NO.2; (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

8. The breeding method of cold-tolerant corn according to claim 7, comprising the following steps: The gene encoding MIEL1 protein is connected to the pBCXUN vector to obtain a recombinant vector pBCXUN-MIEL1 containing the MIEL1 coding sequence, the recombinant vector is transformed into Agrobacterium EHA105 to obtain a recombinant Agrobacterium EHA105 / pBCXUN-MIEL1 containing the recombinant vector pBCXUN-MIEL1, the recombinant Agrobacterium EHA105 / pBCXUN-MIEL1 is infecting corn, and then introduced into a recipient plant to obtain a transgenic plant with increased drought resistance; The amino acid sequence of the MIEL1 protein is any one of the following: (A1) the amino acid sequence shown in SEQ ID NO.2; (A2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.2 obtained by replacing, deleting or inserting one or more amino acid residues, and having the same functional protein.

9. The method for breeding cold-tolerant corn according to claim 8, characterized in that: The primers for detecting the ZmMYB121 gene are shown in SEQ ID No.5-SEQ ID No.

6.

10. The method for breeding cold-tolerant corn according to claim 7, characterized in that: The method comprises the steps of using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation and Agrobacterium-mediated method to introduce a recombinant expression vector containing the corn MIEL1 gene into corn to obtain a transgenic corn strain.

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