Application of HAG18 protein and its coding gene in improving cold tolerance of maize

Abstract

The present application relates to the field of biotechnology, and particularly relates to application of HAG18 protein and its coding gene in improving cold tolerance of corn. It is found in the present application that overexpression of HAG18 gene in corn can enhance the low-temperature tolerance of plants, and further, application of corn HAG18 protein or its coding gene or biological material containing the coding gene in any of the following aspects is proposed: (1) changing the cold tolerance of plants; (2) improving the survival rate of plants in a low-temperature environment; (3) breeding transgenic plants with improved cold tolerance; (4) improving plant cold-tolerant germplasm resources. The present application provides a new gene resource for breeding new low-temperature tolerant plant varieties.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of HAG18 protein and its encoding gene in improving the cold tolerance of corn. Background Art

[0002] Maize (Zeamays L.) is a commercial crop originating in tropical low-latitude regions. Although domesticated and cultivated, it has gradually spread to temperate regions at higher latitudes and altitudes, maize remains highly sensitive to cold damage. Therefore, the development of cold-tolerant and cold-resistant maize is necessary to expand maize cultivation in temperate regions. Maize's sensitivity to low temperatures is primarily due to reduced photosynthetic capacity and metabolic disturbances. Short-term exposure of maize seedlings to low temperatures reduces photosynthetic activity, subsequently engaging dissipative mechanisms and antioxidant systems, and impairing the transport of assimilates.

[0003] Maize HAG18 shares the highest homology with GNAT4 from Arabidopsis thaliana, but no cold-resistant phenotype has been found in Arabidopsis thaliana. Existing transgenic technology can introduce plant stress-tolerance genes into the genetic material of maize plants in need of improvement, resulting in stable inherited stress tolerance in offspring and providing superior cultivar resources for agricultural production. Identifying new genes regulating maize cold tolerance is of great significance for maize breeding and subsequent production. Summary of the Invention

[0004] In response to the deficiencies in the prior art, the present invention aims to provide an application of HAG18 protein and its encoding gene in improving the cold tolerance of corn. By screening a corn library of transgenic overexpression lines and observing the phenotypes, it was found that the phenotypes of different overexpression gene lines were different. Several lines overexpressing the HAG18 gene showed a significant cold tolerance phenotype compared to wild-type plants.

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

[0006] Use of HAG18 protein or its encoding gene, or biological materials 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 HAG18 protein in corn, the cold tolerance of corn is improved.

[0012] Preferably,

[0013] The amino acid sequence of the corn HAG18 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 HAG18 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 of SEQ ID NO. 1 and the nucleotide sequence expresses a protein with the same function;

[0020] (B3) A nucleotide sequence 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 corn cold-resistant HAG18 gene sequence or fragments thereof, host cells containing the vectors, and transformed plant cells and transgenic plants containing the gene sequence or specific fragments thereof. The overexpression vector containing the HAG18 gene is a pBCXUN vector containing the Ubi promoter.

[0024] The nucleotide sequences of the primers for amplifying the cDNA of the HAG18 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 the HAG18 protein, a plant with improved cold resistance is obtained; the amino acid sequence of the HAG18 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 for cold-tolerant corn comprises the following steps:

[0030] (1) Amplifying the full-length cDNA sequence of the HAG18 gene (as shown in SEQ ID NO. 1);

[0031] (2) Construction of an overexpression vector for the HAG18 gene;

[0032] (3) constructing a recombinant Agrobacterium containing an overexpression vector of the HAG18 gene;

[0033] (4) Using the Agrobacterium infection method, transgenic plants overexpressing the HAG18 gene were constructed.

[0034] Specifically, the gene encoding the HAG18 protein is connected to the pBCXUN vector to obtain a recombinant vector pBCXUN-HAG18 containing the HAG18 coding sequence, the recombinant vector is transformed into Agrobacterium EHA105 to obtain a recombinant Agrobacterium EHA105 / pBCXUN-HAG18 containing the recombinant vector pBCXUN-HAG18, the recombinant Agrobacterium EHA105 / pBCXUN-HAG18 is infected with corn, and then introduced into a recipient plant to obtain a transgenic plant with increased drought resistance;

[0035] The amino acid sequence of the HAG18 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 to 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 methods to introduce a recombinant expression vector containing the corn HAG18 gene into corn to obtain a transgenic corn strain.

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

[0043] This study cloned the HAG18 gene, constructed transgenic plants and Crispr / Cas9 mutants overexpressing the HAG18 gene, and verified that the HAG18 gene is involved in regulating maize cold tolerance. Overexpression of the HAG18 gene can enhance maize's low-temperature tolerance. This study provides a new genetic resource for breeding new cold-tolerant plant varieties and lays a theoretical foundation for studying the mechanisms by which maize responds 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 HAG18 gene overexpression test results for the WT group and the corn overexpression strain in Example 2 of the present invention; in the graph, *** represents P<0.001.

[0046] Figure 2 These are photos of plant growth in the WT group and the corn overexpression line 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 the corn overexpression line in Example 3 of the present invention; in the figure, * represents P<0.05. DETAILED DESCRIPTION

[0048] The following describes preferred embodiments of the present invention in conjunction with the accompanying drawings. It should be understood that the following embodiments are provided for illustrative purposes only 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 use of the HAG18 protein and its encoding gene for improving the cold tolerance of corn. Overexpressing corn populations were screened, and a preliminary screening for low-temperature phenotypes was performed using the relative leaf injury area as an indicator. Overexpressing strains that showed phenotypes in the initial screening were rescreened to determine their cold-related phenotypes. By consulting an overexpression information table, the gene number of the overexpressed gene in this strain was found to be GRMZM2G055141. The gene was further identified as the corn histone acetyltransferase HAG18 based on gene annotations on the MaizeGDB website. The present invention determined that the HAG18 gene may be a key gene for cold tolerance and cold resistance in corn. By overexpressing the HAG18 gene in corn, cold-tolerant and cold-resistant transgenic plants were obtained.

[0050] The cDNA sequence of the corn HAG18 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 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;

[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 HAG18 gene consists of 1360 bases, the sequence of which is shown in SEQ ID No. 1. The gene reading frame consists of four exons. The amino acid sequence of the protein encoded by the maize HAG18 gene is shown in SEQ ID No. 2.

[0055] The corn HAG18 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 provided to illustrate the present invention but are not intended to limit the scope of the invention. Unless otherwise specified, the examples were performed according to conventional experimental conditions, such as those in Sambrook et al. (Sambrook J & Russell DW, Molecular cloning: a laboratory manual, 21), or according to 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 biological companies such as NEB and Toyobo; dNTPs purchased from Genestar; plasmid minipreparation kit and agarose gel recovery kit purchased from Shanghai Jierui Bioengineering Co., Ltd.; 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 etc.; the reagents used for real-time quantitative PCR were purchased from TaKaRa, and various other chemical reagents used in the examples were all imported or domestic analytical reagents. The primers used in the examples were synthesized by Liuhe Huada Company and the relevant sequencing was performed.

[0061] Example 1 Construction and detection of HAG18 gene overexpression vector

[0062] Total RNA was extracted from B73 maize (Zea mays L.) and reverse transcribed to obtain cDNA. The HAG18 gene was amplified using the cDNA as a template and primers F and R. The primers contained restriction sites and were then ligated to the overexpression vector. The HAG18 gene overexpression vector was constructed as follows:

[0063] (1) Total RNA from B73 maize was extracted using the RNA extraction kit from Magen. The specific steps were as per the kit instructions.

[0064] (2) Use the reverse transcription kit from Thermo Corporation to reverse transcribe RNA into cDNA. For specific steps, refer to the kit instructions.

[0065] (3) Using corn cDNA as a template and primers F and R, the cDNA of the HAG18 gene (shown as SEQ ID No. 1, and its encoded amino acid sequence is shown as SEQ ID No. 2) was amplified. The amplified product was run on electrophoresis and gel-cut to recover the product. The recovery method was referred to the kit instructions of Tiangen Company.

[0066] The primer sequences used to amplify the HAG18 gene cDNA are:

[0067] Upstream primer F: 5′-ATGCGAGGGCCCTG-3′ (SEQ ID No. 3);

[0068] Downstream primer R: 5'-AGCCTTGGGCACTAGC-3' (SEQ ID No. 4).

[0069] (4) The recovered HAG18 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 enzyme digestion-ligation system. The HAG18 gene was ligated to the pBCXUN vector to prepare a HAG18 gene overexpression vector, and the expression of the HAG18 gene was driven by the Ubi promoter. The enzyme digestion and ligation system was referred to the instructions of the Takara kit.

[0070] The pBCXUN vector is based on the commercial vector pCAMBIA1300, into which the hygromycin resistance gene is ligated (Guo et al., 2018. Stepwise cis-regulatory changes in ZCN8 contribute to maize flowering-time adaptation. Current Bio. 28, 3005–3015). The promoter of the maize ubiquitin gene Ubi was cloned into the vector via enzyme ligation to drive transcription of the downstream overexpression gene.

[0071] (5) Take 5 μL of the product from the enzyme digestion-ligation system and transform competent E. coli. 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 obtained with correct sequencing is named pBCXUN-HAG18.

[0072] The obtained plasmid vector pBCXUN-HAG18 was digested with enzymes and then subjected to electrophoresis detection. The specific method was as follows: pBCXUN-HAG18 was digested with Xba I and Cla I, electrophoresed on a 1% agarose gel at 120 V and 50 mA, and scanned with a UVP Gel Documentation gel analysis system.

[0073] Example 2 Construction and detection of HAG18 gene overexpressing plants

[0074] The HAG18-pBCXUN vector prepared in Example 1 was transformed into Agrobacterium tumefaciens strain EHA105, which was then used to infect corn LH244 callus to obtain transgenic seedlings.

[0075] The specific method is as follows: Agrobacterium EHA105 containing the HAG18-pBCXUN vector is inoculated into 100 mL of LB triple-antibody liquid culture medium (Kan 50 μg / mL, Rif 50 μg / mL, Gen 50 μg / mL), cultured with shaking at 28°C overnight, and harvested by centrifugation at 50g for 15 minutes at room temperature until the OD600 value reaches 1.0-2.0. The cells are then suspended in 2 mL of transformation solution (1 / 2 MS, 5% sucrose, 40 μL Silwet L-77). Corn callus tissue is then immersed in the Agrobacterium transformation solution and sealed. The cells are then placed on a light-treated culture rack and grown normally until plants emerge. The resulting seeds are then screened and subjected to a low-temperature stress experiment.

[0076] In this example, the overexpression strains OE-1 and OE-2 with high expression levels of the HAG18 gene were isolated. Real-time quantitative PCR was used to detect the expression of HAG18 in the overexpression strains OE-1 and OE-2. The specific method is as follows:

[0077] 1) Extract total RNA from plants and reverse transcribe it to obtain cDNA.

[0078] 2) The reverse-transcribed cDNA was diluted 5-fold and subjected to real-time quantitative PCR using a Takara kit. The reaction system used included: 2× SYBR Premix ExTaq buffer, 0.2 μL DyII, 0.4 μL Primer (F1 / R1), and 2 μL cDNA template. The total volume was made up to 20 μL with ddH2O. After thorough mixing, the DNA was amplified in an ABI PRISM 75 real-time quantitative PCR instrument using a two-step PCR method. The reaction conditions were: 95°C for 30 s; 95°C for 5 s; 60°C for 40 s; and 40 cycles.

[0079] The sequences of primers F1 and R1 (primers for qRT-PCR) are as follows:

[0080] F1: 5'-TTGGTCCAAGAAGCAGAGGC-3' (SEQ ID No. 5);

[0081] R1: 5'-CCACGTTGTTTACGTCGCAG-3' (SEQ ID No. 6).

[0082] After the PCR reaction is completed, according to 2 -Δ(ΔCt) The relative expression between the wild type (WT group) and the overexpression strain (OE) was calculated and analyzed graphically. Three biological replicates were performed, and the three 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.

[0083] Example 3 Detection of low temperature resistance of plants overexpressing the HAG18 gene

[0084] First, seeds of the WT group (wild-type corn) and OE-1 and OE-2 obtained in Example 2 were sown in small pots 10 cm long, 10 cm wide, and 10 cm high containing black soil, imported soil, and vermiculite (mass ratio 1:1:1). Five seeds were placed in each pot, covered with 2 cm of soil, and placed in a tray. Water was applied until the soil was completely moistened and placed in a 23°C culture room 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 that, they were taken out and placed in a 23°C culture room to recover for two days. After that, they were photographed and samples were collected for ion leakage rate statistics. Three seedlings of each overexpression line (OE) and wild type (WT) were taken for measurement, and three biological replicates were performed.

[0085] The growth of the WT group and the corn overexpression lines after recovery from low temperature treatment is as follows Figure 2 As shown (the left picture is the control group without low temperature treatment, and the right picture is the experimental group after recovery from low temperature treatment). The results showed that the leaves of the wild-type WT severely wilted, dried up, and even could not 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 resistance phenotype.

[0086] In this example, ion leakage rates were calculated by measuring the relative conductivity of the leaves (L = (S1 - S0) / (S2 - S0) * 100%). An entire corn plant, after low-temperature treatment, was placed in a 15ml centrifuge tube containing 10ml of distilled water. The tube was evacuated with a vacuum pump for 30 minutes and then shaken at room temperature for 1 hour. The initial conductivity was measured using a conductivity meter, yielding S1. The sample was then placed in a boiling water bath for 15 minutes, removed from the tube, shaken for 2 hours, and the conductivity measured again, recording it as S2. S0 represents the conductivity of the distilled water blank.

[0087] 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 40.01% and 38.73%, 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 HAG18 gene can enhance the cold resistance of corn.

[0088] Table 1 Ion leakage rate values ​​(%) of three independent experiments

[0089] WT OE-1 OE-2 70.43996 33.47131 35.812445 77.50863 37.89532 40.302065 76.35223 32.88975 32.005677

[0090] The contents not described in detail in this specification belong to the prior art known to those skilled in the art.

[0091] Finally, it should be noted that the above description is merely a preferred embodiment 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 will be able to modify the technical solutions described in the aforementioned embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

Claims

1. Use of a HAG18 protein or a gene encoding the same, or a biological material containing the gene encoding the same, in any of the following aspects, achieved by increasing the expression level of the HAG18 protein in corn, wherein the amino acid sequence of the HAG18 protein is the amino acid sequence shown in SEQ ID NO. 2; (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 The cDNA nucleotide sequence of the HAG18 protein is the nucleotide sequence shown in SEQ ID NO.

1.

3. The use according to claim 1, characterized in that The biological materials are expression cassettes, vectors, and host cells.

4. A method for breeding cold-tolerant corn, characterized in that: By increasing the expression level of the HAG18 protein, a plant with improved cold tolerance is obtained; the amino acid sequence of the HAG18 protein is the amino acid sequence shown in SEQ ID NO.

2.

5. The breeding method for cold-tolerant corn according to claim 4, characterized in that: Detection ZmMYB121 The primers of the gene are shown as SEQ ID No.5-SEQ ID No.

6.

6. The breeding method for cold-tolerant corn according to claim 4, characterized in that: The corn is transformed by using Ti plasmid, plant virus vector, direct DNA transformation, microinjection, gene gun, electroporation, Agrobacterium-mediated method. HAG18 The recombinant expression vector of the gene is introduced into corn to obtain a transgenic corn strain.

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