Application of corn ZmERF4 gene in saline-alkaline stress tolerance

Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses an application of a corn gene ZmERF4 in saline-alkaline stress tolerance, the amino acid sequence of a protein coded by the ZmERF4 gene is shown as SEQ ID NO. 1, and the nucleotide sequence of the ZmERF4 gene is shown as SEQ ID NO. 2. When the ZmERF4 gene is over-expressed in corn, the salt stress resistance of the corn can be improved, and the ZmERF4 gene has a wide application prospect in the field of corn breeding.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a type of corn. ZmERF4 Application of genes in salt and alkali stress tolerance. Background Technology

[0002] Salt-alkali stress is one of the major environmental factors limiting crop growth and productivity. Globally, approximately 50% of irrigated land and 20% of arable land are affected by salt-alkali stress. The salt-alkali tolerance response mainly involves two aspects: deionization and osmotic regulation. When growing under salt stress conditions, plant roots absorb excessive sodium. + At the same time, suppress K + Absorption leads to K in the tissue + / Na + An imbalance in the ratio leads to ion poisoning, which in turn causes osmotic stress. Therefore, maintaining Na+ is crucial. + K + Homeostasis is of great significance for the formation of salt and alkali tolerance in plants.

[0003] Maize accounts for 40% of China's grain output and is an important crop for food, feed, and industrial raw materials. It is sensitive to salt stress. Previous studies have shown that natural maize populations possess rich genetic diversity, and differences in salt tolerance exist among different maize inbred lines, indicating abundant genetic variation within natural maize populations that can be applied to salt tolerance genetic improvement. With the increasing application of genome-wide association studies (GWAS) and quantitative trait genomics (QTL) analysis methods in the discovery of maize salt tolerance genes, a series of genetic loci associated with salt tolerance variations have been identified. However, to date, only a limited number of maize salt tolerance genes have been cloned, and the development and application of maize salt tolerance molecular markers are also relatively limited. Therefore, discovering and cloning high-quality salt tolerance gene resources and elucidating their salt tolerance mechanisms, developing new salt tolerance molecular markers, and applying them to salt tolerance breeding of maize have significant genetic resource support and application value for salt tolerance maize breeding. Discovering high-quality salt tolerance gene resources and breeding new salt tolerance maize varieties is of great significance to global food security and sustainable agricultural development. Summary of the Invention

[0004] To address at least one deficiency or improvement need in the prior art, the present invention provides a corn ZmERF4 The application of genes in salt and alkali tolerance stress ZmERF4 Genes can positively regulate the salt and alkali stress tolerance of maize. To achieve the above objective, according to one aspect of the present invention, a maize... ZmERF4 The application of genes in salt and alkali tolerance, the aforementioned ZmERF4 The amino acid sequence encoded by the gene is shown in SEQ ID NO.1.

[0005] Furthermore, the aforementioned ZmERF4 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0006] Furthermore, the aforementioned ZmERF4 Genes can improve maize's tolerance to carbonate stress.

[0007] According to another aspect of the invention, a gene expression cassette is also provided, the gene expression cassette comprising the aforementioned gene.

[0008] According to another aspect of the present invention, a recombinant expression vector is also provided, the recombinant expression vector comprising the gene expression cassette described above.

[0009] According to another aspect of the invention, a host cell is also provided, the host cell containing the recombinant expression vector, the host comprising any one of Escherichia coli, Agrobacterium tumefaciens, or non-renewable plant parts.

[0010] According to another aspect of the invention, a method for improving the salt and alkali stress tolerance of maize is also provided, comprising increasing the corn's... ZmERF4 The steps for determining gene expression levels, as described ZmERF4 The gene nucleotide sequence is shown in SEQ ID NO.2.

[0011] Furthermore, the method includes the following steps: (a) Introducing a gene encoding the protein of claim 1 into a plant cell, thereby causing the plant cell to express the protein of claim 1; (b) Regenerate a plant from the plant cells in step (a); wherein the plant is corn.

[0012] In another preferred embodiment, the plant is selected from the group consisting of: corn, rice, soybean, wheat, sorghum, or combinations thereof.

[0013] According to another aspect of the invention, a kit for improving the salt and alkali stress tolerance of plants is also provided, the kit comprising a vector or expression cassette expressing the gene; wherein the plant is maize.

[0014] According to another aspect of the present invention, the application of the described biomaterial, the described expression cassette, the described expression vector, the described host cell, and the method described therein in maize breeding is also provided.

[0015] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: This application provides a corn... ZmERF4 The application of genes in salt and alkali tolerance has proven ZmERF4 Genes can positively regulate the salt and alkali stress tolerance of maize, as shown by [the study / information]. ZmERF4It can increase the plant's tolerance to salt and alkali, and conversely, knocking out... ZmERF4 It will reduce the plant's tolerance to salt and alkali, as indicated by the presence of certain substances in the plant. ZmERF4 The gene can produce plants with excellent agronomic traits that can tolerate salt stress, providing new germplasm resources and breeding ideas for maize breeding, which is of great significance to global food security and sustainable agricultural development. Attached Figure Description

[0016] Figure 1 This is a diagram showing the results of key gene mining related to alkaline salt stress provided in the embodiments of the present invention; (A): Stress plasma model diagram; (B): Cluster diagram of differentially expressed genes; (C): Venn diagram of differentially expressed genes; (D): Diagram of differentially expressed gene significance analysis; (E): ZmERF4、ZmEREB111 and ZmEREB134 Gene expression levels; Figure 2 This is provided by the embodiments of the present invention. ZmERF4 Schematic diagram of the overexpression vector pXG011-ZmERF4-T01; Figure 3 This is provided by the embodiments of the present invention. ZmERF4 Schematic diagram of gene editing vector architecture; Figure 4 This is provided by the embodiments of the present invention. ZmERF4 Image of PCR detection results for overexpression strains; M: DL2000 Marker; 1: Plasmid control; 2-12: ZmERF4 Overexpression lines; Figure 5 This is provided by the embodiments of the present invention. ZmERF4 Image showing PCR detection results of gene-edited strains; M: DL2000 Marker; 1: Plasmid control; 1-6: Editing type 1; 6-12: Editing type 2 Figure 6 This is provided by the embodiments of the present invention. ZmERF4 Image showing the results of gene-edited strain editing; A: ZmERF4 Deoxyribonucleic acid (DNA) sequence editing status of gene-edited strains; B: ZmERF4 Amino acid sequence editing status of gene-edited strains; WT: wild type; KO-1: 81bp deletion genotype; KO-2: 82bp deletion genotype.

[0017] Figure 7 The wild-type corn provided in the embodiments of the present invention, ZmERF4 Overexpression lines and ZmERF4Phenotypic diagram of gene-edited lines growing under salt stress conditions; Figure 8 The wild-type corn provided in the embodiments of the present invention, ZmERF4 Overexpression lines and ZmERF4 Statistical analysis of biomass data of gene-edited strains under salt stress conditions. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0019] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0020] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0021] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0022] Example 1 Construction ZmERF4 Gene overexpression vectors and gene editing vectors 1. Discover and obtain key genes for salt stress tolerance in maize. To identify key genes in maize seedlings responding to salt-alkali stress, the inventors selected the three-leaf stage maize inbred line B73 and treated it with NaCl, NaOH, Na2CO3 stress solutions, and a control solution of water. The specific ion concentrations were as follows: Figure 1Figure A shows the settings based on the plasma model. Samples were taken after 5 h of stress treatment in the above solution for transcriptome sequencing analysis.

[0023] In all three stress conditions, water treatment was used as a control. Differentially expressed genes (DEGs) were screened based on FDR ≤ 0.01 and |log2Ratio| ≥ 1. The results showed that there were 3916 overlapping DEGs across the three stress conditions. Figure 1 C). We performed K-means clustering analysis on these DEGs, classifying them into 6 expression patterns. To further identify genes specifically responsive to alkaline salt stress (Na2CO3), we focused on Cluster 1: these genes maintained low expression levels under NaCl and NaOH stress treatments, but were significantly upregulated under Na2CO3 stress, comprising 678 DEGs ( Figure 1 B).

[0024] Because AP2 / ERF family members have been frequently reported in plant stress responses, the inventors further screened for three AP2 / ERF family genes in Cluster 1, namely... ZmERF4、ZmEREB134 and ZmEREB111 ( Figure 1 D). Expression profiles show that, ZmERF4 The expression pattern of this gene is most specific: it is significantly downregulated under NaCl and NaOH stress, and significantly upregulated under Na2CO3 stress. Figure 1 E). Therefore, we infer ZmERF4 Genes may be key candidate genes for specific responses to Na2CO3 alkaline salt stress.

[0025] In this invention ZmERF4 The gene nucleotide sequence, as shown in SEQ ID NO.1, is 723 bp in length and encodes a protein containing 241 amino acids, the amino acid sequence of which is shown in SEQ ID NO.2. Based on the above sequence information, the nucleotide sequence was artificially synthesized, and the full sequence synthesis was commissioned to Sangon Biotech (Shanghai) Co., Ltd.

[0026] 2. Construction of overexpression vectors and gene editing vectors To further verify ZmERF4 The functions of genes, this invention constructs respectively ZmERF4 Overexpression vectors and gene editing vectors, respectively, have effects on maize ZmERF4 Overexpression and gene knockout were performed to observe its ability to withstand salt and alkali stress.

[0027] 2.1 The overexpression vector in this invention is pXG011- ZmERF4 -T01, its carrier spectrum is as follows Figure 2As shown. The basic framework of this vector is pXG011. In one example, the construction process is roughly as follows: pXG011 plasmid was used BamH I, Hindi III. Double enzyme digestion to recover the vector fragment; in artificially synthesized... ZmERF4 Design on the fragment BamH I, Christmas I restriction site, amplified using the following primer pair. ZmERF4 Excerpt: Upstream primer F: GATGATGATAAAGGATCCATGGCGCCGCGCGTGGCC Downstream primer R: AACATCGTATGGGTACCCGGGGCTGCCGCCGGAGTTCTC Amplified ZmERF4 The fragments were respectively linked into the vector pXG011 BamH I, Hindi At site III, a plant overexpression vector was constructed. The recombinant plasmid was named pXG011- ZmERF4 -T01. The plant expression vector was modified from pXG011, and the plasmid contains a single independent T-DNA region, which includes... ZmERF4 and at least Multiple gene expression cassettes. at least The gene is driven by the p35s promoter. ZmERF4 Driven by the ubiquitin promoter Ubi (containing the ubiquitin 5' UTR region and the first intron) from maize. The recombinant plasmid is transmitted via... BamH I, Hindi III. Double enzyme digestion verification. Recombinant plasmid pXG011- ZmERF4 -T01 was transformed into Agrobacterium EHA105 for subsequent Agrobacterium-mediated transformation of maize embryos and callus tissue.

[0028] 2.2 In order to create corn ZmERF4 Gene editing materials were obtained through the website http: / / crispr.hzau.edu.cn / CRISPR2 / , and two gene editing targets were successfully designed using a dual-target editing strategy. ZmERF4 The target sites on the gene, with sequences as follows: 5'-CCTCGATCTCTTCCACCGGG-3' and 5'-GTAGAGGAAGTACTGATGTG-3'.

[0029] The target sequence was synthesized in its entirety by Sangon Biotech (Shanghai) Co., Ltd.

[0030] ZmERF4Construction of the gene editing vector: In this invention, the gene editing vector is pXG051-ZmERF4-KO, and its vector map is shown below. Figure 3 As shown. The basic framework of this vector is pXG051. In one example, the construction process is roughly as follows: The synthesized sgRNA was annealed using forward and reverse primers to form a double-stranded DNA fragment. The pXG051 plasmid was linearized, and the vector fragment was recovered. The annealed sgRNA double-stranded fragment was ligated into the pXG051 vector using T4 DNA ligase to construct a plant gene editing vector. The plant expression vector pXG051 is a binary vector containing a Cas9 gene, an sgRNA expression cassette, and a bar selection marker gene. The bar gene is driven by the P35s promoter, the Cas9 gene by the ubiquitin promoter Ubi (containing the ubiquitin 5'UTR region and the first intron) from maize, and the sgRNA by the U6 promoter. The recombinant plasmid was sequenced correctly using Sanger sequencing. The recombinant plasmid pXG051-ZmERF4-KO was transformed into Agrobacterium EHA105 for subsequent Agrobacterium-mediated transformation experiments of maize immature embryos and callus tissue.

[0031] 3. Construction ZmERF4 Transgenic maize with overexpression and gene editing This invention employs an Agrobacterium-mediated genetic transformation method to transform overexpression vectors and gene editing vectors into maize KN5585, respectively. The specific methods and procedures are as follows: 3.1 Callus preparation This type of embryogenic callus is characterized by rapid growth, soft texture, loose and brittle structure, and bright color. It can be subcultured for extended periods and retain its embryogenic capacity for a considerable time.

[0032] 3.2 Preparation of Agrobacterium 1) Before infection, Agrobacterium EHA105 containing pXG011-ZmERF4-T01 was spread on YEP solid medium and incubated in the dark at 28°C for 1-3 days. The cultured Agrobacterium was scraped off the plate and resuspended, and the OD550 was adjusted to 0.3 to prepare the infection solution for later use; 3) Activate Agrobacterium by shaking the prepared infection solution on a shaker at 28°C and 200 rpm for 2 hours for infection.

[0033] 3.3 Infection 1) Take corn ears of KN5585 9-12 days after pollination, remove the husks, disinfect with 75% alcohol for 10 minutes, and remove the embryos into centrifuge tubes containing 2mL of resuspension, 100 embryos per tube, for later use. 2) During infection, discard the resuspended solution in the centrifuge tube, add 2 mL of infection solution to the centrifuge tube, gently invert the centrifuge tube several times to mix, and let it stand in the dark at room temperature for 5 min. After infection, inoculate the embryos with the scutellum facing upwards into the co-culture medium and incubate in the dark at 20°C for 3 days; 3.4 Screening Transfer to resting medium and incubate in the dark at 28°C for 7 days. Then transfer to selection medium S1 containing 1.5 mg / L dialamidophos and incubate in the dark at 28°C for 2 weeks. If initial callus tissue has been obtained, transfer it to selection medium S2 containing 3 mg / L dialamidophos, and change the S2 medium every two weeks thereafter.

[0034] 3.5 Differentiation and Rooting When the selected resistant callus proliferates to about 2 cm in diameter, it is transferred to dark differentiation medium and cultured in the dark at 25°C for 2-3 weeks. The differentiated coleoptiles are then transferred to light differentiation medium and cultured in the light at 25°C for 2 weeks. Once the coleoptiles have formed complete seedlings and roots, the seedlings are transferred to culture bottles to promote root growth and seedling development.

[0035] 3.6 Hardening off seedlings and transplanting Ten days later, transplant the seedlings into nutrient pots and cultivate them in an indoor greenhouse. Once the seedlings have grown 1-2 new leaves, transplant them into large flowerpots and move them to a large greenhouse. Thereafter, manage them according to standard methods. After the male inflorescence sheds its pollen, perform self-pollination.

[0036] For positive seedlings, a small number of leaves were taken to extract genomic DNA using the TPS method. Genotyping of the knockout lines was performed by PCR and sequencing to identify mutant plants.

[0037] 4. Overexpression and gene editing of maize plants ZmERF4 Molecular detection of genes When the overexpression and gene-edited transgenic maize plants reached the 5-6 leaf stage, genomic DNA was extracted from the leaves of the plants. PCR amplification was performed using specific primers, and the PCR products were analyzed by agarose gel electrophoresis to detect bands. The overexpression plants were then used... ZmERF4 -OE primer detection, use of gene-edited plants ZmERF4 -KO primer detection.

[0038] Overexpression corn ZmERF4 The PCR amplification conditions for gene molecular detection are as follows: 95℃, 8 min; 95℃, 30 sec, 56℃, 30sec, 72℃, 45sec, a total of 38 cycles; 72℃, 5 min.

[0039] Gene-edited corn ZmERF4The PCR amplification conditions for gene molecular detection were: 95℃, 5 min; 95℃, 30 sec, 56℃, 30 sec, 72℃, 60 sec, for a total of 38 cycles; 72℃, 5 min.

[0040] The amplification products were electrophoresed on a 1.2% agarose gel, and the PCR amplification results were observed using a gel imaging system. The results are as follows: Figure 4 and Figure 5 As shown, ZmERF4 The expected bands were obtained by PCR electrophoresis of both overexpressing maize and gene-edited maize.

[0041] Will ZmERF4 The PCR products of gene-edited maize were recovered and sent to Sanger sequencing at Sangon Biotech (Shanghai) Co., Ltd. Analysis of the sequencing results revealed that... ZmERF4 Gene-edited corn has produced two types of gene editing, as follows: Figure 6 As shown, two gene editing types deleted 81bp and 82bp between the two target sites, respectively. After amino acid alignment of the edited sequences, editing type one (81bp deletion) produced a large amino acid deletion, while editing type two (182bp deletion) produced a frameshift mutation. Example 2 ZmERF4 Application of overexpression and gene-edited maize materials in salt stress tolerance To verify ZmERF4 The present invention will improve the salt stress tolerance of overexpressed and gene-edited maize materials. ZmERF4 Overexpression and gene-edited maize materials, as well as the control wild-type maize KN5585, were grown in the greenhouse of the Institute of Biotechnology, Jilin Academy of Agricultural Sciences, Gongzhuling City, Jilin Province.

[0042] Salt-alkali stress experiments were conducted on maize seedlings (three-leaf stage). Seeds were sown in seedling trays and germinated in a greenhouse at 25°C and 16 hours of light. When the maize seedlings had two leaves, wild-type, overexpression, and gene-edited lines with uniform growth were transplanted into pots and cultured until the three-leaf stage, where they were subjected to salt-alkali stress. A prepared 75 mmol / L Na₂CO₃ stress solution was applied to wild-type, overexpression, and gene-edited lines. ZmERF4 Overexpression and gene-edited maize materials were subjected to stress irrigation. After stress, the seedlings were weighed and photographed. Results are as follows: Figure 7 and Figure 8 As shown, from Figure 7 wild type, ZmERF4 The phenotypic diagrams of overexpression and gene-edited maize materials growing under salt stress conditions show that... ZmERF4 Overexpression-treated maize materials showed the best growth, while gene-edited maize materials showed the worst growth; Figure 8Biomass statistics after moderate stress show that ZmERF4 The overexpression maize material had the highest biomass, superior to the wild type, while ZmERF4 Gene-edited maize materials had the worst biomass. These results suggest that, compared to the wild type, the over-edited maize material... ZmERF4 It can increase corn's tolerance to salt and alkali, and conversely, knockout can increase its tolerance. ZmERF4 This reduces corn's tolerance to salinity and alkalinity, as proven by [the study / information]. ZmERF4 The gene has the ability to positively regulate maize's tolerance to salt and alkali stress.

[0043] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0044] SEQ ID NO.1 MAPRVADKSPLPLATGLKLGVGGGVGGMGLGPHYRGVRKRPWGRYAAEIRDPAKKSRVWLGTYDTAEEAAKAYDVAAREFRGAKAKTNFPFPLAVAVAGGAGSPSSDSTTLESSCGGSGC GVEAPVQAAMPLTPALDLDLFHRAAAVSAVTTGGMPPFFKAFPVVRPTPHQYFLYNQAAAAAAAGYRMFKVASAPVTVAAVAQSDSDSSSVVDRTCSPPAVTAKKEVSFELDLNWPPPAEN SEQ ID NO.2 ATGGCGCCGCGCGTGGCCGACAAGAGCCCCCTCCCGCTGGCCACCGGCCTCAAGCTGGGCGTGGGCGGCGGCGTGGGCGGCATGGGCCTGGGCCCCCACTACCGCGGCGTCCGCAAGAGGCCGTGGGGCCGCTACGCCGCGGAGATCCGCGACCCGGCCAAGAAGTCCAGGGTGTGGCTGGGCACCTACGACACGGCGGAGGAGGCTGCCAAGGCGTACGACGTGGCGGCGCGCGAGTTCAGGGGCGCCAAGGCGAAGACGAACTTCCCATTCCCCCTGGCGGTGGCCGTGGCTGGCGGCGCCGGCAGCCCCTCCAGCGACAGCACCACCCTGGAGTCCTCCTGCGGCGGCAGCGGCTGCGGCGTGGAGGCCCCGGTCCAGGCCGCGATGCCCCTGACGCCGGCCCTCGACCTGGACCTCTTCCACAGGGCCGCGGCCGTGTCCGCGGTCACCACGGGCGGCATGCCCTTCCCGTTCAAGGCGTTCCCGGTGGTCCGCCCCACCCCGCACCAGTACTTCCTCTACAACCAGGCGGCCGCGGCCGCGGCCGCGGGCTACAGGATGTTCAAGGTGGCGAGCGCCCCCGTGACCGTGGCCGCGGTCGCCCAGAGCGACTCCGACTCCTCCTCCGTGGTGGACCGCACCTGCTCCCCGCCGGCCGTGACCGCCAAGAAGGAGGTGTCCTTCGAGCTGGACCTCAACTGGCCGCCCCCGGCCGAGAAC

Claims

1. Corn ZmERF4 The application of genes in salt and alkali tolerance is characterized by, The ZmERF4 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The ZmERF4 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

3. The application as described in claim 1 or 2, characterized in that, The ZmERF4 Genes enhance maize's tolerance to carbonate stress.

4. A gene expression cassette, characterized in that, The gene expression cassette includes the genes described in claims 1 to 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector includes the gene expression cassette as described in claim 4.

6. A host cell containing the recombinant expression vector of claim 5, wherein the host includes any one of Escherichia coli, Agrobacterium tumefaciens, or a non-renewable plant part.

7. A method for improving the salt and alkali stress tolerance of maize, characterized in that, Including increasing the corn ZmERF4 The steps for determining gene expression levels, as described ZmERF4 The gene nucleotide sequence is shown in SEQ ID NO.

2.

8. The method as described in claim 7, characterized in that, The method includes the following steps: (a) Introducing a gene encoding the protein of claim 1 into a plant cell, thereby causing the plant cell to express the protein of claim 1; (b) Regenerate a plant from the plant cells in step (a); wherein the plant is corn.

9. A kit for improving the salt and alkali stress tolerance of plants, characterized in that, The kit includes a vector or expression cassette for expressing the gene as described in claims 1-3; wherein the plant is maize.

10. The application of the biomaterial as described in claim 3, the expression cassette as described in claim 4, the expression vector as described in claim 5, the host cell as described in claim 6, and the method as described in claims 7-8 in maize breeding.

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