Application of ZmDIR11 gene in regulation and control of salt stress resistance of corn

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a ZmDIR11 gene in regulating and controlling salt stress resistance of corn. After salt stress treatment, it is found that the ZmDIR11 gene mutant corn plant growth vigor and material accumulation are significantly lower than those of a wild type B73 material, an oxidative metabolism regulation path, proline accumulation, chlorophyll synthesis, lignan accumulation and a Na < + > / K < + > transport pathway are inhibited, and related metabolic indexes are significantly lower than those of the wild type B73 material. It is shown that the ZmDIR11 gene positively regulates and controls the salt-resistant metabolic pathway of the corn in the corn, and the salt tolerance of the corn is reduced after the ZmDIR11 gene is mutated. The research provides a new gene resource for corn salt-tolerant breeding by utilizing gene engineering.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and specifically relates to the application of the ZmDIR11 gene in regulating salt stress resistance in maize. Background Art

[0002] Salt stress refers to the high osmotic potential impact on plants when growing in a high salinity environment, including the inhibitory effects of free minerals in the soil such as Na + , K + , Mg 2+ , Cl ﹣ and other ions on the growth and development of plants, also known as soil salinization. In saline soil, the growth hindrance of crops is mainly affected by oxidative stress, ion damage, and osmotic stress, etc., which can lead to serious reduction in crop yields or even crop failures. Maize is one of the three major food crops in China, and its yield plays a crucial role in the country's food security and sustainable agricultural development. Therefore, exploring maize salt-tolerant genes, analyzing the maize salt-tolerant mechanism, and improving the salt tolerance of maize are very important for achieving the goals of food security and sustainable development of future agricultural production. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of the ZmDIR11 gene in regulating salt stress resistance in maize. ZmDIR11 positively regulates the salt stress resistance metabolic pathway in maize, providing a new gene resource for maize salt-tolerant breeding using genetic engineering.

[0004] The present invention provides the application of the ZmDIR11 gene in regulating salt stress resistance in maize, and the accession number of the ZmDIR11 gene is Zm00001d012432.

[0005] As a preferred embodiment, the regulation includes overexpressing the ZmDIR11 gene to improve the salt stress resistance ability of maize; inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene to reduce the salt stress resistance ability of maize.

[0006] As a preferred embodiment, the maize includes the maize B73 inbred line.

[0007] The present invention also provides a biological material capable of regulating the expression level of the ZmDIR11 gene, and the accession number of the ZmDIR11 gene is Zm00001d012432.

[0008] As a preferred embodiment, it includes biological materials overexpressing the ZmDIR11 gene. The biological materials overexpressing the ZmDIR11 gene include one or more of a primer set for amplifying the ZmDIR11 gene, a recombinant expression vector containing the ZmDIR11 gene, and a recombinant microorganism containing the ZmDIR11 gene.

[0009] As a preferred embodiment, it includes biological materials for inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene. The method of inhibition includes silencing.

[0010] The present invention also provides the application of the said biological materials in maize germplasm improvement or creation of new germplasms.

[0011] As a preferred embodiment, the creation of new germplasms includes creating maize with improved salt stress resistance and / or creating a maize model with reduced salt stress resistance.

[0012] The present invention also provides a method for creating maize with improved salt stress resistance, which includes the following steps: overexpressing the ZmDIR11 gene in the target maize genome to obtain the maize with improved salt stress resistance. The accession number of the ZmDIR11 gene is Zm00001d012432.

[0013] The present invention also provides a method for creating a maize model with reduced salt stress resistance, which includes the following steps: knocking down the expression level of the ZmDIR11 gene in the target maize genome to obtain the maize model with reduced salt stress resistance. The accession number of the ZmDIR11 gene is Zm00001d012432.

[0014] Beneficial effects: The present invention provides the application of the ZmDIR11 gene in regulating salt stress resistance in maize. After salt stress treatment, it was found that the growth potential and material accumulation of ZmDIR11 gene mutant maize plants were significantly lower than those of the wild-type B73 material, and the oxidative metabolism regulation pathway, proline accumulation, chlorophyll synthesis, lignin accumulation, Na + / K + transport pathway was inhibited, and the related metabolic indexes were significantly lower than those of the wild-type B73 material. It shows that ZmDIR11 positively regulates the salt stress resistance metabolic pathway in maize, and the salt tolerance of maize is reduced after being mutated. This study provides a new gene resource for maize salt tolerance breeding using genetic engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required to be used in the embodiments.

[0016] Figure 1Basic information of ZmDIR11 in Example 1, where A is that the ZmDIR11 protein contains 1 Dirigent domain; B is the protein structure prediction model of ZmDIR11; C is the schematic diagram of the analysis of abiotic stress-related promoter elements in the promoter region of ZmDIR11;

[0017] Figure 2 Basic information of ZmDIR11 in Example 1, where A is the alignment result of ZmDIR11 and partial maize DIR protein sequences; B is the predicted interaction network of the ZmDIR11 protein, with red representing direct interactions and white representing indirect interactions;

[0018] Figure 3 Phylogenetic analysis of DIR in maize (ZmDIRs), Arabidopsis (AtDIRs), rice (OsDIRs), and sugarcane (ScDIRs) in Example 1;

[0019] Figure 4 GO enrichment analysis of ZmDIR11 regarding Biological Process in Example 1;

[0020] Figure 5 Expression pattern of the ZmDIR11 gene in maize in Example 2, where A is the tissue expression pattern of ZmDIR11; B is the induced expression pattern of ZmDIR11 in maize leaves at different time points under salt stress. Wild-type B73 seedlings were treated with 200 mM NaCl at different times (0 h, 1 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h) for 12 days, and ZmActin1 was used as an internal control. Error bars represent ±SD (n = 3); C is the expression of the ZmDIR11 protein localized in the endoplasmic reticulum. The fusion vectors ZmDIR11-GFP, AtH2B-RFP, and AtWAK-HDEL-RFP were transiently expressed in tobacco leaves. GFP fluorescence is green, RFP fluorescence is red, and the Merge figure is the superimposed figure of green and red fluorescence;

[0021] Figure 6 DNA sequence and amino acid sequence alignment recognition map between the wild-type and mutant of ZmDIR11 in Example 3, where Figure A is the gene sequence alignment; Figure B is the amino acid sequence alignment;

[0022] Figure 7The mutation of ZmDIR11 in Example 3 reduces the salt tolerance of maize. Among them, A-E are the phenotypes and phenotypic data of WT and ZmDIR11 ems mutant maize plants under salt stress, including shoot length, root length, fresh weight, and dry weight data. 12-day-old maize seedlings were treated with 200 mM NaCl for 7 days. The error bars in B-E represent ±SD (n = 3). According to two-way ANOVA, ns indicates no significant difference, * indicates a significant difference at P < 0.05, and ** indicates a highly significant difference at P < 0.01;

[0023] Figure 8 ZmDIR11 in Example 4 enhances the antioxidant capacity of maize plants. Among them, A-C are the relative expression analysis of antioxidant metabolism-related genes ZmCAT3 (A), ZmSOD1 (B), and ZmPOD1 (C) before and after salt stress treatment; D-F are the contents of MDA (D), H 2 O 2 (E), and O 2- (F) in maize leaves under stress; G-I are the activities of SOD (G), CAT (H), and POD (I) in maize plants under salt stress. The error bars in A-I represent ±SD (n = 3). According to two-way ANOVA, ns indicates no significant difference, * indicates a significant difference at P < 0.05, and ** indicates a highly significant difference at P < 0.01;

[0024] Figure 9 The mutation of ZmDIR11 under salt stress in Example 5 reduces the accumulation of resistance-related metabolites. Among them, A is the content of proline (Pro); B is the content of total lignans; C is the content of chlorophyll. Seedlings treated with nutrient solution without NaCl under the same conditions were used as controls. The error bars in A-C represent ±SD (n = 3). According to two-way ANOVA, ns indicates no significant difference, * indicates a significant difference at P < 0.05, and ** indicates a highly significant difference at P < 0.01;

[0025] Figure 10 ZmDIR11 in Example 6 affects the transport of Na + and K + ions. Among them, A-C are the contents of Na + and K + and the ratio of Na + / K + . Maize seedlings were treated with 200 mM NaCl for 6 days, and the second true leaf was taken and dried to measure Na + and K +Content, with seedlings treated with nutrient solution without NaCl under the same conditions as the control; D - I are differentially expressed ion - transport - related protein genes in ZmDIR11 ems plants; the error bars in A - I represent ±SD (n = 3). According to two - way ANOVA, ns indicates no significant difference, * indicates a significant difference at P < 0.05, and ** indicates a highly significant difference at P < 0.01. Specific implementation manners

[0026] The present invention provides the application of the ZmDIR11 gene in regulating salt stress resistance in maize, and the accession number of the ZmDIR11 gene is Zm00001d012432.

[0027] As a specific implementation manner, the regulation includes overexpressing the ZmDIR11 gene to improve the salt stress resistance of maize; inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene to reduce the salt stress resistance of maize. As a specific implementation manner, the maize includes the maize B73 inbred line.

[0028] The present invention also provides a biological material capable of regulating the expression level of the ZmDIR11 gene, and the accession number of the ZmDIR11 gene is Zm00001d012432.

[0029] The biological material of the present invention has the function of regulating the expression level of the ZmDIR11 gene, such as overexpressing the ZmDIR11 gene, and inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene. As a specific implementation manner, the biological material for overexpressing the ZmDIR11 gene includes one or more of a primer set for amplifying the ZmDIR11 gene, a recombinant expression vector containing the ZmDIR11 gene, and a recombinant microorganism containing the ZmDIR11 gene. As a specific implementation manner, the recombinant expression vector containing the ZmDIR11 gene includes inserting the ZmDIR11 gene into an overexpression vector to obtain a recombinant overexpression vector. As an implementation manner, the recombinant microorganism containing the ZmDIR11 gene includes Agrobacterium containing the ZmDIR11 gene.

[0030] As a specific implementation manner, the biological material for inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene, the method of inhibition includes silencing. As a specific implementation manner, the vector for knocking out is a recombinant knockout expression vector obtained by inserting the ZmDIR11 gene knockout target into a knockout expression vector, and the recombinant microorganism includes Agrobacterium containing the recombinant knockout expression vector.

[0031] The present invention also provides the use of the said biological material in maize germplasm improvement or the creation of new germplasms. As a specific embodiment, the creation of new germplasms includes creating maize with improved salt stress resistance and / or creating a maize model with reduced salt stress resistance.

[0032] The present invention also provides a method for creating maize with improved salt stress resistance, comprising the following steps: overexpressing the ZmDIR11 gene in the target maize genome to obtain the maize with improved salt stress resistance, and the accession number of the ZmDIR11 gene is Zm00001d012432.

[0033] The present invention also provides a method for creating a maize model with reduced salt stress resistance, comprising the following steps: knocking down the expression level of the ZmDIR11 gene in the target maize genome to obtain the maize model with reduced salt stress resistance, and the accession number of the ZmDIR11 gene is Zm00001d012432. In the examples of the present invention, ZmDIR11 mutants ZmDIR11 ems-1 and ZmDIR11 ems-2 were obtained from the maize EMS mutant library (https: / / elabcaas.cn / ). As a specific embodiment, the mutation of ZmDIR11 caused the growth inhibition of the plant under salt stress to be significantly higher than that of the wild-type B73 material, and the mutation of ZmDIR11 affected the growth and dry matter accumulation of maize seedlings. After the mutation of ZmDIR11, the enzyme activities of CAT, SOD, and POD in the mutant materials were all weaker than those of the wild-type B73, indicating that ZmDIR11 enhances the ability of maize to resist salt stress by stimulating the function of antioxidant defense enzymes and reducing the level of reactive oxygen species.

[0034] As a specific embodiment, after salt stress treatment, it was found that the growth potential and material accumulation of the mutant maize plants were significantly lower than those of the wild-type B73 material, and the oxidative metabolism regulation pathway, proline accumulation, chlorophyll synthesis, lignin accumulation, Na + / K + transport pathway was inhibited, and the related metabolic indexes were significantly lower than those of the wild-type B73 material. The results showed that ZmDIR11 positively regulates the salt resistance metabolic pathway in maize, and the salt tolerance of maize is reduced after mutation.

[0035] To further illustrate the present invention, the following examples are used to describe in detail the application of the ZmDIR11 gene provided by the present invention in regulating maize salt stress resistance, but they should not be construed as limiting the protection scope of the present invention.

[0036] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0037] In the embodiments of the present invention, the test data was statistically analyzed using GraphPad Prism 8.0 software (GraphPad Software, USA), and the Tukey multiple comparison test method was used to test one-way or two-way analysis of variance to determine statistical significance. A difference with a P-value less than 0.05 was considered significant and marked as *; a difference with a P-value less than 0.01 was considered extremely significant and marked as **.

[0038] Example 1 Cloning and bioinformatics analysis of the ZmDIR11 gene

[0039] The gene and protein information of ZmDIR11 (Zm00001d012432) and other ZmDIRs (ZmDIR1, ZmDIR4, ZmDIR22, and ZmDIR-like6) was queried through MaizeGDB, and multiple alignments of the protein and nucleotide sequences were performed using DNAMAN software. The ZmDIR11 protein sequence was predicted for its model using the ExPASy online website (https: / / web.expasy.org / ). The schematic diagrams of the ZmDIR11 gene structure and protein domains were drawn using the IBS 2.0 online website (https: / / www.ibs.renlab.org / # / home). Additionally, the interaction network of the predicted DIR11 protein and GO enrichment analysis were queried in the STRING protein database (https: / / cn.string-db.org / ). Secondly, the 2000 bp sequence before the start codon of ZmDIR11 on the genome was retrieved, and the promoter elements of the ZmDIR11 promoter region were analyzed using the PlantCare online website (http: / / bioinformatics.psb.ugent.be / webtools / plantcare / html / ), and visual prediction was performed using Tbtools software. The DIRs sequences of other species were obtained by querying the NCBI (National Center for Biotechnology Information) database, sequence alignment was performed using MEGA X software, and the phylogenetic tree was constructed using Tbtools software.

[0040] In the early stage of this study, a gene Zm00001d012432 that was up-regulated by salt stress was obtained from the transcriptome data of fresh corn under salt stress, and it was named ZmDIR11. Through bioinformatics analysis, it was found that this gene is located on chromosome 8 of maize, and its gene structure contains only one exon, which is the classical structure of DIRs genes. After amplifying and sequencing the full-length gene sequence, it was found that the full length of this gene is 639bp, encoding a Dirigent protein composed of 212 amino acids. This protein contains a Dirigent domain, located in the peptide segment of amino acids 99-186 ( Figure 1 in A). Its protein prediction model also has the typical structural characteristics of Dirigent proteins ( Figure 1 in B). In addition, promoter element analysis showed ( Figure 1 in C) that there are 6 abiotic stress-related promoters including ARE, MYB, MYC, STRE, LTR and MBS in the promoter region of ZmDIR11. Among them, there are a total of 9 MYB promoter elements, indicating that this element can be specifically recognized and bound by MYB transcription factors. Research has shown that MYB transcription factors are the largest class of transcription factor families in plants, and one of their important functions is to participate in the response of crops to abiotic stress. In this study, the protein sequences of other partial maize ZmDIRs (ZmDIR1, ZmDIR4, ZmDIR22 and ZmDIR-like6) genes were also retrieved from the NCBI database, and they were aligned with the ZmDIR11 protein sequence, and it was found that they have similar conserved amino acid regions ( Figure 2 in A). In order to further explore the homology of ZmDIR11, the phylogenetic tree of ZmDIR11 was constructed using tbtools software and compared with other classical plant species (Arabidopsis thaliana, rice, sugarcane), and it was found that ZmDIR11 is highly homologous to OsDIR44 ( Figure 3 ). In addition, by searching the STRING protein database, the prediction showed that the ZmDIR11 protein may interact with three other maize DIR proteins (Zm00001d024969, Zm00001d004753 and Zm00001d026546), indicating that ZmDIR11 may function together with other DIR proteins ( Figure 2 in B). In addition, the GO enrichment analysis of the STRING database showed that the biological functions of ZmDIR11 are relatively concentrated in plant defense responses and phenylpropanoid biosynthesis processes ( Figure 4 ).

[0041] Example 2 Expression pattern and subcellular localization of ZmDIR11

[0042] Reverse transcription qPCR analysis: The maize (Zea mays L) material used in this study was the B73 inbred line (the B73 inbred line is a well-known inbred line in maize and has been widely used in genetic research and breeding due to its rich genetic background and high level of genetic recombination. The B73 inbred line originated in the early 20th century and was initially selected by the Connecticut Agricultural Experiment Station in the United States from an open-pollinated population named Lancaster Surecrop. After continuous self-crossing for many years, B73 has maintained the homozygosity and stability of genes and has become a standard reference inbred line for maize genetics and genomics research). Maize plants were planted in 10-cubic-centimeter black square pots filled with a mixture of peat soil, vermiculite, and perlite (volume ratio 1:1:1) and cultivated with 1 / 2 Hogland nutrient solution. In the salt stress experiment, when the maize reached the three-leaf stage, the control group was treated with 1 / 2 Hogland nutrient solution, and the stress group was treated with a 200 mmol / L NaCl solution prepared with 1 / 2 Hogland. Young leaves, leaves, roots, stems, silk, and pollen of maize without salt treatment were collected, and maize at different salt treatment time points (0 h, 1 h, 6 h, 12 h, 18 h, 24 h, 36 h, and 48 h) was collected. The second fully expanded true leaf from the top of the maize seedling stage was collected. Total RNA of maize leaves was extracted using the Tiangen TRIZon Total RNA Extraction Kit (DP405), and then the total RNA of maize was reverse transcribed into cDNA using the HiScriptⅢ All-in-one RT Supermix (Vazyme, Nanjing, China) kit. RT-qPCR was performed using the ChamQ SYBR color qPCR Master Mix kit (Vazyme). RT-qPCR analysis was completed using the Applied Biosystems fluorescence quantitative PCR analyzer (QuantStudioTM 6Flex) of Applied Biosystems, USA. ZmDIR11-RT-qPCR-F: 5'-TCGCACTCGCATCCTCAAGCAAGC-3' (as shown in SEQ ID NO.1), ZmDIR11-RT-qPCR-R: 5'-TCCCATCCAGAGGCAGCCCGAACT-3' (as shown in SEQ ID NO.2), and ZmACTIN1 was used as an internal reference gene.

[0043] Subcellular localization: The ZmDIR11 sequence was amplified and cloned into the vector pCAMBIA1305-GFP (Prutin (Beijing) Biotechnology Co., Ltd.; 8600612) (between the BamHI and SmaI sites). The AtH2B (At5g22880) sequence and the AtWAK-HDEL (At1g21250) sequence were respectively cloned into the vector pCAMBIA1305-RFP (Prutin (Beijing) Biotechnology Co., Ltd.; 8604537) (between the BamHI and SmaI sites). After constructing the vectors, the plasmids were extracted, transfected into competent Agrobacterium tumefaciens GV3101, and a mixture was prepared and injected into tobacco leaves that had grown to 4 weeks old. After 36 h of dark culture of the tobacco, laser confocal observation and photography were carried out. The method for constructing the subcellular localization experimental vector was referred to the literature of Sun Yu (Sun Yu. Functional analysis of maize ZmCCR-like5 in regulating salt tolerance [D]. Yangzhou University, 2023. DOI: 10.27441 / d.cnki.gyzdu.2023.000707.). The method for plasmid extraction was referred to the instruction manual of the plasmid mini extraction kit of Beijing Tiangen Biotech Co., Ltd. (product number DP103). The localization control protein H2B is a histone structure with nuclear localization (WOZNY M, SCHATTAT M H, MATHUR N, et al. Color Recovery after Photoconversion of H2B::mEosFP Allows Detection of Increased Nuclear DNA Content in Developing Plant Cells [J]. Plant Physiology, 2012, 158(1): 95-106.). The WAK-HDEL protein is localized in the endoplasmic reticulum. WAK is a cell wall-associated kinase protein (VERICA J A, HE Z H. The Cell Wall-Associated Kinase (WAK) and WAK-Like Kinase Gene Family [J]. Plant Physiology, 2002, 129(2): 455-459.). HDEL is a special endoplasmic reticulum retention polypeptide signal (TEASDALER D, JACKSON M R. SIGNAL-MEDIATED SORTING OF MEMBRANE PROTEINS BETWEEN THE ENDOPLASMIC RETICULUM AND THE GOLGI APPARATUS [J]. Annual Reviews Inc, 1996(12): 27-54.).

[0044] To better explore the function of the ZmDIR11 gene, the present invention used RT-qPCR to detect the tissue expression pattern of ZmDIR11. The results showed that ZmDIR11 was expressed to varying degrees in tissues such as maize young leaves, leaves, roots, stems, and pollen filaments, among which the transcriptional level was the highest in young roots and the lowest in stems ( Figure 5 in A). At the same time, we detected the expression of ZmDIR11 in the treated leaves under different salt stress treatment times (0 h, 1 h, 6 h, 12 h, 24 h, 36 h, and 48 h). As Figure 5 shown in B, the expression level of ZmDIR11 remained unchanged at each time point without treatment. However, when subjected to salt stress, the expression of ZmDIR11 increased over time, reached the highest level at 36 h, and decreased after 48 h.

[0045] Subcellular localization results represent the possible functional positions of proteins. To study the localization of ZmDIR11 in cells, the present invention performed transient protein expression operations using Nicotiana benthamiana leaves ( Figure 5 in C). In the existing literature, the control fusion protein AtWAK-HDEL-RFP was localized to the endoplasmic reticulum structure (ER) of tobacco epidermal cells (GOMORD V, DENMATL, FITCHETTE- A, et al. The C-terminal HDEL sequence is sufficient for retentionof secretory proteins in the endoplasmic reticulum(ER)but promotes vacuolartargeting ofproteins that escape the ER[J]. The Plant Journal, 1997, 11(2):313-325.). In CASTORINA G, DOMERGUE F, CHIARA M, et al. Drought-Responsive ZmFDL1 / MYB94 Regulates CuticleBiosynthesis and Cuticle-Dependent Leaf Permeability[J]. Plant Physiology, 2020, 184(1):266-282., the control fusion protein AtH2B-RFP was localized to the nucleus of tobacco epidermal cells. The results showed that the protein ZmDIR11 co-localized with AtWAK-HDEL-RFP on the endoplasmic reticulum ( Figure 5In (C). The endoplasmic reticulum is one of the organelles for metabolite synthesis in plant cells, indicating that ZmDIR11 may be involved in metabolite synthesis behavior.

[0046] Example 3 ZmDIR11 Positively Regulates Salt Tolerance in Maize

[0047] ZmDIR11 ems Establishment of mutant lines: The first-generation seeds of the ZmDIR11 mutant were obtained from the maize EMS mutant library (https: / / elabcaas.cn / ). The two mutants used in this experiment were EMS4-3f0544 (ZmDIR11 ems-1 ) and EMS4-15dd8f (ZmDIR11 ems-2 ). The detailed information table of gene mutations of the ZmDIR11-EMS mutant is shown in Table 1. The alignment of the ZmDIR11 gene sequence and protein sequence between the mutant and the wild type is shown in Figure 6 . After obtaining the mutant seeds, they were hybridized with the wild type B73 to purify the material background for two generations, and then self-crossed for two generations. The positive mutation sites were identified by PCR-agarose gel electrophoresis for each generation of mutation sites. The homozygous lines of each mutant were selected for salt stress phenotype analysis.

[0048] Table 1 Detailed Information Table of Gene Mutations of the ZmDIR11-EMS Mutant

[0049]

[0050]

[0051] To study the salt tolerance regulation mechanism of ZmDIR11 in maize, the present invention used 200 mM NaCl solution to treat the B73 wild-type seedlings and ZmDIR11 ems mutant seedlings (the treatment method was the same as the salt stress experiment treatment method in Example 2), and the control group was treated with 1 / 2 Hogland nutrient solution. Under normal growth conditions, there was no significant difference in the seedling growth characteristics between the wild type and the mutant materials. Under salt stress treatment, the wilting and chlorosis degree of the ZmDIR11 ems plants was higher ([[]] Figure 7 In A). At the same time, the growth indexes of various plants were measured, including the length of the above-ground part, root length, fresh weight and dry weight ([[]] Figure 7 In B-E) to analyze the regulation mode of ZmDIR11 in the process of maize salt stress tolerance.

[0052] Under the condition of no salt treatment in the control group, the wild type and ZmDIR11 ems-1 , ZmDIR11 ems-2The average above-ground part lengths of the mutant plants were 34.6 cm, 34.1 cm, and 33.5 cm respectively, and there was no difference between the wild-type and mutant plants. Under salt treatment conditions, the overall growth of the plants was inhibited. The average above-ground part length of the wild-type plants was 23.0 cm. In contrast, ZmDIR11 ems-1 and ZmDIR11 ems-2 The average above-ground part lengths of the mutant plants were only 15.1 cm and 14.6 cm respectively, and there was a significant difference between the wild-type plants and the mutant plants ( Figure 7 in B). Similarly, in the untreated control group, the average root lengths of the wild-type and ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants were 34.9 cm, 33.0 cm, and 35.2 cm respectively, and there was no significant difference in root length between the mutant and wild-type plants. After treatment, the root growth of the mutant plants was more severely inhibited. The average root lengths of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants were only 16.9 cm and 16.4 cm respectively, while the average root length of the wild-type plants was still 24.1 cm, which was significantly longer than that of the mutant plants ( Figure 7 in C). In addition, in the untreated control group, there was little difference in the average fresh weight between the wild-type and mutant plants. The average fresh weight of the wild-type was 4.81 g, and the ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants were 4.69 g and 4.75 g respectively. In the treatment group, the average fresh weight of the wild-type plants was 3.75 g. Compared with the 1.67 g and 1.32 g of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants, the difference was extremely significant. The mutant plants were more severely inhibited in growth under salt stress, and the accumulation of substances in the plants was slower ( Figure 7 in D). Similarly, there was little difference in the average dry weight between the wild-type and mutant lines in the control group. Under salt stress, the average dry weights of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants were 0.19 g and 0.21 g respectively, while the average dry weight of the wild-type plants was 0.36 g. At this time, the difference in the average dry weight between the mutant and wild-type was extremely significant ( Figure 7 in E). The above results show that the mutation of ZmDIR11 makes the growth inhibition of the plants under salt stress significantly higher than that of the wild-type B73 material, and the mutation of ZmDIR11 affects the growth and dry matter accumulation of maize seedlings.

[0053] Example 4 ZmDIR11 enhanced the antioxidant capacity of maize plants

[0054] Treat B73 wild-type seedlings and ZmDIR11 mutant seedlings at the three-leaf stage with a 200 mM NaCl solution (the treatment method is the same as the salt stress experiment treatment method in Example 2), and treat the control group with 1 / 2 Hogland nutrient solution. Detect the expression levels of the ZmCAT3, ZmSOD1, and ZmPOD1 genes (the detection method refers to the RT-qPCR method in Example 2). Measure the contents of MDA (malondialdehyde), Pro (proline), H ems O 2 O 2 (hydrogen peroxide), O 2- (superoxide anion) in maize leaves, as well as the activities of three enzymes, SOD (superoxide dismutase), CAT (catalase), and POD (peroxidase) in maize leaves. After treating the three-leaf stage seedlings with a 200 mM NaCl solution for 3 days (the control material is 1 / 2 Hogland nutrient solution), take the second true leaf and detect it according to the following method. All index detections are carried out using kits produced by Solarbio Science & Technology Co., Ltd., and the method is the micro-method, and an enzyme-linked immunosorbent assay (ELISA) reader is used for detection (the product number of the MDA detection kit is BC0025, the product number of the H 2 O 2 detection kit is BC3595, the product number of the superoxide anion content detection kit is BC1295, the product number of the SOD detection kit is BC0175, the product number of the CAT detection kit is BC0205, and the product number of the POD detection kit is BC0095).

[0055] Salt stress can also cause harm to plants such as oxidative damage and oxidative stress. To understand whether ZmDIR11 can regulate the oxidative damage metabolism of maize under salt stress, this study used RT-qPCR to analyze the relative expression levels of ZmCAT3, ZmSOD1, and ZmPOD1 (involved in the antioxidant enzyme defense pathway in maize) in wild-type B73 plants and ZmDIR11 ems plants before and after salt stress treatment, and measured the oxidative-related physiological indexes. The primer sequences are shown in Table 2. As Figure 8 shown in A-C, under normal treatment, there was no significant difference in the relative expression levels of ZmCAT3, ZmSOD1, and ZmPOD1 in the leaves of wild-type and mutant plants. After salt treatment, the relative gene expression levels of ZmCAT1, ZmCAT3, ZmSOD1, and ZmPOD1 in the antioxidant enzyme defense pathway in the leaves of mutant plants were extremely significantly lower than those of wild-type plants. The excessive accumulation of MDA, H 2 O 2 and O 2- will damage the cell membrane, affect plant growth, and weaken the plant's response ability to environmental stress. As Figure 8 shown in D-F, the MDA and H 2 O2 and O 2- accumulation was significantly higher than that of the wild type. Measuring the MDA content of the untreated control group showed that the MDA content of wild-type plants was 80.89 nmol / g, which was not significantly different from that of 80.23 nmol / g in ZmDIR11 ems-1 mutant plants and 75.23 nmol / g in ZmDIR11 ems-2 mutant plants. After salt stress treatment, the MDA content in ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants increased to 201.70 nmol / g and 226.32 nmol / g respectively, while the MDA content in wild-type plants increased to 159.07 nmol / g. The difference in MDA content between mutant plants and wild-type plants was significant after salt stress treatment ( Figure 8 in D). Similarly, measuring the H 2 O 2 content of the untreated control group found that the content in wild-type plant leaves was 2.38 μmol / g, and the contents in ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plant leaves were 2.42 μmol / g and 2.07 μmol / g respectively, with no significant difference. After salt stress treatment, the H ems-1 and ZmDIR11 ems-2 contents in mutant plants increased to 28.81 μmol / g and 30.41 μmol / g respectively. Compared with the H 2 O 2 content of 18.29 μmol / g in wild-type plant leaves, the difference in this index between mutant plants and wild-type plants was extremely significant after salt treatment ( 2 O 2 in E). Subsequently, we continued to measure the superoxide anion (O Figure 8 ) content in the leaves of plants in the untreated control group and the salt-treated group, and found that without salt treatment, the O 2- contents in wild-type and ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plant leaves were 2.16 μmol / g, 1.95 μmol / g and 2.05 μmol / g respectively, and there was no significant difference in O 2- content between wild-type plants and mutant plants. After salt treatment, the O 2- contents in ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plant leaves increased to 6.73 μmol / g and 6.95 μmol / g respectively. Compared with the O 2- content in wild-type plant leaves after treatment2- The content was 4.12 μmol / g. At this time, the difference in this index between the mutant and wild-type plants was extremely significant ( Figure 8 in F). These situations indicate that the mutation of ZmDIR11 led to an aggravated degree of damage to the plasma membrane, thereby increasing the oxidative damage suffered by maize. Oxidative damage caused by salt stress leads to the accumulation of reactive oxygen species (ROS). Three key enzymes, superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD), play important roles in scavenging excessive ROS. Therefore, we detected the activities of these three key enzymes. As Figure 8 shown in G-I, under normal conditions, the activities of CAT, SOD, and POD in the seedling leaves of wild-type and mutant plants were similar, and the differences were not significant. Among them, the CAT enzyme activity in the leaves of wild-type plants was 81.25 U / g, and the SOD enzyme activities of ZmDIR11 ems-1 and ZmDIR11 ems-2 were 88.91 U / g and 79.23 U / g respectively. The SOD enzyme activity in the leaves of wild-type plants was 45.75 U / g, and the SOD enzyme activities of ZmDIR11 ems-1 and ZmDIR11 ems-2 were 40.85 U / g and 37.28 U / g respectively. The POD enzyme activity in the leaves of wild-type plants was 44.76 U / g, and the SOD enzyme activities of ZmDIR11 ems-1 and ZmDIR11 ems-2 were 48.21 U / g and 51.28 U / g respectively ( Figure 8 in G-I). However, after salt treatment, the CAT enzyme activity of wild-type plants reached 422.56 U / g after treatment, while those of ZmDIR11 ems-1 , ZmDIR11 ems-2 mutant plants were only 330.17 U / g and 370.98 U / g respectively; the SOD enzyme activity of wild-type plants reached 104.23 U / g, while those of ZmDIR11 ems-1 , ZmDIR11 ems-2 mutants were 79.35 U / g and 76.24 U / g respectively; the POD enzyme activity of wild-type plants reached 1361.03 U / g after treatment, while those of ZmDIR11 ems-1 , ZmDIR11 ems-2The mutant plants only reached 950.43 U / g and 883.67 U / g respectively. Data tests showed that after salt treatment, the differences in the activities of these three enzymes between the mutant and the wild type were all extremely significant. After the mutation of ZmDIR11, the enzyme activities of CAT, SOD, and POD in the mutant materials were all weaker than those of the wild type B73. This part of the results indicates that ZmDIR11 enhances the ability of maize to resist salt stress by stimulating the function of antioxidant defense enzymes and reducing the level of reactive oxygen species, thereby enhancing the antioxidant damage of cells.

[0056] Example 5 ZmDIR11 regulates the accumulation of metabolites against salt stress

[0057] Take the control materials and the materials of the treatment group in Example 4. For the extraction of chlorophyll, use the second fresh leaf of maize seedlings. Cut the leaf into pieces, weigh 0.1 g of fresh leaf for extraction. The method selected is the organic solvent extraction method. The extraction reagent (acetone) is purchased from Shanghai Yuanye Company (product number: R30054). Take fresh plant leaves, wash them with distilled water, dry the surface moisture, remove the midrib, weigh about 0.1 g, cut into pieces and put them into a mortar. Add extraction powder (calcium carbonate + quartz sand + buffer salt, the volume ratio of the three is 1:1:1) (10 mg) and 1 mL of extraction solution (anhydrous ethanol and acetone are mixed at a volume ratio of 1:2), and grind thoroughly under dark or low-light conditions, then transfer it to a 10 mL test tube. Rinse the mortar with the extraction solution, transfer all the rinsing solutions into the 10 mL tube, make up the volume to 10 mL with the extraction solution, and soak it under dark conditions for 3 h. Observe that the bottom tissue residue is close to white, indicating complete extraction. If the tissue residue is not completely white, continue to soak until the tissue residue color is close to white. Then filter it through a 4-layer white gauze filter, take 200 μL of the supernatant after centrifugation and detect it on an enzyme-labeling instrument. Measure the absorbance values at A663 nm and A645 nm on the enzyme-labeling instrument, and use the formula: total chlorophyll content (mg / g) = (20.2A645 nm + 8.02A663 nm) × V / (1000 × W) to calculate the chlorophyll content (A663 nm and A645 nm represent the absorbance values at wavelengths of 663 nm and 645 nm; V is the volume of the extraction solution for different treatments, unit mL; W is the weight of fresh maize leaves, unit g). For the determination of the total lignan content, weigh 0.1 g of the second true leaf of maize frozen in liquid nitrogen, use methanol as the lignan extraction solvent, the solid-liquid ratio is 1:20 (g: mL), set the power of the ultrasonic extraction instrument to 250 W, and extract at 40 °C for 1 h. Then centrifuge and take the supernatant, and use a plant total lignan ELISA detection kit (Chemical Book, product number CB21405974) for detection. Finally, measure the absorbance value at 450 nm on the enzyme-labeling instrument. The content is calculated according to the standard curve regression equation (y = 0.235x + 0.0239, R 2Calculated as (n = 3, mean ± SD, p < 0.05, R² = 0.996). Proline (Pro) was detected according to the kit instructions (Solarbio; BC0295).

[0058] Under salt stress conditions, the accumulation of plant metabolites changes with the degree of salt stress, regulating the osmotic potential of its own cells, or enhancing related physiological activities to enhance the background resistance of the plant. Free proline (Pro), as a common osmotic regulator, its accumulation can help plants maintain the osmotic balance of cells and improve the stress resistance of plants. In this study, by measuring the experimental plants in the untreated control group, it was found that the Pro content in the leaves of wild-type plants was 19.23 μg / g, and the Pro content in the leaves of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants were 20.56 μg / g and 23.78 μg / g respectively, and there was no significant difference between the wild type and the mutant. After measuring the Pro content after salt stress treatment, it was found that the Pro content in the leaves of wild-type plants accumulated to 167.97 μg / g, while the Pro content in ZmDIR11 ems-1 and ZmDIR11 ems-2 mutant plants were 121.48 μg / g and 115.29 μg / g respectively, and the difference in the accumulation level of this metabolite between mutant plants and wild-type plants after salt treatment was extremely significant. As shown in Figure 9 A, compared with the wild type, the accumulation of Pro in mutant plants was significantly less. In addition, as shown in Figure 9 B, under normal treatment, the wild type and ZmDIR11 ems-1 and ZmDIR11 ems-2 mutants had similar accumulations of total lignan content, which were 35.47 mg / g, 32.65 mg / g, and 30.67 mg / g respectively. Under salt treatment, the total lignan content in all maize leaves increased, and the total lignan content in the leaves of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutants increased to 43.13 mg / g and 47.28 mg / g respectively. Compared with 56.28 mg / g in the leaves of wild-type plants, the total lignan content of mutant lines was significantly lower than that of the wild type. This may be because ZmDIR11 is involved in the synthesis of lignin and uses the antioxidant activity of lignin to enhance the salt tolerance of maize. At the same time, the chlorophyll level in the leaves of mutant plants decreased more after treatment ( Figure 9 C). The wild-type plants and ZmDIR11 ems-1 and ZmDIR11 ems-2The chlorophyll contents of the mutant plant leaves were 1.43 mg / g, 1.38 mg / g, and 1.41 mg / g respectively, with no significant difference. After treatment, the chlorophyll contents of both the wild type and the mutants decreased. The chlorophyll content in the wild type plant leaves decreased to 0.93 mg / g, while ZmDIR11 ems-1 and ZmDIR11 ems -2 The chlorophyll contents of the mutant plant leaves were 0.73 mg / g and 0.65 mg / g respectively. There was a significant difference in chlorophyll content between the mutants and the wild type after treatment. The above results indicate that in maize, ZmDIR11 positively regulates the metabolic accumulation processes of proline and lignin, weakens the loss of chlorophyll in maize leaves, and positively regulates the salt stress resistance of maize.

[0059] Example 6 ZmDIR11 affects the transport of Na + and K + and the expression of related ion transporter genes

[0060] Take the control materials and treatment group materials in Example 4. For the determination of Na + and K + , use the second true leaf of maize seedlings at the seedling stage. After drying at 70 °C in an oven for 2 days, grind and weigh 0.1 g of the sample and use flame photometry to determine the content. Grind each sample into powder and weigh about 0.1 g of each sample and add it to a 15 mL centrifuge tube. After all the samples are ready, add 10 mL of 100 mmol / L acetic acid, then place it in a water bath at 90 °C for 2 h, shake the test tube once every 10 min, then take out the sample and let it stand at room temperature. After the solution cools, filter the liquid through filter paper into a 25 mL volumetric flask and make up to 25 mL with deionized water; prepare standard solutions of Na + and K + with concentrations of 1 μg / mL, 5 μg / mL, 10 μg / mL, 50 μg / mL, and 100 μg / mL respectively. Finally, determine the concentrations of Na + and K + on a flame spectrophotometer.

[0061] Ion toxicity damage is the direct effect of salt stress on plants. The accumulation of Na + will damage the permeability of cell membranes. When plants are dealing with a high-salt environment, they usually pump Na + into the intracellular vacuoles for storage. At the same time, in order to cope with the disruption of the internal charge balance caused by the excessive accumulation of ions, plants will also absorb K + and H +To maintain charge balance. To explore whether ZmDIR11 regulates the salt tolerance of maize by affecting the contents of sodium and potassium ions, the sodium and potassium ion contents in the dry weight of maize leaves were measured. In this study, it was found through measurement that the ZmDIR11 mutant accumulated more sodium ions than the wild-type plants under salt stress. The sodium ion content in the leaves of the wild-type plants in the untreated group was 2.57 mg / g, and the sodium ion contents in the leaves of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutants were 2.61 mg / g and 2.49 mg / g respectively, and the difference was not significant at this time. After salt treatment, it was found through measurement that the sodium ion contents in the leaves of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutants increased to 46.43 mg / g and 51.51 mg / g respectively. Compared with the sodium ion content of 31.73 mg / g in the leaves of the wild-type plants after treatment, the difference between the mutants and the wild-type was extremely significant after treatment ( Figure 10 Figure A). On the contrary, the ZmDIR11 mutants had lower K + contents than the wild-type plants under salt stress ( Figure 10 Figure B). Under normal conditions, the potassium ion contents in the leaves of the wild-type and ZmDIR11 ems-1 and ZmDIR11 ems-2 mutants were 48.37 mg / g, 46.88 mg / g and 50.66 mg / g respectively. After salt stress treatment, the potassium ion content in the leaves of the wild-type plants decreased to 35.16 mg / g, and the potassium ion contents in the leaves of ZmDIR11 ems-1 and ZmDIR11 ems-2 mutants decreased to 27.14 mg / g and 25.14 mg / g respectively. At this time, the difference between the mutants and the wild-type was extremely significant after treatment. As shown in Figure 10 Figure C, the mutation of ZmDIR11 increased the ratio of Na + content to K + content in the mutant maize seedlings ( Figure 10 Figure C). ZmSOS1, ZmSOS2, ZmNHX1, ZmHAK4, ZmHKT1 and ZmHKT1;2 are a series of membrane transporter protein genes encoding the transport of Na + , K + and other ions. At the same time, primers were designed (see Table 2) to perform RT-qPCR to analyze the differences in the relative gene expression levels of these genes under normal treatment and salt stress treatment. It was found that under normal treatment, the relative expression levels of ZmSOS1, ZmSOS2, ZmNHX1, ZmHKT1, ZmHKT1;2 and ZmHAK4 were basically not different. Under salt treatment, the relative expression levels of each gene in the leaves of the mutant plants and the wild-type plants were extremely significantly different (Figure 10 In (D-I). This indicates that ZmDIR11 is involved in regulating ion transport modes to improve the salt stress resistance of plants.

[0062] Table 2 Information table of primers used in RT-qPCR

[0063]

[0064]

[0065] Thus, it can be seen that in this study, the ZmDIR11 gene was cloned, its gene characteristics and protein information were preliminarily analyzed, and the function of ZmDIR11 in maize salt stress tolerance was verified. Salt stress can rapidly induce the expression of ZmDIR11, and this gene can enhance the antioxidant enzyme activity, reduce the accumulation of MDA, H 2 O 2 and Na + , increase the contents of Pro and total lignans, reduce the degradation of chlorophyll, enhance the expression of related antioxidant enzymes and salt-resistant genes, so as to improve the salt tolerance of maize. This study provides a new gene resource for maize salt-tolerant breeding using genetic engineering.

[0066] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments without creative efforts based on this embodiment, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of ZmDIR11 gene in regulating corn salt stress resistance, characterized in that: The accession number of the ZmDIR11 gene is Zm00001d012432.

2. The use according to claim 1, characterized in that: The regulation includes overexpressing the ZmDIR11 gene to improve the salt stress resistance of corn; inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene to reduce the salt stress resistance of corn.

3. The use according to claim 1, characterized in that: The corn includes the corn B73 inbred line.

4. A biomaterial capable of regulating the expression of the ZmDIR11 gene, characterized in that: The accession number of the ZmDIR11 gene is Zm00001d012432.

5. The biomaterial according to claim 4, characterized in that The biological material overexpressing the ZmDIR11 gene comprises one or more of a primer set for amplifying the ZmDIR11 gene, a recombinant expression vector containing the ZmDIR11 gene, and a recombinant microorganism containing the ZmDIR11 gene.

6. The biomaterial according to claim 4, characterized in that The invention comprises biological materials for inhibiting the expression of the ZmDIR11 gene or knocking out the ZmDIR11 gene, wherein the inhibition method comprises silencing.

7. Use of the biological material according to any one of claims 4 to 6 in improving corn germplasm or creating new germplasm.

8. The use according to claim 7, characterized in that: The creation of new germplasm includes creating corn with improved resistance to salt stress and / or creating a corn model with reduced resistance to salt stress.

9. A method for producing corn with improved salt stress resistance, characterized in that: The method comprises the following steps: over-expressing the ZmDIR11 gene in the target corn genome to obtain the corn with improved salt stress resistance, wherein the accession number of the ZmDIR11 gene is Zm00001d012432.

10. A method for creating a corn model with reduced salt stress resistance, characterized in that: The method comprises the following steps: knocking down the expression level of the ZmDIR11 gene in the target corn genome to obtain the corn model with reduced salt stress resistance, and the accession number of the ZmDIR11 gene is Zm00001d012432.

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