Corn nadph quinone oxidoreductase 1 coding gene zm nqo1 and application thereof

Abstract

The application discloses a corn NADPH quinone oxidoreductase 1 coding gene ZmNQO1 and application thereof, and belongs to the technical field of genetic engineering. The application first discovers that the corn NADPH quinone oxidoreductase 1 coding gene ZmNQO1 regulates the salt stress tolerance of corn, so that ZmNQO1 as a target, the gene is transformed into the genome of corn by constructing a plant expression vector for overexpression of the gene, can drive the overexpression of the target protein, so that the corn plant can grow better under salt stress, and finally promotes the formation of crop yield under salt stress. The gene and the related application can be used for cultivating new corn salt-tolerant breeding materials, and provide a theoretical basis and key gene resources for further corn stress-resistant breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to a gene encoding maize NADPH quinone oxidoreductase 1. ZmNQO1 And its applications. Background Technology

[0002] Corn is one of the most widely cultivated and economically valuable crops globally, serving as a food, feed, and industrial raw material. Its kernels are not only a crucial component of human staple food but also a core feed source for livestock, and are widely used in numerous industrial sectors such as starch production, fuel ethanol, and bio-based materials. Against the backdrop of continuous global population growth and ever-increasing food demand, ensuring high and stable crop yields is of paramount importance for maintaining food security.

[0003] Global agricultural production is currently facing a severe abiotic stress challenge: soil salinization. Statistics show that the global area of ​​salinized land has exceeded 1 billion hectares, and due to factors such as improper irrigation, excessive fertilizer use, and climate change, this area continues to expand at a rate of approximately 10 million hectares per year, with the problem being particularly prominent in arid and semi-arid regions. Soil salinization directly leads to a reduction in arable land area, becoming a key bottleneck restricting crop yield increases.

[0004] Salt stress causes multidimensional and systemic damage to the growth and development of maize. Its harmful mechanisms are mainly manifested in the following three aspects: First, osmotic stress damage: High concentrations of sodium and chloride ions in the soil significantly reduce soil water potential, disrupting the osmotic pressure balance between plant roots and soil, leading to difficulty in root water absorption, cell shrinkage due to water loss, and physiological drought, thereby inhibiting seed germination, seedling rooting, and vegetative growth. Second, ion toxicity: Excessive salt ions are passively absorbed into the plant through the roots and accumulate in large quantities in cells, interfering with the homeostasis of essential minerals such as potassium and calcium, inhibiting the activity of various enzymes, damaging cell membrane integrity and fluidity, and causing cellular metabolic disorders. Third, oxidative stress damage: Salt stress induces a burst of reactive oxygen species (ROS) production in plant cells, including superoxide anions, hydrogen peroxide, and hydroxyl radicals. These ROS attack biomolecules such as nucleic acids, proteins, and lipids, causing DNA damage. Cellular fracture, protein denaturation, and cell membrane lipid peroxidation ultimately lead to cell structure damage and loss of function, resulting in yellowing, wilting, and stunted growth in plants, and in severe cases, direct plant death. In field production, corn under salt stress often exhibits reduced plant height, decreased biomass, reduced seed setting rate, and small, shriveled kernels, ultimately causing significant yield reductions and severely hindering the planting and promotion of corn in saline-alkali areas.

[0005] NADPH quinone oxidoreductase 1 (NQO1) is a key flavoprotein protease whose core function is to catalyze the two-electron reduction of quinone compounds using NADPH as an electron donor, generating hydroquinone, which is non-toxic to cells. It can participate in regulating important physiological processes such as apoptosis and stress signal transduction by modulating intracellular redox potential. Current research on plant NQO1 is mostly limited to gene expression; however, the functions of homologous genes may differ significantly or even be redundant across different species, and the specific biological role of NQO1 in gramineous plants, especially maize, remains unclear. Therefore, cloning and functionally validating NQO1 in maize is crucial. NQO1 Genes are of great theoretical and practical significance for enriching the salt tolerance gene resources of maize and for using these gene resources to breed new salt tolerance maize varieties. Summary of the Invention

[0006] In view of the current research status, the purpose of this invention is to provide the gene encoding NADPH quinone oxidoreductase 1. ZmNQO1 Its application in regulating salt tolerance in maize, overexpression ZmNQO1 The gene can significantly enhance the salt tolerance of maize, significantly improve the maize plant's tolerance to salt stress, effectively alleviate the inhibitory effect of salt stress on maize growth and development, ensure that maize maintains normal growth and achieves stable yield under salt stress, and thus provide key gene resources and core technology support for the breeding of new salt-tolerant maize varieties.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides corn ZmNQO1 The application of genes in the following (1) or (2): (1) Improve the salt tolerance of corn; (2) Breed salt-tolerant maize varieties; The corn ZmNQO1 A gene is a DNA molecule as shown in i), ii), or iii): i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 2; iii) A DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.2.

[0008] The term "identity" used here refers to sequence similarity to native nucleic acid sequences. Identity can be evaluated using computer software, such as the BLAST algorithm (Altschul). et al.1990. Journal of Molecular Biology 215:403-410; Karlin and Altschul. 1993. Proceedings of the National Academy of Sciences 90:5873-5877).

[0009] In the aforementioned nucleic acid molecules, the 90% or more identity can be at least 90%, 92%, 93%, 95%, 96%, 98%, or 99% identity.

[0010] In a second aspect, the present invention provides corn ZmNQO1 Application of gene-encoded proteins in improving salt tolerance in maize; the maize ZmNQO1 The protein encoded by the gene is the protein shown in either (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

[0011] In the aforementioned proteins, the protein tag refers to a polypeptide or protein fused with the target protein using in vitro DNA recombination technology for expression, detection, tracing, and / or purification of the target protein. The protein tag may be a Flag tag, His tag, MBP tag, HA tag, MYC tag, GST tag, and / or SUMO tag, etc.

[0012] Preferably, the improvement of corn salt tolerance specifically refers to any one or more of the following (1) to (4): (1) Under salt stress, it promotes the increase of maize plant height and biomass accumulation; (2) Reduce the level of reactive oxygen species accumulation in maize plants under salt stress; (3) Improve the growth phenotype of mature maize under salt stress, reduce the degree of leaf wilting and the degree of plant height inhibition; (4) Reduce the inhibition of salt stress on maize yield traits, resulting in better ear development, higher seed setting rate, and increased yield per plant.

[0013] In a third aspect, the present invention provides a method for promoting corn growth. ZmNQO1 Substances that enhance gene expression or improve corn ZmNQO1 The application of substances that encode protein activity and / or content in the following (1) or (2): (1) Improve the salt tolerance of corn; (2) Cultivate salt-tolerant maize varieties.

[0014] Preferably, the corn-promoting ZmNQO1 The substance used for gene expression is any one of the following: C1) contains ZmNQO1 Gene expression cassettes; C2) contains ZmNQO1 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains ZmNQO1 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains ZmNQO1 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains ZmNQO1 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1).

[0015] Existing plant expression vectors can be used to construct structures containing... ZmNQO1 Gene recombination vectors. Used ZmNQO1 When constructing recombinant vectors using genes, any type of enhancing or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter and the maize ubiquitin promoter. These can be used alone or in combination with other plant promoters. Furthermore, when constructing plant expression vectors using the genes of this invention, enhancers, including translational enhancers or transcriptional enhancers, can also be used. These enhancer regions can be ATG start codons or adjacent start codons, but they must be identical to the reading frame of the coding sequence to ensure correct translation of the entire sequence. Translation. The translation initiation region can originate from the transcription initiation region or a structural gene.

[0016] In a fourth aspect, the present invention provides a method for improving the salt tolerance of maize, comprising: inducing salt concentration in maize plants... ZmNQO1 The steps of gene overexpression.

[0017] In the above method, the corn plant is made to... ZmNQO1 Gene overexpression is achieved through the following methods: The exogenous transfer of the above-mentioned components into maize ZmNQO1 Recombinant gene expression vectors; or the introduction of genes that can activate or enhance maize... ZmNQO1 DNA fragments at the transcriptional level of genes.

[0018] In a fifth aspect, the present invention provides a method for breeding salt-tolerant maize varieties, comprising the following steps: Transition to wild-type maizeZmNQO1 Genes were used to obtain transgenic maize, which has a higher salt tolerance than wild-type maize; Salt-tolerant corn varieties can be bred by self-pollinating genetically modified corn with enhanced salt tolerance or by crossing it with other salt-tolerant corn varieties.

[0019] The beneficial effects of this invention are: This invention is the first to discover and verify the gene encoding maize NADPH quinone oxidoreductase 1. ZmNQO1 It has the function of positively regulating salt tolerance in maize. This is achieved through the construction of... ZmNQO1 Transgenic lines overexpressing the vector and transforming maize exhibited comprehensive growth advantages under salt stress: seedling height and biomass increased by up to 12.1% and 40.5%, respectively, with significantly lower reactive oxygen species accumulation levels than the wild type; in mature plants, under continuous salt stress, the overexpressing lines showed less growth inhibition and stable yield traits. Conversely, ZmNQO1 Gene knockout mutants exhibited stronger salt sensitivity. The aforementioned positive and negative experimental evidence collectively confirms this. ZmNQO1 The core role of genes is to provide key gene targets with clearly defined functions, proven and effective technical pathways, and a genetic material basis with potential breeding value for breeding new salt-tolerant maize varieties through molecular breeding methods (including transgenic and gene editing). Attached Figure Description

[0020] Figure 1 for ZmNQO1 Gene expression at 0, 1, 2, 3, 4, 5, and 6 h after salt stress.

[0021] Figure 2 for ZmNQO1 Gene editing, protein sequence alignment, and overexpression vector construction; among which, (A) using CRISPR / Cas9 technology for gene editing, protein sequence alignment, and overexpression vector construction. ZmNQO1 A schematic diagram of targeted gene mutation; the red and gray bars represent exons and introns, respectively, and the red letters indicate PAM sites, showcasing transgenic lines. ZmNQO1 KO1 and ZmNQO1 KO2 (A) Sequence alignment results with wild type; (B) Amino acid sequence alignment of ZmNQO1 protein in wild type and two knockout lines, with red asterisks indicating premature translation termination positions; (C) ZmNQO1 A schematic diagram of the construction of a gene overexpression vector.

[0022] Figure 3 for ZmNQO1 Expression levels in gene overexpression lines.

[0023] Figure 4 for ZmNQO1Phenotypic analysis of seedlings under salt stress; (A) Comparison of growth status of wild-type, overexpressed and knockout lines under salt stress; (B) Statistical results of plant height; (C) Statistical results of aboveground fresh weight (biomass).

[0024] Figure 5 for ZmNQO1 Accumulation of reactive oxygen species in mutant strains under salt treatment.

[0025] Figure 6 for ZmNQO1 Analysis of oxidative stress indices in mutant strains under salt stress; among which Figure 6 In Figure A, the change in hydrogen peroxide content under salt stress conditions is represented; where... Figure 6 B represents the change in malondialdehyde content under salt stress conditions.

[0026] Figure 7 for ZmNQO1 Morphological changes in mature plants of gene mutant lines under salt stress; among which Figure 7 In section A, plant morphology is compared between wild-type, knockout mutant and overexpression lines under continuous salt stress. Figure 7 Figure B represents the statistical results of plant height under continuous salt stress for wild-type, knockout mutant, and overexpression lines.

[0027] Figure 8 Under salt stress ZmNQO1 Analysis of ear phenotype and yield of mature mutant plants; (A) comparison of ear morphology between wild type and mutant lines after salt stress treatment; (B) statistical analysis of single ear yield. Detailed Implementation

[0028] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0029] As mentioned earlier, improving the salt stress tolerance of maize is one of the urgent problems to be solved. Plant NADPH quinone oxidoreductases (NQOs) are widely involved in processes such as redox balance regulation and abiotic stress response. Based on this, this invention conducted an in-depth study on maize NADPH quinone oxidoreductase-related genes, and the results showed that the gene encoding maize NADPH quinone oxidoreductase 1... ZmNQO1 Positive regulation of maize's tolerance to salt stress.

[0030] The gene encoding NADPH quinone oxidoreductase 1 ZmNQO1Located on chromosome 9, the gene is 782 bp in length and consists of 3 exons and 2 introns. Sequence analysis shows that the coding region (CDS) sequence of this gene is as shown in SEQ ID NO.1, as follows: .

[0031] ZmNQO1 The encoded protein contains 200 amino acids, and its amino acid sequence is shown in SEQ ID NO.2; specifically as follows: MEAPAAKRVLRVAAISGSIRKASWHSGLIRAAAEVCEDCIPGLRVDHLDVADLPMLNTDLETNGGTGFPPAVEAFRAKVRHADCFLFASPEYNYSITSPLKNALDWASRGVNCWADKPAAIVSAGGNFGGGRSSYHLRQVGVFLDIHFINKPELFVFAFFEPAKFFDSDGNLIDADTRERLKQVLLSLEAFTRRLQNNKD.

[0032] Conserved domain analysis of the NQO1 protein was performed using SMART (http: / / mart.embl-heidelberg.de / ), and the results showed that the NQO1 protein has an FMN-red domain at amino acid positions 10-160.

[0033] For research ZmNQO1 The inventors constructed the functional framework using pBUE411-2gR as the carrier. ZmNQO1 Gene knockout vectors were used to genetically transform maize immature embryos via Agrobacterium-mediated transformation, resulting in two... ZmNQO1 Transgenic mutant lines. Compared to wild type, ZmNQO1 KO1 The mutant strain had 11 bases removed from the first PAM site and 2 bases removed from the first PAM site, resulting in a mutation in the encoded protein from amino acid position 59 to 100, and premature termination of translation at amino acid position 100. ZmNQO1 KO2 The mutant strain had 2 bases removed from the first PAM site and 2 bases added to the first PAM site, resulting in a mutation in the encoded protein from amino acid position 61 to 103, and premature termination of translation at amino acid position 103.

[0034] These two ZmNQO1 The amino acid sequences of the NADPH quinone oxidoreductase ZmNQO1 in the gene mutant line are shown in SEQ ID NO.3 and SEQ ID NO.4, respectively; specifically as follows: ZmNQO1 KO1 (SEQ ID NO.3): MEAPAAKRVLRVAAISGSIRKASWHSGLIRAAAEVCECDCIPGLRVDHLDVADLPMLNTDRWYGLPARRRGVPRQGPPCRLLPLRLARIQLLHHQPTEERA.

[0035] ZmNQO1KO2 (SEQ ID NO.4): MEAPAAKRVLRVAAISGSIRKASWHSGLIRAAAEVCECDCIPGLRVDHLDVADLPMLNTDLERWYGLPARRRGVPRQGPPCRLLPLRLARIQLLHHQPTEERA.

[0036] ZmNQO1 KO1 and ZmNQO1 KO2 The mutation caused premature termination of protein translation, which disrupted the FMN-red domain of the ZmNQO1 protein. This demonstrated, from a genetic manipulation perspective, that the integrity of the ZmNQO1 protein and its FMN-red domain play a crucial role in the regulation of salt tolerance in maize.

[0037] Functional validation experiments further support the above conclusions. Under salt stress conditions, compared with the wild type, ZmNQO1 The overexpression lines showed significantly increased plant height and biomass, indicating enhanced salt tolerance. Simultaneously, physiological analysis revealed significantly lower levels of reactive oxygen species (ROS) accumulation in the overexpression lines compared to the wild type, suggesting... ZmNQO1 It is possible that this can alleviate oxidative damage caused by salt stress by enhancing the antioxidant capacity of cells. Correspondingly, the aforementioned mutants lacking the FMN-red domain ( ZmNQO1 KO1 , ZmNQO1 KO2 Under salt stress, the wild type exhibits more severe growth inhibition and ROS accumulation, indicating enhanced salt sensitivity. Both positive and negative genetic and phenotypic evidence confirm this. ZmNQO1 This gene plays a key positive regulatory role in regulating salt tolerance in maize. Salt stress experiments at the mature plant stage also showed that overexpressed lines maintained better growth and yield traits, further highlighting the potential of this gene in maize stress resistance breeding.

[0038] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0039] All experimental materials used in the embodiments of this invention are conventional experimental materials in the art and can be purchased through commercial channels. This invention introduces expression vectors into plant cells using methods well-known to those skilled in the art, including but not limited to: Agrobacterium-mediated transformation, gene gun method, electroporation method, ovary injection method, etc. Experimental operations not described in detail in this invention are all prior art techniques.

[0040] The maize genetic transformation material used in this invention is the B104 inbred line.

[0041] Example 1: The gene encoding NADPH quinone oxidoreductase 1 ZmNQO1 Cloning according to ZmNQO1 The gene sequence number Zm00001eb394340 was retrieved from the MaizeGDB database (https: / / www.maizegdb.org / ) to obtain the cDNA sequence of the gene, which was used for primer design and screening for gene cloning.

[0042] Primers for PCR amplification were designed using PRIMER 5.0 software. ZmNQO1 The gene is located on chromosome 9, with a total length of 782 bp, consisting of 3 exons and 2 introns. Sequence analysis showed that the coding region of the gene is 603 bp in length, as shown in SEQ ID No. 1. The encoded ZmNQO1 protein contains 200 amino acids, and its amino acid sequence is shown in SEQ ID No. 2. SMART analysis of the conserved domains of the ZmNQO1 protein (http: / / mart.embl-heidelberg.de / ) revealed the presence of an FMN-red domain at amino acids 10-160.

[0043] ZmNQO1 -CDS-F:5'ATGGAAGCACCGGCTGCGAAG3'; (SEQ ID NO.5) ZmNQO1 -CDS-R:5'TCAGTCCTTGTTGTTCTGGAGC3'. (SEQ ID NO.6) Example 2: ZmNQO1 Analysis of gene expression patterns under salt stress (1) Material treatment: Select plump corn kernels of similar size and diameter, and sow them in small pots filled with vermiculite, with 5 corn kernels sown in each pot at a depth of about 1.5-2 cm. After watering thoroughly, cultivate them in an artificial climate chamber (16 h of light, 8 h of darkness, temperature 27℃, relative humidity 54%). After two weeks of natural growth of wild-type corn plants, salt-alkali stress treatment was applied. Excess water in the bottom tray was poured out and the plants were thoroughly watered with a 150 mM NaCl:Na2SO4=9:1 solution. Corn roots were collected at 1 h, 2 h, 3 h, 4 h, 5 h and 6 h after stress treatment, and real-time fluorescence quantitative PCR experiments were performed.

[0044] (2) qRT-PCR analysis: ZmNQO1Gene expression levels were detected using quantitative real-time immunoassay. The detection method was as follows: The SuperRealPreMix Plus (SYBR Green) real-time PCR reagent from Mona Biotechnology Co., Ltd. was used. The reaction system preparation process must be carried out in the dark. Three biological replicates and three technical replicates were set up for each sample to ensure the reliability of the experimental data. Samples were placed in a Roche LightCycler 96 real-time PCR instrument. The primers for quantitative detection were: ZmNQO1 -qRTPCR-F: 5'-CTTGACTGGGCTTCTAGAGGAGT-3'; (SEQ ID NO.7) ZmNQO1 -qRTPCR-R: 5'-TACGAACAGTTCCGGCTTGT-3'. (SEQ ID NO.8) (3) Results: The results show that, ZmNQO1 The gene expression level was relatively high before salt stress treatment, but within 0-1 h after salt stress treatment, ZmNQO1 Gene expression was significantly downregulated within 2-6 hours after salt stress treatment, compared to before treatment. ZmNQO1 Gene expression levels continue to increase ( Figure 1 ).

[0045] Example 3: Creation ZmNQO1 Mutant strains Using maize B104 inbred line recipient material, to pBUE411-2gR Construction of the carrier skeleton ZmNQO1 Gene CRISPR / Cas9 knockout vectors were used for genetic transformation of maize immature embryos via Agrobacterium-mediated transformation, resulting in... ZmNQO1 Gene mutation strain.

[0046] 1. Construction of CRISPR / Cas9 knockout vector: The construction of CRISPR / Cas9 vectors mainly follows the methods described in the literature (Xing et al., 2014), and the steps are as follows: (1) Target design: Select a 19bp specific sequence from the CDS sequence of the target gene, and use the sequence to predict the target on the website (http: / / crispor.tefor.nev) to select a suitable target (PAM1 sequence: 5'-CCGACCTCGAGACGAACGGTGG-3', PAM2 sequence: 5'-TTCTAGAGGAGTGAACTGCTGG-3'). (2) Design primers based on the target sequence according to the following structure: CAP-F: aagagttgtgcagatgatccgtGGCGCCGACCTCGAGACGAACGG; (SEQ ID NO.9) MT1T2-F: GCCGACCTCGAGACGAACGGGTTTTAGAGCTAGAAATAGC; (SEQ ID NO.10) MT1T2-R: GCAGTTCACTCCTCTAGAACGCTTCTTGGTGCC; (SEQ ID NO.11) CAP-R: taacttgctatttctagctctaAAACGCAGTTCACTCCTCTAGAA; (SEQ ID NO. 12) (3) PCR amplification: Using pCBC-MT1T2 as a template, four primers were used for amplification; (4) After the PCR product is recovered and purified, enzyme digestion and ligation are performed according to the reaction system shown in Table 1. The prepared reaction system mixture is gently inverted and mixed, and then centrifuged briefly. Table 1: Enzyme digestion and ligation reaction system (5) The reaction mixture was incubated at 37°C for 5 hours, at 50°C for 5 minutes, and at 80°C for 10 minutes. (6) Take 5 μl of the reaction solution to transform Escherichia coli, and screen and identify positive clones; (7) Extract plasmids and perform sequencing verification.

[0047] 2. Genetic transformation of maize immature embryos using Agrobacterium-mediated transformation. (1) Preparation of Agrobacterium competent cells: Take 50 μL of Agrobacterium EHA105 competent cells from a -80°C freezer and thaw them in ice for 5 min. Add 5 μL of the plasmid that has been verified to be correct for sequencing to a centrifuge tube, gently tap to mix, and let stand on ice for 5 min. Quick freeze with liquid nitrogen for 5 min, then treat in a 37°C water bath for 5 min, and quickly immerse in ice for 5 min. Transfer 1 mL of YEP to a centrifuge tube and incubate in a 28°C shaker for 1 h at a speed of 180 rpm. Then centrifuge at 5000 rpm for 1 min. In a clean bench, discard most of the supernatant, resuspend the bacterial culture, and transfer it to a pre-prepared YEP plate containing the corresponding antibiotic. Spread evenly, seal the plate, and invert it in a 28°C incubator for 48 h-72 h before identification.

[0048] (2) Using maize inbred line B104 as material, Agrobacterium-mediated genetic transformation was carried out.

[0049] T0 transformants were screened to identify CRISPR / Cas9-induced target gene mutations. Self-pollination was performed from T0 to T2 generations. Herbicide screening was conducted. DNA was extracted from leaves of transplanted plants and analyzed by PCR. The upstream and downstream primers were: ZmNQO1 -cas9JD-F:5'ACATCCGCCATATTCAGGCAT3'; (SEQ ID NO.13) ZmNQO1 -cas9JD-R:5' TACGAACAGTTCCGGCTTGT 3'. (SEQ ID NO.14) Ultimately, two different editing types were obtained, and both lines stably inherited CRISPR / Cas9 transgene knockout lines lacking the Cas9 vector backbone were named [names omitted]. ZmNQO1 KO1 and ZmNQO1 KO2 Compared to the wild type, ZmNQO1 KO1 The transgenic knockout line produced a 10-base deletion near the NGG site of PAM1 (Protospacer-adjacent motif) and a 2-base deletion near the TGG site of PAM2, resulting in a mutation in the encoded protein from amino acid position 60 to 100, and premature termination of translation at amino acid position 100. ZmNQO1 KO2 The transgenic knockout line produced a two-base deletion near NGG at the PAM1 site and a one-base insertion near TGG at the PAM2 site, resulting in a mutation from amino acids 62 to 103 of the encoded protein, and premature termination of translation at amino acid 103. This indicates two types of editing. ZmNQO1 Structural alterations and loss of function of the ZmNQO1 protein in transgenic knockout lines (see...) Figure 2 AB).

[0050] Example 4: Creation ZmNQO1 Gene overexpression lines Using maize root cDNA as a template, 2×Taq Plus Master Mix II was used to... ZmNQO1 Amplify the coding sequence (CDS) by adding the following reaction components to the PCR tube in the order shown in the table; briefly centrifuge the prepared reaction system to mix, and then amplify according to the reaction procedure in Table 2. Table 2 PCR reaction system (3) After the reaction, the PCR product was removed and detected by a 1% (v / w) agarose gel. The PCR product was purified using the Cisco gel recovery PCR product purification kit. According to the reaction system shown in Table 3, the reaction solution was added to the PCR tube in sequence, gently mixed, and placed in a 37℃ water bath for 45 min. The overexpression vector was double-digested with enzymes. After the reaction, the digested products were detected by electrophoresis and the gel was cut and recovered. Table 3 Enzyme digestion system Using maize B104 inbred line recipient material, to pCAMBIA 3300 Construction of the carrier skeleton ZmNQO1 Gene overexpression vectors were used for genetic transformation of maize immature embryos via Agrobacterium-mediated transformation, resulting in... ZmNQO1 Gene overexpression lines.

[0051] Use the Ubiquitin starter driver ZmNQO1 Constitutive overexpression of CDS. The cauliflower mosaic virus (CaMV) 35S promoter drives bar gene expression as a herbicide resistance selection marker (see...). Figure 2 C). Purified ZmNQO1 The CDS fragment was ligated to the overexpression backbone vector digested with SmaI enzyme. The ligation product was transformed into competent *E. coli* cells, and positive clones were screened on LB agar plates containing kanamycin. The recombinant overexpression plasmid was obtained by colony PCR and sequencing verification. *Agrobacterium*-mediated maize genetic transformation was performed according to the method described in Example 3. Detection ZmNQO1 Overexpression plants ZmNQO1 Gene expression levels were measured using the same method as in Example 2. Results showed that the gene expression levels created using the recombinant vector... ZmNQO1 OE-1 and ZmNQO1 OE-2 The plant, its ZmNQO1 Gene expression levels increased more than 3-fold (see...) Figure 3 ).

[0052] Example 5: ZmNQO1 Identification of salt tolerance traits in seedlings of transgenic lines Based on the laboratory standards for identifying salt tolerance in maize seedlings, a 150 mM NaCl:Na₂SO₄ solution of 9:1 was used. To observe the growth status of plants under stress, plastic trays filled with an equal weight of a mixture of nutrient soil and vermiculite were irrigated with the salt solution until the soil was saturated. Overexpression lines of maize with similar growth after 3 days of germination were selected. ZmNQO1 OE-1 , ZmNQO1 OE-2 , ZmNQO1 KO-1 ,ZmNQO1 KO-2 Wild type (B104) was planted evenly in plastic trays at a depth of 2-3cm, with 12 plants per tray, and cultivated in an artificial climate greenhouse for 10 days.

[0053] Under normal conditions, wild type and ZmNQO1 There was no significant difference in growth among the transgenic lines. However, under salt stress, the wild-type was significantly smaller, and the overexpression lines showed significantly better growth than the wild-type and knockout lines. Figure 4 The plant height of the overexpressing lines increased by 12.1% and 8.5% compared to the wild type, respectively. In contrast, the plant height of the knockout lines decreased by 19.0% and 17.6% compared to the wild type, respectively. Furthermore, compared to the wild type, the biomass of the overexpressing lines under salt stress increased by 40.5% and 32.4%, respectively. In contrast, the biomass of the knockout lines decreased by 48.6% and 47% compared to the wild type, respectively. This indicates that overexpression... ZmNQO1 Genes can significantly improve the salt and alkali tolerance of corn.

[0054] Example 6: ZmNQO1 Accumulation of reactive oxygen species in transgenic lines Because reactive oxygen species accumulate in plant cells under salt stress, we studied the accumulation of hydrogen peroxide (DAB staining) and superoxide anion (NBT staining) in wild-type and... ZmNQO1 Reactive oxygen species levels in roots of transgenic lines under normal and salt stress conditions.

[0055] I. ROS accumulation in histochemical staining analysis 1. DAB staining method (1) Select plump and uniformly sized corn kernels and plant them in a plant growth chamber under long-day conditions (28℃, 16 h light / 8 h dark). After normal watering and 7 days of growth, treat with 150mM NaCl:Na2SO4=9:1 for 12 h. Then take the second corn leaf for subsequent staining. (2) Prepare 0.1 M phosphate buffer (pH 6.5), add 5 mL of phosphate buffer to a 10 mL centrifuge tube, weigh 5 mg of DAB powder and dissolve it in the phosphate buffer (mg / mL), wrap the centrifuge tube with aluminum foil, vortex to mix, and soak the corn leaves in the phosphate buffer containing DAB powder. (3) Vacuum was applied for 30 min at a pressure of -0.06 MPa, and then the mixture was kept at room temperature in the dark for 12 h. (4) Use 95% (v / v) ethanol to decolorize the leaves by boiling them three times for 10 minutes each time. The leaves can be decolorized multiple times. (5) After decolorization, the leaves were completely immersed in 95% (v / v) ethanol, and the stained root tips and leaves were photographed and observed using an Olympus microscope.

[0056] 2. NBT staining (1) Prepare 0.05 mol phosphate buffer (pH 7.5), add 5 mL of phosphate buffer to a 10 mL centrifuge tube, weigh 2.5 mg NBT powder and dissolve it in phosphate buffer (0.5 mg / mL), wrap the centrifuge tube with aluminum foil, vortex to mix, and soak the corn leaves in phosphate buffer containing DAB powder. (2) Vacuum was applied for 30 min at a pressure of -0.06 MPa, and then the mixture was kept at room temperature in the dark for 6 h. (3) Use 95% (v / v) ethanol to decolorize the leaves by boiling them 3 times for 10 minutes each time. The leaves can be decolorized multiple times. (4) After decolorization, the leaves were completely immersed in 95% (v / v) ethanol, and the stained root tips and leaves were photographed and observed using an Olympus microscope.

[0057] Under normal growth conditions, wild type, ZmNQO1 Neither the knockout nor the overexpression lines showed significant brownish / blue-purple staining with DAB or NBT staining, indicating that ROS accumulation levels were low across all genotypes. After salt stress treatment, DAB staining results showed that the wild-type and... ZmNQO1 The leaves of the knockout strains showed a distinct brownish-red coloration, with the knockout strains exhibiting a deeper brownish hue; while ZmNQO1 The leaves of the overexpression lines showed only a slight brownish hue, significantly weaker staining than the wild-type and knockout lines. NBT staining results showed that the wild-type and... ZmNQO1 The leaves of the knockout strains showed a distinct bluish-purple hue, with a more dense distribution of this coloration; while ZmNQO1 The leaves of the overexpression lines showed a significant reduction in blue-purple coloration, with only a few light blue-purple spots remaining. Figure 5 ).

[0058] II. Quantitative Analysis of Physiological and Biochemical Indicators 1. Hydrogen peroxide content detection This experiment used the hydrogen peroxide content detection kit provided by Greens Biotechnology Co., Ltd., and the specific steps are as follows: (1) Sample preparation: Select corn kernels of uniform size and plant them in a plant growth chamber under long-day conditions. After normal watering for 3 days, treat with 150mM NaCl:Na2SO4=9:1. After one week of treatment, detect the hydrogen peroxide content. Weigh about 0.1 g of corn leaf tissue and put it into a 1.5 mL centrifuge tube pre-loaded with steel balls (0.5 g of leaves with sufficient moisture can be taken). Quick freeze with liquid nitrogen for 40 s. Place the centrifuge tube in a high-throughput tissue homogenizer and homogenize at 55 Hz for 60 s. Immediately place on ice after homogenization. Add 1 mL of extraction solution to the centrifuge tube, vortex to mix, and place on ice for 10-15 min. Vortex once every 5 min. Centrifuge at 12000 rpm and 4℃ for 12 min. Take the supernatant and place it on ice for testing. (2) Preheat the microplate reader for 30 minutes and adjust the wavelength to 510 nm; (3) Add the reaction system sequentially to the 96-well plate according to Table 4: (4) Mix thoroughly, let stand at room temperature for 5 min, and then read the absorbance at 510 nm. △A = A_determination - A_blank; Table 4. Reaction system for determining H2O2 activity (4) Calculation of results H2O2 content (μmol / g fresh weight) = [(△A + 0.0115) ÷ 14.429] ÷ (W × V1 ÷ V) × D = 0.69 × (△A + 0.0115) ÷ W × D V: Volume of extract added, 1 mL; V1: Volume of sample added to the reaction system, 0.1 mL; W: Sample mass, g; D: Dilution factor, 1 for undiluted.

[0059] 2. Malondialdehyde (MDA) content detection This experiment used the malondialdehyde (MDA) content detection kit provided by Greens Biotechnology Co., Ltd., and the specific steps are as follows: (1) Sample preparation: Select corn kernels of uniform size and plant them in a plant growth chamber under long-day conditions. After normal watering for 3 days, treat with 150mM NaCl:Na2SO4=9:1. After one week of treatment, malondialdehyde content was detected. Weigh about 0.1g of corn leaves and put them into a 1.5 mL centrifuge tube pre-loaded with steel balls (0.5g of leaves with sufficient moisture can be taken). Quick freeze with liquid nitrogen for 40 s. Place the centrifuge tube in a high-throughput tissue homogenizer and homogenize at 55 Hz for 60 s. Immediately place on ice after homogenization. Add 1 mL of extraction solution to the centrifuge tube, mix well, and place on ice for 10-15 min. Vortex once every 5 min. Centrifuge at 12000 rpm and 4℃ for 12 min. Take the supernatant and place it on ice for testing. (2) Preheat the spectrophotometer for 30 minutes in advance, zero it with distilled water, and heat it to 90-95℃ in a water bath at the same time. (3) Add the reaction system to the EP tube in sequence according to Table 5: Table 5. Reaction system for determining MDA activity After inverting the sample, incubate it in a water bath at 90-95℃ for 30 min, then immediately place it on ice for 1 min. Centrifuge at 12000 rpm for 10 min at 25℃. Transfer all supernatant to a 1 mL glass cuvette and read the absorbance A at 532 nm and 600 nm, respectively. ΔA = A 532 -A 600 ; (4) Calculation of results: MDA content (nmol / g fresh weight) = [△A ÷ (ε × d) × V² × 10] 9 ]÷(W×V1÷V)= 16.1×△A÷W V: Total volume of sample extract, 1 mL; V1: Volume of sample added to the reaction system, 0.4 mL; V2: Total reaction volume of sample extract and working solution, 1 × 10⁻⁶ -3 L; d: optical path length of the cuvette, 1 cm; ε: molar extinction coefficient of MDA, 155 × 10⁻⁶ 3 L / mol / cm; W: sample mass, g.

[0060] Under normal conditions, wild type, ZmNQO1 There were no significant differences in hydrogen peroxide (H2O2) and malondialdehyde (MDA) content between the knockout and overexpression lines. Salt stress treatment significantly increased the H2O2 and MDA content in the wild-type line. ZmNQO1 The knockout strains showed further increases in H2O2 and MDA content (significantly higher than the wild type); while ZmNQO1 The H2O2 and MDA contents of the overexpression lines were significantly lower than those of the wild-type and knockout lines, indicating that ROS accumulation in the overexpression lines was effectively suppressed. Figure 6 ).

[0061] Under saline-alkali stress, both wild-type and transgenic lines showed reactive oxygen species (ROS) accumulation. However, compared with the wild-type, the knockout lines had higher ROS accumulation levels, while the overexpression lines had lower ROS levels. The results indicate that... ZmNQO1 Genes may improve the salt tolerance of maize by scavenging reactive oxygen species produced after salt stress.

[0062] Example 8: Salt stress on ZmNQO1 Effects of transgenic lines on yield-related traits and growth period Under normal growth conditions, wild-type, ZmNQO1 KO-1 and ZmNQO1 OE-1 There were no significant differences in ear traits and yield among the wild-type plants. After salt stress treatment, the seed setting rate of the wild-type plants decreased significantly (obvious barren tips on the ears) and the grain yield per plant decreased significantly. ZmNQO1 KO-1 The ear length further decreased, the seed setting rate was almost lost (the ears were sparsely populated), and the yield per plant dropped to an extremely low level; while ZmNQO1 OE-1 The spike length was significantly longer than that of wild-type and knockout lines, the seed setting rate remained at a high level, and the loss of yield per plant was significantly reduced. Figure 8 ).

[0063] The above results indicate that ZmNQO1 Overexpression significantly alleviated the inhibitory effects of salt stress on maize ear development, yield formation, and growth period coordination. ZmNQO1 Functional loss increases the plant's sensitivity to salt stress. ZmNQO1 The gene has a positive regulatory function on the yield stability and growth period regulation of maize under salt stress.

[0064] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions and improvements should all be included within the scope of protection of this application.

Claims

1. Corn ZmNQO1 The application of genes in the following (1) or (2): (1) Improve the salt tolerance of corn; (2) Breed salt-tolerant maize varieties; The corn ZmNQO1 A gene is a DNA molecule as shown in i), ii), or iii): i) The nucleotide sequence is the DNA molecule shown in SEQ ID NO.1; ii) DNA molecules other than i) encoding the amino acid sequence shown in SEQ ID NO. 2; iii) A DNA molecule that has 90% or more identity with the DNA fragment defined in i) or ii) and encodes a protein that is functionally equivalent to the protein shown in SEQ ID NO.

2.

2. Corn ZmNQO1 Application of gene-encoded proteins in improving salt tolerance in maize; the maize ZmNQO1 The protein encoded by the gene is the protein shown in either (A1) or (A2) below: (A1) A protein with the amino acid sequence shown in SEQ ID NO.2; (A2) The protein obtained by attaching a protein tag to the N-terminus and / or C-terminus of the protein defined in (A1).

3. The application according to claim 1 or 2, characterized in that, The improvement of corn salt tolerance specifically refers to any one or more of the following (1) to (4): (1) Under salt stress, maize plants increase in height and biomass accumulation; (2) Reduce the level of reactive oxygen species accumulation in maize plants under salt stress; (3) Improve the growth phenotype of mature maize under salt stress, reduce the degree of leaf wilting and the degree of plant height inhibition; (4) Reduce the inhibition of salt stress on maize yield traits, resulting in better ear development, higher seed setting rate, and increased yield per plant.

4. Promote corn ZmNQO1 Substances that enhance gene expression or improve corn ZmNQO1 The application of substances that encode protein activity and / or content in the following (1) or (2): (1) Improve the salt tolerance of corn; (2) Cultivate salt-tolerant maize varieties.

5. The application according to claim 4, characterized in that, Promote corn ZmNQO1 The substance used for gene expression is any one of the following: C1) contains ZmNQO1 Gene expression cassettes; C2) contains ZmNQO1 Recombinant vectors of genes, or recombinant vectors containing the expression cassette described in C1); C3) contains ZmNQO1 Recombinant microorganisms containing genes, or recombinant microorganisms containing the expression cassette described in C1), or recombinant microorganisms containing the recombinant vector described in C2); C4) contains ZmNQO1 Transgenic plant cell lines containing the gene, or transgenic plant cell lines containing the expression cassette described in C1); C5) contains ZmNQO1 Transgenic plant tissue containing the gene, or transgenic plant tissue containing the expression cassette described in C1).

6. A method for improving the salt tolerance of corn, characterized in that, include: In corn plants ZmNQO1 The steps of gene overexpression.

7. The method according to claim 6, characterized in that, In corn plants ZmNQO1 Gene overexpression is achieved through the following methods: The exogenous transfer of the above-mentioned components into maize ZmNQO1 Recombinant gene expression vectors; or the introduction of genes that can activate or enhance maize... ZmNQO1 DNA fragments at the gene transcription level.

8. A method for breeding a salt-tolerant maize variety, characterized in that, Includes the following steps: Transition to wild-type maize ZmNQO1 Genes were used to obtain transgenic maize, which has a higher salt tolerance than wild-type maize; Salt-tolerant corn varieties can be bred by self-pollinating genetically modified corn with enhanced salt tolerance or by crossing it with other salt-tolerant corn varieties.

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