Application of RDS gene in regulating maize response to abiotic stress

Abstract

The application discloses RDS The application belongs to the technical field of biotechnology and particularly relates to application of a gene in regulating corn response to abiotic stress RDS The protein, a substance for regulating expression of the coding gene of the protein, or a substance for regulating activity or content of the protein can be applied as follows: A1) application in regulating plant abiotic stress resistance and / or application in preparing a product for regulating plant abiotic stress resistance; A2) application in plant breeding and / or application in preparing a product for plant breeding; the amino acid sequence of the protein is SEQ ID No: 3. The method of the application can enhance plant abiotic stress resistance and provides a basis for plant breeding with enhanced abiotic stress resistance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to RDS Application of a gene in regulating maize response to abiotic stress. BACKGROUND

[0002] Plants inevitably encounter various abiotic stresses during their life cycle, which pose a serious challenge to their survival and reproduction. With global climate change and environmental degradation, problems such as low temperature, drought, and soil salinization are becoming increasingly severe, seriously threatening agricultural production and food security. These abiotic stress factors, acting alone or in combination, affect physiological processes such as photosynthesis, respiration, water balance, and ion balance in plants, thereby limiting their growth and development. Therefore, in-depth study of the mechanisms of plant response to abiotic stress is of great significance for improving crop stress resistance and yield.

[0003] Plants have developed a series of complex and sophisticated mechanisms to cope with abiotic stress during long-term evolution. When adversity strikes, plants usually respond by osmotic adjustment—accumulating osmotic adjustment substances such as proline and betaine to maintain cell turgor pressure and water balance; by activating antioxidant systems—activating antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) to scavenge reactive oxygen species (ROS) and reduce oxidative damage; by ion balance regulation—depending on ion channels and transporters to regulate ion absorption, transport, and distribution to maintain intracellular ion balance; and by various hormone pathways to adapt to adversity. Among them, gene regulation and expression, protein modification, and subcellular localization play a key role in the formation of plant stress resistance.

[0004] In recent years, with the rapid development of molecular biology and genetic engineering technology, significant progress has been made in the study of stress-resistant genes. Researchers have continuously identified and characterized new stress-resistant genes through gene cloning, functional analysis, and transgenic verification, and have further explored their mechanisms of action. For example, the DREB / CBF gene family plays an important role in transcriptional regulation in plant response to drought and cold stress; the SOS gene is involved in ion balance regulation in plants under saline conditions; and the HSP gene helps plants maintain protein stability under high temperature conditions.

[0005] At present, stress-resistant genes have been applied to plant genetic improvement. By introducing stress-resistant genes into crops through genetic engineering technology, some varieties with enhanced stress resistance have been successfully bred. However, the application of existing stress-resistant genes still has some limitations. First, the introduction of a single stress-resistant gene can only improve the resistance of plants to a specific stress, and the comprehensive resistance to multiple stresses is limited. Second, genetic engineering technology is limited by public concerns about its safety and environmental impact, which restricts its large-scale application. In addition, the expression regulation mechanism of stress-resistant genes is not clear enough, which makes it difficult to control the expression level and stability of the genes in transgenic plants, affecting the improvement effect of stress resistance.

[0006] Remorin proteins are a class of oligomeric filamentous proteins anchored in the plasma membrane / membrane raft of terrestrial plants, which are mainly located in cells through the C-terminal conserved coiled-coil domain. Most studies focus on the function of Remorin in responding to biological stress such as pathogenic bacteria. In recent years, researchers have also found that it can also participate in the regulation of abiotic stress. Corn is the world's largest food crop and plays an important role in food security and agricultural economic development. However, abiotic stress such as drought, low temperature and salinity seriously restricts the yield and quality of corn. In-depth study of the mechanism of corn responding to abiotic stress is of great significance to improve the stress resistance of corn and ensure the sustainable development of agriculture. However, there is no report on the involvement of Remorin in regulating abiotic stress in corn. The present invention will clarify the function of corn Remorin-like genes RDS in response to abiotic stress, providing potential genetic resources for breeding stress-resistant and stable crops. SUMMARY

[0007] The technical problem solved by the present invention is how to regulate the abiotic stress resistance of plants, especially corn.

[0008] To solve the above problems, the present invention provides the related application of a protein, a substance for regulating the expression of the coding gene of the protein, or a substance for regulating the activity or content of the protein.

[0009] The protein, the substance for regulating the expression of the coding gene of the protein, or the substance for regulating the activity or content of the protein provided by the present invention is applied in any one of the following:

[0010] 1) in regulating the abiotic stress resistance of plants;

[0011] 2) in preparing products for regulating the abiotic stress resistance of plants;

[0012] 3) in breeding plants with altered abiotic stress resistance;

[0013] 4) use in the manufacture of a product for breeding a plant altered in abiotic stress resistance;

[0014] 5) use in plant breeding.

[0015] The protein is any one of the following proteins:

[0016] a1) a protein having an amino acid sequence of SEQ ID No: 3;

[0017] a2) a protein having an amino acid sequence of SEQ ID No: 3 with substitution and / or deletion and / or addition of one or several amino acid residues and having the same function;

[0018] a3) a protein having an amino acid sequence of any one of a1) or (a2) with 80% or more identity and having the same function;

[0019] a4) a fusion protein obtained by linking a terminal tag to the protein defined in any one of a1) to (a3).

[0020] In the above protein, the protein tag refers to a polypeptide or protein fused and expressed with the target protein by DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and / or purification of the target protein. The protein tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0021] In the above protein, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using the homology search site on the Internet, such as the BLAST webpage of the NCBI homepage website. For example, the identity of a pair of amino acid sequences can be calculated by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) respectively, and performing a search in the Advanced BLAST 2.1, and then the value of the identity (%) can be obtained.

[0022] In the above protein, the 80% or more identity can be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0023] Among the above-mentioned proteins, SEQ ID No: 3 consists of 421 amino acid residues. It is named as RDS protein, and its encoding gene is RDS gene.

[0024] In the above-mentioned application, the protein is derived from corn (Zea mays L.). Zea mays L.).

[0025] Herein, the substance that regulates the activity and / or content of the protein can be a substance that regulates the expression of a gene encoding the protein RDS.

[0026] Herein, the substance that regulates the expression of a gene can be a substance that performs at least one of the following six regulations: 1) regulation at the transcription level of the gene; 2) regulation after the transcription of the gene (i.e. regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of the RNA transport of the gene (i.e. regulation of the transport of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; and 6) post-translational regulation of the gene (i.e. regulation of the activity of the protein translated from the gene).

[0027] In the present application, the regulation can be up-regulation or enhancement or increase; the regulation can also be down-regulation or weakening or decrease.

[0028] Herein, the enhancement, increase or up-regulation of the expression of the gene encoding the above-mentioned protein in a recipient plant, or / and the enhancement, increase or up-regulation of the activity and / or content of the gene encoding the above-mentioned protein is achieved by introducing the gene encoding the above-mentioned protein into the recipient plant.

[0029] Herein, the regulation of the expression of the gene encoding the protein can be inhibition or decrease or down-regulation of the expression of the encoding gene. The inhibition or decrease or down-regulation of the expression of the encoding gene can be achieved by gene knockout or gene silencing.

[0030] The gene knock-out refers to the phenomenon of inactivating a specific target gene by gene editing technology. The gene knock-out inactivates a specific target gene through changes in DNA sequences, including but not limited to Zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and CRISPR / Cas system, CRISPR (clustered regulatory interspaced short palindromic repeat) is a site containing multiple short repeat sequences in the genome, and Cas9 protein can cut the target sequence recognized by crRNA-tracrRNA under the mediation of RNA.

[0031] The gene silencing refers to the phenomenon of making a gene not expressed or lowly expressed without damaging the original DNA. The gene silencing takes the premise of not changing the DNA sequence to make the gene not expressed or lowly expressed. The gene silencing can occur at two levels, one is the transcription level gene silencing caused by DNA methylation, heterochromatinization and position effect, and the other is the post-transcriptional gene silencing, that is, the gene is inactivated by specific inhibition of target RNA at the level after gene transcription, including antisense RNA, co-suppression, gene quelling, RNA interference (RNAi) and microRNA (miRNA) mediated translation inhibition, etc.

[0032] In the above applications, the substance for regulating the expression of the coding gene of the protein or the substance for regulating the activity or content of the protein can be a biological material related to the protein described above, and the biological material can be any one of the following:

[0033] c1) a nucleic acid molecule encoding the protein described above;

[0034] c2) an expression cassette containing the nucleic acid molecule of c1);

[0035] c3) a recombinant vector containing the nucleic acid molecule of c1) or an expression cassette of c2);

[0036] c4) a recombinant microorganism containing the nucleic acid molecule of c1), or an expression cassette of c2), or a recombinant vector of c3);

[0037] c5) a transgenic plant cell line comprising the nucleic acid molecule of c1), or a transgenic plant cell line comprising the expression cassette of c2);

[0038] c6) a transgenic plant tissue comprising the nucleic acid molecule of c1), or a transgenic plant tissue comprising the expression cassette of c2);

[0039] c7) a transgenic plant organ comprising the nucleic acid molecule of c1), or a transgenic plant organ comprising the expression cassette of c2);

[0040] e1) a nucleic acid molecule that inhibits or reduces or silences the expression of the protein-encoding gene as defined above;

[0041] e2) an expression cassette comprising the nucleic acid molecule of e1);

[0042] e3) a recombinant vector comprising the nucleic acid molecule of e1), or an expression cassette comprising the nucleic acid molecule of e2);

[0043] e4) a recombinant microorganism comprising the nucleic acid molecule of e1), or an expression cassette comprising the nucleic acid molecule of e2), or a recombinant microorganism comprising the recombinant vector of e3);

[0044] e5) a transgenic plant cell line comprising the nucleic acid molecule of e1), or a transgenic plant cell line comprising the expression cassette of e2);

[0045] e6) a transgenic plant tissue comprising the nucleic acid molecule of e1), or a transgenic plant tissue comprising the expression cassette of e2);

[0046] e7) a transgenic plant organ comprising the nucleic acid molecule of e1), or a transgenic plant organ comprising the expression cassette of e2).

[0047] In the above uses, the nucleic acid molecule of c1) can be any one of the following DNA molecules,

[0048] d1) a DNA molecule whose nucleotide sequence is as set forth in SEQ ID No: 1;

[0049] d2) a DNA molecule whose coding sequence is as set forth in SEQ ID No: 2;

[0050] d3) a DNA molecule having 90% or more identity with the nucleotide sequence defined in d1) or d2), and encoding the protein as defined above;

[0051] d4) a DNA molecule that hybridizes to the nucleotide sequence defined in d1) or d2) under stringent conditions, and encoding the protein as defined above.

[0052] The nucleic acid molecules described herein can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecules can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA, or antisense RNA.

[0053] The vectors described herein are well known to those skilled in the art, and include but are not limited to plasmids, bacteriophages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids, or viral vectors. Specifically, the vector can be pXUE411C-BG.

[0054] The recombinant plant expression vector containing the gene can be constructed using existing plant expression vectors. RDS The plant expression vector includes but is not limited to Agrobacterium binary vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector can also contain the 3' untranslated region of the foreign gene, i.e., a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylate to the 3' end of the mRNA precursor, such as the 3' untranslated region of the Agrobacterium tumefaciens (Ti) plasmid gene (such as the nopaline synthase Nos gene), the plant gene (such as the soybean storage protein gene), etc.

[0055] The recombinant plant expression vector is constructed using the gene. RDS When constructing the recombinant plant expression vector using the gene, any one of the enhancer promoters or constitutive promoters can be added before the transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CAMV) 35S promoter, the maize ubiquitin promoter, which can be used alone or in combination with other plant promoters; in addition, when constructing the plant expression vector using the gene of the present application, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be the ATG initiation codon or the adjacent region initiation codon, etc., but must be the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The source of the translation control signal and the initiation codon is wide, and can be natural or synthetic. The translation initiation region can be from the transcription initiation region or the structural gene.

[0056] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants to produce color changes or luminescent compounds (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.), or chemical reagent-resistant marker genes (such as herbicide-resistant genes), etc. For the safety of transgenic plants, no selective marker gene can be added, and the transformed plants can be directly screened under stress.

[0057] The present application also provides a method for changing the abiotic stress resistance of a plant, comprising the following steps M or P:

[0058] The step M is to inhibit or reduce or silence the activity and / or content of the protein as described above in the plant of interest, or / and to inhibit or reduce or silence the expression of the gene encoding the protein as described above, so as to enhance the abiotic stress resistance of the plant;

[0059] The method comprises the step P, which is to enhance, increase or up-regulate the activity and / or content of the protein as described above in the plant of interest, or / and to enhance, increase or up-regulate the expression of the gene encoding the protein as described above, so as to weaken the abiotic stress resistance of the plant.

[0060] In the above method, the reduction of the expression and / or activity of the gene encoding the protein RDS in the plant of interest can be achieved by using gene mutation, gene knockout, gene editing or gene knockdown technology to reduce or inactivate the activity of the gene encoding the protein RDS in the genome of the plant of interest.

[0061] The present application also provides a method for breeding a plant with enhanced abiotic stress resistance, comprising inhibiting or reducing or silencing the expression of the gene encoding the protein as described above in the plant of interest, and / or the activity and / or content of the protein, so as to obtain a plant with enhanced abiotic stress resistance, wherein the abiotic stress resistance of the plant with enhanced abiotic stress resistance is stronger than that of the plant of interest.

[0062] In a specific embodiment, the inhibition or reduction or silencing of the expression of the gene encoding the protein as described above in the plant comprises introducing the nucleic acid molecule, expression cassette or recombinant vector as described above for inhibiting or reducing or silencing the expression of the gene encoding the protein as described above into the plant of interest, so as to obtain a plant with enhanced abiotic stress resistance.

[0063] In the present application, the knockout can be achieved by the CRISPR / Cas9 system.

[0064] Further, the target point of the CRISPR / Cas9 system gene editing is 1457-1475 of SEQ ID No: 1 or 451-469 of SEQ ID No: 2.

[0065] In the present application, the knockout of the gene encoding the protein of the corn of interest can be at least one mutation of the gene of the protein RDS shown in SEQ ID No: 1 in the corn genome:

[0066] 1) replacing 5'-GACTATGGAGGCCGCCCGC-3' in the coding gene of the protein in the corn genomic DNA with 5'-GACATGGAGGCCGCCCGC-3', so as to knock out the gene encoding the RDS protein;

[0067] 2) replacing 5'-GACTATGGAGGCCGCCCGC-3' in the coding gene of the protein in the corn genomic DNA with 5'-GACTTATGGAGGCCGCCCGC-3', so as to knock out the gene encoding the RDS protein.

[0068] The present application also provides a method for breeding a plant with weakened abiotic stress resistance, comprising enhancing, increasing or up-regulating the expression of the coding gene of the protein as described above in a plant of interest, and / or the activity and / or content of the protein, so as to obtain a plant with weakened abiotic stress resistance, wherein the abiotic stress resistance of the plant with weakened abiotic stress resistance is weaker than that of the plant of interest.

[0069] In a specific embodiment, the enhancing, increasing or up-regulating the expression of the coding gene of the protein as described above in a plant comprises introducing the nucleic acid molecule, the expression cassette or the recombinant vector as described above into the plant of interest, so as to obtain a plant with weakened abiotic stress resistance.

[0070] Herein, the breeding objective comprises breeding a plant with enhanced abiotic stress resistance; and the breeding objective also comprises breeding a plant with weakened abiotic stress resistance.

[0071] Herein, the abiotic stress can be temperature stress, drought stress or / and salt stress.

[0072] The temperature stress is specifically cold stress. The temperature of the cold stress is specifically 4℃.

[0073] Herein, the abiotic stress resistance can be increased survival rate or / and decreased ion permeability.

[0074] Herein, the corn can be corn ND101.

[0075] Herein, the recombinant microorganism can be Agrobacterium EHA105.

[0076] The protein as described above and / or the biological material also belong to the scope of protection of the present application.

[0077] In the above-mentioned application or method, the plant can be any one of the following:

[0078] N1) a monocotyledonous plant:

[0079] N2) a plant of the order Poales;

[0080] N3) Gramineae plants;

[0081] N4) Plants of the genus *Zea*;

[0082] N5) Corn.

[0083] This invention identifies a gene in maize whose expression is significantly downregulated under salt and cold treatment. RDS RDS CRISPR knockout maize was obtained using plant genetic engineering technology, demonstrating that RDS knockout can significantly improve maize's stress resistance.

[0084] The gene involved in this invention was obtained through in-depth research and rigorous screening, and compared with existing stress-resistance genes, it has the following innovations and breakthroughs:

[0085] (1) High-efficiency editing: The advanced gene editing technology CRISPR-Cas9 system can efficiently and accurately knock out the target gene, which greatly improves the success rate and efficiency of gene editing;

[0086] (2) Broad-spectrum resistance: Knocking out this gene can simultaneously confer significant resistance to multiple abiotic stresses such as low temperature, salt, and drought and salt superposition, making up for the lack of single resistance of existing stress resistance genes;

[0087] (3) Stable germplasm: New stress-resistant materials are obtained by gene knockout technology, without the problems of gene expression stability and gene loss;

[0088] (4) Safe and reliable: The method of the present invention does not introduce foreign genes, but only modifies the genes of corn itself, avoiding the potential risks and public concerns that may be brought about by transgenic technology, and conforming to the safety and ethical standards of modern biotechnology. Attached Figure Description

[0089] Figure 1 This study analyzed the expression patterns of RDS in maize under treatment with 150 mM NaCl and at 4℃. A) Analysis of the relative expression levels of RDS in maize under 150 mM NaCl treatment; B) Analysis of the relative expression levels of RDS in maize under 4℃ treatment. Maize ZmActin7 was used as an internal reference in both A and B analyses. The analytical method was one-way ANOVA. ** p < 0.01, *** p < 0.001, **** p < 0.0001.

[0090] Figure 2 This is a schematic diagram of the pXUE411C-RDS knockout vector.

[0091] Figure 3 for RDS Analysis of gRNA target sequences in gene knockout maize. WT represents wild-type maize. rds-1 andrds-2 For RDS Knockout corn material.

[0092] Figure 4 For T2 generation transgenic corn strain seedling stage resistance analysis. Among them, A is the phenotype of WT, RDS knockout corn under salt treatment and salt superimposed drought treatment; B is the plant height statistics of WT, RDS knockout corn under normal conditions and salt treatment; C is the survival rate statistics of WT, RDS knockout corn after salt superimposed drought treatment; D is the phenotype observation of WT, RDS knockout corn under normal and cold treatment; E is the ion permeability measurement of WT, RDS knockout corn after cold treatment. DETAILED DESCRIPTION

[0093] The application will be further described in detail below in conjunction with the specific embodiments. The examples given are only to illustrate the application, and are not intended to limit the scope of the application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the application.

[0094] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0095] Unless otherwise specified, the quantitative tests in the following examples are all set up with three repeated experiments, and the results are averaged.

[0096] ND101 in the following examples has been described in: Wang Y (2022) Functional study of maize GSK3-like kinase ZmSK2 in embryo development. Doctoral dissertation of China Agricultural University. The public can obtain this biological material from the applicant, which is only used for repeating the experiments of the application and cannot be used for other purposes.

[0097] EHA105 in the following examples has been described in: Wang Y (2022) Functional study of maize GSK3-like kinase ZmSK2 in embryo development. Doctoral dissertation of China Agricultural University. The public can obtain this biological material from the applicant, which is only used for repeating the experiments of the application and cannot be used for other purposes.

[0098] The construction method of the pXUE411C-BG starting vector in the following examples is as follows:

[0099] 1) Construct vector pBUE411C, the vector pBUE411C is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 5 between the BsaI sites of the starting vector pBUE411, while keeping other sequences of the vector pBUE411 unchanged. pBUE411 has been described in Xing et al., A CRISPR / Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biol. 2014 Nov 29; 14(1): 327. The biological material is available to the public from the applicant, which is only used for repeating the experiments of the present application and cannot be used for other purposes.

[0100] 2) To further improve the positive rate of transgenic offspring, insert the p35S-CTP2-CP4-EPSPS-TNOS expression frame on the basis of step 1) vector pBUE411C to construct pXUE411C-BG. pXUE411C-BG is a recombinant vector obtained by inserting a DNA fragment with the sequence of SEQ ID No: 6 between the KpnI enzyme cutting sites of the starting vector pBUE411C, while keeping other sequences of the vector pBUE411C unchanged.

[0101] The chemical reagents (analytical pure) in the following examples are purchased from the National Pharmaceutical Group; biological reagents such as T4 ligase and Bas endonuclease I are purchased from the United States NEB (New England Biolabs) company; M-MLV reverse transcriptase, RNase inhibitor and DNase I are purchased from the United States Promega company; fluorescent quantitative PCR 2xUltraSYBR Mix is purchased from Kangwei Century Company; primer synthesis is all in Beijing Qikeng Company.

[0102] The formula of 1 / 4 Hoagland's nutrient solution in the following examples is as follows:

[0103] Macronutrients (100x): take 800 mL of deionized water, add K2SO413.07 g, MgSO4·7H2O 16 g, KCl 0.75 g, KH2PO413.6 g, dissolve and make up to 1 L.

[0104] Micronutrients (1000x): take 800 mL of deionized water, add H3BO30.0618 g, MnSO4·H2O 0.1690 g, CuSO4·5H2O 0.0250 g, ZnSO4·7H2O 0.2875 g, (NH4)6Mo7O 24 ·4H2O 0.0062 g, dissolve and make up to 1 L.

[0105] Iron salt (100x): 800 mL of deionized water was measured, 3.66 g of iron salt of ethylenediaminetetraacetic acid sodium was added, and after fully dissolved, it was diluted to 1 L.

[0106] Ca(NO3)2·4H2O (200x): 800 mL of deionized water was measured, 94.45 g of Ca(NO3)2·4H2O solid was added, and after fully dissolved, it was diluted to 1 L.

[0107] After mixing each component, 4 M NaOH was used to adjust the pH to 6.0.

[0108] The following examples use GraphPad Prism 9 statistical software to process data, and the experimental results are expressed as mean ± standard deviation, One-way ANOVA test is used, P<0.05 (*) indicates significant difference, P<0.01 (**) indicates extremely significant difference, P<0.001 (****) indicates extremely significant difference.

[0109] Example 1, RDS Gene expression pattern analysis

[0110] RDS The coding sequence (CDS) of the gene in corn variety B73 is SEQ ID No: 2, which encodes the amino acid sequence of RDS protein of SEQ ID No: 3. In the genomic DNA of corn variety B73, the genomic gene encoding the RDS protein is shown in SEQ ID No: 1 of the sequence listing.

[0111] 1. Stress treatment of plant material

[0112] Select corn ND101 seedlings with consistent growth for 10 days, place them in 1 / 4 Hoagland's nutrient solution, 28°C, long light (16 h light / 8 h d dark conditions) for three days, then add NaCl to the final concentration of 150 mM to the culture solution or place the corn seedlings in a 4°C incubator for culture. The aboveground part was sampled at 0 h, 0.5 h, 1 h, 3 h, 6 h and 12 h.

[0113] 2. Extraction of total RNA from plant tissues

[0114] Take a certain amount of plant tissue, add liquid nitrogen pre-cooled mortar (pre-treated 180℃ for 4 h to remove RNase), and grind into powder with a pestle. Divide into new 1.5 mL RNase-free centrifuge tubes, about 100-200 mg per tube; add 1 mL of pre-cooled Trizol, vortex for 10 min to mix well; add 200 μL of pre-cooled chloroform to the centrifuge tube, vortex vigorously for 30 s, stand for 2 min, then centrifuge at 12,000 rpm, 4℃ for 15 min; transfer about 500 μL of supernatant to a new RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix well by inverting, stand at room temperature for 10 min, then centrifuge at 12,000 rpm, 4℃ for 10 min; discard the supernatant, add 1 mL of pre-cooled 70% ethanol, mix by tapping, centrifuge at 12,000 rpm, 4℃ for 3 min; discard the supernatant, open the cap and place it on ice for 5 min to completely evaporate the remaining liquid, add 34.5 μL of DEPC ddH2O to dissolve, and prepare the reaction system as shown in the table below for DNase I digestion:

[0115] Table 1, digestion reaction system

[0116]

[0117] After mixing gently, centrifuge immediately, and react at 37℃ for 30 min; add 150 μL of DEPC ddH2O, 100 μL of water-saturated phenol, and 100 μL of chloroform to the above reaction, vortex for 30 s, stand for 2 min, then centrifuge at 12,000 rpm, 4℃ for 10 min; transfer the supernatant to a new RNase-free centrifuge tube, add 1 / 10 volume (about 20 μL) of 3 M NaAc (pH 5.2) and 2.5 times volume (about 500 μL) of pre-cooled anhydrous ethanol, mix well by inverting, and precipitate at -80℃ for 1 h or overnight; centrifuge at 12,000 rpm, 4℃ for 15 min; discard the supernatant, wash with 70% ethanol to remove salt ions, open the cap on ice and blow for 5 min, add 30 μL of DEPC ddH2O to dissolve; take an appropriate amount of RNA for electrophoresis detection, then use for RNA reverse transcription or store at -80℃ for future use.

[0118] 3. RNA reverse transcription to cDNA

[0119] The reverse transcription system is as follows: RNA 4 μg, 50 μM Oligo (dT) 2 μL, DEPC ddH2O to 37.5 μL, mix well, centrifuge immediately, denature at 70℃ for 5 min, and ice bath for 5 min;

[0120]

[0121] 42°C metal bath for 1 h, 70°C for 15 min, and the product was stored in a -20°C refrigerator.

[0122] 4. Real-time fluorescent quantitative PCR

[0123] The primer sequences used were RTZmActin7-F / R and RTRDS-F / R, and the specific information is as follows:

[0124] RTZmActin7-F: 5'-GTTGGGCGTCCTCGTCA-3';

[0125] RTZmActin7-R: 5'-TGGGTCATCTTCTCCCTGTT-3';

[0126] RTRDS-F: 5'-ATGACTCCAATTGCGAGCCA-3';

[0127] RTRDS-R: 5'-GCGGCTATAGTGGTCTTCCC-3'.

[0128] The reaction system was as follows: cDNA (10-50 ng / μL) 1 μL, UltraSYBR Mixture (2x) 10 μL, 5' primer (10 μM) 0.5 μL, 3' primer (10 μM) 0.5 μL, and ddH2O supplemented to 20 μL.

[0129] The amplification procedure was as follows: 95°C for 10 min; 95°C for 10 s, 60°C for 1 min for 40 cycles, fluorescence values were collected, and the balance time was 6 s from 60°C to 95°C.

[0130] 2 -△△CT The relative expression of the gene was calculated, and the expression of ZmActin7 was used as the internal reference.

[0131] It was found that (Results) Figure 1 A and B in the middle): RDS transcription levels were down-regulated after NaCl and 4°C treatment.

[0132] Example 2, obtaining of CRISPR Cas9 gene editing plants

[0133] 1. Construction of CRISPR Cas9 knockout vector pXUE411C-RDS

[0134] Using CRISPOR (http: / / crispor.tefor.net / ), an online sgRNA analysis website that includes maize genome sequences, a suitable target sequence was selected: Target: 5'-GACTATGGAGGCCGCCCGC-3'. The target site of this sgRNA is located at... RDS The nucleotide sequence of the sgRNA target in the third exon of the gene is positions 1457-1475 of SEQ ID No: 1 or positions 451-469 of SEQ ID No: 2.

[0135] A pair of oligonucleotide sequences, RDS-OF / OR, was designed based on the target site sequence, where RDS-OF: 5'-ATTGGCGGGCGGCCTCCATAGTC-3'; and RDS-OR: 5'-AAACGACTATGGAGGCCGCCCGC-3'. Equal volumes of RDS-OF and RDS-OR (100 μmol / L each) were mixed and heated at 95°C for 5 minutes, then cooled to room temperature to generate the DNA insert.

[0136] The vector pXUE411C-BG was constructed using Golden Gate assembly. It was provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University. The reaction system is as follows:

[0137]

[0138] The reaction conditions were: 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min.

[0139] Then, DH5α competent cells were transformed. Sequencing confirmed that the pXUE411C-RDS vector was correctly constructed, and the gene knockout vector pXUE411C-RDS was as follows: Figure 2 As shown.

[0140] The structure of the pXUE411C-RDS carrier is described as follows: it is based on the departure carrier pXUE411C-BG. Bsa The recombinant vector was obtained by inserting a DNA fragment with the sequence 5'-GACTATGGAGGCCGCCCGC-3' between the I restriction sites, while keeping the other sequences of the vector pXUE411C-BG unchanged.

[0141] pXUE411C-RDS contains an sgRNA expression cassette with nucleotide sequences of positions 58-824 of SEQ ID No: 4. The sgRNA is shown as nucleotides 439-457 of SEQ ID No: 4. Nucleotides 58-437 are the promoters that initiate sgRNA transcription, and nucleotides 534-824 are the terminators that terminate sgRNA transcription.

[0142] First, the recombinant gene editing vector pXUE411C-RDS was introduced into Agrobacterium EHA105 strain, and then the recombinant bacteria EHA105 / pXUE411C-RDS successfully entered the corn inbred line ND101 by means of Agrobacterium-mediated transformation.

[0143] Subsequently, the medium containing Basta resistance was used for screening, so as to obtain the T0 generation of transgenic plants. RDS The knockout transgenic seedlings and the corresponding transgenic plants.

[0144] Then, the copy number of the resistance gene Bar of the seedling leaves of the T0 generation of transgenic plants was detected, and the transgenic positive plants with low copy number were selected by screening.

[0145] Finally, the identified T0 generation of transgenic plants was selfed, so as to obtain the T1 generation of gene editing plants, and the T1 generation of gene editing plants was selfed, so as to obtain the T2 generation of gene editing plants.

[0146] 2. Extraction of corn genomic DNA and PCR detection

[0147] Test sample: corn variety ND101 and T2 generation of RDS gene editing plants obtained in step 1.

[0148] 1) CTAB method for extracting plant genomic DNA

[0149] Take fresh corn leaves about 1-2 cm in a 2.0 mL centrifuge tube, add a 6 mm diameter steel ball to each tube, freeze quickly with liquid nitrogen, and grind with a tissue grinder at 1400 rpm for 40 s; add 600 μL of preheated CTAB extraction buffer, shake well, and incubate at 65°C for 30 min; cool to room temperature, add 500 μL of chloroform:isopropyl alcohol (24:1), mix well, and extract on a decolorizing shaker at low speed for 5 min; centrifuge at 12,000 rpm for 15 min at room temperature, and transfer the supernatant (about 600 μL) to a new 1.5 mL centrifuge tube; add 0.7 times the volume of isopropanol, mix gently, and centrifuge at 12,000 rpm for 10 min at room temperature; discard the supernatant, add 1 mL of 70% ethanol to suspend the precipitate, and rinse; centrifuge at 12,000 rpm for 5 min at room temperature, discard the supernatant, and blow dry in a clean bench; add 30-50 μL of ddH2O to dissolve the genomic DNA, and store at 4°C for standby.

[0150] CTAB extraction buffer: 10 g CTAB, 40.9 g NaCl were put into a beaker, 300 mL deionized water was added to dissolve, then 50 mL 1 M Tris-HCl (pH 8.0), 20 mL 0.5M EDTA (pH 8.0) were added, and finally deionized water was added to 500 mL.

[0151] 2) RDS Cutting analysis of gene knockout corn sgRNA target points

[0152] The T0 generation RDS gene knockout corn genomic DNA was extracted, and the primers RDS-cas9-F / RRDS-cas9-F: 5'-CACAGGTCAAGAAACCGTGCCCC-3'; RDS-cas9-R: 5'-CGCCTTCTCCGTCTCCTGCCAAT-3' were designed with the target point as the center.

[0153] 5 μL of the PCR product was taken for 1% agarose gel electrophoresis detection, and the length of the amplified fragment was about 1450 bp. The remaining PCR product was sent to Genescript Biotech Co., Ltd. for sequencing.

[0154] Through sequence alignment analysis of the sequencing results, two types of gene editing mutations were found:

[0155] a. Compared with the wild type genomic DNA, in the rds-1 plant (represented by rds-1), the genes encoding RDS protein in the two homologous chromosomes were both mutated as follows: "5'-GACTATGGAGGCCGCCCGC-3'' (corresponding to positions 1457-1475 of SEQ ID No: 1 or positions 451-469 of SEQ ID No: 2)" was mutated to "5'-GACATGGAGGCCGCCCGC-3'"; that is, one base "T" was deleted, thereby knocking out the gene encoding RDS protein. The sequencing results of the mutation site and the surrounding nucleotides are shown in Figure 3 .

[0156] b. Compared with the wild type genomic DNA, in the rds-2 plant (represented by rds-2), the genes encoding RDS protein in the two homologous chromosomes were both mutated as follows: "5'-GACTATGGAGGCCGCCCGC-3'' (corresponding to positions 1457-1475 of SEQ ID No: 1 or positions 451-469 of SEQ ID No: 2)" was mutated to "5'-GACTTATGGAGGCCGCCCGC-3'"; that is, one base "T" was inserted, thereby knocking out the gene encoding RDS protein. The sequencing results of the mutation site and the surrounding nucleotides are shown in Figure 3 .

[0157] These two knockout materials were named respectively. rds-1 and rds-2 ( Figure 3 ).

[0158] Example 3: Seedling stage stress resistance analysis of T2 generation transgenic maize lines

[0159] Test samples: wild-type ND101 (WT) and T2 generation gene-edited maize materials rds-1 and rds-2 seed.

[0160] 1. T2 generation transgenic maize was treated with 100 mM NaCl and then subjected to drought treatment.

[0161] Wild-type and gene-edited maize seeds were planted in 8 cm × 8 cm × 8 cm square pots, with 4 plants per pot and 2 pots per line, divided into treatment and control groups. They were watered with 100 mM NaCl solution or tap water and placed in a 16 h (light) / 8 h (dark) artificial culture room under normal conditions for about two weeks.

[0162] The results showed that under normal conditions, the plant height of the RDS material was slightly shorter than that of the WT material; however, under salt treatment, the growth of the RDS material was significantly better than that of the WT material. Figure 4 (A and B). Afterwards, watering was stopped for approximately 18 days of drought treatment, followed by two days of rehydration. The survival rate of the RDS material was found to be significantly higher than that of the WT material (see [link to RDS]). Figure 4 (A and C in the middle).

[0163] 2. Cold treatment was applied to T2 generation gene-edited maize.

[0164] Wild-type and gene-edited maize seeds were planted in 8 cm × 8 cm × 8 cm square pots, with 4 plants per pot and 2 pots per line, divided into treatment and control groups. The plants were grown in normal indoor conditions for about two weeks. The treatment groups were subjected to cold treatment (4℃, 4 days) and then recovered at 25℃ for 24 hours.

[0165] Ion permeability measurement method: After cold treatment and recovery, corn leaves were placed in a 15 mL centrifuge tube containing 10 mL of ddH₂O. The ion concentration was measured using a conductivity meter (METTLER TOLEDO FE30) and recorded as S1. The ion concentration of ddH₂O was measured as S0. The sample was then placed in boiling water for 30 minutes, cooled to room temperature, and the ion concentration was measured and recorded as S2. Ion permeability (%) = (S2 - S0) / (S1 - S0).

[0166] The results show that the rds material grows better than WT, the wilting degree of leaf is lighter, and the ion permeability is obviously lower than WT (see Figure 4 Fig. 6 is a graph showing the ion permeability of the rds material and WT.

[0167] The above detailed the present application. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range of equivalent parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including the changes made by the conventional technology known in the art, which is out of the scope disclosed in the present application.

Claims

1. Use of a substance for knocking out a protein-encoding gene or a substance for reducing the activity or content of the protein in any one of the following: 1) use in improving the abiotic stress resistance of a plant; 2) use in the preparation of a product for improving the abiotic stress resistance of a plant; 3) use in breeding a plant with enhanced abiotic stress resistance; 4) use in the preparation of a product for breeding a plant with enhanced abiotic stress resistance; 5) use in plant breeding; the protein is a protein with an amino acid sequence of SEQ ID No: 3; the abiotic stress resistance is cold stress resistance, drought stress resistance and salt stress resistance; the purpose of the breeding is to breed a plant variety with improved cold stress resistance, drought stress resistance and salt stress resistance; the plant is corn.

2. Use according to claim 1, characterized in that, The substance for reducing the activity or content of the protein is a biological material related to the protein in the use of claim 1, and the biological material is any one of the following: e1) a nucleic acid molecule for inhibiting or reducing or silencing the expression of the protein-encoding gene; e2) an expression cassette containing the nucleic acid molecule of e1); e3) a recombinant vector containing the nucleic acid molecule of e1) or containing the expression cassette of e2); e4) a recombinant microorganism containing the nucleic acid molecule of e1) or containing the expression cassette of e2) or containing the recombinant vector of e3); e5) a transgenic plant cell line containing the nucleic acid molecule of e1) or containing the expression cassette of e2).

3. Use according to claim 2, characterized in that: The method comprises inhibiting or reducing or silencing the activity and / or content of the protein in the plant of interest in claim 1, or / and, inhibiting or reducing or down-regulating the expression of the protein-encoding gene in claim 1, to enhance the abiotic stress resistance of the plant; 4. A method of altering abiotic stress resistance in a plant, comprising: the plant is corn; the abiotic stress resistance is cold stress resistance, drought stress resistance and salt stress resistance. The method comprises inhibiting or reducing or silencing the expression of the protein-encoding gene in claim 1 in the plant of interest, and / or, the activity and / or content of the protein, to obtain a plant with enhanced abiotic stress resistance, wherein the abiotic stress resistance of the plant with enhanced abiotic stress resistance is stronger than that of the plant of interest; 5. A method of breeding a plant with enhanced resistance to abiotic stress, characterized in that, the plant is corn; the abiotic stress resistance is cold stress resistance, drought stress resistance and salt stress resistance. The inhibition or reduction or silencing of the expression of the protein-encoding gene in claim 1 in the plant comprises introducing the nucleic acid molecule of e1), the expression cassette of e2) or the recombinant vector of e3) in claim 3 into the plant of interest, to obtain a plant with enhanced abiotic stress resistance.

6. The method of claim 5, wherein, The inhibition or reduction or silencing of the expression of the protein-encoding gene in claim 1 in the plant is achieved by a CRISPR / Cas9 system.

7. The method according to claim 5 or 6, characterized in that, ​

Images