Application of RDS gene in regulation and control of response of corn to abiotic stress

Abstract

The invention discloses an application of an RDS gene in regulating and controlling corn to respond to abiotic stress. The invention belongs to the technical field of biology, and particularly relates to application of an RDS gene in regulation and control of response of corn to abiotic stress. The protein, the substance for regulating and controlling the expression of the coding gene of the protein or the substance for regulating and controlling the activity or content of the protein can be applied as follows: A1) application in regulating and controlling the plant abiotic stress resistance and / or application in preparing a product for regulating and controlling the plant abiotic stress resistance; a2) application in plant breeding and / or application in preparation of products for plant breeding, and the amino acid sequence of the protein is SEQ ID No.3. By means of the method, the abiotic stress resistance of the plants can be enhanced, and a basis is provided for breeding of the abiotic stress resistance enhanced plants.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to RDS Application of genes in regulating maize response to abiotic stress. Background Art

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

[0003] Plants have developed a series of complex and sophisticated mechanisms to cope with abiotic stresses during their long evolution. When adversity comes, plants usually maintain cell turgor and water balance through osmotic regulation - accumulating osmotic regulating substances such as proline and betaine; through antioxidant systems - activating antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT) to remove reactive oxygen species (ROS) and reduce oxidative damage; through ion balance regulation - relying on ion channels and transporters to regulate the absorption, transport and distribution of ions to maintain intracellular ion balance; and through 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, research on stress resistance genes has made significant progress. Through gene cloning, functional analysis and transgenic verification, researchers continue to explore and identify new stress resistance genes and explore their mechanisms of action. For example, the DREB / CBF gene family plays an important role in transcriptional regulation when plants respond to drought and low temperature stress; the SOS gene is involved in the regulation of ion balance in plants under saline-alkali environments; and the HSP gene helps plants maintain protein stability under high temperature conditions.

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

[0006] Remorin protein is a type of oligomeric filamentous protein anchored in the plasma membrane / membrane raft that is unique to terrestrial plants. Most of them are localized in cells through the conserved coiled-coil domain at the C-terminus. Most studies focus on the function of Remorin in responding to biotic stresses such as pathogens. In recent years, some 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 ensuring food security and agricultural economic development. However, abiotic stresses such as drought, low temperature and salinity seriously restrict the yield and quality of corn. In-depth research on the mechanism of corn coping with abiotic stress is of great significance for improving corn's stress resistance and ensuring the sustainable development of agriculture. However, there has been no report on the involvement of Remorin in the regulation of abiotic stress in corn. The present invention will clarify the role of corn Remorin genes in the regulation of abiotic stress. RDS The function of participating in the response to abiotic stresses provides potential genetic resources for breeding stress-resistant and stable-yield crops. Summary of the invention

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

[0008] In order to solve the above problems, the present invention provides related applications of proteins, substances for regulating the expression of genes encoding the proteins, or substances for regulating the activity or content of the proteins.

[0009] Use of the protein provided by the present invention, a substance for regulating the expression of a gene encoding the protein, or a substance for regulating the activity or content of the protein in any of the following: 1) Application in regulating plant abiotic stress resistance; 2) Application in the preparation of products for regulating plant abiotic stress resistance; 3) Application in breeding plants with altered abiotic stress resistance; 4) Use in the preparation of products for breeding plants with altered abiotic stress resistance; 5) Application in plant breeding.

[0010] The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No: 3; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No: 3 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence of at least 80% identity with any of the amino acids specified in a1) or (a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any protein defined in a1) to (a3).

[0011] In the above proteins, the protein tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology 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.

[0012] In the above proteins, identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using a homology search site on the Internet, such as the BLAST page on the NCBI homepage website. For example, in Advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a matrix, setting the Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default values) respectively, and searching for the identity of a pair of amino acid sequences, the identity value (%) can be calculated.

[0013] In the above proteins, the above 80% identity may be at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

[0014] Among the above proteins, SEQ ID No: 3 consists of 421 amino acid residues. It is named RDS protein, and its encoding gene is RDS Gene.

[0015] In the above application, the protein is derived from corn ( Zea mays L.).

[0016] Herein, the substance that regulates the activity and / or content of the protein may be a substance that regulates the expression of a gene encoding the protein RDS.

[0017] In the above, the substance that regulates gene expression may be a substance that performs at least one of the following six types of regulation: 1) regulation at the transcription level of the gene; 2) regulation after transcription of the gene (that is, regulation of the splicing or processing of the primary transcript of the gene); 3) regulation of RNA transport of the gene (that is, 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; 6) post-translational regulation of the gene (that is, regulation of the activity of the protein translated from the gene).

[0018] In the present invention, the regulation may be up-regulation, enhancement or increase; the regulation may also be down-regulation, attenuation or reduction.

[0019] Herein, the enhancement, increase or upregulation of the expression level of the gene encoding the protein mentioned above in the recipient plant, or / and the enhancement, increase or upregulation of the activity and / or content of the gene encoding the above protein is achieved by introducing the gene encoding the above protein into the recipient plant.

[0020] Herein, regulating the expression of the coding gene of the protein may be inhibiting, reducing or down-regulating the expression of the coding gene. Inhibiting, reducing or down-regulating the expression of the coding gene may be achieved by gene knockout or gene silencing.

[0021] The gene knock-out refers to the phenomenon of inactivating a specific target gene through gene editing technology. Gene knock-out inactivates a specific target gene by changing the DNA sequence, 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 clustered regularly spaced short palindromic repeat sequence, which is a site in the genome containing multiple short repeat sequences. Cas9 protein can cut the target sequence recognized by crRNA-tracrRNA under the mediation of RNA.

[0022] The gene silencing refers to the phenomenon that the gene is not expressed or is underexpressed without damaging the original DNA. Gene silencing is based on the premise that the DNA sequence is not changed, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level caused by DNA methylation, heterochromatinization and position effect, and the other is post-transcriptional gene silencing, that is, at the level after gene transcription, the gene is inactivated by specifically inhibiting the target RNA, including antisense RNA, co-suppression, gene repression, RNA interference (RNAi) and microRNA (miRNA)-mediated translation inhibition, etc.

[0023] In the above application, the substance that regulates the expression of the gene encoding the protein or the substance that regulates the activity or content of the protein may be a biological material related to the protein mentioned above, and the biological material may be any of the following: c1) a nucleic acid molecule encoding the protein described above; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2); e1) Nucleic acid molecules that inhibit, reduce or silence the expression of the protein encoding gene mentioned above; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

[0024] In the above application, the nucleic acid molecule in c1) can be any of the following DNA molecules, d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No: 1; d2) the coding sequence is the DNA molecule shown in SEQ ID No: 2; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence specified in d1) or d2) and encodes the protein described above; d4) A DNA molecule that hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described above.

[0025] The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

[0026] The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, phages (such as lambda phage or M13 filamentous phage, etc.), cosmids (i.e., cosmids), Ti plasmids or viral vectors. Specifically, the vector may be pXUE411C-BG.

[0027] Existing plant expression vectors can be used to construct RDS The plant expression vector includes, but is not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also include the 3' non-translated region of the foreign gene, i.e., a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor, such as but not limited to Agrobacterium crown gall induction (Ti) plasmid genes (such as carmine synthase Nos gene), plant genes (such as soybean storage protein gene) 3' end transcription non-translated regions have similar functions.

[0028] use RDSWhen the gene is used to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before the transcription initiation nucleotide, including but not limited to cauliflower mosaic virus (CAMV) 35S promoter and corn ubiquitin promoter, which can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct a plant expression vector, an enhancer can also be used, including a translation enhancer or a transcription enhancer, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The sources of the translation control signal and the start codon are extensive, and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0029] 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 and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of transgenic plants, no selective marker genes can be added, and transformed plants can be directly screened by adversity.

[0030] The present invention also provides a method for changing abiotic stress resistance of plants, the method comprising the following steps M or P: The step M is to inhibit, reduce or silence the activity and / or content of the protein mentioned above in the target plant, or / and, inhibit, reduce or silence the expression of the gene encoding the protein mentioned above, so as to enhance the non-stress resistance of the plant; The method comprises step P, wherein step P is to enhance, increase or upregulate the activity and / or content of the protein mentioned above in the target plant, or / and enhance, increase or upregulate the expression level of the gene encoding the protein mentioned above to weaken the plant's non-stress resistance.

[0031] In the above method, reducing the expression level and / or activity of the gene encoding the protein RDS in the target plant 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 target plant.

[0032] The present invention also provides a method for cultivating plants with enhanced abiotic stress resistance, comprising inhibiting or reducing or silencing the expression level of the gene encoding the protein mentioned above in the target plant, 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 target plant.

[0033] In a specific embodiment, the inhibiting, reducing or silencing the expression of the gene encoding the protein mentioned above in the plant comprises introducing the nucleic acid molecule, expression cassette or recombinant vector for inhibiting, reducing or silencing the expression of the gene encoding the protein mentioned above into the target plant to obtain a plant with enhanced abiotic stress resistance.

[0034] In the present invention, the knockout can be achieved by the CRISPR / Cas9 system.

[0035] Furthermore, the target of gene editing by the CRISPR / Cas9 system is positions 1457-1475 of SEQ ID No: 1 or positions 451-469 of corresponding SEQ ID No: 2.

[0036] In the present invention, the knockout of the coding gene of the protein in the target corn may be to perform at least one of the following mutations on the gene of the protein RDS shown in SEQ ID No: 1 in the corn genome: 1) replacing 5'-GACTATGGAGGCCGCCCGC-3' in the gene encoding the protein in corn genomic DNA with 5'-GACATGGAGGCCGCCCGC-3', thereby knocking out the gene encoding the RDS protein; 2) Replacing 5'-GACTATGGAGGCCGCCCGC-3' in the gene encoding the protein in corn genomic DNA with 5'-GACTTATGGAGGCCGCCCGC-3', thereby knocking out the gene encoding the RDS protein.

[0037] The present invention also provides a method for cultivating plants with reduced abiotic stress resistance, comprising enhancing, increasing or upregulating the expression amount of the gene encoding the protein mentioned above in the target plant, and / or the activity and / or content of the protein to obtain a plant with reduced abiotic stress resistance, wherein the abiotic stress resistance of the plant with reduced abiotic stress resistance is weaker than that of the target plant.

[0038] In a specific embodiment, the enhancing, increasing or upregulating the expression of the gene encoding the protein described above in the plant comprises introducing the nucleic acid molecule, expression cassette or recombinant vector described above into the target plant to obtain a plant with reduced resistance to abiotic stress.

[0039] Herein, the breeding purpose includes breeding plants with enhanced abiotic stress resistance; the breeding purpose also includes breeding plants with reduced abiotic stress resistance.

[0040] Herein, the abiotic stress may be temperature stress, drought stress or / and salt stress.

[0041] The temperature stress is specifically cold stress, and the specific temperature of cold stress is 4°C.

[0042] Herein, the abiotic stress resistance may be an increase in survival rate or / and a decrease in ion permeability.

[0043] Herein, the corn may be corn ND101.

[0044] Herein, the recombinant microorganism may be Agrobacterium EHA105.

[0045] The proteins and / or biological materials mentioned above also fall within the scope of protection claimed by the present invention.

[0046] In the above application or method, the plant may be any of the following: N1) Monocotyledonous plants: N2) Gramineae; N3) Gramineae; N4) Zea mays; N5) Corn.

[0047] The present invention identifies a gene in corn that is significantly downregulated under salt and cold treatments RDS . Plant genetic engineering technology was used to obtain RDS CRISPR knockout corn, proving that knockout of RDS can significantly improve corn's stress resistance.

[0048] The genes involved in the present invention are obtained through in-depth research and strict screening. Compared with existing stress resistance genes, they have the following innovations and breakthroughs: (1) Efficient editing: The advanced gene editing technology CRISPR-Cas9 system can efficiently and accurately knock out the target gene, greatly improving the success rate and efficiency of gene editing; (2) Broad-spectrum resistance: Knockout of this gene can simultaneously endow plants with significant resistance to multiple abiotic stresses, such as low temperature, salt, and the superposition of drought and salt, making up for the shortcomings of the existing single resistance genes; (3) Germplasm stability: Gene knockout technology is used to obtain new stress-resistant materials without problems of gene expression stability and gene loss; (4) Safe and reliable: The method of the present invention does not introduce exogenous genes, but only modifies the genes of corn itself, thus avoiding the potential risks and public concerns that may be brought about by genetic modification technology, and conforming to the safety and ethical standards of modern biotechnology. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1The expression pattern of RDS under 150 mM NaCl and 4°C treatment. A, relative expression analysis of RDS in maize under 150 mM NaCl treatment; B, relative expression analysis of RDS in maize under 4°C treatment. A and B used maize ZmActin7 as an internal reference, and the analysis method was one-way ANOVA, ** p<0.01, *** p<0.001, **** p<0.0001.

[0050] Figure 2 Schematic diagram of the pXUE411C-RDS knockout vector.

[0051] Figure 3 for RDS Analysis of gRNA target sequence of gene knockout maize. WT is wild-type maize, rds-1 and rds-2 for RDS Knockout corn materials.

[0052] Figure 4 This is a stress resistance analysis of the T2 transgenic corn lines at the seedling stage. A is the phenotype of WT and RDS knockout corn under salt treatment and salt superimposed drought treatment; B is the plant height statistics of WT and RDS knockout corn under normal conditions and salt treatment; C is the survival rate statistics of WT and RDS knockout corn after salt superimposed drought treatment; D is the phenotypic observation of WT and RDS knockout corn under normal and cold treatment; E is the ion permeability measurement of WT and RDS knockout corn after cold treatment. DETAILED DESCRIPTION

[0053] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0054] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0055] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0056] ND101 in the following example has been recorded in: Wang Yan (2022). Functional study of maize GSK3-like kinase ZmSK2 in embryo development. Doctoral dissertation of China Agricultural University. The public can obtain the biological material from the applicant, which is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0057] EHA105 in the following example has been recorded in: Wang Yan (2022). Functional study of maize GSK3-like kinase ZmSK2 in embryo development. Doctoral dissertation of China Agricultural University. The public can obtain the biological material from the applicant, which is only used for repeating the experiment of the present invention and cannot be used for other purposes.

[0058] The construction method of the pXUE411C-BG starting vector in the following examples is as follows: 1) Constructing vector pBUE411C, which is a recombinant vector obtained by inserting a DNA fragment with a sequence of SEQ ID No: 5 between the BsaI sites of the starting vector pBUE411, while keeping the other sequences of the vector pBUE411 unchanged. pBUE411 has been described in: Xing et al., A CRISPR / Cas9 toolkit for multiplex genome editing inplants. BMC Plant Biol. 2014 Nov 29;14(1):327. The public can obtain the biological material from the applicant, and the biological material can only be used for repeating the experiment of the present invention and cannot be used for other purposes.

[0059] 2) To further improve the positive rate of transgenic offspring, the p35S-CTP2-CP4-EPSPS-TNOS expression frame was inserted into the vector pBUE411C in step 1) to construct pXUE411C-BG. pXUE411C-BG is a recombinant vector obtained by inserting a DNA fragment with a sequence of SEQ ID No: 6 between the KpnI restriction sites of the starting vector pBUE411C and keeping the other sequences of the vector pBUE411C unchanged.

[0060] The chemical reagents (analytical grade) in the following examples were purchased from Sinopharm Group; biological reagents such as T4 ligase and Bas I endonuclease was purchased from NEB (New England Biolabs), USA; M-MLV reverse transcriptase, RNaseInhibitor and DNaseⅠ were purchased from Promega, USA; fluorescent quantitative PCR 2×UltraSYBR Mix was purchased from Kangwei Century Company; primers were synthesized by Beijing Qingke Company.

[0061] The formula of 1 / 4 Hoagland's nutrient solution in the following examples is as follows: Macroelements (100×): Measure 800 mL of deionized water, add 13.07 g of K2SO4, 16 g of MgSO4·7H2O, 0.75 g of KCl, and 13.6 g of KH2PO4, and adjust the volume to 1 L after dissolving.

[0062] Trace elements (1000×): Measure 800 mL of deionized water, add 0.0618 g H3BO3, 0.1690 g MnSO4·H2O, 0.0250 g CuSO4·5H2O, 0.2875 g ZnSO4·7H2O, and (NH4)6Mo7O 24 4H2O 0.0062 g, dissolve and adjust volume to 1 L.

[0063] Iron salt (100×): Take 800 mL of deionized water, add 3.66 g of sodium ethylenediaminetetraacetic acid iron salt, and dilute to 1 L after fully dissolving.

[0064] Ca(NO3)2·4H2O (200×): Take 800 mL of deionized water, add 94.45 g of Ca(NO3)2·4H2O solid, and adjust the volume to 1 L after it is fully dissolved.

[0065] After mixing all ingredients, adjust the pH to 6.0 with 4 M NaOH.

[0066] In the following examples, GraphPad Prism 9 statistical software was used to process the data. The experimental results were expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicated a significant difference, P < 0.01 (**) indicated a very significant difference, P < 0.001 (***) indicated a very significant difference, and P < 0.001 (****) indicated a very significant difference.

[0067] Example 1: RDS Analysis of gene expression patterns RDS The coding sequence (CDS) of the gene in corn variety B73 is SEQ ID No: 2, and the encoded amino acid sequence is 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.

[0068] 1. Stress treatment of plant materials Select corn ND101 seedlings that have grown for 10 days and are in the same state. After three days of culture in 1 / 4 Hoagland's nutrient solution at 28°C and long photoperiod (16 h daylight / 8 hd dark conditions), add NaCl to the culture solution to a final concentration of 150 mM or culture the corn seedlings in a 4°C incubator. Samples were collected from the aboveground parts at 0 h, 0.5 h, 1 h, 3 h, 6 h, and 12 h of treatment.

[0069] 2. Extraction of total RNA from plant tissues Take a certain amount of plant tissue, add it to a liquid nitrogen pre-cooled mortar (treated at 180℃ for 4 h in advance to remove RNase), quickly grind it into powder with a pestle, and distribute it into new 1.5 mL RNase-free centrifuge tubes, about 100-200 mg per tube; add 1 mL of pre-cooled Trizol, Vortex oscillation and mixing for 10 min to fully lyse the plant tissue; add 200 μL of pre-cooled chloroform to the above centrifuge tube, Vortex oscillation vigorously for 30 s, let it stand for 2 min, and centrifuge it at 12,000 rpm at 4℃ for 15 min; draw about 500 μL of supernatant into a new RNase-free centrifuge tube, add an equal volume of pre-cooled isopropanol, mix it by inversion, let it stand at room temperature for 10 min, and centrifuge it at 12,000 rpm at 4℃ for 10 min; discard the supernatant, add 1 mL of pre-cooled 70% ethanol, flick to mix, and centrifuge it at 12,000 rpm at 4℃ for 3 Discard the supernatant, open the lid and place on ice for 5 min to completely evaporate the remaining liquid, add 34.5 μL DEPC ddH2O to dissolve, and prepare the reaction system according to the table below for DNase I digestion: Table 1. Digestion treatment reaction system

[0070] After gentle mixing, centrifugation was performed instantaneously, and the tube was placed at 37°C for 30 min. 150 μL DEPC ddH2O, 100 μL water-saturated phenol and 100 μL chloroform were added to the above reaction in sequence, and the tube was extracted by Vortex shaking for 30 s. After standing for 2 min, the tube was centrifuged at 12,000 rpm and 4°C for 10 min. The supernatant was transferred to a new RNase-free centrifuge tube, and 1 / 10 volume (about 20 μL) 3 M NaAc (pH 5.2) and 2.5 volumes (about 500 μL) of pre-cooled anhydrous ethanol were added. The tube was mixed by inversion, and precipitated at -80°C for more than 1 h or overnight. The tube was centrifuged at 12,000 rpm and 4°C for 15 min. The supernatant was discarded, and the tube was washed with 70% ethanol to remove salt ions. The tube was blown open on ice for 5 min, and 30 μL DEPC ddH2O was added to dissolve the tube. An appropriate amount of RNA was taken for electrophoresis detection, and then used for RNA reverse transcription or stored at -80°C for later use.

[0071] 3. Reverse transcription of RNA into cDNA The reverse transcription system was as follows: 4 μg RNA, 2 μL 50 μM Oligo (dT), DEPC ddH2O to 37.5 μL, mix well and centrifuge, denature at 70°C for 5 min, and ice bath for 5 min;

[0072] Incubate in a 42℃ metal bath for 1 h, react at 70℃ for 15 min, and store the product in a -20℃ refrigerator.

[0073] 4. Real-time fluorescence quantitative PCR The primer sequences used are RTZmActin7-F / R and RTRDS-F / R. The specific information is as follows: RTZmActin7-F: 5'-GTTGGGCGTCCTCGTCA-3'; RTZmActin7-R: 5'-TGGGTCATCTTCTCCCTGTT-3'; RTRDS-F: 5'-ATGACTCCAATTGCGAGCCA-3'; RTRDS-R: 5'-GCGGCTATAGTGGTCTTCCC-3'.

[0074] The reaction system was as follows: cDNA (10-50 ng / μL) 1μL, UltraSYBR Mixture (2×) 10μL, 5' primer (10 μM) 0.5μL, 3' primer (10 μM) 0.5 μL, and ddH2O was added to 20μL.

[0075] The amplification program was as follows: 95°C for 10 min; 95°C for 10 s, 60°C for 1 min for 40 cycles, collecting fluorescence values, and 60°C-95°C equilibrium time for 6 s.

[0076] Use 2 -△△CT The relative gene expression levels were calculated, and the expression level of ZmActin7 was used as an internal reference.

[0077] turn out( Figure 1 (A and B): RDS transcription level was downregulated after NaCl and 4°C treatment.

[0078] Example 2. Obtaining CRISPR Cas9 gene-edited plants 1. Construction of CRISPR Cas9 knockout vector pXUE411C-RDS The sgRNA online analysis website CRISPOR (http: / / crispor.tefor.net / ) that contains the maize genome was used to select the appropriate target sequence Target: 5'-GACTATGGAGGCCGCCCGC-3'. The target site of this sgRNA is located at RDS The third exon of the gene, the nucleotide sequence of the sgRNA target is positions 1457-1475 of SEQ ID No: 1 or positions 451-469 of SEQ ID No: 2.

[0079] A pair of oligonucleotide sequences RDS-OF / OR were designed according to the target site sequence, wherein RDS-OF: 5'-ATTGGCGGGCGGCCTCCATAGTC-3'; RDS-OR: 5'-AAACGACTATGGAGGCCGCCCGC-3'. RDS-OF and RDS-OR were mixed in equal amounts (100 μmol / L each), heated at 95°C for 5 minutes, and then cooled to room temperature to generate DNA inserts.

[0080] The Golden Gate assembly was used to construct the vector. pXUE411C-BG was provided by the Crop Functional Genomics and Molecular Breeding Research Center of China Agricultural University. The reaction system is as follows:

[0081] The reaction conditions were: 37℃ for 5 h, 50℃ for 5 min, and 80℃ for 10 min.

[0082] Then transform DH5α competent cells. The pXUE411C-RDS vector was constructed correctly by sequencing. The gene knockout vector pXUE411C-RDS was Figure 2 shown.

[0083] The structure of the pXUE411C-RDS vector is described as follows: Bsa A DNA fragment with the sequence 5'-GACTATGGAGGCCGCCCGC-3' was inserted between the restriction sites I and the other sequences of the vector pXUE411C-BG were kept unchanged to obtain a recombinant vector.

[0084] pXUE411C-RDS contains an sgRNA expression cassette whose nucleotide sequence is nucleotides 58-824 of SEQ ID No: 4, the sgRNA is shown as nucleotides 439-457 of SEQ ID No: 4, nucleotides 58-437 are a promoter for initiating sgRNA transcription, and nucleotides 534-824 are a terminator for terminating sgRNA transcription.

[0085] First, the recombinant gene editing vector pXUE411C-RDS was introduced into the Agrobacterium EHA105 strain, and then the recombinant bacteria EHA105 / pXUE411C-RDS was successfully introduced into the corn inbred line ND101 using the Agrobacterium-mediated transformation method.

[0086] Subsequently, the cells were screened using a culture medium containing Basta resistance to obtain the T0 generation. RDS Knockout transgenic seedlings and corresponding transgenic plants.

[0087] Then, the seedling leaves of the T0 generation transgenic plants were selected to detect the copy number of the resistance gene Bar, and the transgenic positive plants with low copies were selected through screening.

[0088] Finally, the identified and confirmed T0 generation transgenic plants are self-pollinated to obtain T1 generation gene-edited plants, and the T1 generation gene-edited plants are then self-pollinated to obtain T2 generation gene-edited plants.

[0089] 2. Extraction and PCR detection of corn genomic DNA Samples to be tested: corn variety ND101 and T2 generation RDS gene-edited plants obtained in step 1.

[0090] 1) Extraction of plant genomic DNA using the CTAB method Take about 1-2 cm of fresh corn leaves in a 2.0 mL centrifuge tube, add 6 mm diameter steel balls to each tube, freeze them quickly with liquid nitrogen, and grind them with a tissue grinder at 1400 rpm for 40 s; add 600 μL of preheated CTAB extraction buffer, shake well, and place in a 65°C water bath for 30 min; after cooling to room temperature, add 500 μL of chloroform:isoamyl alcohol (24:1), mix well, and place on a decolorizing shaker for slow extraction for 5 min; centrifuge at room temperature at 12,000 rpm for 15 min, draw the supernatant (about 600 μL) into a new 1.5 mL centrifuge tube, add 0.7 times the volume of isopropanol, and gently invert to mix; centrifuge at room temperature at 12,000 rpm for 10 min, discard the supernatant, add 1 mL of 70% ethanol to suspend the precipitate for rinsing; centrifuge at room temperature at 12,000 rpm for 5 min, discard the supernatant, and blow dry in a clean bench; add 30-50 μL Dissolve genomic DNA in ddH2O and store at 4°C until use.

[0091] CTAB extraction buffer: 10 g CTAB, 40.9 g NaCl were placed in a beaker and dissolved in 300 mL deionized water. Then 50 mL 1 M Tris-HCl (pH 8.0) and 20 mL 0.5 M EDTA (pH 8.0) were added. Finally, the volume was adjusted to 500 mL with deionized water.

[0092] 2) RDS Cleavage analysis of sgRNA target sites for gene knockout in maize The genomic DNA of T0 generation RDS gene knockout maize was extracted, and the primers RDS-cas9-F / RRDS-cas9-F: 5'-CACAGGTCAAGAAACCGTGCCCC-3'; RDS-cas9-R: 5'-CGCCTTCTCCGTCTCCTGCCAAT-3' were designed based on the target site.

[0093] 5 μL of PCR product was tested by 1% agarose gel electrophoresis, and the length of the amplified fragment was about 1450 bp. The remaining PCR product was sent to Qingke Biotech for sequencing.

[0094] Through sequence comparison analysis of sequencing results, two types of gene editing mutations were found: a. Compared with the genomic DNA of the wild type, the genes encoding the RDS protein in the two homologous chromosomes of the rds-1 plant (denoted by rds-1) have undergone the following mutation: "5'-GACTATGGAGGCCGCCCGC-3'' (corresponding to positions 1457-1475 of SEQ ID No: 1 or positions 451-469 of SEQ ID No: 2)" mutated to "5'-GACATGGAGGCCGCCCGC-3'", that is, a base "T" was deleted, thereby knocking out the gene encoding the RDS protein. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 3 .

[0095] b. Compared with the genomic DNA of the wild type, the genes encoding the RDS protein in the two homologous chromosomes of the rds-2 plant (represented by rds-2) have undergone the following mutations: "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, a base "T" was inserted, thereby knocking out the gene encoding the RDS protein. The sequencing results of the mutation site and its surrounding nucleotides are shown in Figure 3 .

[0096] The two knockout materials were named rds-1 and rds-2 ( Figure 3 ).

[0097] Example 3: Analysis of stress resistance of T2 transgenic maize lines at the seedling stage Samples to be tested: Wild-type ND101 (WT) and T2 gene-edited corn materials rds-1 and rds-2 seed.

[0098] 1. T2 transgenic corn was treated with 100 mM NaCl and superimposed with drought treatment Wild-type and gene-edited corn seeds were planted in 8 cm × 8 cm × 8 cm square pots, 4 plants in each pot, and 2 pots for each strain, divided into treatment group and control group, watered with 100 mM NaCl solution or tap water, and placed in a 16 h (light) / 8 h (dark) artificial culture room for normal growth for about two weeks.

[0099] The results showed that under normal conditions, the plant height of rds material was slightly shorter than that of WT; however, under salt treatment, the growth trend of rds material was significantly better than that of WT ( Figure 4A and B). After that, watering was stopped for about 18 days and drought treatment was carried out. After two days of re-watering, it was found that the survival rate of rds material was significantly higher than that of WT (see Figure 4 A and C).

[0100] 2. Cold treatment of T2 generation gene-edited corn.

[0101] Wild-type and gene-edited corn seeds were planted in 8 cm × 8 cm × 8 cm square pots, 4 plants in each pot, and 2 pots for each strain, divided into treatment group and control group. They were grown under normal conditions in the artificial culture room for about two weeks. The treatment group was cold treated (4℃, 4 d) and recovered at 25℃ for 24 h.

[0102] Ion permeability measurement method: Place the corn leaves that have been cold treated and recovered into a 15 mL centrifuge tube containing 10 mL ddH2O. Measure the ion concentration using a conductivity meter (METTLER TOLEDO FE30) and record the value as S1. The ion concentration of ddH2O is measured as S0. Place the sample in boiling water for 30 minutes, cool to room temperature, measure the ion concentration and record it as S2. Then the ion permeability (%) = (S2 - S0) / (S1 - S0).

[0103] The results showed that the growth state of rds material was better than that of WT, the degree of leaf wilting was less severe, and the ion permeability was significantly lower than that of WT (see Figure 4 D and E).

[0104] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a protein or a substance that regulates the expression of a gene encoding the protein or a substance that regulates the activity or content of the protein in any of the following; 1) Application in regulating plant abiotic stress resistance; 2) Application in the preparation of products for regulating plant abiotic stress resistance; 3) Application in breeding plants with altered abiotic stress resistance; 4) Use in the preparation of products for breeding plants with altered abiotic stress resistance; 5) Application in plant breeding; The protein is any of the following: a1) a protein having an amino acid sequence of SEQ ID No: 3; a2) a protein having the same function as the amino acid sequence shown in SEQ ID No: 3 after one or more amino acid residues are substituted and / or deleted and / or added; a3) a protein with an amino acid sequence identity of more than 80% with that specified in a1) or a2) and having the same function; a4) A fusion protein obtained by connecting a tag to the end of any one of the proteins defined in a1) to a3).

2. The use according to claim 1, characterized in that: The protein is derived from corn.

3. The use according to claim 1 or 2, characterized in that: The substance that regulates gene expression or the substance that regulates the activity or content of the protein is a biological material related to the protein in the application according to claim 1 or 2, and the biological material is any one of the following: c1) a nucleic acid molecule encoding the protein; c2) an expression cassette containing the nucleic acid molecule described in c1); c3) a recombinant vector containing the nucleic acid molecule described in c1), or a recombinant vector containing the expression cassette described in c2); c4) a recombinant microorganism containing the nucleic acid molecule described in c1), or a recombinant microorganism containing the expression cassette described in c2), or a recombinant microorganism containing the recombinant vector described in c3); c5) a transgenic plant cell line containing the nucleic acid molecule described in c1) or a transgenic plant cell line containing the expression cassette described in c2); c6) transgenic plant tissue containing the nucleic acid molecule described in c1) or transgenic plant tissue containing the expression cassette described in c2); c7) a transgenic plant organ containing the nucleic acid molecule described in c1) or a transgenic plant organ containing the expression cassette described in c2); e1) a nucleic acid molecule that inhibits, reduces or silences the expression of the gene encoding the protein; e2) an expression cassette containing the nucleic acid molecule described in e1); e3) a recombinant vector containing the nucleic acid molecule described in e1), or a recombinant vector containing the expression cassette described in e2); e4) a recombinant microorganism containing the nucleic acid molecule described in e1), or a recombinant microorganism containing the expression cassette described in e2), or a recombinant microorganism containing the recombinant vector described in e3); e5) a transgenic plant cell line containing the nucleic acid molecule described in e1), or a transgenic plant cell line containing the expression cassette described in e2); e6) transgenic plant tissue containing the nucleic acid molecule described in e1), or transgenic plant tissue containing the expression cassette described in e2); e7) A transgenic plant organ containing the nucleic acid molecule described in e1) or a transgenic plant organ containing the expression cassette described in e2).

4. The use according to claim 3, characterized in that: c1) The nucleic acid molecule is a DNA molecule as shown below: d1) The nucleotide sequence is the DNA molecule shown in SEQ ID No: 1; d2) the coding sequence is the DNA molecule shown in SEQ ID No: 2; d3) a DNA molecule that has 90% or more identity with the nucleotide sequence defined in d1) or d2) and encodes the protein described in claim 1; d4) A DNA molecule which hybridizes with the nucleotide sequence defined in d1) or d2) under stringent conditions and encodes the protein described in claim 1.

5. A method for altering abiotic stress resistance in plants, characterized in that: The method comprises step M or P, wherein step M is to inhibit, reduce or silence the activity and / or content of the protein of claim 1 or 2 in the target plant, or / and, inhibit, reduce or down-regulate the expression of the gene encoding the protein of claim 1 or 2, so as to enhance the plant's resistance to abiotic stress; The method comprises step P, wherein step P is to enhance, increase or up-regulate the activity and / or content of the protein described in claim 1 or 2 in the target plant, or / and enhance, increase or up-regulate the expression level of the gene encoding the protein described in claim 1 or 2 to weaken the plant's resistance to abiotic stress.

6. A method for cultivating plants with enhanced abiotic stress resistance, characterized in that: The method comprises inhibiting, reducing or silencing the expression level of the gene encoding the protein according to claim 1 or 2 in the target plant, and / or the activity and / or content of the protein, thereby obtaining 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 target plant.

7. The method according to claim 6, characterized in that The inhibition, reduction or silencing of the expression of the gene encoding the protein of claim 1 or 2 in the plant comprises introducing the nucleic acid molecule e1) of claim 4, the expression cassette e2) or the recombinant vector e3) into the target plant to obtain a plant with enhanced abiotic stress resistance.

8. The method according to claim 6 or 7, characterized in that: The inhibition, reduction or silencing of the expression of the gene encoding the protein according to claim 1 or 2 in the plant is achieved by the CRISPR / Cas9 system.

9. The protein according to claim 1 or 2 and / or the biomaterial according to claim 3 or 4.

10. The method according to any one of claims 5 to 8, characterized in that: The plant is any of the following: N1) Monocotyledonous plants: N2) Gramineae; N3) Gramineae; N4) Zea mays; N5) Corn.

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