A method for improving corn resistance to southern rust

Abstract

The present invention relates to a method for improving corn resistance to southern rust, belonging to the field of genetic engineering. The present invention can achieve the technical effect of improving corn resistance to southern rust by inhibiting the corn ZmJAZ12 gene.

Description

Technical Field

[0001] The invention relates to a method for improving corn resistance to southern rust, and belongs to the field of genetic engineering. Background Art

[0002] In recent years, with the continuous expansion of corn planting areas and changes in farming practices, the incidence of corn leaf diseases has become increasingly serious. Southern corn rust is a common fungal disease in corn production, seriously affecting both yield and quality. Southern corn rust is particularly prone to outbreaks in high temperature and humidity environments, resulting in a 20%-30% reduction in corn yield. In severe cases, yield losses can reach as high as 80%, or even total crop failure. Breeding and promoting disease-resistant varieties is a key goal of corn genetics and is the most economical and effective means of preventing and controlling southern corn rust.

[0003] JAZ proteins are a class of proteins unique to plants, belonging to the TIFY protein family and localized in the cell nucleus. JAZ proteins act as core inhibitory factors in the plant hormone jasmonic acid (JA) signaling pathway. Their functions are diverse and redundant. Maize has as many as 39 JAZ protein family members, and the functions of most remain unclear.

[0004] Through association analysis, the present invention found that the maize ZmJAZ12 gene is related to maize drought resistance. After inhibiting the expression of the ZmJAZ12 gene, it was unexpectedly found that the edited maize had enhanced resistance to southern rust. Summary of the Invention

[0005] The object of the present invention is to provide a method for improving resistance to southern rust.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides an application of a corn gene in improving resistance to southern rust of corn, characterized in that the LOC number of the gene in the B73 reference genome is Zm00001d027900 or GRMZM5G838098.

[0008] In some embodiments, the genomic sequence of the above gene is shown as SEQ ID NO.1.

[0009] The present invention also provides a method for improving corn resistance to southern rust, characterized in that the expression and / or activity of the protein encoded by the above gene is inhibited in corn, and corn materials with improved southern rust resistance are selected.

[0010] In some embodiments, the above-mentioned method of inhibiting a gene is to mutate the genomic sequence using gene editing technology.

[0011] In some embodiments, the target sequences selected by the above-mentioned gene editing method are shown as SEQ ID NO.2 and SEQ ID NO.3.

[0012] The present invention also provides a kit, characterized in that it comprises any one of the following:

[0013] (1) RNA molecules capable of recognizing the target sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3;

[0014] (2) a DNA molecule encoding the RNA described in (1);

[0015] (3) a vector for expressing the RNA described in (1);

[0016] In some embodiments, the above-mentioned kit further comprises a Cas9 protein or a nucleic acid molecule encoding a Cas9 protein or a vector expressing a Cas9 protein;

[0017] In some embodiments, the RNA molecule sequence is shown as SEQ ID NO.4 or SEQ ID NO.5.

[0018] The present invention also provides a mutant gene, characterized in that the sequence of the mutant gene is shown as SEQ ID NO.7.

[0019] The present invention also provides the use of the above method, kit, mutant gene, or mutant protein in improving the resistance of corn to southern rust.

[0020] Compared with the existing technology, the beneficial effects of the present invention are: the present invention found that the southern rust resistance of corn was enhanced after the ZmJAZ12 gene was inhibited, and provided a new method for improving the southern rust resistance of corn. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Expression analysis of maize JAZ family members in response to drought stress. A: Analysis of maize JAZ family members and phylogenetic tree. B: Expression analysis of maize JAZ family members in response to drought stress. C: Expression levels of six genes that respond to drought stress. FPKM: Fragments Per Kilobase of exon model per Million mapped fragments, representing gene expression levels. WW (normal watering), DT2 (soil moisture 30-35%), DT3 (soil moisture 20-25%), and DT4 (soil moisture 10-15%).

[0022] Figure 2Gene-edited corn demonstrates resistance to southern rust. A: Disease statistics. SCR indicates southern corn rust; "- / -" indicates pure knockout; "+ / +" indicates pure knockout. B: Plant lesion appearance. DETAILED DESCRIPTION

[0023] The following definitions and methods are provided to better define this application and to guide those skilled in the art in practicing this application. Unless otherwise noted, terms are to be understood according to conventional usage by those skilled in the relevant art. All patent documents, academic papers, industry standards, and other publications cited herein are hereby incorporated by reference in their entirety.

[0024] As used herein, "corn" refers to any corn plant and includes all plant varieties that can be bred with corn, including whole plants, plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant callus, intact plant cells in plants or plant parts, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, stems, roots, root tips, anthers, etc. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in an amino to carboxyl orientation. Amino acids can be represented herein by their commonly known three-letter symbols or by the single-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides can be represented by commonly accepted single-letter codes. Numerical ranges include numbers that define the range. As used herein, "nucleic acid" includes deoxyribonucleotides or ribonucleotide polymers in single-stranded or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the terms "encoding" or "encoded" are used in the context of a specific nucleic acid to refer to a nucleic acid that contains the necessary information to direct the translation of the nucleotide sequence into a specific protein. Codons are used to represent information encoding proteins. As used herein, "full-length sequence" relating to a specific polynucleotide or its encoded protein refers to the entire nucleic acid sequence or the entire amino acid sequence with a natural (non-synthetic) endogenous sequence. A full-length polynucleotide encodes the full-length, catalytically active form of the specific protein. The terms "polypeptide," "polypeptide," and "protein" are used interchangeably herein to refer to polymers of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). The amino acid can be a naturally occurring amino acid and, unless otherwise limited, can include known analogs of a naturally occurring amino acid that can function in a manner similar to the naturally occurring amino acids.

[0025] The term "trait" refers to a physiological, morphological, biochemical or physical characteristic of a plant or a specific plant material or cell. In some cases, this characteristic is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch or oil content of seeds or leaves, or by observing metabolic or physiological processes, for example, by measuring tolerance to water deprivation or specific salt or sugar or nitrogen concentrations, or by observing the expression level of one or more genes, or by agronomic observations such as osmotic stress tolerance or yield.

[0026] "Transgenic" refers to any cell, cell line, callus, tissue, plant part, or plant whose genome is altered by the presence of a heterologous nucleic acid, such as a recombinant DNA construct. As used herein, the term "transgenic" includes those original transgenic events and those generated from the original transgenic events by sexual crosses or asexual propagation, and does not encompass genomic (chromosomal or extrachromosomal) alterations made by conventional plant breeding methods or by naturally occurring events, such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[0027] "Plant" includes reference to whole plants, plant organs, plant tissues, seeds, and plant cells, as well as their progeny. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. "Progeny" includes any subsequent generation of a plant.

[0028] In this application, the words "comprises," "comprising," or variations thereof are to be understood as including, in addition to the described elements, numbers, or steps, other elements, numbers, or steps. A "test plant" or "test plant cell" refers to a plant or plant cell in which a genetic modification has been effected, or a progeny of a plant or cell so modified that contains the modification. A "control," "control plant," or "control plant cell" provides a reference point for measuring phenotypic changes in the test plant or plant cell.

[0029] Negative or control plants can include, for example: (a) wild-type plants or cells, i.e., plants or cells having the same genotype as the genetically modified starting material that produced the test plant or cell; (b) plants or plant cells having the same genotype as the starting material but that have been transformed with an empty construct (i.e., with a construct that has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) plants or plant cells that are non-transformed segregants of the test plant or plant cell; (d) plants or plant cells that are genetically identical to the test plant or plant cell but that have not been exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the test plant or plant cell itself, which is under conditions where the gene of interest is not expressed.

[0030] Those skilled in the art will readily recognize that advances in the field of molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, provide a wide range of appropriate tools and procedures for modifying or engineering the amino acid sequence and underlying gene sequence of proteins of agricultural interest.

[0031] In some embodiments, the nucleotide sequences of the present application can be altered to make conservative amino acid substitutions. The principles and examples of conservative amino acid substitutions are further described below. In certain embodiments, the nucleotide sequences of the present application can be substituted without changing the amino acid sequence according to the disclosed monocot codon preferences, for example, codons encoding the same amino acid sequence can be replaced with codons preferred by monocots without changing the amino acid sequence encoded by the nucleotide sequence. In some embodiments, part of the nucleotide sequence in the present application is replaced with different codons encoding the same amino acid sequence, thereby not changing the amino acid sequence encoded by the nucleotide sequence while changing the nucleotide sequence. Conservative variants include those sequences that encode the amino acid sequence of one of the proteins of the embodiments due to the degeneracy of the genetic code. In some embodiments, part of the nucleotide sequence in the present application is replaced according to the monocot codon preference. Those skilled in the art will recognize that amino acid additions and / or substitutions are generally based on the relative similarity of the amino acid side chain substituents, for example, the hydrophobicity, charge, size, etc. of the substituents. Exemplary amino acid substitution groups with various aforementioned properties are well known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Guidance on appropriate amino acid substitutions that do not affect the biological activity of the protein of interest can be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found., Washington, DC) (incorporated herein by reference). Conservative substitutions such as replacing one amino acid with another amino acid having similar properties can be performed. Sequence identity identification includes hybridization techniques. For example, all or part of a known nucleotide sequence is used as a probe for selective hybridization with other corresponding nucleotide sequences present in cloned genomic DNA fragments or cDNA fragment groups (i.e., genomic libraries or cDNA libraries) from a selected organism. The hybridization probe can be a genomic DNA fragment, a cDNA fragment, an RNA fragment or other oligonucleotide, and can be marked with a detectable group such as 32P or other detectable markers. Thus, for example, a hybridization probe can be prepared by marking a synthetic oligonucleotide based on the embodiment sequence. The method for preparing hybridization probes and building cDNA and genomic libraries is generally known in the art. The hybridization of the sequence can be carried out under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" represents following conditions, i.e., under these conditions, relative to hybridizing with other sequences, the probe will hybridize with its target sequence to a greater extent (e.g., at least 2 times, 5 times or 10 times of background) that can be detected.Stringent conditions are sequence-dependent and vary in different environments. By controlling hybridization stringency and / or controlling washing conditions, a target sequence that is 100% complementary to the probe can be identified (homologous probe method). Alternatively, stringent conditions can be adjusted to allow some sequence mismatches in order to detect lower similarities (heterologous probe method). Typically, the probe length is less than about 1000 or 500 nucleotides. Typically, stringent conditions are those in which the salt concentration is less than about 1.5 M Na ions, typically about 0.01 M to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is: when used for short probes (e.g., 10 to 50 nucleotides), at least about 30°C; when used for long probes (e.g., greater than 50 nucleotides), at least about 60°C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37°C using 30% to 35% formamide buffer, 1M NaCl, 1% SDS (sodium dodecyl sulfate), and washing in 1× to 2× SSC (20× SSC = 3.0M NaCl / 0.3M trisodium citrate) at 50°C to 55°C. Exemplary moderate stringency conditions include hybridization at 37°C in 40% to 45% formamide, 1.0M NaCl, 1% SDS, and washing in 0.5× to 1× SSC at 55°C to 60°C. Exemplary high stringency conditions include hybridization at 37°C in 50% formamide, 1M NaCl, 1% SDS, and a final wash in 0.1× SSC at 60°C to 65°C for at least about 20 minutes. Optionally, the wash buffer may contain about 0.1% to about 1% SDS. Duration of hybridization is typically less than about 24 hours, typically about 4 hours to about 12 hours. Specificity generally depends on post-hybridization washes, with the key factors being the ionic strength and temperature of the final wash solution. The Tm (thermodynamic melting point) of a DNA-DNA hybrid can be approximated by the formula of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm = 81.5°C + 16.6 (log M) + 0.41 (% GC) - 0.61 (% formamide) - 500 / L, where M is the molar concentration of monovalent cations, % GC is the percentage of guanosine and cytosine nucleotides in the DNA, "% formamide" is the percentage of formamide in the hybridization solution, and L is the base pair length of the hybrid. The Tm is the temperature (under defined ionic strength and pH) at which 50% of the complementary target sequence hybridizes to a perfectly matched probe. Washes are typically performed at least until equilibrium is reached and low background levels of hybridization are achieved, such as for 2 hours, 1 hour, or 30 minutes. Each 1% mismatch should reduce the Tm by about 1°C; thus, the Tm, hybridization, and / or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≥90% identity are desired, the Tm can be reduced by 10°C.Generally, stringent conditions are selected to be about 5°C lower than the Tm of the specific sequence and its complement at a defined ionic strength and pH. However, under very stringent conditions, hybridization and / or washing can be performed at 4°C below the Tm; under moderately stringent conditions, hybridization and / or washing can be performed at 6°C below the Tm; and under low stringency conditions, hybridization and / or washing can be performed at 11°C below the Tm.

[0032] In some embodiments, fragments of nucleotide sequences and the amino acid sequences they encode are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide of an embodiment or a portion of the amino acid sequence of a polypeptide. Fragments of nucleotide sequences can encode protein fragments that retain the biological activity of a native or corresponding full-length protein and thus have protein activity. Mutant proteins include biologically active fragments of native proteins that contain contiguous amino acid residues that retain the biological activity of the native protein. Some embodiments also include transformed plant cells or transgenic plants that contain the nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed using an expression vector that contains the nucleotide sequence of at least one embodiment and a promoter that drives expression in plant cells operably linked thereto. Transformed plant cells and transgenic plants refer to plant cells or plants that contain heterologous polynucleotides in their genomes. Generally speaking, the heterologous polynucleotides are stably integrated in the genome of the transformed plant cells or transgenic plants so that the polynucleotides are passed on to future generations. The heterologous polynucleotides can be integrated into the genome individually or as part of an expression vector. In some embodiments, the plants involved in the present application include plant cells, plant protoplasts, plant cell tissue cultures that can regenerate plants, plant calli, plant masses and plant cells, which are complete plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, kernels, ears, cobs, shells, stalks, roots, root tips, anthers, etc. The present application also includes plant cells, protoplasts, tissues, calli, embryos, flowers, stems, fruits, leaves and roots derived from the transgenic plants of the present application or their progeny, and thus at least partially comprising the nucleotide sequence of the present application.

[0033] The term "amplification" in the context of nucleic acid amplification is any process in which additional copies of a selected nucleic acid (or its transcribed form) are produced. Common amplification methods include various polymerase-based replication methods, including polymerase chain reaction (PCR), ligase-mediated methods such as ligase chain reaction (LCR), and RNA polymerase-based amplification (e.g., by transcription) methods.

[0034] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, the modification or replacement of the inventive method, step or condition is within the scope of the present application. Unless otherwise specified, the examples are according to conventional experimental conditions, such as the molecular cloning laboratory manual (Sambrook J & Russell D W, Molecular cloning: alaboratory manual, 2001) by Sambrook et al., or according to the conditions recommended by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are conventional commercial reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

[0035] Example 1 Identification of the ZmJAZ12 gene in maize

[0036] This study used a high-density of SNP markers from a population of maize individuals and drought-treated survival data from 368 maize inbred lines to perform an association analysis of candidate maize drought-resistance genes. Using drought seedling survival as the phenotypic data, a total of 525,105 SNP markers (with a MAF > 0.05) were identified, including several genes implicated in the JAZ family (Table 1).

[0037] The present invention further analyzed the JAZ family member genes of maize by transcriptome analysis of B73 under different drought levels. Different drought levels were selected for treatment: WW (normal watering), DT2 (soil moisture of 30-35%), DT3 (soil moisture of 20-25%) and DT4 (soil moisture of 10-15%) (refer to Plant J, 2019, 98(4): 697-713). It was found that the expression levels of 6 JAZ family members were significantly affected by drought ( Figure 1 ), among which the expression level of ZmJAZ12 gene increased most significantly, indicating that ZmJAZ12 (LOC number in the B73 reference genome is Zm00001d027900 or GRMZM5G838098) is a gene related to maize drought resistance.

[0038] Table 1 Some JAZ genes with significant signals after GWAS analysis

[0039]

[0040] Example 2 ZmJAZ12 gene editing

[0041] To verify the gene function of ZmJAZ12, the inventors designed a gene editing experiment. The knockout site was designed to be in the coding (CDS) region and as close as possible to the front end of the protein or the important functional domain region. The specific target site was 5'-AGCAGGTTCGCGGTGACGTA-3' (SEQ ID NO. 2) 5'-CTTCCCCGCCGAGAAGGCCG-3' (SEQ ID NO. 3), and the editing vector used was p210144bar-pXG001.

[0042] Agrobacterium EHA105 containing the editing vector was spread on YEP solid medium and cultured in the dark at 28°C for 1-3 days. The cultured Agrobacterium was scraped from the plate and resuspended, and the OD550 was adjusted to 0.3 to prepare the infection solution for later use.

[0043] Take the young ears of corn y822 (a corn inbred line bred by Jilin Academy of Agricultural Sciences) 9-12 days after pollination, remove the husks, disinfect with 75% alcohol for 10 minutes, and remove the young embryos into a centrifuge tube containing 2 mL of resuspension solution, with 100 young embryos in each tube, for later use.

[0044] During infection, discard the resuspension, add 2 mL of infection solution, gently invert the centrifuge tube several times to mix thoroughly, and let it rest in the dark at room temperature for 5 minutes. After infection, inoculate the immature embryos scutellum-side up into the co-culture medium and incubate in the dark at 20°C for 3 days. Transfer to resting medium and incubate in the dark at 28°C for 7 days. Then transfer to selective medium S1 containing 1.5 mg / L bialaphos and incubate in the dark at 28°C for 2 weeks. If initial callus tissue has been obtained, transfer it to screening medium S2 containing 3 mg / L bialaphos. Replace the S2 medium every two weeks.

[0045] When the resistant callus obtained by screening proliferates to a diameter of about 2 cm, it is transferred to a dark differentiation medium and cultured in the dark at 25°C for 2-3 weeks. The differentiated coleoptile is transferred to a light differentiation medium and cultured under light at 25°C for 2 weeks. After the coleoptile forms complete seedlings and roots, the seedlings are transferred to a culture bottle to promote root growth and seedling growth. After 10 days, the seedlings are transplanted into nutrient pots and cultured in an indoor greenhouse. After the seedlings grow 1-2 new leaves, they are moved into large flower pots and transferred to a large greenhouse. Daily management can be carried out according to conventional methods. After the male spike sheds pollen, self-pollination is carried out.

[0046] Example 3 Identification of Gene-Edited Corn Traits

[0047] The drought resistance of gene-edited corn was identified, and it was found that the drought resistance of the edited corn did not change significantly.

[0048] The present invention further subjected the gene-edited maize to other biotic and abiotic stresses and unexpectedly discovered that the edited maize exhibited significantly improved resistance to southern rust. Two ZmJAZ12-KO transformed lines (7a and 9a) showed overall improved disease resistance compared to negative segregant controls. One edited line, 9a, exhibited a lower southern rust infection rate than its own negative segregant control.

[0049] Southern rust infection was assessed using the following 1-9 point scale: "1": no disease symptoms or only hypersensitivity; "2": 5-10% of the leaf area was infected; "3": 15-20% of the leaf area was infected; "4": 30-40% of the leaf area was infected; "5": 50% of the leaf area was infected; "6": 60% of the leaf area was infected; "7": 70% of the leaf area was infected; "8": 80-90% of the leaf area was infected; "9": the entire leaf was infected and died.

[0050] Sequencing results of the target sites showed that 7a and 9a had deleted 377 bp and 390 bp, respectively, at the target sites. The genotype sequences after the mutations are shown in SEQ ID NO. 6 and SEQ ID NO. 7, respectively.

[0051] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein.

Claims

1. A method for improving resistance to southern rust in corn, characterized in that: The expression and / or activity of the protein encoded by the gene with the genomic sequence shown in SEQ ID NO. 1 is inhibited in corn, and corn materials with improved resistance to southern rust are selected.

2. The method according to claim 1, characterized in that The method of suppressing genes is to mutate the genome sequence using gene editing technology.

3. The method according to claim 2, characterized in that The target sequences selected by the gene editing method are shown in SEQ ID NO.2 and SEQ ID NO.

3.

4. A kit, characterized in that Includes any of the following: (1) gRNA molecules capable of recognizing the target sequences shown in SEQ ID NO. 2 and SEQ ID NO. 3; (2) A DNA molecule encoding the gRNA described in (1).

5. The kit according to claim 4, characterized in that The kit also includes Cas9 protein or a nucleic acid molecule encoding Cas9 protein or a vector expressing Cas9 protein.

6. The kit according to claim 4, wherein The gRNA molecule sequences are shown in SEQ ID NO.4 and SEQ ID NO.

5.

7. Use of the method according to any one of claims 1 to 3 or the kit according to any one of claims 4 to 6 in improving resistance of corn to southern rust.

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