CN113980919A - DNA sequence for regulating and controlling resistance of maize ear rot, mutant, molecular marker and application thereof - Google Patents

Abstract

The invention discloses a DNA sequence for regulating and controlling resistance of maize ear rot, a mutant, a molecular marker and application thereof. The invention firstly provides a key DNA sequence for regulating and controlling the resistance of the maize ear rot and a mutant thereof, wherein the base sequence is shown as SEQ ID No.1, SEQ ID No.2 and SEQ ID No.14, the key DNA sequence can regulate and control the expression of ZmLOX3 gene in maize ears and grains, and can improve the resistance of the maize ear rot or culture a new maize ear rot resistance variety. The invention provides application of Indel151 or Indel363 serving as a molecular marker of ZmLOX3 gene in corn ear and grain expression, regulation of ZmLOX3 gene in corn expression level and improvement of corn ear rot resistance. The invention further provides a detection primer for detecting the DNA sequence and the mutant variation condition thereof and a detection primer for detecting the ZmLOX3 gene expression level in corn.

Description

DNA sequence for regulating and controlling resistance of maize ear rot, mutant, molecular marker and application thereof

Technical Field

The invention relates to a DNA sequence for regulating and controlling maize ear rot resistance and a mutant thereof, in particular to a key DNA sequence for regulating and controlling a promoter region and an intron region of ZmLOX3 gene expression and a mutant thereof, a molecular marker for ZmLOX3 gene expression and a detection primer thereof, and further relates to application of the DNA sequence and the mutant thereof in regulating and controlling maize ear rot resistance, belonging to the fields of the DNA sequence for regulating and controlling maize ear rot resistance, the mutant thereof and the application thereof.

Background

Corn is an important food, feed and industrial raw material, and is also the first crop in China at present. The healthy and safe production of the corn plays a very important role in guaranteeing the grain safety and the agricultural development of China.

Maize ear rot (ear rot), also known as kernel and ear rot, is a type of fungal disease that commonly occurs in maize production areas worldwide. More than 40 species of pathogenic fungi causing ear rot have been identified so far, among which the following are the most common pathogenic fungi: fusarium verticillioides, f.moniliforme, f.graminearum, f.oxysporum, f.proliferum, f.culmorum flavum, f.avenacum, f.equiseti, f.seminudum, a. flavus, a. ochracea, etc. Generally, the composition of pathogenic bacteria varies greatly from place to place, and different nosocomial fungi can cause the occurrence of the corn ear rot by single or compound infection.

The incidence of the maize ear rot is generally in close relation with the genetic background of the material, and is also influenced by different environments and climatic conditions, and the occurrence of the maize ear rot is easily aggravated in the high-temperature and high-humidity environment.

The occurrence of the head rot can cause the rotting of the seeds, and the yield loss is up to 30-40%. Besides directly causing yield loss, pathogenic fungi such as fusarium and aspergillus of the ear rot can generate various toxins (for example, fusarium can generate mycotoxins such as trichothecene, fumonisin and zearalenone, aflatoxin and ochratoxin generated by aspergillus), and the safe storage and eating safety of corns are influenced. Mycotoxins not only affect the extent of infestation and destruction of the host by fungi, but also have serious toxic effects on vertebrates, including humans and livestock. The different types of toxins cause different degrees of damage and symptoms to host plants and animals. Fumonisins are sphingolipid compounds, which competitively inhibit the biosynthesis of sphingolipids in animal and plant cells, thereby causing accumulation of sphingosine in the cells, causing necrosis of host tissues and causing pig pulmonary edema, equine leukomalacia, rat liver cancer, human esophageal cancer and the like; nivalenol and deoxynivalenol have the ability of combining with eukaryotic 60S ribosome subunit, resulting in the inhibition of protein synthesis and the induction of apoptosis, and produce the symptoms of wilting, chlorosis or necrosis, causing the symptoms of human and animal vomiting, diarrhea, food refusal or estrogen poisoning, etc.; aflatoxins are important carcinogens that inhibit the synthesis of white matter in host plants and animals, causing severe damage to animal and plant tissues. On the whole, the corn ear rot not only causes the reduction of the yield and the quality of the corn, but also brings great hidden danger to the safety of food and feed and directly threatens the health of people and livestock, so the control of the occurrence and the harm of the corn ear rot is of great significance.

The excavation and regulation of the resistance gene of the maize ear rot is a key measure for improving the resistance of the maize ear rot, cultivating disease-resistant varieties and ensuring the safe production of the maize. Previous studies found that lipoxygenase genes (ZmLOXs) are important signal exchange molecules for the interaction between host and pathogenic fungi, and lipoxygenase genes derived from host maize play an important role in regulating and controlling the resistance of maize head rot. Among them, the maize 9-LOX gene ZmLOX3 may be utilized as a pathogenic factor by Fusarium verticillium (F. verticillioides) to cause resistance to stem/ear rot. Complete knockout of the ZmLOX3 gene increases resistance of maize to fusarium granulosis, but shows susceptibility to aspergillus flavus-induced granulosis; the complexity of the ZmLOX3 gene in the regulation of the resistance to the maize ear rot is shown, and the direct knockout of the gene cannot be directly used for the resistance improvement of the maize ear rot.

In view of the antagonistic action of the ZmLOX3 gene on the regulation and control of fusarium and aspergillus flavus resistance, a strategy for ensuring the protein function of the gene but properly reducing the expression level of the gene can possibly achieve the effect of resisting fusarium and aspergillus flavus simultaneously, thereby realizing the comprehensive improvement of the resistance of maize ear rot. In the evolution process of nearly ten thousand years, corn accumulates abundant natural variation, and the natural variation which does not change the function of ZmLOX3 protein but can properly reduce the expression abundance of the ZmLOX3 protein is likely to exist in nature. The excavation of the natural variation is crucial to the improvement of the resistance of the corn ear rot, and has wide application prospect in corn breeding.

Disclosure of Invention

One of the purposes of the invention is to provide a key DNA sequence for regulating and controlling the maize ear rot;

the other purpose of the invention is to provide a mutant of the key DNA sequence for regulating and controlling the maize ear rot;

the invention also aims to provide a molecular marker for regulating the expression of the ZmLOX3 gene in corn ears and grains;

the fourth purpose of the invention is to provide a method for reducing the risk of the maize to generate the ear rot or improving the resistance of the ear rot or a method for cultivating a new maize variety with resistance to the ear rot;

the fifth purpose of the invention is to provide a detection primer for detecting the variation condition of the molecular marker;

the sixth purpose of the invention is to provide a detection primer for detecting the ZmLOX3 gene expression level in corn;

the above object of the present invention is achieved by the following technical solutions:

the invention provides a key DNA sequence for regulating and controlling the resistance of maize ear rot, and the polynucleotide is shown in (a), (b), (c) or (d):

(a) the polynucleotide sequence shown as SEQ ID No.1, or

(b) A polynucleotide sequence capable of hybridizing to the complement of SEQ ID No.1 under stringent hybridization conditions;

(c) a polynucleotide sequence having at least 90% or more homology with the polynucleotide shown in SEQ ID No. 1; or

(d) Mutant with deletion, substitution or insertion of one or more basic groups on the basis of the polynucleotide sequence shown in SEQ ID No.1, and the mutant still has the function or activity of regulating and controlling maize ear rot.

The invention discovers that the key DNA sequence can regulate and control the expression quantity change of the ZmLOX3 gene in corn ears and grains so as to influence the resistance of corn to ear rot or ear rot.

The 'ear rot' or 'ear granule rot' in the invention is the same disease.

In the context of the present invention, the term "mutant" is a DNA sequence containing alterations in which one or more nucleotides of the original sequence are deleted, added and/or substituted, preferably while substantially maintaining the DNA sequence. For example, one or more base pairs may be deleted from the 5 'or 3' end of a DNA sequence to produce a "truncated" DNA sequence; one or more base pairs may also be inserted, deleted or substituted within the DNA sequence. Variant DNA sequences may be generated, for example, by standard DNA mutagenesis techniques or by chemical synthesis of variant DNA sequences or portions thereof. Mutant polynucleotides also include polynucleotides of synthetic origin, e.g., mutants obtained by site-directed mutagenesis, or by recombinant means (e.g., DNA shuffling), or by natural selection.

As a specific embodiment of the mutant, the invention provides a mutant of a key DNA sequence for regulating and controlling the resistance of the maize head rot, and the polynucleotide sequence of the mutant is shown as SEQ ID No. 2; the mutant is characterized in that 151bp base insertion is caused at a promoter region Indel151 of a ZmLOX3 gene, and the inserted sequence is shown as SEQ ID No. 3; 361bp or 363bp base is deleted at Indel363 of a second intron region of the ZmLOX3 gene, and the deleted polynucleotide sequence is shown as SEQ ID No.4 or SEQ ID No. 5; the mutant can increase the expression quantity of the ZmLOX3 gene in corn ears and corn grains, and shows that the risk of generating the ear rot is increased.

As a preferred embodiment of the mutant, the invention provides a mutant of a key DNA sequence for regulating and controlling the resistance of the maize head rot, and the polynucleotide sequence of the mutant is shown as SEQ ID No. 14; the mutant is characterized in that 151bp base is deleted at Indel151 of a ZmLOX3 gene promoter region, and the deleted sequence is shown as SEQ ID No. 3; 363bp bases are inserted into a second intron region Indel363 of the ZmLOX3 gene, and the inserted polynucleotide sequence is shown as SEQ ID No. 5; the mutant can reduce the expression quantity of the ZmLOX3 gene in corn ears and grains, and shows that the risk of generating the ear rot is obviously reduced.

The invention determines that the corn material which lacks the 151bp segment shown in SEQ ID No.3 in the promoter region Indel151 of the ZmLOX3 gene or inserts the base segment shown in SEQ ID No.4 or SEQ ID No.5 in the second intron region Indel363 of the ZmLOX3 gene generally has lower ZmLOX3 gene expression quantity, and further shows resistance to the ear rot caused by Fusarium verticillium and reduced risk of the occurrence of the ear rot; on the contrary, the corn material which is inserted with the 151bp segment shown in SEQ ID No.3 in the promoter region Indel151 of the ZmLOX3 gene or is deleted with the base segment shown in SEQ ID No.4 or SEQ ID No.5 in the second intron region Indel363 of the ZmLOX3 gene generally has higher ZmLOX3 gene expression quantity, shows that the corn material is susceptible to the ear rot caused by Fusarium rotavirus and has higher risk of generating the ear rot.

Thus, the present invention provides a method of reducing the risk of developing ear rot or providing resistance to ear rot in corn, comprising: deletion of a 151bp fragment shown in SEQ ID No.3 in an Indel151 promoter region of a ZmLOX3 gene; or inserting a base fragment shown in SEQ ID No.4 or SEQ ID No.5 at the second intron region Indel363 of the ZmLOX3 gene; or a 151bp fragment shown in SEQ ID No.3 is deleted in the promoter region Indel151 of the ZmLOX3 gene and a base fragment shown in SEQ ID No.4 or SEQ ID No.5 is inserted in the second intron region Indel363 of the ZmLOX3 gene.

The invention also provides a method for cultivating a new corn variety with resistance to the ear rot, which comprises the steps of reducing the expression quantity of the ZmLOX3 gene in corn ears and grains, and further improving the resistance of the ear rot; therefore, the method for improving the resistance of the corn ear rot by reducing the expression of the ZmLOX3 gene in the corn ears and grains belongs to the protection scope of the invention; comprises a method for driving the expression of ZmLOX3 gene in corn ears and grains by using other constitutive or tissue-specific promoters and an expression cassette constructed by ZmLOX3, or changing the expression of ZmLOX3 gene in corn ears and grains by using other natural variation, or reducing the expression of ZmLOX3 gene in corn ears and grains by RNAi.

As a preferred embodiment of this aspect, the method for reducing the expression level of ZmLOX3 gene in corn ear and kernel may be: deletion of a 151bp fragment shown in SEQ ID No.3 in an Indel151 promoter region of a ZmLOX3 gene; or inserting a base fragment shown in SEQ ID No.4 or SEQ ID No.5 at the second intron region Indel363 of the ZmLOX3 gene; or a 151bp fragment shown in SEQ ID No.3 is deleted in the promoter region Indel151 of the ZmLOX3 gene and a base fragment shown in SEQ ID No.4 or SEQ ID No.5 is inserted in the second intron region Indel363 of the ZmLOX3 gene.

Furthermore, the expression cassette of the mutant containing part or all of the DNA sequence shown in SEQ ID No.1, the DNA sequence shown in SEQ ID No.2 or SEQ ID No.14, the recombinant plant expression vector containing the expression cassette, the transgenic cell line and the host bacterium belong to the protection scope of the invention.

The recombinant plant expression vector is constructed by the expression cassette and a plasmid or an expression vector and can be transferred into plant host cells, tissues or organs.

The DNA sequences of the invention or mutants thereof can be used for the production of transgenic plants. For example, a recombinant plant expression vector containing the DNA sequence or a mutant thereof is introduced into a plant cell, tissue or organ by Agrobacterium-mediated or biolistic methods, and the transformed plant cell, tissue or organ is cultured to a plant to obtain a transgenic plant. The starting vector for constructing the plant expression vector can be any binary vector for transforming the plant by agrobacterium or a vector for plant microprojectile bombardment and the like.

Conventional compositions and methods for making and using plant expression vectors and host cells are well known to those skilled in the art for practicing the present invention, and specific methods can be found in, for example, Sambrook et al.

The recombinant plant expression vector may also contain a selectable marker gene for selection of transformed cells. Selectable marker genes are used to select transformed cells or tissues. The marker gene comprises: genes encoding antibiotic resistance, genes conferring resistance to herbicidal compounds, and the like. In addition, the marker gene also comprises phenotypic markers, such as beta-galactosidase, fluorescent protein and the like.

In conclusion, the DNA sequence, the mutant of the sequence, the molecular marker Indel151 or Indel363, the specific detection primer for detecting the variation condition of the molecular marker, the specific detection primer for detecting the ZmLOX3 gene expression level and the like provided by the invention can be applied to the cultivation of new corn varieties with disease resistance, particularly the improvement of the resistance to the corn ear rot and the like.

For reference, the present invention provides a method of modulating resistance to ear rot in corn, comprising: the expression of ZmLOX3 gene in corn is regulated by the DNA sequence shown in SEQ ID No.1, the DNA sequence shown in SEQ ID No.2 or the mutant of the DNA sequence shown in SEQ ID No. 14.

The transformation protocol described in the present invention and the protocol for introducing the polynucleotide or polypeptide into a plant may vary depending on the type of plant (monocot or dicot) or plant cell used for transformation. Suitable methods for introducing the polynucleotide or polypeptide into a plant cell include: microinjection, electroporation, agrobacterium-mediated transformation, direct gene transfer, and high-speed ballistic bombardment, among others. In particular embodiments, the expression cassettes of the invention can be provided to plants using a variety of transient transformation methods. The transformed cells can be regenerated into stably transformed plants using conventional methods (McCormick et al plant Cell reports.1986.5: 81-84).

The present invention can be used to transform any plant species, including but not limited to monocots or dicots, preferably maize.

The invention further determines that Indel151 or Indel363 can be used as a molecular marker for regulating the expression of the ZmLOX3 gene in corn ears and grains. The invention also discovers that Indel151 and Indel363 are almost completely linked together, and any one of the variations can be independently used as a molecular marker for detecting the expression of the ZmLOX3 gene in corn ears and grains.

The invention further provides a detection primer for detecting Indel151 variation; as a preferred embodiment, the nucleotide sequences of the detection primers are respectively shown as SEQ ID No.6 and SEQ ID No. 7; the specific detection primer can be used for detecting the variation condition of Indel151 in the corn variety, so that the primer is used for molecular assisted breeding of corn.

The invention further provides a detection primer for detecting Indel363 variation; as a preferred embodiment, the nucleotide sequences of the detection primers are respectively shown as SEQ ID No.8 and SEQ ID No. 9; the specific detection primer can be used for detecting the variation condition of Indel363 in corn varieties, so that the primer is used for molecular assisted breeding of corn.

In addition, other primers designed according to SEQ ID No.1, SEQ ID No.2 or SEQ ID No.14, or primers designed by other methods that can be used to detect Indel151 or Indel363 mutations are also within the scope of the present invention.

The invention also further provides a specific amplification primer for detecting the ZmLOX3 gene expression quantity, and the specific detection primer sequences are respectively shown as SEQ ID No.10 and SEQ ID No. 11; the specific detection primer can be used for detecting the ZmLOX3 gene expression quantity of a corn variety, and further provides reference for research on ZmLOX3 genes in corn breeding.

The nucleotide sequence of the coding region of the ZmLOX3 gene is shown in SEQ ID No.12, and the coding protein of the ZmLOX3 gene is shown in SEQ ID No. 13.

The DNA sequence or mutant for regulating and controlling the maize ear rot provided by the invention can regulate and control the expression of the ZmLOX3 gene in maize ears or grains, has important significance for improving the maize ear rot, and can be further applied to breeding new maize varieties. The invention further provides a molecular marker Indel151 or Indel363 for regulating the expression of the ZmLOX3 gene, a specific detection primer for detecting the variation condition of the molecular marker and a specific detection primer for detecting the expression level of the ZmLOX3 gene in the corn, which can be directly applied to directionally improving the resistance of the maize to the ear rot and have important application potential for breeding new maize disease-resistant varieties.

Definitions of terms to which the invention relates

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

The term "polynucleotide" or "nucleotide" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single-or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless otherwise specifically limited, the term also means oligonucleotide analogs, which include PNAs (peptide nucleic acids), DNA analogs used in antisense technology (phosphorothioates, phosphoramidates, and the like). Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly specified. In particular, degenerate codon substitutions may be achieved by generating sequences in which the 3 rd position of one or more selected (or all) codons is substituted with mixed base and/or deoxyinosine residues (Batzer et al, Nucleic Acid Res.19:5081 (1991); Ohtsuka et al, J.biol.chem.260: 2605-S2608 (1985); and Cassol et al (1992); Rossolini et al, Mol cell. probes 8:91-98 (1994)).

The term "homology" refers to the level of similarity or percent identity between polynucleotide sequences in terms of percent nucleotide position identity (i.e., sequence similarity or identity). The term homology, as used herein, also refers to the concept of similar functional properties between different polynucleotide molecules, e.g., promoters with similar functions may have homologous cis-elements. Polynucleotide molecules are homologous when they specifically hybridize under specific conditions to form duplex molecules. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule can be used as a probe or primer for identifying another polynucleotide molecule that shares homology.

The "stringent hybridization conditions" as used herein means conditions of low ionic strength and high temperature known in the art. Typically, a probe hybridizes to its target sequence to a greater extent than it can hybridize to other sequences under stringent conditions (e.g., at least 2-fold over backgroundThe hybridization conditions are sequence dependent and will be different under different environmental conditions, with longer sequences hybridizing specifically at higher temperatures. Target sequences that are 100% complementary to the probe can be identified by controlling the stringency of hybridization or wash conditions. For an exhaustive guidance of Nucleic acid Hybridization, reference is made to the literature (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic acids Probes, "Overview of principles of Hybridization and the" protocol of Nucleic acid assays. 1993). More specifically, the stringent conditions are generally selected to be below the thermal melting point (T) of the specific sequence at a defined ionic strength pH_m) About 5-10 ℃. T is_mIs the temperature (at a given ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (at T because the target sequence is present in excess_mAt equilibrium 50% of the probes are occupied). Stringent conditions may be as follows: wherein the salt concentration is less than about 1.0M sodium ion concentration, typically about 0.01 to 1.0M sodium ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature is at least about 30 ℃ for short probes (including but not limited to 10 to 50 nucleotides) and at least about 60 ℃ for long probes (including but not limited to greater than 50 nucleotides). Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal can be at least two times background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions may be as follows: 50% formamide, 5 XSSC and 1% SDS, incubated at 42 ℃; or 5 XSSC, 1% SDS, incubated at 65 ℃, washed in 0.2 XSSC and washed in 0.1% SDS at 65 ℃. The washing may be for 5, 15, 30, 60, 120 minutes or more.

The term "plurality" as used herein generally means 2 to 8, preferably 2 to 4; the "substitution" refers to the substitution of one or more amino acid residues with different amino acid residues, respectively; the term "deletion" refers to a reduction in the number of amino acid residues, i.e., the absence of one or more amino acid residues, respectively; by "insertion" is meant a change in the sequence of amino acid residues that results in the addition of one or more amino acid residues relative to the native molecule.

The term "coding sequence": a nucleic acid sequence transcribed into RNA.

The term "promoter" refers to a polynucleotide molecule that is located upstream or 5' of the translation initiation codon of the open reading frame (or protein coding region) in its native state and that is involved in the recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.

The term "plant promoter" is a native or non-native promoter that is functional in a plant cell. Constitutive plant promoters function in most or all tissues throughout plant development. Any plant promoter can be used as a 5' regulatory element for regulating the expression of one or more specific genes operably linked thereto. When operably linked to a transcribable polynucleotide molecule, a promoter typically causes transcription of the transcribable polynucleotide molecule in a manner similar to the transcription of the transcribable polynucleotide molecule to which the promoter is typically linked. Plant promoters may include promoters produced by manipulating known promoters to produce artificial, chimeric, or hybrid promoters. Such promoters may also be combined with cis-elements from one or more promoters, for example, by adding heterologous regulatory elements to an active promoter having some or all of its own regulatory elements.

The term "cis-element" refers to a cis-acting transcriptional regulatory element that confers an aspect of overall control over gene expression. The cis-element may function to bind transcription factors, trans-acting protein factors, which regulate transcription. Some cis-elements bind more than one transcription factor, and transcription factors may interact with more than one cis-element with different affinities.

The term "operably linked" refers to the linkage of a first polynucleotide molecule (e.g., a promoter) to a second transcribable polynucleotide molecule (e.g., a gene of interest), wherein the polynucleotide molecules are arranged such that the first polynucleotide molecule affects the function of the second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single contiguous polynucleotide molecule, and more preferably are contiguous. For example, a promoter is operably linked to a gene of interest if it regulates or mediates transcription of the gene of interest within the cell.

The term "transcribable polynucleotide molecule" refers to any polynucleotide molecule capable of being transcribed into an RNA molecule. Methods are known for introducing constructs into cells in such a way that transcribable polynucleotide molecules are transcribed into functional mRNA molecules which are translated and thereby expressed as protein products. Constructs capable of expressing antisense RNA molecules can also be constructed in order to inhibit translation of a particular RNA molecule of interest.

The term "recombinant plant expression vector": one or more DNA vectors for effecting plant transformation; these vectors are often referred to in the art as binary vectors. Binary vectors, together with vectors with helper plasmids, are most commonly used for agrobacterium-mediated transformation. Binary vectors typically include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be capable of expression in plant cells, heterologous DNA sequences to be transcribed, and the like.

The term "conversion": a method for introducing a heterologous DNA sequence into a host cell or organism.

The term "expression": transcription and/or translation of endogenous genes or transgenes in plant cells.

The term "recombinant host cell strain" or "host cell" means a cell comprising a polynucleotide of the present invention, regardless of the method used for insertion to produce the recombinant host cell, e.g., direct uptake, transduction, f-pairing, or other methods known in the art. The exogenous polynucleotide may remain as a non-integrating vector, such as a plasmid, or may integrate into the host genome. The host cell may be a prokaryotic cell or a eukaryotic cell, and the host cell may also be a monocotyledonous or dicotyledonous plant cell.

Drawings

FIG. 1A shows that there is an Indel natural variation (Indel) in each of the promoter and second intron regions of ZmLOX3 by a sequencing method; indel151, Indel of 151bp of the promoter region; indel363, Indel of 363bp of the second intronic region. The right side of the panel is the expression of the ZmLOX3 gene in ovules on day of pollination (Ovule — 0DAP) and kernels 5 days after pollination (Kernel — 5DAP) of the 12 inbred lines sequenced. Overall, the expression level of ZmLOX3 gene in inbred lines with Indel151 insertion or Indel363 deletion was higher; b is GWAS analysis, which shows that Indel151 and Indel363 are obviously related to the expression level of ZmLOX3 gene, and the lower part of a Manhattan diagram is a linkage disequilibrium module (LD-Block) diagram of all markers; c is the relation between different genotypes of Indel151 and the expression level of the ZmLOX3 gene in the inbred line, and when Indel151 is inserted, the expression level of the ZmLOX3 gene is higher; d is the allelic frequency variation of different genotypes of Indel151 between maize varieties of different ages, wherein the genotype corresponding to the reduced expression of the ZmLOX3 gene is selected during breeding; e is the allelic frequency distribution of the two genotypes at Indel151 between different heterosis population materials.

FIG. 2 shows the results of genotyping Indel151 in 12 inbred lines.

FIG. 3 shows the results of genotype identification of Indel363 from 12 inbred lines.

FIG. 4 shows the inoculation results of corn kernels from 10 inbred lines; the 5 inbred lines with Indel151 inserted and deleted at Indel363 were significantly more susceptible than the remaining inbred lines of different genotypes.

FIG. 5 shows the expression level of ZmLOX3 gene for different Indel151 genotypes in the hybrids (left panel) and the allelic frequency variation between Indel151 genotypes across the hybrids of different ages (right panel).

Detailed Description

The invention is further described below in conjunction with specific embodiments, the advantages and features of which will become apparent from the description. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be within the scope of the invention.

The inbred lines used in the following examples can obtain relevant information from the "Chinese crop germplasm information network" and apply for obtaining corresponding seeds.

Example 1 experiment of insertion deletion (Indel) variation of promoter region and second intron region of ZmLOX3 Gene regulating the variation of expression level of ZmLOX3 Gene

1. Discovery of ZmLOX3 gene promoter region and second intron region variation site

The 12 maize backbone inbred line parents widely used worldwide were genome-wide assembled using on average >55 × PacBio SMRT reads data, >94 × second generation Illumina 150bp double-end sequencing data, and >139 × BioNano monomolecular data. The 12 inbred lines were: PH207, A632, OH43, Zheng58, Ye478, Dan340, Huanggzaosi, Chang7-2, Jing92, Jing724, Xu178, S37; together with the published B73 and Mo17 genomes, these 14 high quality genomes cover almost all the core representative inbred lines of the heterosis population on maize breeding.

The genomic sequences of 10kb upstream and downstream of the ZmLOX3 gene in 14 core inbred lines were extracted and subjected to Blast alignment of DNA sequences, and it was found that there was an insertion-deletion variation (Indel, A in FIG. 1) in each of the promoter and the second intron of the ZmLOX3 gene in 14 inbred lines. Wherein the Indel length of the promoter region is 151bp, and the sequence is SEQ ID No. 3; the Indel length of the second exon region is 363bp, and the base sequence thereof is shown in SEQ ID No.5, or the Indel length of the exon region is 361bp, and the base sequence thereof is shown in SEQ ID No. 4.

2. Significant association between Indel151 and Indel363 and the variation in expression level of ZmLOX3 gene

China historically experienced 6 times of corn variety (hybrid) updating, and the inventor collected and combined 116 hybrids widely used in the variety updating process of different ages and 137 parent inbred lines corresponding to the hybrids (some hybrids have shared parents). In summer of 2019, female ears in the spinning period of the 253 materials (116 hybrid and 137 inbred lines) are sampled (3 times of sampling for each material), and then RNA-Seq analysis is carried out, so that the expression quantity data of 20000 genes in 253 material ears is obtained. In the invention, the expression data of the ZmLOX3 gene is extracted separately for subsequent analysis.

25,320,664 single nucleotide polymorphism molecular markers (SNPs) and 4,319,510 insertion deletion polymorphism molecular markers (indels) were mined by combining 137 parts of data of deep (> 10X) second-generation re-sequencing of maize inbred lines with published maize B73V 3 genome. In addition, genotypes of Indel151 (FIG. 2) and Indel363 (FIG. 3) in 137 maize inbred lines were identified using the developed PCR primers LOX3-2 (SEQ ID No.6 and SEQ ID No.7) and LOX3-3 (SEQ ID No.8 and SEQ ID No. 9).

The population structure and the genetic relationship of 137 maize inbred lines are estimated by using the mined molecular markers, and then the detected expression data of the ZmLOX3 gene is used as a phenotype for genome-wide association analysis (GWAS). Wherein one significant association signal was identified near the ZmLOX3 gene, with the most significant signal falling on the Indel151 marker (p-value 1.40E-9) and Indel363 being the second significant association marker (fig. 1B), indicating that Indel151 and Indel363 are significantly associated with the variation in expression of the ZmLOX3 gene.

In the test, 137 parts of materials are further divided into two types according to the genotype at Indel151, wherein one type is the genotype material with a 151bp deletion sequence at Indel151, and the total amount is 95 parts; the other is genotype material with 151bp sequence at Indel151, and the total number is 33; comparing the expression level of ZmLOX3 gene between the two types of materials, the expression level of ZmLOX3 gene was found to be lower in the material lacking Indel151 (FIG. 1C), indicating that Indel151 can positively regulate the expression of ZmLOX3 gene. Linkage disequilibrium analysis also showed that Indel151 and Indel363 are located within the same LD-block (linkage disequilibrium block) (fig. 1B); and sequence analysis in 14 inbred lines also showed that these two variations always appeared linked together in a phase-repulsion fashion, i.e. when the genotype for deletion of 151bp sequence at Indel151 in one material, often the genotype for insertion of 363bp or 361bp sequence at Indel363 (FIG. 1A). Indicating that it is possible to infer the genotype at the other Indel by the genotype of either; however, Indel151 has a stronger correlation with the expression level of the ZmLOX3 gene, so Indel151 can be used as a more excellent molecular marker for detecting the expression level of the ZmLOX3 gene.

Example 2 Indel151 and Indel363 influence resistance to maize head rot by regulating the expression level of ZmLOX3 gene

The expression level of the ZmLOX3 gene in the seeds is properly reduced, but the protein function of the seeds is not changed, and the effects of resisting fusarium and aspergillus flavus are possibly achieved.

The results of previous studies by the present inventors showed that the material lacking Indel151 or having Indel363 inserted therein often had a low expression level of ZmLOX3 gene. To further verify the relationship between Indel151 and Indel363 and the expression level of ZmLOX3 gene, 12 representative inbred lines (PH207, A632, OH43, ZHEN 58, Ye478, Dan340, Huangzaosi, Chang7-2, Jing92, Jing724, Xu178, S37) were planted in the field, and the ovules in the spinning stage and the kernels after pollination for 5 days were sampled and liquid nitrogen snap frozen. Each 5 individual samples were mixed into one sample, 3 biological replicates were taken from each inbred line, RNA was extracted by the TRIzol method, and the expression level of ZmLOX3 gene was measured using specific primers (SEQ ID No.10 and SEQ ID No. 11). It was found that, as in the previous GWAS analysis, the material lacking Indel151 or inserted Indel363 (PH207, A632, ZHEN 58, Ye478, lacing 724, Xu178, S37) generally had lower ZmLOX3 gene expression levels (A in FIG. 1) in either unpolished ovules or 5-day pollinated kernels, and the relationship between Indel151 and Indel363 and ZmLOX3 gene expression levels was again verified.

Further verticillides inoculation experiments were performed on 10 material corn kernels from 12 representative inbred lines (fig. 4). The results show that 5 inbred lines with low ZmLOX3 gene expression (B73, Ye478, Mo17, PH207, A632, deletion Indel151, insertion Indel363) are generally more resistant to panicle rot caused by Fusarium verticillarum than 5 inbred lines with high ZmLOX3 gene expression (Jing92, Dan340, Huanggzaosi, OH43, Chang7-2, insertion Indel151, deletion Indel 363). These results indicate that Indel151 and Indel363 can influence the resistance to maize head rot by regulating the expression level of ZmLOX3 gene.

Example 3 Excellent alleles at Indel151 were strongly artificially selected in modern maize breeding

The inventor previously collected 350 parts of maize inbred line material from different breeding stages in China and the United states, including 163 parts of American early (Public-US) and near-modern (Ex-PVP) breeding material, 187 parts of Chinese early (CN1960&70s), middle (CN1980&90s) and current (CN2000&10s) main planting maize breeding material. Analysis of the frequency distribution of Indel151 genotypes (Indel363 is closely linked to Indel151 and can be represented by Indel151 genotypes) in these materials shows that, with the advancement of the breeding period, the genotype frequencies of the deletion Indel151 (corresponding to a lower ZmLOX3 gene expression level and higher resistance to panicle rot) genotypes are significantly increased in the breeding process of corn in China and America (FIG. 1D), which indicates that the deletion Indel151 genotypes are subjected to strong manual selection in the modern corn breeding process.

In addition, 350 parts of maize inbred line material can be divided into 8 heterosis groups, namely SS, PA, X, NSS, TSPT, IDT, PB and Mixed. Previous reports show that TSPT germplasm is generally more susceptible to the ear rot, wherein Huangzaosi, Chang7-2 and Jing92 in the previous paragraphs are typical TSPT germplasm, and the inoculation experiment of the invention also proves that the TSPT germplasm is really more susceptible to the ear rot (figure 4), which proves the previous reports. The invention also discovers that the inserting frequency of indels 151 in TSPT germplasm is higher, and the inserting of indels 151 corresponds to higher ZmLOX3 gene expression level, so that the TSPT germplasm is susceptible to the ear rot, and discloses the reason of the TSPT germplasm susceptibility to the ear rot. Meanwhile, the important potential of Indel151 and Indel363 in the aspect of improvement of TSPT germplasm spike rot resistance is also predicted.

Furthermore, the present invention also analyzed the expression of ZmLOX3 gene and the distribution of Indel151 genes in 116 different generations of hybrids. Analysis revealed that ZmLOX3 was expressed in significantly lower amounts in hybrid species in the material lacking Indel151 than in the material heterozygous and inserted for Indel151 (left panel in FIG. 5). The low expression level of ZmLOX3 corresponds to resistance to ear rot, so the genotype of deletion Indel151 is an excellent genotype, and the genotype of insertion Indel151 is an unfavorable genotype. Allele frequency analysis found that homozygous good alleles of Indel151 rose gradually during breeding of the hybrids, while homozygous unfavorable genotypes almost disappeared in the hybrids after 2000 years; and as the breeding age advances, the proportion of heterozygous genotypes is lower and lower, further proving that Indel151 is strongly selected in the breeding process. These hybrids, all of which are varieties used directly in maize production, were selected among the hybrids, and the importance of this natural variation (Indel151 and its closely linked Indel363) was further confirmed.

SEQUENCE LISTING

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> DNA sequence for regulating and controlling resistance of maize ear rot, mutant, molecular marker and application thereof

<130> BJ-2002-210706A

<160> 14

<170> PatentIn version 3.5

<210> 1

<211> 6655

<212> DNA

<213> Zea mays L.

<400> 1

ggcgaactcc gctccgcccg accccagggc tcggactcgg gctaagaccc ggaagacggc 60

gaactccgct ccgcccgacc ccagggctcg gactcgggct aagacccgga agacggcgaa 120

ctctgctccg cccgacccca gggctcggac tcgggctaag acccggaaga cggcgaatct 180

ccgcctcgcc cgaccccagg gctcagactc cgccctggcc tcggccaaac gatctccgcc 240

tcgcccgacc ccagggctcg gactccgccc tagcctcggc caaacgatct ccgcctcgcc 300

cgacccgggg gctcgggctc ggcctcggca acggaaggca gactcgacct cgacttcgga 360

ggagccccca cgtcgccctg cctagggcac aggtccgcca cgtcaacagg aagcgccatc 420

accaacctac cccgagccga cttgggacac gaaggacaag accggcgtcc catctggcca 480

gctccgccgg atgggcaatg atggcgcccc ccgagctctg tgacgacggc ggctcttagc 540

tctcttacgg cagcagagcg acgtcagcaa ggactcgacc gctccaacag ctgtccctcc 600

gtcaggctcc gtcgctcctc cgacagccac gacatcacgc cagcaaggtg ccaagacctc 660

tccggctgcc acattggcat gtacccaggg cgttagctct ctctctctcc gctagacacg 720

tagcactctg ctacccccgt tgtacacctg gatcctctcc ttacgactat aaaaggaagg 780

accagggcct tcttagagaa ggttggccgc gcgggaccga ggacgggaca ggcgctctct 840

tggggccgct cgcttccctc acccgcgtgg acgcttgtaa cccccctact gcaagcgcac 900

ctgacctggg cgcgggacga acacgaaggc cgcgggactt ccacctctct cacgctcgac 960

tctggccacc tcgcctctcc ccccttcgcg ctcgcccacg cgctcgaccc atctgggctg 1020

gggcacgcag cacactcact cgtcggctta gggacccccc tgtctcgaaa cgccgacaat 1080

aagcttatct cgaattcatg tggtggagga ttggaaatga tttttatgta ttagtagaat 1140

ttgtttctac tctgtaaatt acatgaccct cttcgtctca ctcctctata gtaaaaatat 1200

agcacataaa tatctccgac atcttgctaa taatagtata caaatatatt tttcatcaaa 1260

ccgaattaac ttaattgata tatgtctaaa ttactgttat tagaatggaa ttcaattcca 1320

atgaaccaaa cggggcgtaa gtgatttctt agtgaggtgg cacctatgga aatcacttag 1380

tagtttctgg gttgtaattg ctcttatggc atgaagaaaa ttttgcattc gagctcgaac 1440

gctacaaggt cgttttagaa gctacatcag taatgcttcg agtatgacaa acaacatcac 1500

aagtaacatc tacctatatg caccgaatct ccaccaccac cacaatatcg atgtcagaag 1560

gctagggaag gacgaccaat aatatagaag acacataggc cacactcaat tcaatcaaca 1620

tcacgacaat ctacttgatg tcctatgtag ccatgatctc tacgagcaat caatatcaat 1680

gcactagaat cccaactacg tagagtatta ttggacgaac gagtaaaaca ccaaagagca 1740

gccactggac gactttcttg catctccaca aggataaatt ctctcagata gtcagatgcc 1800

taatgaaatt caagtccatc aatattgaca tgtgcgaagg caagaaggat ccaaattagt 1860

tgattcgaca ctacttggtt accatcaatc tcaagggagg aaacaaagat atcaaagcat 1920

catgctttca aatgtactca aagctactct agtgacttgg cttgaatagc tcccaagagg 1980

ctacatcgat aacaggctag cactctccaa ggaattaatg gacaacttcc aaagtgcatg 2040

gcctcgttcg gacaaccggt acaatcttca aaatggaagt agaagattgg tgaaagcata 2100

tgcaactagt acaagctctt gatcgaaatt tgatgctaca ggcctaacta cttcgacgat 2160

gatcatgggg aaaagttcta gtatcagaat ttcataaaaa caaaccaaaa acaattcatg 2220

agttctaaca catgatcaac aactgaatga atgtcgaaga cacagtacga gataagtttg 2280

acaattgaaa ccgtagatat aacaataaag ataacattca tagagacaac aagggcagct 2340

tatcggataa gaagtgaggg aaagataaca ctatgataag cactcgcatc aaagaagggt 2400

ggcaagaaca aatcattcaa ttagcaactc tttgagcaac acccatacca cctacatcat 2460

aaatcataag tgctctacca ctaaatgcat gcaactcaag aactttggca tcatcttagc 2520

tcggaagcaa gacaaaacca aacagagttg ataaaaaata ttaggaaaaa tagaataatg 2580

gagacttcta gagggccaca aacatagtta acctcatctt caaaggagca tcttccttaa 2640

tacctaagag gaagactaaa cttacccttt tgtgagatga tggttgttga accatcacct 2700

acaacatata tgtgatggtt cgagatccca attcaattta taagggagga ttagtttctc 2760

aaacatagga tgctatggtc tagtcctggc cccgatggtg gcaggacttc aacttagtaa 2820

ggaaggtgaa ttaccaatat gttcaaaagg atggggtaaa aggaaggtga attatgcaat 2880

ctatttttcg cactttttca ttaatcaaaa cctatatgga taaccaatcg ttcatatgtg 2940

caactaaggt tttgactaag tgttgctatc tctaccgtaa aaggagtttt gctaccccaa 3000

acctatcaac tagtctatga ctaagctaag aagataaatc acacaaccac aagtaacaat 3060

ataaatgtgg aattttaaat atggtagaga tacaaactct cgttgatgtg tcagtatttt 3120

tactggagta tcaagaaacg cgcaagcttc ttactaatcc ttcttagagc ctcacgcaag 3180

gctaagctcc cgctcaggta accctgtata gatacaaacc ctaccaatga ctatagtaac 3240

ttttatgaag atatagggaa acacacaagt tttaccctag ttctcgttgg agcctcttat 3300

aaacatgtcc acaaggggat gaagctaaga gtcgagtaag ggtttgtttg atttctttag 3360

tcttaatgac taaaactaag caaagagctt taattattgt gcaaattgat tacattatcc 3420

ctaattaatg ctatcttcga ctactgttta cccacacgca gagctactgt tcgtgcgcat 3480

tcacaatgct gcatgcgtgc acgttgcttt ggggaggagc ctacttcatg cgcgttggtt 3540

agggagggca tatgaagtaa aaatgtcact ttatggttag tttgacaccc tcattttttt 3600

caagggattg tattttcaca aaagaaatta atttattttt cttgaaaaat aggaatccct 3660

tagaaaaaaa tagagttgtc aaactaaccc ttattcattt tagtcactct tttggtaatt 3720

agaggactat aatttagttg gaggatttta gtcacactgt gttgattctt tagtgactaa 3780

aaatgactaa aatttaatca attaaatgta gtcacccaaa ccaaacagag tcgcgcgtcc 3840

gacgtgcctc ctcaaccggc gcgtgggagt aggtctggaa aaaagctcaa gctcgatgag 3900

ttggcttggg ctcacggtaa cttgggtcgg ctcgacgctc aaaacgagtc caagtcctat 3960

ttttgtggct cgtgataagt gtgagctagc tcggctcggc tcacgaaccg acaacaattt 4020

actacataaa attctgatta gcatataatg ttagtaccaa atatacaatt agtatatgtt 4080

atttgatatt tatatacaaa attaatttat ttctatgtta tattagtact ataattattt 4140

attaatttaa gagatgtaat ttgattattt ttgatatttt atgttataat ttgtaacgta 4200

agctggtgct tgtggttaga ctctctacta gtcgagtttt cacgctcgtc aaaggaccga 4260

gccgagacga ggcaagctca acacattacc gagccgaccg gctcgtttcc gccacgtggt 4320

agagcggcag cacccttccc agatctcgtg tcccgcgcgc cgtgcgcaca gccgcgcaca 4380

cacacggact gccgcgtccg ggcgcgatcc gttctcgcga acacaccggt cgtagccgtc 4440

cgtaatttgt actagtatgg cacgcggaac acgagtcggc gccagcaccg gcacgtcacc 4500

atcggggccg ctggccccat acgggcagcc aattaataaa tccttgctct gcgacagacg 4560

cgcaggggca cgtcacgcag ccgtagtcga tcggtgcacg gtgctcgcgc ccccgttgca 4620

gcgcgcagct ctcccgcgct ataaatgccc cggctcggcc tcgctcccac agccacagcc 4680

tcacacagac accaacgcca ctgcactgca aaagcaagag cagctagcta gtaaagatgc 4740

tgagcgggat catcgacggg ctgacggggg cgaacaagca tgcgcggctc aagggcacgg 4800

tggtgctcat gcgcaagaac gtgctggacc tcaacgactt cggcgccacc gtcgttgaca 4860

gcatcagcga gttcctcggc aagggggtca cctgccagct catcagctcc accctcgtcg 4920

acgccagtga gtaccgcgcc gcgccgccgg cacctctccg atctgcgctt ccccatgtcg 4980

atcgatctcg atctctctag gctctagagc tatagctctc tcggccccca ctttttacct 5040

ttgcaagcat tttccctgca tgcgaaacaa gcgatagttt actatttggg cggccatgct 5100

gctgctgctt cggctacctt gcctccgtca tctttgacgg gacatggaaa gaaagaaaga 5160

atagagagag agacagagag agagagagag caaacaccga gaaaaagaca gcaaagctag 5220

ttgtagcctg ggcgcagaac acagcaccag atgctggcta gctcgtgaca aagtaaaaaa 5280

agagaggaac gaacacagta gtaccaagag atcagggacg agacctttct actttgaact 5340

ggattatata ttggattctt tatcagtaac ttactgctgc tagtataccc ctaccctagt 5400

ctcggggcga cgtgcctgcg tgcatgcccg acgcgtacga aacacatcga cggactcaca 5460

tgggccaccg cgcgcgcgcg tgcctgcttt aactttcgct gtgcagacaa cggcaaccgc 5520

gggcgggtcg gggcggaggc gaacctggag cagtggctga cgagcctgcc gtcgctgacg 5580

accggcgagt ccaagttcgg cgtcacgttc gactgggagg tggagaagct gggagtgccg 5640

ggggccgtcg tcgtcaagaa caaccacgcc gccgagttct tcctcaagac aatcaccctc 5700

gacgacgtgc ccggccgcgg cgccgtcacc ttcgtcgcca actcctgggt ctaccccgcg 5760

ggcaagtacc gctacaaccg cgtcttcttc tccaacgatg tgagtccttt ctcgatagat 5820

cattatgttt gtttgttatt ttgtttcttg gtatcatgta ggcatgtagc tagccgccat 5880

gtcgtcagtt ggagtgcagt aggtaggaaa aaggacgaca tgggatggga gtggttaaga 5940

aaatccatgc aagtgggact agtgtgtaac tggtagtata gctgaagaat ctagtggtag 6000

aatgatcttg tacgtgaata atgtttctga cgctgagcgc tgaggctatc cgcaaccgtt 6060

aaccctaaat ttttccctct atatcatttt ttcctctatt ttcctcccta ttttttcatc 6120

tcccgcagcg gttcccccta aatactcccc ctatatctca ctaccactat aaaatattat 6180

tttctatacc aattatcaat tttttatcta ctaacaatta ctcgtggacc cacagcacag 6240

tgtttagggt gatgaacagt gacacgctag atctgaaggg agagagaagg ggaccgacac 6300

gtagggagcc tgtagagggc accgctgcgg ccgtagggtg ctccctacgc gccgcataca 6360

aggggagggg ggagaggcag cggtaaccgc tgcgcacagc ctgagggcga ggcatgtgag 6420

ttcaccacgt gagtagcagc aaaaggaaac aacccttctt cacccggcta tcatctaacg 6480

tatcgccccg ggagaatcaa taactctaac gagatgacga aaagtcaaaa ataaagtcgt 6540

gtgatggcca tgaaagtcag tcaagcaaat cagctgctaa cacgtgtccc ttatctacag 6600

gtgtaagaga gtagagtctt gtcaatcaac ctgggttgtt ttctatctgc gtttt 6655

<210> 2

<211> 6434

<212> DNA

<213> Zea mays L.

<400> 2

ggcgaactcc gctccgcccg accccagggc tcggactcgg gctaagaccc ggaagacggc 60

gaactccgct ccgcccgacc ccagggctcg gactcgggct aagacccgga agacggcgaa 120

ctctgctccg cccgacccca gggctcggac tcgggctaag acccggaaga cggcgaatct 180

ccgcctcgcc cgaccccagg gctcagactc cgccctggcc tcggccaaac gatctccgcc 240

tcgcccgacc ccagggctcg gactccgccc tagcctcggc caaacgatct ccgcctcgcc 300

cgacccgggg gctcgggctc ggcctcggca acggaaggca gactcgacct cgacttcgga 360

ggagccccca cgtcgccctg cctagggcac aggtccgcca cgtcaacagg aagcgccatc 420

accaacctac cccgagccga cttgggacac gaaggacaag accggcgtcc catctggcca 480

gctccgccgg atgggcaatg atggcgcccc ccgagctctg tgacgacggc ggctcttagc 540

tctcttacgg cagcagagcg acgtcagcaa ggactcgacc gctccaacag ctgtccctcc 600

gccaggctcc gtcgctcctc cgacagccac gacatcacgc cagcaaggtg ccaagacctc 660

tccggctgcc acattggcat gtacccaggg cgttagctct ctctctctcc gctagacacg 720

tagcactctg ctaccccccg ttgtacacct ggatcctctc cttacgacta taaaaggaag 780

gaccagggcc ttcttagaga aggttggccg cgcgggaccg aggacgggac aggcgctctc 840

ttggggccgc tcgcttccct cacccgcgtg gacgcttgta acccccctac tgcaagcgca 900

cctgacctgg gcgcgggacg aacacgaagg ccgcgggact tccacctctc tcacgctcga 960

ctccggccac ctcgcctctc cccccttcgc gctcgcccac gcgctcgacc catctgggct 1020

ggggcacgca gcacactcac tcgtcggctt agggaccccc ctgtctcgaa acgccgacaa 1080

taagcttatc tcgaattcat ggggtggagg attggaaatg atttttatgt attagtagaa 1140

tttgtttcta ctctgtaaat tacatgaccc tcttcgtctc actcctctat agtaaaaata 1200

tagcacataa atatctccga catcttgcta ataatagtat acaaatatat ttttcatcaa 1260

accgaattaa cttaattgat atatgtctaa attactgtta ttagaatgga attcaattcc 1320

aatgaaccaa acggggcgta agtgatttct tagtgaggtg gcacctatgg aaatcactta 1380

gtagtttctg ggttgtaatt gctcttatgg catgaagaaa attttgcatt cgagctcgaa 1440

cgctacaagg tcgttttaga agctacatca gtaatgcttc gagtatgaca aacaacatca 1500

caagtaacat ctacctatat gcaccgaatc tccaccacca ccacaatatc gatgtcagaa 1560

ggctagggaa ggacgaccaa taatatagaa gacacatagg ccacactcaa ttcaatcaac 1620

atcacgacaa tctacttgat gtcctatgta gccatgatct ctacgagcaa tcaatatcaa 1680

tgcactagaa tcccaactac gtagagtatt attggacgaa cgagtaaaac accaaagagc 1740

agccactgga cgactttctt gcatctccac aaggataaat tctctcagat agtcagatgc 1800

ctaatgaaat tcaagtccat caatattgac atgtgcgaag gcaagaagga tccaaattag 1860

ttgattcgac actacttggt taccatcaat ctcaagggag gaaacaaaga tatcaaagca 1920

tcatgctttc aaatgtactc aaagctactc tagtgacttg gcttgaatag ctcccaagag 1980

gctacatcga taacaggcta gcactctcca aggaattaat ggacaacttc caaagtgcat 2040

ggcctcgttc ggacaaccgg tacaatcttc aaaatggaag tagaagattg gtgaaagcat 2100

atgcaactag tacaagctct tgatcgaaat ttgatgctac aggcctaact acttcgacga 2160

tgatcatggg gaaaagttct agtatcagaa tttcataaaa acaaaccaaa aacaattcat 2220

gagttctaac acatgatcaa caactgaatg aatgtcgaag acacagtacg agataagttt 2280

gacaattgaa accgtagata taacaataaa gataacattc atagagacaa caagggcagc 2340

ttatcggata agaagtgagg gaaagataac actatgataa gcactcgcat caaagaaggg 2400

tggcaagaac aaatcattca attagcaact ctttgagcaa cacccatacc acctacatca 2460

taaatcataa gtgctctacc actaaatgca tgcaactcaa gaactttggc atcatcttag 2520

ctcggaagca agacaaaacc aaacagagtt gataaaaaat attaggaaaa atagaataat 2580

ggagacttct agagggccac aaacatagtt aacctcatct tcaaaggagc atcttcctta 2640

atacctaaga ggaagactaa acttaccctt ttgtgagatg atggttgttg aaccatcacc 2700

tacaacatat atgtgatggt tcgagatccc aattcaattt ataagggagg attagtttct 2760

caaacatagg atgctatggt ctagtcctgg ccccgatggt ggcaggactt caacttagta 2820

aggaaggtga attaccaata tgttcaaaag gatggggtaa aaggaaggtg aattatgcaa 2880

tctatttttc gcactttttc attaatcaaa acctatatgg ataaccaatc gttcatatgt 2940

gcaactaagg ttttgactaa gtgttgctat ctctaccgta aaaggagttt tgctacccca 3000

aacctatcaa ctagtctatg actaagctaa gaagataaat cacacaacca caagtaacaa 3060

tataaatgtg gaattttaaa tatggtagag atacaaactc tcgttgatgt gtcagtattt 3120

ttactggagt atcaagaaac gcgcaagctt cttactaatc cttcttagag cctcacgcaa 3180

ggctaagctc ccgctcaggt aaccctgtat agatacaaac cctaccaatg actatagtaa 3240

cttttatgaa gatataggga aacacacaag ttttacccta gttctcgttg gagcctctta 3300

taaacatgtc cacaagggga tgaagctaag agtcgagtaa gggtttgttt gatttcttta 3360

gtcttaatga ctaaaactaa gcaaagagct ttaattattg tgcaaattga ttacattatc 3420

cctaattaat gctatcttcg actactgttt acccacacgc agagctactg ttcgtgcgca 3480

ttcacaatgc tgcatgcgtg cacgttgctt tggggaggag cctacttcat gcgcgttggt 3540

tagggagggc atatgaagta aaaatgtcac tttatggtta gtttgacacc ctcatttttt 3600

tcaagggatt gtattttcac aaaagaaatt aatttatttt tcttgaaaaa taggaatccc 3660

ttagaaaaaa atagagttgt caaactaacc cttattcatt ttagtcactc ttttggtaat 3720

tagaggacta taatttagtt ggaggatttt agtcacactg tgttgattct ttagtgacta 3780

aaaatgacta aaatttaatc aattaaatgt agtcacccaa accaaacaga gtcgcgcgtc 3840

cgacgtgcct cctcaaccgg cgcgtgggag taggtctgga aaaaagctcg agctcgatga 3900

gttggcttgg gctcacggta acttgggtcg gctcgacgct caaaacgagt ccaagtccta 3960

tttttgtggc tcgtgataag tgtgagctag ctcggctcgg ctcacgaacc gacaacaatt 4020

tactacataa aattctgatt agcatataat gttagtacca aatatacaat tagtatatgt 4080

tatttgatat ttatatacaa aattaattta tttctatgtt atattagtac tataattatt 4140

tattaattta agagatgtaa tttgattatt tttgatattt tatgttataa tttgtaacgt 4200

aagctggtgc ttgtggttag actctctact agtcgagttt tcacgctcgt caaaggaccg 4260

agccgagacg aggcaagctc aacacattac cgagccgacc ggctcgtttc cgccacgtgg 4320

tagagcggca gcacccttcc cagatctcgt gtcccgcgcg ccgtgcgcac agccgcgcac 4380

acacacggac tgccgcgtcc gggcgcgatc cgttctcgcg aacacaccgg tcgtagccgt 4440

ccgtaatttg tactagtatg gcacgcggaa cacgagtcgg cgccagcacc ggcacgtcac 4500

catcggggcc gctggcccca tacgggcagc caattaataa aagagccgaa gcgaccgacg 4560

gcgcaggtac acgcaggcgc ctcctgcacc attcaccatt cacgtctctg tggcccaata 4620

ataaaaccgc attaattacg ctcgcgcaga cacggtagca cactggccgc cacagtgcca 4680

cccacccacc aatccttgct ctgcgacaga cgcgcagggg cacgtcacgc agccgtagtc 4740

gatcggtgca cggtgctcgc gcccccgttg cagcgcgcag ctctcccgcg ctataaatgc 4800

cccggctcgg cctcgctccc acagccacag cctcacacag acaccaacgc cactgcactg 4860

caaaagcaag agcagctagc tagtaaagat gctgagcggg atcatcgacg ggctgacggg 4920

ggcgaacaag catgcgcggc tcaagggcac ggtggtgctc atgcgcaaga acgtgctgga 4980

cctcaacgac ttcggcgcca ccgtcgttga cagcatcagc gagttcctcg gcaagggggt 5040

cacctgccag ctcatcagct ccaccctcgt cgacgccagt gagtaccgcg ccgcgccgcc 5100

ggcacctctc cgatctgcgc ttccccatgt cgatcgatct cgatctctct aggctctaga 5160

gctatagctc tctcggcccc cactttttac ctttgcaagc attttccctg catgcgaaac 5220

aagcgatagt ttactatttg ggcggccatg ctgctgctgc ttcggctacc ttgcctccgt 5280

catctttgac gggacatgga aagaaagaaa gaatagagag agagacagag agagagagag 5340

agcaaacacc gagaaaaaga cagcaaagct agttgtagcc tgggcgcaga acacagcacc 5400

agatgctggc tagctcgtga caaagtaaaa aaagagagga acgaacacag tagtaccaag 5460

agatcaggga cgagaccttt ctactttgaa ctggattata tattggattc tttatcagta 5520

acttactgct gctagtatac ccctacccta gtctcggggc gacgtgcctg cgtgcatgcc 5580

cgacgcgtac gaaacacatc gacggactca catgggccac cgcgcgcgcg cgtgcctgct 5640

ttaactttcg ctgtgcagac aacggcaacc gcgggcgggt cggggcggag gcgaacctgg 5700

agcagtggct gacgagcctg ccgtcgctga cgaccggcga gtccaagttc ggcgtcacgt 5760

tcgactggga ggtggagaag ctgggagtgc cgggggccgt cgtcgtcaag aacaaccacg 5820

ccgccgagtt cttcctcaag acaatcaccc tcgacgacgt gcccggccgc ggcgccgtca 5880

ccttcgtcgc caactcctgg gtctaccccg cgggcaagta ccgctacaac cgcgtcttct 5940

tctccaacga tgtgagtcct ttctcgatag atcattatgt ttgtttgttt attggtatca 6000

tgtagctagc cgccatgtcg tcagttggag tgcagtaggt aggaaaaagg acgacatggg 6060

atgggagtgg ttaagaaaat ccatgcaagt gggactagtg tgtaactggt agtatagctg 6120

aagaatctag tggtagaatg atcttgtacg tgaataatgt ttctgacgct gagcgctgag 6180

ggcgaggcat gtgagttcac cacgtgagta gcagcaaaag gaaacgaccc ttcttcaccc 6240

ggctatcatc taacgtatcg ccccgggaga atcaataact ttattaacga gatgacgaaa 6300

agtcaaaaaa aaaagtcgtg tgatggccat gatagtcagt caagcaaatc agcgtccaac 6360

acgtgtccct tatctacagg tgtaagagag tagagtcttg tcaatcaacc tgggttgttt 6420

tctatctgcg tttt 6434

<210> 3

<211> 151

<212> DNA

<213> Zea mays L.

<400> 3

aaagagccga agcgaccgac ggcgcaggta cacgcaggcg cctcctgcac cattcaccat 60

tcacgtctct gtggcccaat aataaaaccg cattaattac gctcgcgcag acacggtagc 120

acactggccg ccacagtgcc acccacccac c 151

<210> 4

<211> 361

<212> DNA

<213> Zea mays L.

<400> 4

ctgaggctat ccgcaaccgt taaccctaaa tttttccctc tatatcattt tttcctctat 60

tttcctccct attttttcat ctcccgcagc ggttccccct aaatactccc cctatatctc 120

actaccacta taaaatatta ttttctatac caattatcaa ttttttatct actaacaatt 180

actcgtggac ccacagcaca gtgtttaggg tgatgaacag tgacacgcta gatctgaagg 240

gagagagaag gggaccgaca cgtagggagc ctgtagaggg caccgctgcg gccgtagggt 300

gctccctacg cgccgcatac aaggggaggg gggagaggca gcggtaaccg ctgcgcacag 360

c 361

<210> 5

<211> 363

<212> DNA

<213> Zea mays L.

<400> 5

ctgaggctat ccgcaaccgt taaccctaaa tttttccctc tatatcattt tttcccctat 60

tttcctccct attttttcat ctcccgcagc ggttccccct aaatactccc cctatatccc 120

actaccacta taaaatatta ttttctatac caattatcaa ttttttatct actaacaatt 180

actcgtggac ccacagcaca gtgtttaggg tgatgaacag tgacacgcta gatctgaagg 240

gagagagaag gggaccgaca cgtagggagc ctgtagaggg caccgctgcg gccgtagggt 300

gctccctacg cgccgcatac aaggggaggg gggggagagg cagcggtaac cgctgcgcac 360

agc 363

<210> 6

<211> 20

<212> DNA

<213> Artifical sequence

<400> 6

gctcacgaac cgacaacaat 20

<210> 7

<211> 21

<212> DNA

<213> Artifical sequence

<400> 7

tcccgctcag catctttact a 21

<210> 8

<211> 20

<212> DNA

<213> Artifical sequence

<400> 8

tacgaaacac atcgacggac 20

<210> 9

<211> 20

<212> DNA

<213> Artifical sequence

<400> 9

gttattgatt ctcccggggc 20

<210> 10

<211> 22

<212> DNA

<213> Artifical sequence

<400> 10

gttcttcctc aagacaatca cc 22

<210> 11

<211> 21

<212> DNA

<213> Artifical sequence

<400> 11

gagaagaaga cgcggttgta g 21

<210> 12

<211> 2604

<212> DNA

<213> Zea mays L.

<400> 12

atgctgagcg ggatcatcga cgggctgacg ggggcgaaca agcatgcgcg gctcaagggc 60

acggtggtgc tcatgcgcaa gaacgtgctg gacctcaacg acttcggcgc caccgtcgtt 120

gacagcatca gcgagttcct cggcaagggg gtcacctgcc agctcatcag ctccaccctc 180

gtcgacgcca acaacggcaa ccgcgggcgg gtcggggcgg aggcgaacct ggagcagtgg 240

ctgacgagcc tgccgtcgct gacgaccggc gagtccaagt tcggcgtcac gttcgactgg 300

gaggtggaga agctgggagt gccgggggcc gtcgtcgtca agaacaacca cgccgccgag 360

ttcttcctca agacaatcac cctcgacgac gtgcccggcc gcggcgccgt caccttcgtc 420

gccaactcct gggtctaccc cgcgggcaag taccgctaca accgcgtctt cttctccaac 480

gatacgtacc tgccaagcca gatgccggcg gcgctgaagc cgtaccgcga cgacgagctc 540

cgcaacctcc gcggcgacga ccagcagggc ccctaccagg agcacgaccg cgtgtaccgc 600

tacgacgtct acaacgacct cggcgagccc gacggcggca acccgcgccc catcctcggc 660

ggctccgccg accacccgta cccgcgccgc tgccgcacgg gccgcaagcc caccaaaacc 720

gaccccaact cggagagccg actgtcgctg gtggagcaga tctacgtgcc gcgggacgag 780

cgcttcggcc acctcaagat gtccgacttc ctgggctact ccatcaaggc catcacgcag 840

ggcatcatcc cggcggtgcg cacgtacgtg gacaccaccc cgggcgagtt cgactccttc 900

caggacatca tcaacctgta cgagggcggg atcaagctgc ccaagatcca ggcgctcgag 960

gacatgcgca agctcttccc gctccagctc gtcaaggacc tcctccccgc cggcggggac 1020

tacctgctca agctccccat cccacagatc atccaaggca cgtcacagga caagaacgcg 1080

tggaggaccg acgaggagtt cgcgcgggag gtgctcgccg gcgtcaaccc gatggtgatc 1140

acgcgcctca cggagttccc gcccaagagc acgctggacc ccagcaagta cggcgaccac 1200

accagcacga tcacggcgga gcacatcgag aagaacctcg agggcctcac ggtgcagcag 1260

gcgctggacg gcaacaggct ctacatcctg gaccaccacg accgcttcat gccgttcctc 1320

atcgacgtca acaacctgga gggcaacttc atctacgcca ccaggacgct cttcttcctg 1380

cgcggcgacg gcaggctcgc gcccctcgcc atcgagctca gcgagccgta catcgacggg 1440

gacctcaccg tggccaagag caaggtctac acgccggcgt ccagcggcgt cgaggcctgg 1500

gtgtggcagc tcgccaaggc ctatgtcgcc gtcaacgact ctggctggca ccaactcgtc 1560

agccactggc tgaacaccca cgcggtgatg gagccgttcg tgatcgcgac gaaccggcag 1620

ctgagcgtga cgcacccggt gcacaagctc ctgagctcgc acttccgcga caccatgacc 1680

atcaacgcgc tggcgcggca gacgctcatc aacggcggcg gcatcttcga gatgaccgtc 1740

ttcccgggca agtacgcgct gggcatgtcc tccgtggtgt acaagagctg gaacttcacc 1800

gagcagggcc tccccgccga cctcgtcaag aggggcgtgg cggtggcgga cccgtccagc 1860

ccgtacaagg tgcggctgct gatcgaggac tacccgtacg cgagcgacgg gctggccatc 1920

tggcacgcca tcgagcagtg ggtgggcgag tacctggcca tctactaccc cgacgacggc 1980

gcgctgcggg gcgacgagga gctgcaggcg tggtggaagg aggtgcgcga ggtcgggcac 2040

ggcgaccaca aggacgcgcc ctggtggccc aagatgcagg ccgtgtcgga gctcgccagc 2100

gcctgcacca ccatcatctg gatcgcgtcg gcgctccacg ccgccgtcaa cttcggccag 2160

tacccgtacg cggggtacct cccgaacagg cccacggtga gccggcgccg gatgccggag 2220

cccggcagca aggagtacga ggagctggag cgcgacccgg agcgcggctt catccacacc 2280

atcacgagcc agatccagac catcatcggc atctcgctca tcgagatcct ctccaagcac 2340

tcctccgacg aggtgtacct cggccagcgc gacacccccg agtggacctc cgacgcccgg 2400

gcgctggcgg cgttcaagag gttcagcgac gcgctggtca agatcgaggg caaggtggtg 2460

ggcgagaacc gcgacccgca gctgaggaac aggaacggcc ccgccgagtt cccctacatg 2520

ctgctctatc ccaacacctc tgaccacagt ggcgccgccg cagggctcac tgccaagggc 2580

atccccaaca gcatctccat ctga 2604

<210> 13

<211> 867

<212> PRT

<213> Zea mays L.

<400> 13

Met Leu Ser Gly Ile Ile Asp Gly Leu Thr Gly Ala Asn Lys His Ala

1 5 10 15

Arg Leu Lys Gly Thr Val Val Leu Met Arg Lys Asn Val Leu Asp Leu

20 25 30

Asn Asp Phe Gly Ala Thr Val Val Asp Ser Ile Ser Glu Phe Leu Gly

35 40 45

Lys Gly Val Thr Cys Gln Leu Ile Ser Ser Thr Leu Val Asp Ala Asn

50 55 60

Asn Gly Asn Arg Gly Arg Val Gly Ala Glu Ala Asn Leu Glu Gln Trp

65 70 75 80

Leu Thr Ser Leu Pro Ser Leu Thr Thr Gly Glu Ser Lys Phe Gly Val

85 90 95

Thr Phe Asp Trp Glu Val Glu Lys Leu Gly Val Pro Gly Ala Val Val

100 105 110

Val Lys Asn Asn His Ala Ala Glu Phe Phe Leu Lys Thr Ile Thr Leu

115 120 125

Asp Asp Val Pro Gly Arg Gly Ala Val Thr Phe Val Ala Asn Ser Trp

130 135 140

Val Tyr Pro Ala Gly Lys Tyr Arg Tyr Asn Arg Val Phe Phe Ser Asn

145 150 155 160

Asp Thr Tyr Leu Pro Ser Gln Met Pro Ala Ala Leu Lys Pro Tyr Arg

165 170 175

Asp Asp Glu Leu Arg Asn Leu Arg Gly Asp Asp Gln Gln Gly Pro Tyr

180 185 190

Gln Glu His Asp Arg Val Tyr Arg Tyr Asp Val Tyr Asn Asp Leu Gly

195 200 205

Glu Pro Asp Gly Gly Asn Pro Arg Pro Ile Leu Gly Gly Ser Ala Asp

210 215 220

His Pro Tyr Pro Arg Arg Cys Arg Thr Gly Arg Lys Pro Thr Lys Thr

225 230 235 240

Asp Pro Asn Ser Glu Ser Arg Leu Ser Leu Val Glu Gln Ile Tyr Val

245 250 255

Pro Arg Asp Glu Arg Phe Gly His Leu Lys Met Ser Asp Phe Leu Gly

260 265 270

Tyr Ser Ile Lys Ala Ile Thr Gln Gly Ile Ile Pro Ala Val Arg Thr

275 280 285

Tyr Val Asp Thr Thr Pro Gly Glu Phe Asp Ser Phe Gln Asp Ile Ile

290 295 300

Asn Leu Tyr Glu Gly Gly Ile Lys Leu Pro Lys Ile Gln Ala Leu Glu

305 310 315 320

Asp Met Arg Lys Leu Phe Pro Leu Gln Leu Val Lys Asp Leu Leu Pro

325 330 335

Ala Gly Gly Asp Tyr Leu Leu Lys Leu Pro Ile Pro Gln Ile Ile Gln

340 345 350

Gly Thr Ser Gln Asp Lys Asn Ala Trp Arg Thr Asp Glu Glu Phe Ala

355 360 365

Arg Glu Val Leu Ala Gly Val Asn Pro Met Val Ile Thr Arg Leu Thr

370 375 380

Glu Phe Pro Pro Lys Ser Thr Leu Asp Pro Ser Lys Tyr Gly Asp His

385 390 395 400

Thr Ser Thr Ile Thr Ala Glu His Ile Glu Lys Asn Leu Glu Gly Leu

405 410 415

Thr Val Gln Gln Ala Leu Asp Gly Asn Arg Leu Tyr Ile Leu Asp His

420 425 430

His Asp Arg Phe Met Pro Phe Leu Ile Asp Val Asn Asn Leu Glu Gly

435 440 445

Asn Phe Ile Tyr Ala Thr Arg Thr Leu Phe Phe Leu Arg Gly Asp Gly

450 455 460

Arg Leu Ala Pro Leu Ala Ile Glu Leu Ser Glu Pro Tyr Ile Asp Gly

465 470 475 480

Asp Leu Thr Val Ala Lys Ser Lys Val Tyr Thr Pro Ala Ser Ser Gly

485 490 495

Val Glu Ala Trp Val Trp Gln Leu Ala Lys Ala Tyr Val Ala Val Asn

500 505 510

Asp Ser Gly Trp His Gln Leu Val Ser His Trp Leu Asn Thr His Ala

515 520 525

Val Met Glu Pro Phe Val Ile Ala Thr Asn Arg Gln Leu Ser Val Thr

530 535 540

His Pro Val His Lys Leu Leu Ser Ser His Phe Arg Asp Thr Met Thr

545 550 555 560

Ile Asn Ala Leu Ala Arg Gln Thr Leu Ile Asn Gly Gly Gly Ile Phe

565 570 575

Glu Met Thr Val Phe Pro Gly Lys Tyr Ala Leu Gly Met Ser Ser Val

580 585 590

Val Tyr Lys Ser Trp Asn Phe Thr Glu Gln Gly Leu Pro Ala Asp Leu

595 600 605

Val Lys Arg Gly Val Ala Val Ala Asp Pro Ser Ser Pro Tyr Lys Val

610 615 620

Arg Leu Leu Ile Glu Asp Tyr Pro Tyr Ala Ser Asp Gly Leu Ala Ile

625 630 635 640

Trp His Ala Ile Glu Gln Trp Val Gly Glu Tyr Leu Ala Ile Tyr Tyr

645 650 655

Pro Asp Asp Gly Ala Leu Arg Gly Asp Glu Glu Leu Gln Ala Trp Trp

660 665 670

Lys Glu Val Arg Glu Val Gly His Gly Asp His Lys Asp Ala Pro Trp

675 680 685

Trp Pro Lys Met Gln Ala Val Ser Glu Leu Ala Ser Ala Cys Thr Thr

690 695 700

Ile Ile Trp Ile Ala Ser Ala Leu His Ala Ala Val Asn Phe Gly Gln

705 710 715 720

Tyr Pro Tyr Ala Gly Tyr Leu Pro Asn Arg Pro Thr Val Ser Arg Arg

725 730 735

Arg Met Pro Glu Pro Gly Ser Lys Glu Tyr Glu Glu Leu Glu Arg Asp

740 745 750

Pro Glu Arg Gly Phe Ile His Thr Ile Thr Ser Gln Ile Gln Thr Ile

755 760 765

Ile Gly Ile Ser Leu Ile Glu Ile Leu Ser Lys His Ser Ser Asp Glu

770 775 780

Val Tyr Leu Gly Gln Arg Asp Thr Pro Glu Trp Thr Ser Asp Ala Arg

785 790 795 800

Ala Leu Ala Ala Phe Lys Arg Phe Ser Asp Ala Leu Val Lys Ile Glu

805 810 815

Gly Lys Val Val Gly Glu Asn Arg Asp Pro Gln Leu Arg Asn Arg Asn

820 825 830

Gly Pro Ala Glu Phe Pro Tyr Met Leu Leu Tyr Pro Asn Thr Ser Asp

835 840 845

His Ser Gly Ala Ala Ala Gly Leu Thr Ala Lys Gly Ile Pro Asn Ser

850 855 860

Ile Ser Ile

865

<210> 14

<211> 6642

<212> DNA

<213> Zea mays L.

<400> 14

ggcgaactcc gctccgcccg accccagggc tcggactcgg gctaacaccc ggaagacggc 60

gaactccgct ccgcccgacc ccagggctcg gactcgggct aagacccgga agacggcgaa 120

ctctgctccg cccgacccca gggctcggac tcgggctaag acccggaaga cggcgaatct 180

ccgcctcgcc cgaccccagg gctcagactc cgccctggcc tcggccaaac gatctccgcc 240

tcgcccgacc ccagggctcg gactccgccc tagcctcggc caaacgatct ccgcctcgcc 300

cgacccgggg gctcgggctc ggcctcggca acggaaggca gactcgacct cgacttcgga 360

ggagccccca cgtcgccctg cctagggcac aggtccgcca cgtcaacagg aagcgccatc 420

accaacctac cccgagccga cttgggacac gaaggacaag accggcgtcc catctggcca 480

gctccgccgg atgggcaatg atggcgcccc ccgagctctg tgacgacggc ggctcttagc 540

tctcttacgg cagcagagcg acgtcagcaa ggactcgacc gctccaacag ctgtccctcc 600

gccaggctcc gtcgctcctc cgacagccac gacatcacgc cagcaaggtg ccaagacctc 660

tccggctgcc acattggcat gtacccaggg cgttagctct ctctctctcc gctagacacg 720

tagcactctg ctaccccccg ttgtacacct ggatcctctc cttacgacta taaaaggaag 780

gaccagggcc ttcttagaga aggttggccg cgcgggaccg aggacgggac aggcgctctc 840

ttggggccgc tcgcttccct cacccgcgtg gacgcttgta acccccctac tgcaagcgca 900

cctgacctgg gcgcgggacg aacacgaagg ccgcgggact tccacctctc tcacgctcga 960

ctccggccac ctcgcctctc cccccttcgc gctcgcccac gcgctcgacc catctgggct 1020

ggggcacgca gcacactcac tcgtcggctt agggaccccc ctgtctcgaa acgccgacaa 1080

taagcttatc tcgaattcat ggggtggagg attggaaatg atttttatgt attagtagaa 1140

tttgtttcta ctctgtaaat tacatgaccc tcttcgtctc actcctctat agtaaaaata 1200

tagcacataa atatctccga catcttgcta ataatagtat acaaatatat ttttcatcaa 1260

accgaattaa cttaattgat atatgtctaa attactgtta ttagaatgga attcaattcc 1320

aatgaaccaa acggggcgta agtgatttct tagtgaggtg gcacctatgg aaatcactta 1380

gtagtttctg ggttgtaatt gctcttatgg catgaagaaa attttgcatt cgagctcgaa 1440

cgctacaagg tcgttttaga agctacatca gtaatgcttc gagtatgaca aacaacatca 1500

caagtaacat ctacctatat gcaccgaatc tccaccacca ccacaatatc gatgtcagaa 1560

ggctagggaa ggacgaccaa taatatagaa gacacatagg ccacactcaa ttcaatcaac 1620

atcacgacaa tctacttgat gtcctatgta gccatgatct ctacgagcaa tcaatatcaa 1680

tgcactagaa tcccaactac gtagagtatt attggacgaa cgagtaaaac accaaagagc 1740

agccactgga cgactttctt gcatctccac aaggataaat tctctcagat agtcagatgc 1800

ctaatgaaat tcaagtccat caatattgac atgtgcgaag gcaagaaggg tccaaattag 1860

ttgattcgac actacttggt taccatcaat ctcaagggag gaaacaaaga tatcaaagca 1920

tcatgctttc aaatgtactc aaagctactc tagtgacttg gcttgaatag ctcccaagag 1980

gctacatcga taacaggcta gcactctcca aggaattaat ggacaacttc caaagtgcat 2040

ggcctcgttc ggacaaccgg tacaatcttc aaaatggaag tagaagattg gtgaaagcat 2100

atgcaactag tacaagctct tgatcgaaat ttgatgctac aggcctaact acttcgacga 2160

tgatcatggg gaaaagttct agtatcagaa tttcataaaa acaaaccaaa aacaattcat 2220

gagttctaac acatgatcaa caactgaatg aatgtcgaag acacagtacg agataagttt 2280

gacaattgaa accgtagata taacaataaa gataacattc atagagacaa caagggcagc 2340

ttatcggata agaagtgagg gaaagataac actatgataa gcactcgcat caaagaaggg 2400

tggcaagaac aaatcattca attagcaact ctttgagcaa cacccatacc acctacatca 2460

taaatcataa gtgctctacc actaaatgca tgcaactcaa gaactttggc atcatcttag 2520

ctcggaagca agacaaaacc aaacagagtt gataaaaaat attaggaaaa atagaataat 2580

ggagacttct agagggccac aaacatagtt aacctcatct tcaaaggagc atcttcctta 2640

atacctaaga ggaagactaa acttaccctt ttgtgagatg atggttgttg aaccatcacc 2700

tacaacatat atgtgatggt tcgagatccc aattcaattt ataagggagg attagtttct 2760

caaacatagg atgctatggt ctagtcctgg ccccgatggt ggcaggactt caacttagta 2820

aggaaggtga attaccaata tgttcaaaag gatggggtaa aaggaaggtg aattatgcaa 2880

tctatttttc gcactttttc attaatcaaa acctatatgg ataaccaatc gttcatatgt 2940

gcaactaagg ttttgactaa gtgttgctat ctctaccgta aaaggagttt tgctacccca 3000

aacctatcaa ctagtctatg actaagctaa gaagataaat cacacaacca caagtaacaa 3060

tataaatgtg gaattttaaa tatggtagag atacaaactc tcgttgatgt gtcagtattt 3120

ttactggagt atcaagaaac gcgcaagctt cttactaatc cttcttagag cctcacgcaa 3180

ggctaagctc ccgctcaggt aaccctgtat agatacaaac cctaccaatg actatagtaa 3240

cttttatgaa gatataggga aacacacaag ttttacccta gttctcgttg gagcctctta 3300

taaacatgtc cacaagggga tgaagctaag agtcgagtaa gggtttgttt gatttcttta 3360

gtcttaatga ctaaaactaa gcaaagagct ttaattattg tgcaaattga ttacattatc 3420

cctaattaat gctatcttcg actactgttt acccacacgc agagctactg ttcgtgcgca 3480

ttcacaatgc tgcatgcgtg cacgttgctt tggggaggag cctacttcat gcgcgttggt 3540

tagggagggc atatgaagta aaaatgtcac tttatggtta gtttgacacc ctcatttttt 3600

tcaagggatt gtattttcac aaaagaaatt aatttatttt tcttgaaaaa taggaatccc 3660

ttagaaaaaa atagagttgt caaactaacc cttattcatt ttagtcactc ttttggtaat 3720

tagaggacta taatttagtt ggaggatttt agtcacactg tgttgattct ttagtgacta 3780

aaaatgacta aaatttaatc aattaaatgt agtcacccaa accaaacaga gtcgcgcgtc 3840

cgacgtgcct cctcaaccgg cgcgtgggag taggtctgga aaaaagctcg agctcgatga 3900

gttggcttgg gctcacggta acttgggtcg gctcgacgct caaaacgagt ccaagtccta 3960

tttttgtggc tcgtgataag tgtgagctag ctcggctcgg ctcacgaacc gacaacaatt 4020

tactacataa aattctgatt agcatataat gttagtacca aatatacaat tagtatatgt 4080

tatttgatat ttatatacaa aattaattta tttctatgtt atattagtac tataattatt 4140

tattaattta agagatgtaa tttgattatt tttgatattt tatgttataa tttgtaacgt 4200

aagctggtgc ttgtggttag actctctact agtcgagttt tcacgctcgt caaaggaccg 4260

agccgagacg aggcaagctc aacacattac cgagccgacc ggctcgtttc cgccacgtgg 4320

tagagcggca gcacccttcc cagatctcgt gtcccgcgcg ccgtgcgcac agccgcgcac 4380

acacacggac tgccgcgtcc gggcgcgatc cgttctcgcg aacacaccgg tcgtagccgt 4440

ccgtaatttg tactagtatg gcacgcggaa cacgagtcgg cgccagcacc ggcacgtcac 4500

catcggggcc gctggcccca tacgggcagc caattaataa atccttgctc tgcgacagac 4560

gcgcaggggc acgtcacgca gccgtagtcg atcggtgcac ggtgctcgcg cccccgttgc 4620

agcgcgcagc tctcccgcgc tataaatgcc ccggctcggc ctcgctccca cagccacagc 4680

ctcacacaga caccaacgcc actgcactgc aaaagcaaga gcagctagct agtaaagatg 4740

ctgagcggga tcatcgacgg gctgacgggg gcgaacaagc atgcgcggct caagggcacg 4800

gtggtgctca tgcgcaagaa cgtgctggac ctcaacgact tcggcgccac cgtcgttgac 4860

agcatcagcg agttcctcgg caagggggtc acctgccagc tcatcagctc caccctcgtc 4920

gacgccagtg agtaccgcgc cgcgccgccg gcacctctcc gatctgcgct tccccatgtc 4980

gatcgatctc gatctctcta ggctctagag ctatagctct ctcggccccc actttttacc 5040

tttgcaagca ttttccctgc atgcgaaaca agcgatagtt tactatttgg gcggccatgc 5100

tgctgctgct tcggctacct tgcctccgtc atctttgacg ggacatggaa agaaagaaag 5160

aatagagaga gagacagaga gagagagaga gcaaacaccg agaaaaagac agcaaagcta 5220

gttgtagcct gggcgcagaa cacagcacca gatgctggct agctcgtgac aaagtaaaaa 5280

aagagaggaa cgaacacagt agtaccaaga gatcagggac gagacctttc tactttgaac 5340

tggattatat attggattct ttatcagtaa cttactgctg ctagtatacc cctaccctag 5400

tctcggggcg acgtgcctgc gtgcatgccc gacgcgtacg aaacacatcg acggactcac 5460

atgggccacc gcgcgcgcgc gtgcctgctt taactttcgc tgtgcagaca acggcaaccg 5520

cgggcgggtc ggggcggagg cgaacctgga gcagtggctg acgagcctgc cgtcgctgac 5580

gaccggcgag tccaagttcg gcgtcacgtt cgactgggag gtggagaagc tgggagtgcc 5640

gggggccgtc gtcgtcaaga acaaccacgc cgccgagttc ttcctcaaga ccatcaccct 5700

cgacgacgtg cccggccgcg gcgccgtcac cttcgtcgcc aactcctggg tctaccccgc 5760

gggcaagtac cgctacaacc gcgtcttctt ctccaacgat gtgagtcctt tctcgataga 5820

tcattatgtt tgtttgttta ttggtatcat gtagctagcc gccatgtcgt cagttggagt 5880

gcagtaggta ggaaaaagga cgacatggga tgggagtggt taagaaaatc catgcaagtg 5940

ggactagtgt gtaactggta gtatagctga agaatctagt ggtagaatga tcttgtacgt 6000

gaataatgtt tctgacgctg agcgctgagg ctatccgcaa ccgttaaccc taaatttttc 6060

cctctatatc attttttccc ctattttcct ccctattttt tcatctcccg cagcggttcc 6120

ccctaaatac tccccctata tcccactacc actataaaat attattttct ataccaatta 6180

tcaatttttt atctactaac aattactcgt ggacccacag cacagtgttt agggtgatga 6240

acagtgacac gctagatctg aagggagaga gaaggggacc gacacgtagg gagcctgtag 6300

agggcaccgc tgcggccgta gggtgctccc tacgcgccgc atacaagggg agggggggga 6360

gaggcagcgg taaccgctgc gcacagcctg agggcgaggc atgtgagttc accacgtgag 6420

tagcagcaaa aggaaacaac ccttcttcac ccggctatca tctaacgtat cgccccggga 6480

gaatcaataa ctttaacgag atgacgaaaa gtcaaaaata aagtcgtgtg atggccatga 6540

aagtcagtca agcaaatcag ctgctaacac gtgtccctta tctacaggtg taagagagta 6600

gagtcttgtc aatcaacctg ggttgttttc tatctgcgtt tt 6642

Claims (10)

1. A key DNA sequence for regulating and controlling the resistance of corn ear rot disease, which is characterized in that the polynucleotide is shown as (a), (b), (c) or (d):

(a) the polynucleotide sequence shown in SEQ ID No. 1; (b) a polynucleotide capable of hybridizing to the complement of SEQ ID No.1 under stringent hybridization conditions; (c) a polynucleotide having at least 90% or more homology with the polynucleotide represented by SEQ ID No. 1; or (d) a mutant which is subjected to deletion, substitution or insertion of one or more bases on the basis of the polynucleotide shown in SEQ ID No.1, and the mutant still has the function or activity of regulating and controlling the maize ear rot.

2. Mutant of the key DNA sequence according to claim 1, characterized in that its polynucleotide sequence is shown as SEQ ID No.2 or SEQ ID No. 14.

3. Use of the key DNA sequence of claim 1 or a mutant of the key DNA sequence of claim 2 for regulating the expression level of ZmLOX3 gene in maize or improving maize head rot resistance.

4. A specific primer for detecting the key DNA sequence of claim 1; preferably, the nucleotide sequences of the specific primers are respectively shown as SEQ ID No.10 and SEQ ID No.11

5. A method of reducing the risk of developing ear rot or increasing resistance to ear rot in corn, comprising: deletion of a 151bp fragment shown in SEQ ID No.3 in an Indel151 promoter region of a ZmLOX3 gene; or inserting a base fragment shown in SEQ ID No.4 or SEQ ID No.5 at the second intron region Indel363 of the ZmLOX3 gene; or a 151bp fragment shown in SEQ ID No.3 is deleted in the promoter region Indel151 of the ZmLOX3 gene and a base fragment shown in SEQ ID No.4 or SEQ ID No.5 is inserted in the second intron region Indel363 of the ZmLOX3 gene.

6. A method for cultivating a new corn variety with resistance to ear rot is characterized by comprising the steps of reducing the expression quantity of ZmLOX3 gene in corn ears and grains, thereby improving the resistance of the ear rot;

preferably, the method for reducing the expression level of the ZmLOX3 gene in the corn ears and grains comprises the following steps: deletion of a 151bp fragment shown in SEQ ID No.3 in an Indel151 promoter region of a ZmLOX3 gene; or inserting a base fragment shown in SEQ ID No.4 or SEQ ID No.5 at the second intron region Indel363 of the ZmLOX3 gene; or a 151bp fragment shown in SEQ ID No.3 is deleted in the promoter region Indel151 of the ZmLOX3 gene and a base fragment shown in SEQ ID No.4 or SEQ ID No.5 is inserted in the second intron region Indel363 of the ZmLOX3 gene.

Use of Indel151 or Indel363 as a molecular marker for expression of the ZmLOX3 gene in corn ears and grains; wherein the DNA sequence of Indel151 is shown as SEQ ID No.3, and the DNA sequence of Indel363 is shown as SEQ ID No.4 or SEQ ID No. 5.

The application of Indel151 or Indel363 in regulating the expression level of ZmLOX3 gene in corn, or in regulating the resistance of corn to ear rot and cultivating new corn varieties with resistance to ear rot; wherein the DNA sequence of Indel151 is shown as SEQ ID No.3, and the DNA sequence of Indel363 is shown as SEQ ID No.4 or SEQ ID No. 5.

9. The use of claim 8, wherein the modulation of the expression level of the ZmLOX3 gene in maize is a reduction in the expression level of the ZmLOX3 gene in maize; the regulation and control of the resistance to the ear rot of the corn is to improve the resistance to the ear rot of the corn.

10. A detection primer for detecting Indel151 or Indel363 variation; wherein the DNA sequence of Indel151 is shown as SEQ ID No.3, and the DNA sequence of Indel363 is shown as SEQ ID No.4 or SEQ ID No. 5; preferably, the nucleotide sequences of the detection primers for detecting Indel151 variation are respectively shown as SEQ ID No.6 and SEQ ID No. 7; the nucleotide sequences of the detection primers for detecting the Indel363 variation condition are respectively shown as SEQ ID No.8 and SEQ ID No. 9.

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