CN113980919B - DNA sequence for regulating and controlling corn ear rot resistance, mutant, molecular marker and application thereof - Google Patents

Abstract

The invention discloses a DNA sequence for regulating and controlling corn ear rot resistance, a mutant, a molecular marker and application thereof. The invention firstly provides a key DNA sequence for regulating and controlling corn ear rot resistance and mutants thereof, wherein the base sequences of the key DNA sequence are 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 genes in corn ears and seeds, and the key DNA sequence can improve the corn ear rot resistance or cultivate new varieties of corn ear rot resistance. The invention provides a molecular marker of Indel151 or Indel363 as ZmLOX3 gene expressed in corn ears and kernels, and application of ZmLOX3 gene in regulating and controlling corn expression and improving corn ear rot resistance. The invention further provides a detection primer for detecting the mutation condition of the DNA sequence and the mutant thereof and a detection primer for detecting the ZmLOX3 gene expression in corn.

Description

DNA sequences regulating corn ear rot resistance and their mutants, molecular markers and applications

Technical field

The present invention relates to DNA sequences that regulate corn ear rot resistance and mutants thereof, in particular to key DNA sequences that regulate the promoter region and intron region of ZmLOX3 gene expression and mutants thereof, as well as molecular markers and detection of ZmLOX3 gene expression. Primers, the present invention further relates to their application in regulating corn ear rot resistance, and belongs to the field of DNA sequences regulating corn ear rot resistance, mutants thereof and their application fields.

Background technique

Corn is an important food, feed and industrial raw material, and is currently China's largest crop. The healthy and safe production of corn plays a decisive role in ensuring my country's food security and agricultural development.

Corn ear rot, also called kernel and ear rot, is a common fungal disease that occurs in corn producing areas around the world. So far, more than 40 pathogenic fungi causing corn ear rot have been identified, among which the common pathogenic bacteria are mainly the following: F. verticillioides, F. moniliforme, F. graminearum .graminearum, F.oxysporum, F.proliferatum, F.culmorum, F.avenaceum, F.equiseti, F.semitectum, Aspergillus flavus A .flavus, A.ochraceus, etc. Generally, the composition of pathogenic bacteria varies greatly from place to place, and fungi in different hospitals can cause single or compound infection to cause corn ear rot.

The incidence of corn ear rot is usually closely related to the genetic background of the material, and is also affected by different environmental and climatic conditions. High temperature and high humidity environments can easily aggravate the occurrence of corn ear rot.

The occurrence of ear rot can cause grain rot, resulting in yield losses of up to 30%-40%. In addition to directly causing yield losses, pathogenic fungi such as Fusarium and Aspergillus that cause ear rot can produce a variety of toxins (for example, Fusarium can produce mycotoxins such as trichothecenes, fumonisins, and zearalenone). , aflatoxins and ochratoxins produced by Aspergillus, etc.), also affect the safe storage and eating safety of corn. Mycotoxins can not only affect the degree of infection and damage caused by fungi to the host, but also have serious toxic effects on vertebrates including humans, livestock and poultry. Different types of toxins cause different degrees of damage and symptoms to host plants, humans and animals. Fumonisins are a type of sphingolipid compounds that competitively inhibit the biosynthesis of sphingolipids in animal and plant cells, causing accumulation of sphingosine in cells, leading to host tissue necrosis, and can cause pulmonary edema in pigs and brain damage in horses. Leukomalacia, rat liver cancer, human esophageal cancer, etc.; nivalenol and deoxynivalenol have the ability to bind to the eukaryotic 60S ribosomal subunit, leading to the inhibition of protein synthesis and induction of apoptosis. , producing symptoms of wilting, chlorosis or necrosis, causing vomiting, diarrhea, food refusal or estrogen poisoning in humans and animals; aflatoxin is an important carcinogen, which can inhibit the synthesis of white matter in host plants and humans and animals, and infect animal and plant tissues. causing serious damage. Overall, corn ear rot not only reduces corn yield and quality, but also poses major hidden dangers to the safety of food and feed and directly threatens human and animal health. Therefore, it is of great significance to control the occurrence and harm of corn ear rot. .

Discovering resistance genes that regulate corn ear rot is a key measure to improve corn ear rot resistance, cultivate disease-resistant varieties, and ensure safe corn production. Previous studies have found that lipoxygenase genes (ZmLOXs) are important signal communication molecules in the interaction between the host and pathogenic fungi. The lipoxygenase genes derived from the host corn play a role in regulating resistance to corn ear rot. important role. Among them, the maize 9-LOX gene ZmLOX3 may be used as a pathogenic factor by F. verticillioides to cause stem/ear rot resistance. Complete knockout of the ZmLOX3 gene will increase the resistance of corn to grain rot caused by Fusarium, but show susceptibility to grain rot caused by Aspergillus flavus; indicating the complexity of the ZmLOX3 gene in the regulation of corn ear rot resistance. It also shows that direct knockout of this gene cannot be directly used to improve resistance to corn ear rot.

In view of the antagonistic effect of the ZmLOX3 gene in regulating resistance to Fusarium and Aspergillus aflatoxin, a strategy that ensures the protein function of the gene but appropriately reduces its expression level may be able to achieve resistance to both Fusarium and Aspergillus aflatoxin. function, thereby comprehensively improving corn ear rot resistance. During the evolutionary process of nearly 10,000 years, corn has accumulated abundant natural variation. It is likely that there is such a natural variation in nature that does not change the function of ZmLOX3 protein, but can appropriately reduce its expression abundance. The mining of this type of natural variation is crucial to the improvement of corn ear rot resistance and will have broad application prospects in corn breeding.

Contents of the invention

One of the purposes of the present invention is to provide a key DNA sequence for regulating corn ear rot;

The second object of the present invention is to provide a mutant of the key DNA sequence for regulating corn ear rot;

The third object of the present invention is to provide a molecular marker for regulating the expression of ZmLOX3 gene in corn ears and grains;

The fourth object of the present invention is to provide a method for reducing the risk of ear rot in corn or improving resistance to ear rot, or a method for cultivating new corn varieties resistant to ear rot;

The fifth object of the present invention is to provide a detection primer for detecting the mutation of the molecular marker;

The sixth object of the present invention is to provide detection primers for detecting ZmLOX3 gene expression in corn;

The above objects of the present invention are achieved through the following technical solutions:

In one aspect, the present invention provides a key DNA sequence for regulating corn ear rot resistance, and its polynucleotide is represented by (a), (b), (c) or (d):

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

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

(c) A polynucleotide sequence that is at least 90% or more homologous to the polynucleotide shown in SEQ ID No. 1; or

(d) A mutant in which one or more bases are deleted, substituted or inserted based on the polynucleotide sequence shown in SEQ ID No. 1, and the mutant still has the function of regulating corn ear rot; or active.

The present invention found that the above-mentioned key DNA sequence can regulate the expression changes of the ZmLOX3 gene in corn ears and grains, thereby affecting the resistance of corn to ear rot or ear rot.

The "ear rot" or "ear rot" mentioned in the present invention are the same disease.

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

As a specific embodiment of the mutant, the present invention provides a mutant of a key DNA sequence that regulates corn ear rot resistance, and its polynucleotide sequence is shown in SEQ ID No. 2; the mutation The body causes a 151bp base insertion at Indel151 in the ZmLOX3 gene promoter region, and the inserted sequence is SEQ ID No. 3; a 361bp or 363bp base deletion at Indel363 in the second intron region of the ZmLOX3 gene, and its deletion The polynucleotide sequence is shown in SEQ ID No. 4 or SEQ ID No. 5; this mutant can increase the expression of ZmLOX3 gene in corn ears and grains, showing an increased risk of ear rot.

As a preferred specific embodiment of the mutant, the present invention provides a mutant of a key DNA sequence that regulates corn ear rot resistance, and its polynucleotide sequence is shown in SEQ ID No. 14; The mutant is a 151bp base deleted at Indel151 in the promoter region of the ZmLOX3 gene, and the deleted sequence is shown in SEQ ID No. 3; a 363bp base is inserted at Indel363, the second intron region of the ZmLOX3 gene, and its insertion The polynucleotide sequence is shown in SEQ ID No. 5; this mutant can reduce the expression of ZmLOX3 gene in corn ears and grains, showing a significant reduction in the risk of ear rot.

The present invention therefore determines that the 151 bp fragment shown in SEQ ID No. 3 is deleted from Indel151 in the ZmLOX3 gene promoter region or that SEQ ID No. 4 or SEQ ID No. 5 is inserted into the second intron region Indel363 of the ZmLOX3 gene. Corn materials with base fragments generally have lower ZmLOX3 gene expression levels, and thus show resistance to ear rot caused by Fusarium verticilliformis, and reduce the risk of ear rot; on the contrary, SEQ is inserted into Indel151 in the promoter region of the ZmLOX3 gene The 151 bp fragment shown in ID No. 3 or the maize material lacking the base fragment shown in SEQ ID No. 4 or SEQ ID No. 5 at Indel363, the second intron region of the ZmLOX3 gene, generally has higher ZmLOX3 The gene expression level shows susceptibility to ear rot caused by Fusarium verticilliformis, and the risk of ear rot is increased.

Therefore, the present invention provides a method for reducing the risk of ear rot in corn or providing resistance to ear rot, including: deleting the 151 bp fragment shown in SEQ ID No. 3 in Indel151, the ZmLOX3 gene promoter region; or, Insert the base fragment shown in SEQ ID No.4 or SEQ ID No.5 at Indel363, the second intron region of the ZmLOX3 gene; or delete the 151 bp sequence shown in SEQ ID No.3 at Indel151 in the promoter region of the ZmLOX3 gene. fragment and insert the base fragment shown in SEQ ID No. 4 or SEQ ID No. 5 at Indel363, the second intron region of the ZmLOX3 gene.

The present invention also provides a method for cultivating new corn varieties resistant to ear rot, which includes reducing the expression level of the ZmLOX3 gene in corn ears and grains, thereby improving the resistance to corn ear rot; therefore, by reducing the expression of the ZmLOX3 gene in corn ears and grains, Methods for improving corn ear rot resistance by increasing the expression level in corn ears and grains all fall within the scope of the present invention; including using other constitutive or tissue-specific promoters and expression cassettes constructed from ZmLOX3 to drive the ZmLOX3 gene in Expression in corn ears and grains, or using other natural variations to change the expression of ZmLOX3 gene in corn ears and grains, or using RNAi to reduce the expression of ZmLOX3 gene in corn ears and grains.

As a preferred specific embodiment of this aspect, the method for reducing the expression level of the ZmLOX3 gene in corn ears and grains can be: deleting the 151 bp fragment shown in SEQ ID No. 3 in Indel151, the promoter region of the ZmLOX3 gene; or, Insert the base fragment shown in SEQ ID No.4 or SEQ ID No.5 at Indel363, the second intron region of the ZmLOX3 gene; or delete the 151 bp sequence shown in SEQ ID No.3 at Indel151 in the promoter region of the ZmLOX3 gene. fragment and insert the base fragment shown in SEQ ID No. 4 or SEQ ID No. 5 at Indel363, the second intron region of the ZmLOX3 gene.

Further, an expression cassette containing a mutant of part or all of the DNA sequence shown in SEQ ID No. 1, part or all of the DNA sequence shown in SEQ ID No. 2 or SEQ ID No. 14, and an expression cassette containing the same. The recombinant plant expression vectors, transgenic cell lines and host bacteria all fall within the protection scope of the present invention.

The recombinant plant expression vector is a recombinant plant expression vector constructed by combining the expression cassette with a plasmid or expression vector and can transfer the expression cassette into plant host cells, tissues or organs.

The DNA sequence of the invention or its mutants can be used to prepare transgenic plants. For example, the recombinant plant expression vector containing the DNA sequence or its mutant is introduced into plant cells, tissues or organs through methods such as Agrobacterium-mediated or biolistic methods, and then the transformed plant cells, tissues or organs are cultivated into plants. , to obtain transgenic plants. The starting vector used to construct the plant expression vector can be any binary vector used to transform plants with Agrobacterium or a vector that can be used for plant microprojectile bombardment, etc.

In order to implement the present invention, conventional compositions and methods for preparing and using plant expression vectors and host cells are well known to those skilled in the art. Specific methods can be referred to, for example, Sambrook et al.

The recombinant plant expression vector may also contain a selectable marker gene for selecting transformed cells. Selectable marker genes are used to select transformed cells or tissues. The marker genes include genes encoding antibiotic resistance and genes conferring resistance to herbicidal compounds. In addition, the marker genes also include phenotypic markers, such as β-galactosidase and fluorescent proteins.

In short, the DNA sequence provided by the present invention, mutants of the sequence, molecular markers Indel151 or Indel363, specific detection primers for detecting the variation of the molecular markers, and specific detection primers for detecting the expression of the ZmLOX3 gene can be used to cultivate antibodies. New corn varieties with disease, especially used in improving corn ear rot resistance.

For reference, the present invention provides a method for regulating ear rot resistance of corn, including: using the DNA sequence shown in SEQ ID No. 1, the DNA sequence shown in SEQ ID No. 2 or the DNA sequence shown in SEQ ID No. 14. Mutants of the DNA sequences shown regulate expression of the ZmLOX3 gene in maize.

The transformation protocols described in this invention and the protocols for introducing the polynucleotides or polypeptides into plants may vary depending on the type of plant (monocotyledonous or dicotous) or plant cell used for transformation. Suitable methods for introducing the polynucleotide or polypeptide into plant cells include microinjection, electroporation, Agrobacterium-mediated transformation, direct gene transfer, high-speed ballistic bombardment, and the like. In certain embodiments, a variety of transient transformation methods can be used to provide expression cassettes of the invention to plants. Stable transformed plants can be regenerated from transformed cells 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 present invention further determines that Indel151 or Indel363 can be used as a molecular marker to regulate the expression of ZmLOX3 gene in corn ears and grains. The present invention also found that Indel151 and Indel363 are almost completely linked together, and any one of the mutations can be used alone as a molecular marker to detect the expression of ZmLOX3 gene in corn ears and grains.

The present invention further provides detection primers for detecting Indel151 mutations; as a preferred specific embodiment, the nucleotide sequences of the detection primers are shown in SEQ ID No. 6 and SEQ ID No. 7 respectively; The specific detection primer can be used to detect the variation of Indel151 in corn varieties, thereby being used for molecular-assisted breeding of corn.

The present invention further provides detection primers for detecting Indel363 mutation; as a preferred embodiment, the nucleotide sequences of the detection primers are shown in SEQ ID No. 8 and SEQ ID No. 9 respectively; using The specific detection primer can detect the variation of Indel363 in corn varieties, and can be 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 should also belong to the protection of the present invention. scope.

The present invention further provides specific amplification primers for detecting the expression level of the ZmLOX3 gene. The specific detection primer sequences are shown in SEQ ID No. 10 and SEQ ID No. 11 respectively; the specific detection primers can be used to detect The expression level of ZmLOX3 gene in corn varieties was detected to provide reference for the research on ZmLOX3 gene in corn breeding.

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

The DNA sequence for regulating corn ear rot or its mutant provided by the present invention can regulate the expression of ZmLOX3 gene in corn ears or grains, which is of great significance for improving corn ear rot, and can be further applied to new corns. Breeding of varieties. The present invention further provides molecular markers Indel151 or Indel363 used to regulate the expression of the ZmLOX3 gene, specific detection primers for detecting the variation of the molecular markers, and specific detection primers for detecting the expression level of the ZmLOX3 gene in corn, all of which can be directly used. For targeted improvement of ear rot resistance of corn, it also has important application potential for the breeding of new disease-resistant corn varieties.

Definitions of terms involved in this invention

Unless otherwise defined, 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 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 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 specifically limited otherwise, the term also means oligonucleotide analogs, which include PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioates, phosphoamidates, etc.) . 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 explicitly specified sequences. Specifically, degenerate codon substitution can be achieved by generating a sequence in which position 3 of one or more selected (or all) codons is substituted with a mixed base and/or a deoxyinosine residue (Batzer et al. , Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (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 (ie, sequence similarity or identity). The term homology, as used herein, also refers to the concept of similar functional properties between different polynucleotide molecules, for example, promoters with similar functions may have homologous cis-elements. Polynucleotide molecules are homologous when they hybridize specifically under specific conditions to form duplex molecules. Under these conditions (called stringent hybridization conditions) one polynucleotide molecule can be used as a probe or primer to identify another polynucleotide molecule that shares homology.

"Stringent hybridization conditions" as used in the present invention refer to conditions of low ionic strength and high temperature known in the art. Typically, under stringent conditions, a probe hybridizes to its target sequence to a more detectable extent (e.g., at least 2-fold above background) than to other sequences. Stringent hybridization conditions are sequence-dependent and vary in different environments. Conditions will vary, 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 washing conditions. Detailed guidance on nucleic acid hybridization can be found in the relevant literature. (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays. 1993). More specifically, the stringent conditions are usually chosen to be lower than the specified ion strength for the specific sequence. The thermal melting point ( _Tm ) at pH is about 5-10°C. _Tm is the temperature at which a probe complementary to the target is 50% hybridized to the target sequence at equilibrium (under specified ionic strength, pH and nucleic acid concentration ) (50% of the probe is occupied at equilibrium at T _m because the target sequence is present in excess). Stringent conditions may be conditions where the salt concentration is less than about 1.0 M sodium ion concentration at pH 7.0 to 8.3 , typically about 0.01 to 1.0 M sodium ion concentration (or other salt), and a temperature of at least about 30°C for short probes (including (but not limited to) 10 to 50 nucleotides) and at least about 30°C for long probes. For selective or specific hybridization, it is at least about 60° C. The signal can be at least two times background hybridization, and optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5×SSC, and 1% SDS, incubated at 42°C; or 5×SSC, 1% SDS, incubate at 65°C, wash in 0.2×SSC and wash in 0.1% SDS at 65°C. The washes can be performed for 5, 15, 30, 60, 120 minutes or longer.

The "multiple" mentioned in the present invention usually means 2-8, preferably 2-4; the "replacement" refers to replacing one or more amino acid residues with different amino acid residues; The "deletion" mentioned above refers to the reduction in the number of amino acid residues, that is, the lack of one or more amino acid residues; the "insertion" refers to the change in the sequence of amino acid residues. Compared with natural molecules, The alteration results in the addition of one or more amino acid residues.

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

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

The term "plant promoter" refers to a natural or non-native promoter that is functional in plant cells. 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 to which it is operably linked. When operably linked to a transcribable polynucleotide molecule, a promoter generally causes the transcription of the transcribable polynucleotide molecule in a manner that is consistent with the transcription of the transcribable polynucleotide molecule to which the promoter is normally linked. similar. Plant promoters may include promoters generated by manipulating known promoters to produce artificial, chimeric or hybrid promoters. Such promoters may also combine cis-elements from one or more promoters by, for example, adding heterologous regulatory elements to an active promoter with some or all of its own regulatory elements.

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

The term "operably linked" refers to the connection 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 A polynucleotide molecule affects the function of a second polynucleotide molecule. Preferably, the two polynucleotide molecules are part of a single contiguous polynucleotide molecule, and more preferably are adjacent. For example, a promoter is operably linked to a gene of interest if the promoter regulates or mediates the transcription of the gene of interest within the cell.

The term "transcriptable polynucleotide molecule" refers to any polynucleotide molecule capable of being transcribed into an RNA molecule. Methods are known for introducing a construct into a cell in such a way that a transcribable polynucleotide molecule is transcribed into a functional mRNA molecule, which is translated and thus expressed as a protein product. In order to inhibit the translation of a specific RNA molecule of interest, a construct capable of expressing an antisense RNA molecule can also be constructed.

The term "recombinant plant expression vector": one or more DNA vectors used to effect 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 usually include: cis-acting sequences required for T-DNA transfer, selectable markers engineered to be expressed in plant cells, heterologous DNA sequences to be transcribed, etc.

The term "transformation": A method of introducing heterologous DNA sequences into a host cell or organism.

The term "expression": the transcription and/or translation of an endogenous gene or transgene in plant cells.

The term "recombinant host cell strain" or "host cell" means a cell containing a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or any other method known in the art. Other methods are known. The exogenous polynucleotide can be maintained as a non-integrating vector, such as a plasmid, or can be integrated into the host genome. The host cell can be a prokaryotic cell or a eukaryotic cell, and the host cell can also be a monocotyledonous or dicotyledonous plant cell.

Description of drawings

Figure 1A shows that through sequencing methods, it was found that the promoter and second intron region of ZmLOX3 each have an insertion-deletion natural variation (Indel); Indel151, a 151bp Indel in the promoter region; Indel363, a 151-bp Indel in the second intron region 363bp Indel. The right side of the picture shows the expression of the ZmLOX3 gene in the ovules on the day of pollination (Ovule_0DAP) and the kernels 5 days after pollination (Kernel_5DAP) of the 12 inbred lines sequenced. Overall, the expression level of ZmLOX3 gene is higher in inbred lines with Indel151 insertion or Indel363 deletion; B is GWAS analysis showing that Indel151 and Indel363 are significantly associated with the expression level of ZmLOX3 gene. The lower part of the Manhattan plot shows the linkage disequilibrium of all markers. Module (LD-Block) diagram; C is the relationship between different genotypes of Indel151 and ZmLOX3 gene expression in inbred lines. When there is an insertion of Indel151, the expression of ZmLOX3 gene is higher; D is the different genes of Indel151 Changes in allelic frequency among maize varieties of different ages, among which the genotype corresponding to reduced ZmLOX3 gene expression was selected during the breeding process; E is the variation of the two genotypes at Indel151 between different heterotic group materials Allele frequency distribution.

Figure 2 shows the Indel151 genotype identification results of 12 inbred lines.

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

Figure 4 shows the results of corn kernel inoculation of 10 inbred lines; among them, 5 inbred lines with Indel151 insertion and Indel363 deletion are significantly more susceptible to the disease than the other inbred lines with different genotypes.

Figure 5 shows the expression levels of the ZmLOX3 gene corresponding to different Indel151 genotypes in hybrids (left picture) and the allele frequency changes of different Indel151 genotypes in hybrids of different generations (right picture).

Detailed ways

The present invention will be further described below in conjunction with specific embodiments, and the advantages and features of the present invention will become clearer with the description. However, these embodiments are only exemplary and do not constitute any limitation on the scope of the present invention. Those skilled in the art should understand that the details and forms of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and substitutions all fall within the protection scope of the present invention.

For the inbred lines used in the following examples, relevant information and corresponding seeds can be obtained from the "China Crop Germplasm Information Network".

Example 1 Experiment on how insertion and deletion (Indel) variation in the ZmLOX3 gene promoter region and the second intron region regulates changes in the expression level of the ZmLOX3 gene

1. Discovery of mutation sites in the ZmLOX3 gene promoter region and the second intron region

Using average >55× PacBio SMRT reads data, >94× second-generation Illumina 150bp paired-end sequencing data, and >139× BioNano single-molecule data, 12 maize backbone inbred line parents widely used around the world were analyzed. Whole genome assembly. These 12 inbred lines are: PH207, A632, OH43, Zheng58, Ye478, Dan340, Huangzaosi, Chang7-2, Jing92, Jing724, Xu178, S37; plus the published B73 and Mo17 genomes, these 14 high-quality genomes The core representative inbred lines cover almost all heterotic groups in corn breeding.

The 10 kb genome sequence upstream and downstream of the ZmLOX3 gene in the 14 core inbred lines was retrieved, and Blast comparison of the DNA sequences was performed. It was found that in the 14 inbred lines, in the promoter and second intron of the ZmLOX3 gene There is one indel variant (Indel, A in Figure 1) each. The Indel length of the promoter region is 151 bp, and its sequence is SEQ ID No. 3; the Indel length of the second exon region is 363 bp, and its base sequence is SEQ ID No. 5, or the exon region The Indel length is 361bp, and its base sequence is shown in SEQ ID No. 4.

2. Indel151 and Indel363 are significantly associated with the expression variation of ZmLOX3 gene

China has experienced six times of upgrading of corn varieties (hybrids) in history. In the early stage, the inventor collected and assembled 116 hybrids widely used in the variety upgrading process of different eras and their corresponding 137 parental inbred lines (partially Hybrids have shared parents). In the summer of 2019, the ears of these 253 materials (116 hybrids and 137 inbred lines) were sampled during the silking stage (3 replicates were sampled for each material), and then RNA-Seq analysis was performed, resulting in more than 20,000 Gene expression data in the ears of 253 materials. Among them, in the present invention, the expression data of the ZmLOX3 gene are extracted separately for subsequent analysis.

Using deep (>10×) second-generation resequencing data of 137 maize inbred lines combined with the published maize B73V3 genome, 25,320,664 single nucleotide polymorphism molecular markers (SNPs) and 4,319,510 indel polymorphisms were discovered. Morphic molecular markers (Indel). In addition, 137 maize inbred lines were identified using the developed PCR primers LOX3-2 (sequences are SEQ ID No. 6 and SEQ ID No. 7) and LOX3-3 sequences are SEQ ID No. 8 and SEQ ID No. 9. Genotypes of Indel151 (Fig. 2) and Indel363 (Fig. 3).

These mined molecular markers were used to estimate the population structure and genetic relationship of 137 maize inbred lines, and then the detected expression data of the ZmLOX3 gene were used as phenotypes for genome-wide association analysis (GWAS). Among them, a significant association signal was identified near the ZmLOX3 gene. The most significant signal fell on the Indel151 marker (p-value=1.40E-9), and Indel363 was the second most significant association marker (Figure 1B), which indicated that Indel151 and Indel363 is significantly associated with the expression variation of the ZmLOX3 gene.

This experiment further divided 137 materials into two categories based on the genotype of Indel151. One type is the genotype materials with a 151bp sequence missing at Indel151, a total of 95 materials; the other type is a genotype material with a 151bp sequence present at Indel151, a total of 33 materials. portion; comparing the ZmLOX3 gene expression between the two types of materials, it was found that the ZmLOX3 gene expression in the material lacking Indel151 was lower (Figure 1C), indicating that Indel151 can positively regulate the expression of the ZmLOX3 gene. Linkage disequilibrium analysis also showed that Indel151 and Indel363 are located in the same LD-block (linkage disequilibrium module) (Figure 1B); and sequence analysis in 14 inbred lines also showed that these two mutations always repel each other. Phase forms appear linked together, that is, when a material has a genotype with a 151bp deletion at Indel151, it often has a genotype with a 363bp or 361bp insertion sequence at Indel363 (Figure 1A). This indicates that it is possible to infer the genotype of the other Indel from the genotype of one of the two; however, the correlation between Indel151 and the expression level of the ZmLOX3 gene is stronger, so Indel151 can be used as a better molecular marker for detecting the expression level of the ZmLOX3 gene. .

Example 2 Indel151 and Indel363 affect corn ear rot resistance by regulating ZmLOX3 gene expression

Appropriately reducing the expression of ZmLOX3 gene in grains without changing its protein function may play a role in resisting Fusarium and Aspergillus aflatoxin.

The inventor's preliminary research results show that materials lacking Indel151 or inserting Indel363 often have lower ZmLOX3 gene expression. In order to further verify the relationship between Indel151 and Indel363 and ZmLOX3 gene expression, 12 representative inbred lines mentioned above (PH207, A632, OH43, Zheng58, Ye478, Dan340, Huangzaosi, Chang7-2, Jing92, Jing724, Xu178 , S37), were planted in the field, and the ovules at the silking stage and the grains 5 days after pollination were sampled and quick-frozen in liquid nitrogen. Samples from every five individual plants were mixed into one sample, and three biological replicates were taken from each inbred line. RNA was then extracted using the TRIzol method, and specific primers (SEQ ID No. 10 and SEQ ID No. 11) were used to detect the ZmLOX3 gene. amount of expression. It was found that the results of the previous GWAS analysis were the same as that of the materials lacking Indel151 or inserting Indel363 (PH207, A632, Zheng58, Ye478, Jing724, Xu178, S37), whether in unpollinated ovules or in grains 5 days after pollination, generally higher The low ZmLOX3 gene expression (A in Figure 1) once again verified the relationship between Indel151 and Indel363 and ZmLOX3 gene expression.

Further experiments were conducted on the corn kernels of 10 materials from 12 representative inbred lines for inoculation with F. verticillioides (Figure 4). The results showed that the five inbred lines with low ZmLOX3 gene expression (B73, Ye478, Mo17, PH207, A632, deletion of Indel151, and insertion of Indel363) were generally better than the five inbred lines with high ZmLOX3 gene expression (Jing92, Dan340, Huangzaosi, OH43 , Chang7-2, Indel151 insertion, Indel363 deletion) are more resistant to ear rot caused by Fusarium verticillioides. These results indicate that Indel151 and Indel363 can affect corn ear rot resistance by regulating the expression of ZmLOX3 gene.

Example 3 The excellent allele at Indel151 has been subject to strong artificial selection during modern corn breeding.

The inventor collected 350 corn inbred line materials from different breeding periods in China and the United States in the early stage, including 163 early American (Public-US) and modern (Ex-PVP) breeding materials, early Chinese (CN1960&70s) , 187 copies of main corn breeding materials in the mid-term (CN1980&90s) and current (CN2000&10s). Analysis of the frequency distribution of the Indel151 genotype (Indel363 is closely linked to Indel151 and can be represented by the Indel151 genotype) in these materials found that as the breeding period progresses, Indel151 is missing (corresponding to lower ZmLOX3 gene expression) and higher ear rot resistance) genotype frequencies have significantly increased during the corn breeding process in China and the United States (Figure 1D), indicating that genotypes lacking the Indel151 class have been strongly affected in the modern corn breeding process. of artificial selection.

In addition, 350 corn inbred line materials can also be divided into eight heterotic groups, namely SS, PA, X, NSS, TSPT, IDT, PB, and Mixed. Previous reports have shown that TSPT germplasm is generally more susceptible to ear rot. Among them, Huangzaosi, Chang7-2, and Jing92 mentioned above are all typical TSPT germplasms. The inoculation experiment of the present invention also proves that they are indeed more susceptible to ear rot. rot (Figure 4), confirming previous reports. The present invention also found that in TSPT germplasm, the frequency of genotypes inserted into the Indel151 class is higher, and the insertion of Indel151 corresponds to a higher ZmLOX3 gene expression level, which makes the TSPT germplasm susceptible to ear rot, revealing that the TSPT germplasm is susceptible to ear rot. The cause of the disease. At the same time, it also indicates the important potential of Indel151 and Indel363 in improving ear rot resistance of TSPT germplasm.

Furthermore, the present invention also analyzed the expression of ZmLOX3 gene and the distribution of different genotypes of Indel151 in 116 hybrids of different ages. The analysis found that in the hybrids, the expression level of ZmLOX3 in the Indel151-deleted material was significantly lower than that of the heterozygous and Indel151-inserted materials (left picture in Figure 5). The low expression level of ZmLOX3 corresponds to resistance to ear rot, so the genotype lacking Indel151 is an excellent genotype, and the genotype with Indel151 insertion is an unfavorable genotype. Allele frequency analysis found that the homozygous superior allele type of Indel151 gradually increased during the breeding process of hybrids, while the homozygous unfavorable genotype almost disappeared in hybrids after 2000; and as the breeding years progressed , the proportion of heterozygous genotypes is also getting lower and lower, further proving that Indel151 has been subject to strong selection during the breeding process. These hybrids are all directly cultivated and utilized in corn production, and are selected among the hybrids, further confirming the importance of this natural variation (Indel151 and its closely linked Indel363).

SEQUENCE LISTING

<110> Institute of Biotechnology, Chinese Academy of Agricultural Sciences

<120> DNA sequences regulating corn ear rot resistance and their mutants, molecular markers and applications

<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

accagggccttcttagagaa 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 gggaccccccc tgtctcgaaa cgccgacaat 1080

aagctttatct 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 ctctttatggc 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 ttaactaatcc ttcttagagc ctcacgcaag 3180

gctaagctcc cgctcaggta accctgtata gatacaaacc ctaccaatga ctatagtaac 3240

ttttatgaag atatagggaa acacacaagt tttaccctag ttctcgttgg agcctctttat 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 ggggagaggagc 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

gggccgtcg tcgtcaagaa caaccacgcc gccgagttct tcctcaagac aatcaccctc 5700

gacgacgtgc ccggccgcgg cgccgtcacc ttcgtcgcca actcctgggt ctaccccgcg 5760

ggcaagtacc gctacaaccg cgtcttcttc tccaacgatg tgagtccttt ctcgatagat 5820

cattatgttt gtttgttatttgtttcttg 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 tcgcttcct 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 acaaatatttttcatcaa 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 acttacccttttgtgagatg 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 acccaacacgc agagctactg ttcgtgcgca 3480

ttcacaatgc tgcatgcgtg cacgttgctt tggggagaggag 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 ctggggagtgc 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

accactggccg ccacagtgcc acccaccccac 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 gggacccgaca 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 gggacccgaca cgtagggagc ctgtagaggg caccgctgcg gccgtagggt 300

gctccctacg cgccgcatac aaggggaggg gggggagagg cagcggtaac cgctgcgcac 360

agc 363

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

gctcacgaac cgacaacaat 20

<210> 7

<211> 21

<212> DNA

<213> Artificial sequence

<400> 7

tcccgctcag catctttact a 21

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

tacgaaacac atcgacggac 20

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence

<400> 9

gttattgattctcccggggc 20

<210> 10

<211> 22

<212> DNA

<213> Artificial sequence

<400> 10

gttcttcctc aagacaatca cc 22

<210> 11

<211> 21

<212> DNA

<213> Artificial 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 tcgcttcct 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 acaaatatttttcatcaa 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 acttacccttttgtgagatg 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 acccaacacgc agagctactg ttcgtgcgca 3480

ttcacaatgc tgcatgcgtg cacgttgctt tggggagaggag 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 tccccactacc 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 (7)

1. A key DNA for regulating and controlling corn ear rot resistance is characterized in that the polynucleotide is shown as SEQ ID No. 1.

2. The mutant of the key DNA according to claim 1, wherein the polynucleotide sequence is shown in SEQ ID No.2 or SEQ ID No. 14.

3. The critical DNA of claim 1 or the mutant of the critical DNA of claim 2 is in regulationZmLOX3The application of the gene in the expression level of corn or the regulation of the corn cob rot resistance; the saidZmLOX3The amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 13.

4. A method of reducing the risk of corn for developing or increasing resistance to ear rot comprising: at the position ofZmLOX3A fragment of 151bp shown in SEQ ID No.3 is deleted in a gene promoter region Indel 151; or in the presence ofZmLOX3The second intron Indel363 of the gene is inserted with the base fragment shown in SEQ ID No.4 or SEQ ID No. 5; or in the presence ofZmLOX3151bp fragment shown in SEQ ID No.3 deleted in gene promoter region Indel151 and its use inZmLOX3The second intron Indel363 of the gene is inserted with the base fragment shown in SEQ ID No.4 or SEQ ID No. 5; the saidZmLOX3The amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 13.

Indel151 or Indel363 asZmLOX3The application of the molecular marker expressed by the genes in corn ears and kernels; 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 saidZmLOX3The amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 13.

6. Indel151 deleted Agents are regulatedZmLOX3Gene expression in corn or regulation of corn resistanceThe application of the corn with spike rot resistance in cultivating new varieties of corn with spike rot resistance; or Indel363 is regulatingZmLOX3The application of the gene in the expression quantity of corn or in regulating and controlling the corn spike rot resistance and cultivating new corn varieties with spike rot resistance; 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; said controllingZmLOX3The expression level of the gene in corn is reducedZmLOX3The expression level of the gene in corn; the regulation of the corn ear rot resistance is to improve the corn ear rot resistance; the saidZmLOX3The amino acid sequence of the protein coded by the gene is shown as SEQ ID No. 13.

7. A method for detecting whether Indel151 or Indel363 exist in corn materials by using a detection primer, which is characterized in that 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; wherein, the nucleotide sequences of the detection primers for detecting whether Indel151 exists in the corn material are respectively shown in SEQ ID No.6 and SEQ ID No. 7; the nucleotide sequences of the detection primers used to detect whether Indel363 is present in corn material are shown as SEQ ID No.8 and SEQ ID No.9, respectively.