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

DNA sequence for regulating and controlling corn ear rot resistance, mutant, molecular marker and application thereof Download PDF

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CN113980919B
CN113980919B CN202111065285.3A CN202111065285A CN113980919B CN 113980919 B CN113980919 B CN 113980919B CN 202111065285 A CN202111065285 A CN 202111065285A CN 113980919 B CN113980919 B CN 113980919B
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王宝宝
王海洋
赵斌斌
廖新阳
李长育
候美
李耀耀
谢钰容
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South China Agricultural University
Biotechnology Research Institute of CAAS
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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 sequence for regulating and controlling corn ear rot resistance, mutant, molecular marker and application thereof
Technical Field
The invention relates to a DNA sequence for regulating and controlling corn 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 and a detection primer of ZmLOX3 gene expression, and further relates to application of the key DNA sequence in regulating and controlling corn ear rot resistance, belonging to the field of a corn ear rot resistance regulating DNA sequence, a mutant thereof and application thereof.
Background
Corn is an important grain, feed and industrial raw material, and is also the first crop in china today. The healthy and safe production of the corn plays a significant role in guaranteeing the grain safety and agricultural development of China.
Ear rot (ear rot), also known as ear grain rot (kernel and ear rot), is a type of fungal disease that commonly occurs in corn producing areas worldwide. More than 40 pathogenic fungi have been identified to date that cause corn ear rot, among which the following are the most common pathogenic bacteria: fusarium rotavatum F.verillioides, fusarium verticillium F.moniliforme, fusarium graminearum F.oxysporum, fusarium oxysporum F.progranatum, fusarium flavum F.culmorum, fusarium avenaceum, fusarium equiseti F.semitectum, fusarium flavum A.flavus, aspergillus ochraceus A.ocher, etc. In general, pathogenic bacteria vary widely in composition from place to place, and different hospital fungi can be infected individually or in combination to cause corn ear rot.
The incidence rate of the corn ear rot is usually closely related to the genetic background of materials, and is also influenced by different environments and climatic conditions, and the occurrence of the corn ear grain rot is easily aggravated in the high-temperature and high-humidity environment.
The occurrence of spike rot can cause kernel rot, resulting in yield loss as high as 30% -40%. In addition to directly causing yield loss, pathogenic fungi such as fusarium and aspergillus for the spike rot can produce various toxins (such as fusarium can produce mycotoxins such as trichothecene, fumonisins and zearalenone, aflatoxins produced by aspergillus and ochratoxins), and the safety storage and eating safety of corn are also affected. Mycotoxins are not only capable of affecting the extent of infection and destruction of a host by fungi, but also have serious deleterious effects on vertebrates including humans and livestock. The degree of injury and the symptoms caused by different types of toxins to host plants and humans and animals vary. Fumonisins are a type of sphingolipid compounds, and in animal and plant cells, the fumonisins competitively inhibit the biosynthesis of sphingolipids, so that sphingosine is accumulated in the cells, and host tissue necrosis is caused, and pig pulmonary edema, horse leukomalacia, rat liver cancer, human esophageal cancer and the like can be caused; fusarium nivalenol and deoxynivalenol have the ability to bind to eukaryotic 60S ribosomal subunits, resulting in inhibition of protein synthesis and induction of apoptosis, and produce symptoms of wilting, chlorosis or necrosis, resulting in symptoms of vomiting, diarrhea, antifeedant or estrogenic poisoning of humans and animals; aflatoxins are important carcinogens that inhibit the synthesis of white matter in host plants and humans and animals, causing serious damage to animal and plant tissues. In the whole, corn ear rot causes not only the reduction of corn yield and quality, but also brings great hidden danger to the safety of foods and feeds and directly threatens the health of people and livestock, so that the control of the generation and hazard of corn ear rot has great significance.
The digging of resistance gene for regulating and controlling corn ear rot is a key measure for improving corn ear rot resistance and cultivating disease-resistant variety and ensuring safe production of corn. Previous studies have found that lipoxygenase genes (ZmLOXs) are important signaling molecules for the interaction between the host and pathogenic fungi, and that lipoxygenase genes derived from the host maize play an important role in regulating resistance to corn ear rot. Among them, the maize 9-LOX gene ZmLOX3 may be utilized as a causative agent by Fusarium verticillium (F.verticillioides) to cause stem/ear rot resistance. Complete knockout of the ZmLOX3 gene increases maize resistance to fusarium-induced granulosis but exhibits susceptibility to aspergillus flavus-induced granulosis; the complexity of ZmLOX3 gene in the aspect of corn ear rot resistance regulation is shown, and the direct knockout of the gene is not directly applicable to the resistance improvement of corn ear rot.
In view of antagonism of ZmLOX3 gene in fusarium and aspergillus flavus resistance regulation, a strategy for ensuring the protein function of the gene, but properly reducing the expression quantity of the gene, the fusarium resistance and the aspergillus flavus resistance can be achieved, so that the comprehensive improvement of the corn ear rot resistance is realized. During the evolution process of corn for nearly ten thousand years, the 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 is likely to exist in the natural world. The excavation of the natural variation is important for improving the resistance of 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 corn ear rot;
the second object of the invention is to provide mutants 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 expression of ZmLOX3 gene in corn ear and grain;
the fourth object of the invention is to provide a method for reducing the risk of corn to develop ear rot or improving ear rot resistance or a method for cultivating new variety of corn with ear rot resistance;
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 a primer for detecting the expression level of ZmLOX3 gene in corn;
the above object of the present invention is achieved by the following technical solutions:
in one aspect, the invention provides a key DNA sequence for regulating and controlling corn ear rot resistance, wherein the polynucleotide is shown in (a), (b), (c) or (d):
(a) The polynucleotide sequence shown in SEQ ID No.1, or
(b) A polynucleotide sequence capable of hybridizing under stringent hybridization conditions to the complement of SEQ ID No. 1;
(c) A polynucleotide sequence having at least 90% or more homology to the polynucleotide shown in SEQ ID No. 1; or (b)
(d) A mutant with deletion, substitution or insertion of one or more bases based on the polynucleotide sequence shown in SEQ ID No.1, and the mutant still has the function or activity of regulating corn ear rot.
The key DNA sequence can regulate and control the expression quantity change of ZmLOX3 gene in corn ears and seeds so as to influence the resistance of corn to ear rot or ear grain rot.
The spike rot or the spike grain rot in the invention is the same disease.
In the context of the present invention, the term "mutant" is a DNA sequence containing a change 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, such as mutants obtained by site-directed mutagenesis, or mutants obtained by recombinant means (e.g., DNA shuffling), or mutants obtained 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 corn ear rot, and the polynucleotide sequence of the mutant is shown as SEQ ID No. 2; the mutant is characterized in that 151bp of base is inserted into a ZmLOX3 gene promoter region Indel151, and the inserted sequence is shown as SEQ ID No.3; deletion of 361bp or 363bp base at the second intron Indel363 of ZmLOX3 gene with the deleted polynucleotide sequence shown as SEQ ID No.4 or SEQ ID No. 5; the mutant can raise the expression level of ZmLOX3 gene in corn ear and seed grain, and has raised risk of producing ear rot.
As a preferred specific embodiment of the mutant, the invention provides a mutant of a key DNA sequence for regulating and controlling the resistance of corn ear rot, and the polynucleotide sequence of the mutant is shown as SEQ ID No. 14; the mutant is obtained by deleting 151bp of base at a ZmLOX3 gene promoter region Indel151, and the deleted sequence is shown as SEQ ID No.3; 363bp base is inserted into the second intron Indel363 of ZmLOX3 gene, whose inserted polynucleotide sequence is shown in SEQ ID No. 5; the mutant can reduce the expression quantity of ZmLOX3 genes in corn ears and kernels, and the risk of occurrence of ear rot is obviously reduced.
The invention thus determines that corn materials with a 151bp fragment shown in SEQ ID No.3 deleted in a ZmLOX3 gene promoter region Indel151 or a base fragment shown in SEQ ID No.4 or SEQ ID No.5 inserted in a ZmLOX3 gene second intron region Indel363 generally have lower expression level of ZmLOX3 genes, and further show resistance to Fusarium-induced spike rot, and lower risk of spike rot; in contrast, corn materials with a 151bp fragment shown in SEQ ID No.3 inserted into the ZmLOX3 gene promoter region Indel151 or a base fragment shown in SEQ ID No.4 or SEQ ID No.5 deleted at the second intron region Indel363 of the ZmLOX3 gene generally have higher expression levels of the ZmLOX3 gene, show susceptibility to Fusarium-induced spike rot and have an increased risk of spike rot.
Thus, the present invention provides a method of reducing the risk of corn for developing or providing resistance to ear rot, comprising: deleting a fragment of 151bp shown in SEQ ID No.3 in a ZmLOX3 gene promoter region Indel 151; alternatively, the base fragment shown in SEQ ID No.4 or SEQ ID No.5 is inserted at the second intron Indel363 of the ZmLOX3 gene; or a fragment of 151bp shown in SEQ ID No.3 was deleted in the ZmLOX3 gene promoter region Indel151 and a base fragment shown in SEQ ID No.4 or SEQ ID No.5 was inserted at the ZmLOX3 gene second intron region Indel 363.
The invention also provides a method for cultivating the new corn variety with spike rot resistance, which comprises the steps of reducing the expression quantity of ZmLOX3 genes in corn ears and seeds, and further improving the resistance of the corn spike rot; therefore, the method for improving the corn ear rot resistance by reducing the expression quantity of ZmLOX3 genes in corn ears and kernels belongs to the protection scope of the invention; methods include driving expression of the ZmLOX3 gene in corn ears and kernels using other constitutive or tissue-specific promoters and expression cassettes for ZmLOX3, or using other natural variations to alter expression of the ZmLOX3 gene in corn ears and kernels, or reducing expression of the ZmLOX3 gene in corn ears and kernels by RNAi.
As a preferred embodiment of the present invention, the method for reducing the expression level of ZmLOX3 gene in corn ears and kernels may be: deleting a fragment of 151bp shown in SEQ ID No.3 in a ZmLOX3 gene promoter region Indel 151; alternatively, the base fragment shown in SEQ ID No.4 or SEQ ID No.5 is inserted at the second intron Indel363 of the ZmLOX3 gene; or a fragment of 151bp shown in SEQ ID No.3 was deleted in the ZmLOX3 gene promoter region Indel151 and a base fragment shown in SEQ ID No.4 or SEQ ID No.5 was inserted at the ZmLOX3 gene second intron region Indel 363.
Furthermore, the expression cassette containing the partial or whole DNA sequence shown in SEQ ID No.1, the mutant of the partial or whole 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 bacteria are all within the protection scope of the invention.
The recombinant plant expression vector is a recombinant plant expression vector constructed by the expression cassette and plasmid or expression vector and can be transferred into a plant host cell, tissue or organ.
The DNA sequences of the invention or mutants thereof can be used for the preparation 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, gene gun method or the like, and the transformed plant cell, tissue or organ is cultivated into a plant, thereby obtaining a transgenic plant. The starting vector used for constructing the plant expression vector can be any binary vector used for agrobacterium transformation of plants or a vector used for plant microprojectile bombardment, and the like.
For the practice of the present invention, conventional compositions and methods for making and using plant expression vectors and host cells are well known to those skilled in the art, and specific methods can be referenced, for example, by 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 includes phenotypic markers such as beta-galactosidase and fluorescent protein.
In a word, 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 quantity and the like provided by the invention can be applied to the cultivation of new disease-resistant corn varieties, in particular to the improvement of corn ear rot resistance and the like.
For reference, the invention provides a method for regulating and controlling the ear rot resistance of corn, which comprises the following steps: the expression of ZmLOX3 gene in corn is regulated by using 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, high-velocity ballistic bombardment, and the like. 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 by conventional methods (McCormick et al plant Cell reports 1986.5:81-84).
The invention may be used to transform any plant species including, but not limited to, monocotyledonous or dicotyledonous plants, preferably maize.
The invention further determines that Indel151 or Indel363 can be used as a molecular marker for regulating expression of ZmLOX3 gene in corn ears and kernels. The invention also finds that Indel151 and Indel363 are almost completely linked together, and any one of the variants can be used alone as a molecular marker for detecting the expression of ZmLOX3 gene in corn ears and kernels.
The invention further provides a detection primer for detecting Indel151 mutation; as a preferred 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 for detecting the variation condition of Indel151 in corn varieties, so that the primer is used for molecular auxiliary breeding of corn.
The invention further provides a detection primer for detecting the mutation condition of Indel 363; as a preferred embodiment, the nucleotide sequences of the detection primers are shown in SEQ ID No.8 and SEQ ID No.9 respectively; 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 auxiliary breeding of corn.
In addition, other primers designed according to SEQ ID No.1, SEQ ID No.2 or SEQ ID No.14, or by other methods, which can be used to detect Indel151 or Indel363 mutation, shall also fall within the scope of the present invention.
The invention further provides a specific amplification primer for detecting the ZmLOX3 gene expression quantity, wherein 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 of corn varieties, so that reference is provided for researching ZmLOX3 genes in corn breeding.
The nucleotide sequence of the coding region of the ZmLOX3 gene is shown as SEQ ID No.12, and the coding protein is shown as SEQ ID No. 13.
The DNA sequence for regulating and controlling the corn ear rot or the mutant thereof provided by the invention can regulate and control the expression of the ZmLOX3 gene in corn ears or seeds, has important significance for improving the corn ear rot, and can be further applied to the breeding of new corn varieties. The invention further provides a molecular marker Indel151 or Indel363 used for regulating and controlling ZmLOX3 gene expression, a specific detection primer for detecting the mutation condition of the molecular marker and a specific detection primer for detecting the expression quantity of the ZmLOX3 gene in corn, which can be directly applied to directionally improving the ear rot resistance of corn and have important application potential for breeding new disease-resistant varieties of corn.
Definition of terms in connection with the present invention
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 reference nucleic acids 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, phosphoroamidites, 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 sequence explicitly indicated. 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 bases and/or deoxyinosine residues (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 (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 hybridize specifically under specific conditions to form duplex molecules. Under these conditions (referred to as stringent hybridization conditions) one polynucleotide molecule may be used as a probe or primer to identify another polynucleotide molecule that shares homology.
The term "stringent hybridization conditions" as used herein means conditions of low ionic strength and high temperature known in the art. In general, the probe hybridizes to its target sequence to a greater degree than to other sequences (e.g., at least 2-fold over background. Stringent hybridization conditions are sequence dependent and will differ under different environmental conditions, longer sequences will hybridize specifically at higher temperatures. Target sequences 100% complementary to the probe can be identified by controlling the stringency or wash conditions of hybridization. Detailed guidance for 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 assys.1993.) more particularly, the stringent conditions are generally selected to be below the thermal melting point (T m ) About 5-10 deg.c. T (T) m Is the temperature at which a probe that is 50% complementary to the target in the equilibrium state hybridizes to the target sequence (at a given ionic strength, pH and nucleic acid concentration) (at T because the target sequence is present in excess) m 50% of the probes are occupied in the equilibrium state). Stringent conditions may be the following conditions: 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 with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least twice background hybridization, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can be as follows: 50% formamide, 5 XSSC and 1% SDS, at 42 ℃; or 5 XSSC, 1% SDS, at 65℃in 0.2 XSSC and at 65℃in 0.1% SDS. The washing may be performed for 5, 15, 30, 60, 120 minutes or more.
The term "plurality" as used herein generally means 2 to 8, preferably 2 to 4; "substitution" refers to the substitution of one or more amino acid residues with different amino acid residues, respectively; by "deletion" is meant a reduction in the number of amino acid residues, i.e., the absence of one or more amino acid residues therein, respectively; by "insertion" is meant an alteration 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 in its natural state upstream or 5' to the translation initiation codon of the open reading frame (or protein coding region) and is involved in recognition and binding of RNA polymerase II and other proteins (trans-acting transcription factors) to initiate transcription.
The term "plant promoter" is a natural or non-natural promoter that is functional in plant cells. Constitutive plant promoters function in most or all tissues throughout plant development. Any plant promoter may 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 generally causes transcription of the transcribable polynucleotide molecule in a manner similar to that of the transcribable polynucleotide molecule to which the promoter is normally linked. Plant promoters may include promoters produced by manipulation of known promoters to produce artificial, chimeric or hybrid promoters. Such promoters may also incorporate cis-elements from one or more promoters by, for example, 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 of gene expression. The cis-element may function to bind transcription factors that regulate transcription, trans-acting protein factors. Some cis-elements bind more than one transcription factor, and a transcription factor 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 the promoter regulates or mediates transcription of the gene of interest in a 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 a transcribable polynucleotide molecule is transcribed into a functional mRNA molecule, which is translated and thus expressed as a protein product. Constructs capable of expressing antisense RNA molecules may 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 "transformation": methods of introducing heterologous DNA sequences into host cells or organisms.
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 invention, regardless of the method used to insert to produce a recombinant host cell, such as 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 the promoter and the second intron region of ZmLOX3 each have an Indel natural variation (Indel) by sequencing; indel151, indel of 151bp of promoter region; indel363, 363bp Indel of the second intron region. The right side of the figure is the expression of ZmLOX3 gene in Ovule on the day of pollination (ovule_0dap) and grain 5 days after pollination (kernel_5dap) in the 12 inbred lines sequenced. Overall, the expression level of ZmLOX3 gene was high in the inbred line with Indel151 insertion or Indel363 deletion; b is GWAS analysis, which shows that Indel151 and Indel363 are obviously related to the expression level of ZmLOX3 genes, and the lower part of the Manhattan diagram is a diagram of all marked linkage disequilibrium modules (LD-blocks); c is the relation between different genotypes of Indel151 and the ZmLOX3 gene expression quantity in the inbred line, when Indel151 is inserted, the ZmLOX3 gene expression quantity is higher; the different genotypes of which D is Indel151 are selected in the breeding process under the condition of allele frequency variation among corn varieties in different ages, wherein the genotype corresponding to the reduced ZmLOX3 gene expression; e is the distribution of allele frequencies between different heterosis materials for the two genotypes at Indel 151.
FIG. 2 shows the results of Indel151 genotyping of 12 inbred lines.
FIG. 3 shows the genotyping results for Indel363 from 12 inbred lines.
FIG. 4 shows the maize grain inoculation results for 10 inbred lines; among them, 5 inbred lines with Indel151 insertion deleted at Indel363 were significantly more susceptible than the remaining different genotype inbred lines.
FIG. 5 shows the expression level of ZmLOX3 gene corresponding to the different Indel151 genotypes in the hybrid (left panel) and the allele frequency variation of the different Indel151 genotypes among the different year hybrids (right panel).
Detailed Description
The invention will be further described with reference to specific embodiments, and advantages and features of the invention will become apparent from the description. These examples are merely exemplary and do not limit the scope of the invention in any way. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
The inbred lines used in the examples below can obtain relevant information from the "chinese crop germplasm information net" and apply for obtaining the corresponding seeds.
EXAMPLE 1 test for controlling changes in expression level of ZmLOX3 Gene by Indel (Indel) mutation of promoter region and second intron region of ZmLOX3 Gene
1. Development of mutation sites of ZmLOX3 Gene promoter region and second intron region
Full genome assembly was performed on 12 maize backbone inbred parents widely used worldwide using average >55 x PacBio SMRT reads data, >94 x second generation Illumina 150bp double-ended sequencing data, and >139 x BioNano single-molecule data. The 12 inbred lines are: PH207, A632, OH43, zheng58, ye478, dan340, huangzaosi, chang7-2, sting 92, sting 724, 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 in maize breeding.
A10 kb genomic sequence was taken up upstream and downstream of the ZmLOX3 gene in the 14 core inbred lines, blast alignment of the DNA sequences was performed, and it was found that in the 14 inbred lines, one Indel variation was present in each of the promoter and the second intron of the ZmLOX3 gene (Indel, FIG. 1A). Wherein the Indel length of the promoter region is 151bp, and the sequence of the Indel length is SEQ ID No.3; the Indel length of the second exon region is 363bp, the base sequence of which is shown as SEQ ID No.5, or the Indel length of the second exon region is 361bp, and the base sequence of which is shown as SEQ ID No. 4.
2. Indel151 and Indel363 are significantly correlated with the variation in the expression level of ZmLOX3 gene
The present inventors have collected and assembled 116 varieties of different ages and their corresponding 137 parental inbred lines (some of them have shared parents) widely used in the process of updating the varieties. In summer 2019, samples (3 replicates per material) were taken from the female ears of 253 parts of material (116 hybrids and 137 inbred lines) during the silking period, and then RNA-Seq analysis was performed to obtain data on the expression levels of 20000 genes in 253 parts of material ears. Wherein, the expression quantity data of ZmLOX3 gene are independently extracted for subsequent analysis.
25,320,664 single nucleotide polymorphism molecular markers (SNPs) and 4,319,510 Indel polymorphism molecular markers (indels) were mined using data from deep (> 10×) second generation resequencing of 137 maize inbred lines in combination with published maize B73V 3 genome. In addition, genotypes of Indel151 (FIG. 2) and Indel363 (FIG. 3) in 137 parts of maize inbred line 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 kindred relationship of 137 maize inbred lines are estimated by using the mined molecular markers, and then genome-wide association analysis (GWAS) is performed using the detected expression amount data of the ZmLOX3 gene as a phenotype. Wherein a significant association signal was identified near the ZmLOX3 gene, the most significant signal falling on the Indel151 marker (p-value=1.40E-9), indel363 being the second significant association marker (fig. 1B), indicating that Indel151 and Indel363 are significantly associated with the variation in expression level of the ZmLOX3 gene.
The test further divides 137 parts of materials into two types according to genotypes at Indel151, wherein one type is the genotype materials with a deletion 151bp sequence at Indel151, and the total is 95 parts; the other class is genotype materials with 151bp sequence at Indel151, which is 33 shares in total; comparing the expression level of ZmLOX3 gene between the two types of materials, it was found that the expression level of ZmLOX3 gene in the material lacking Indel151 was lower (FIG. 1C), indicating that Indel151 can positively regulate the expression of ZmLOX3 gene. Linkage disequilibrium analysis also indicated that Indel151 and Indel363 are located within the same LD-block (linkage disequilibrium module) (fig. 1B); and sequence analysis in 14 inbred lines also showed that these two variants always appeared linked together in a repulsive phase, i.e. when there was a genotype of deletion 151bp sequence at Indel151 in one material, there was always a genotype of insertion 363bp or 361bp sequence at Indel363 (fig. 1A). It was shown that it is possible to infer the genotype at the other Indel by the genotype of one of the two; however, indel151 has a stronger correlation with the expression level of ZmLOX3 gene, so Indel151 can be used as a molecular marker for detecting the expression level of ZmLOX3 gene more preferably.
Example 2 Indel151 and Indel363 influence on corn ear rot resistance by modulation of ZmLOX3 Gene expression
The expression level of ZmLOX3 gene in the seeds is properly reduced, but the protein function is not changed, so that the Fusarium-resistant and aspergillus flavus-resistant functions can be realized.
The previous results of the present inventors showed that materials lacking Indel151 or inserted Indel363 tend to have lower ZmLOX3 gene expression levels. To further verify the relationship between Indel151 and Indel363 and ZmLOX3 gene expression levels, 12 representative inbred lines (PH 207, a632, OH43, zheng58, ye478, dan340, huangzaosi, chang7-2, jing92, jing724, xu178, S37) as above were respectively planted in the field, ovules during their laying period and kernels after 5 days pollination were sampled and snap frozen with liquid nitrogen. Samples of every 5 individuals were mixed into one sample, 3 biological replicates were taken for each inbred line, and then RNA was extracted by TRIzol method, and the expression level of ZmLOX3 gene was detected using specific primers (SEQ ID No.10 and SEQ ID No. 11). As a result of the foregoing GWAS analysis, it was found that the material deleted from Indel151 or inserted into Indel363 (PH 207, A632, zheng58, ye478, jin 724, xu178, S37) had a low expression level of ZmLOX3 gene in the ovule which was not pollinated, or in the kernel after 5 days of pollination (FIG. 1A), and the relationship between Indel151 and Indel363 and the expression level of ZmLOX3 gene was again verified.
Further Fusarium rotavailing F.verrucosa inoculation experiments were performed on 10 material corn kernels in 12 representative inbred lines (FIG. 4). The results showed that 5 inbred lines with low ZmLOX3 gene expression (B73, ye478, mo17, PH207, A632, deletion Indel151, insertion Indel 363) are generally more resistant to Fusarium rotati caused ear rot than 5 inbred lines with higher ZmLOX3 gene expression (Jing 92, dan340, huangzaosi, OH, chang7-2, insertion Indel151, deletion Indel 363). These results indicate that Indel151 and Indel363 can affect corn ear rot resistance by regulating the ZmLOX3 gene expression level.
Example 3 excellent alleles at indel151 are subjected to strong artificial selection during modern maize breeding
The present inventors have previously collected 350 parts of maize inbred material from different breeding stages in China and the United states, including 163 parts of early American (Public-US) and near modern (Ex-PVP) breeding materials, 187 parts of early China (CN 1960&70 s), mid-term (CN 1980&90 s) and current (CN 2000&10 s) main cultivated maize breeding materials. Analysis of the frequency distribution of Indel151 genotypes (Indel 363 is closely linked to Indel151 and can be represented by Indel151 genotypes) in these materials revealed that with the advancement of breeding period, the genotype frequency of the deletion Indel151 (corresponding to lower ZmLOX3 gene expression and higher ear rot resistance) class was significantly improved during both chinese and us maize breeding (fig. 1D), indicating that the deletion Indel151 class genotypes were strongly selected manually during modern maize breeding.
In addition, 350 parts of maize inbred material can be divided into 8 heterosis groups, SS, PA, X, NSS, TSPT, IDT, PB, mixed respectively. The former report shows that TSPT germplasm is generally susceptible to spike rot, wherein Huangzaosi, chang-2 and sting 92 are typical TSPT germplasm, and the inoculation experiment of the invention also proves that the TSPT germplasm is indeed susceptible to spike rot (figure 4), thus proving the report of the former. The invention also discovers that the genotype frequency of the Indel151 type inserted in the TSPT germplasm is higher, and the insertion of Indel151 corresponds to higher ZmLOX3 gene expression quantity, so that the TSPT germplasm is susceptible to the spike rot, and the reason of the TSPT germplasm susceptibility to the spike rot is revealed. Simultaneously, the important potential of Indel151 and Indel363 in improving the resistance of TSPT germplasm ear rot is also predicted.
Furthermore, the invention also analyzes the expression condition of ZmLOX3 genes and the distribution condition of Indel151 different genotypes in 116 different-year hybrid seeds. Analysis found that in the hybrid, the amount of ZmLOX3 expressed in the Indel151 deleted material was significantly lower than in the heterozygous and Indel151 inserted material (left panel in fig. 5). The low expression level of ZmLOX3 corresponds to the resistance to ear rot, so that the genotype of deletion Indel151 is an excellent genotype, and the genotype of insertion Indel151 is a disadvantageous genotype. Allele frequency analysis revealed that the homozygous superior allele of Indel151 gradually increased during the breeding of the hybrid, whereas the homozygous adverse genotype was almost lost in the hybrid after 2000; and along with the promotion of breeding ages, the proportion of heterozygous genotypes is also lower and lower, and further proves that Indel151 is strongly selected in the breeding process. All of these hybrid species were used directly in corn production, and selection among the hybrid species was performed, and the importance of the natural variation (Indel 151 and closely linked Indel 363) was further confirmed.
SEQUENCE LISTING
<110> institute of biotechnology of national academy of agricultural sciences
<120> DNA sequence for regulating and controlling corn ear rot resistance, 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
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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
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tcacgacaat ctacttgatg tcctatgtag ccatgatctc tacgagcaat caatatcaat 1680
gcactagaat cccaactacg tagagtatta ttggacgaac gagtaaaaca ccaaagagca 1740
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gcctcgttcg gacaaccggt acaatcttca aaatggaagt agaagattgg tgaaagcata 2100
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tcggaagcaa gacaaaacca aacagagttg ataaaaaata ttaggaaaaa tagaataatg 2580
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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
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ataaatgtgg aattttaaat atggtagaga tacaaactct cgttgatgtg tcagtatttt 3120
tactggagta tcaagaaacg cgcaagcttc ttactaatcc ttcttagagc ctcacgcaag 3180
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tcacacagac accaacgcca ctgcactgca aaagcaagag cagctagcta gtaaagatgc 4740
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ggattatata ttggattctt tatcagtaac ttactgctgc tagtataccc ctaccctagt 5400
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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
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aaatccatgc aagtgggact agtgtgtaac tggtagtata gctgaagaat ctagtggtag 6000
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tcccgcagcg gttcccccta aatactcccc ctatatctca ctaccactat aaaatattat 6180
tttctatacc aattatcaat tttttatcta ctaacaatta ctcgtggacc cacagcacag 6240
tgtttagggt gatgaacagt gacacgctag atctgaaggg agagagaagg ggaccgacac 6300
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<211> 6434
<212> DNA
<213> Zea mays L.
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ctctgctccg cccgacccca gggctcggac tcgggctaag acccggaaga cggcgaatct 180
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tcgcccgacc ccagggctcg gactccgccc tagcctcggc caaacgatct ccgcctcgcc 300
cgacccgggg gctcgggctc ggcctcggca acggaaggca gactcgacct cgacttcgga 360
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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 (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.
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Disruption of a Maize 9-Lipoxygenase Results in Increased Resistance to Fungal Pathogens and Reduced Levels of Contamination with Mycotoxin Fumonisin;Xiquan Gao等;MPMI;第20卷(第8期);摘要，第922页第1段 *

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