Corn cDNAs encoding corn Mlo proteins

This is a continuation-in-part of Application Ser. No. 09/483,545 filed Jan. 14, 2000, now abandoned, which claims priority benefit of divisional Application 09/183,959 filed Nov. 2, 1998, now U.S. Pat. No. 6,303,332, which claims priority benefit of Provisional Application No. 60/064,492, filed Nov. 5, 1997.

This invention is in the field of plant molecular biology. More specifically, this invention pertains to isolated nucleic acid fragments encoding proteins with similarities to the barley Mlo protein and involved in corn ( Zea mays ) resistance to disease.

Southern or Maydis Leaf Blight is a serious disease of corn caused by Helminthosporium maydis Nisik. ( Cochliobolus heterostrophus (Drechs)). While corn hybrids with normal cytoplasm are resistant to Race T of the pathogen, Race O shows no differential reaction between plants with different cytoplasms; however, sources of resistance are available (Shurtleff (1973) Compendium of Corn Diseases). Chlorotic lesion resistance to Race O was identified in an East African line of corn (Jeweus and Daniel-Kalio (1968) Plant Dis. Rep . 52:134-136). The resistant plants inhibit fungal sporulation and are distinguished by the presence of small chlorotic lesions. Significantly, the resistance is recessive (Smith and Hooker (1973) Crop Science 13:330-331). It was reported that disease is controlled by two linked recessive genes.

It is well established that resistance to many diseases in plants is mediated by the interaction of plant genes referred to as “R” genes with corresponding Avr genes expressed by the pathogen (Hammond-Kosack, K. E. and Jones, J. D. G. (1997) Ann Rev. Plant Phusiol Plant Mol. Bio . 48:1-39). This interaction leads to the activation of plant responses which in turn results in increased resistance to disease. This resistance is frequently mediated by the “hypersensitive response”, a localized cell death phenomenon that occurs in the areas of plant tissue invaded by the pathogen. This Avr/R interaction is race-specific; i.e., particular alleles of the plant R gene respond only to specific races of the pathogen which express a corresponding Avr gene (Flor, H. (1971) Ann. Rev. Phytopathol . 9:275-296).

Most of the R genes characterized to date are dominant; i.e., resistance is exhibited by plants carrying the appropriate R allele in either an heterozygous or homozygous state. Some R genes have been isolated by map-based cloning. Many homologs of R genes have also been identified by sequence similarity. Because of the rapid evolution of plant resistance to disease, homology of a cDNA or genomic DNA sequence to a known R gene from a different plant species does not allow one to predict the pathogen specificity of that particular R gene. No general methods for such prediction exist today.

Genes involved in resistance of plants to plant pathogens may be used to engineer disease resistance into plants normally sensitive to disease using several different approaches. Transgenic plants containing alleles of R genes demonstrate resistance to corresponding races of the pathogen (Wang, G. -L. et al. (1996) Mol. Plant - Microbe Interact . 9(9):850-855). The resistance genes may also be engineered to respond to non-native signals derived from the pathogen. In addition, pathogen-derived Avr genes may be expressed in a controlled manner in plants to strengthen the response to a pathogen. Genes further downstream from the R genes in the signal transduction pathways that transmit signals to the disease response effector genes may be engineered to directly respond to pathogen infection, thereby shortening the response pathway.

The process of the hypersensitive response to a pathogen, and the signaling networks involved, are poorly understood. In no case have all of the genes involved in transmitting the signal from the pathogen to the site of the initiation of the hypersensitive response been identified.

In a few cases, the functioning of a disease resistance mechanism different from the Avr/R interaction described above results in a recessive, rather than dominant pattern of resistance. One such example is the Mlo gene of barley, which conveys resistance to Erysiphe graminis f sp. hordei. The barley gene has been recently isolated by a positional cloning approach (Bueschges, R. et al. (1997) Cell 88:695-705). The dominant (sensitive) allele (Mlo) is thought to encode a protein involved in regulation of leaf cell death and in the onset of pathogen defense. The partial or complete inactivation of Mlo results in the priming of the disease-resistance response even in the absence of the pathogen, and leads to increased resistance to E. graminis.

The available scientific data concerning Mlo-mediated disease resistance in barley points towards another approach to controlling disease: priming the pathogen response pathway by diminishing the effectiveness of negative regulation of the hypersensitive response. Appropriately engineered plants may show increased pathogen resistance at the expense of expressing some pathogen response-related genes even in the absence of pathogen. Sense or antisense inhibition or targeted gene disruption of Mlo and Mlo-related genes may have such an effect. Resistance to other pathogens may also be increased using this approach.

Mlo-related cDNA clones and DNA segments of genomic DNA, and their homologs and derivatives, may also be used as molecular probes to track inheritance of corresponding loci in genetic crosses, and thus facilitate the plant breeding process. Moreover, these DNA sequences may also be used as probes to isolate, identify and genetically map Mlo and other closely related disease resistance genes.

The instant invention relates to isolated nucleic acid fragments encoding corn homologs of the barley Mlo protein. Specifically, the instant invention relates to isolated nucleic acid fragments encoding plant proteins involved in the regulation of plant cell death response in corn which is associated with disease resistance. In addition, this invention relates to nucleic acid fragments that are complementary to nucleic acid fragments encoding a corn homolog of the barley Mlo protein.

An additional embodiment of the instant invention pertains to a polypeptide encoding all or a substantial portion of a corn Mlo protein.

In another embodiment, the instant invention relates to a chimeric gene that comprises a nucleic acid fragment encoding the corn Mlo or to a chimeric gene that comprises a nucleic acid fragment that is complementary to the nucleic acid fragment encoding the corn Mlo, operably linked to suitable regulatory sequences, wherein expression of the chimeric gene results in production of levels of a corn Mlo in transformed host cells that are altered (i.e., increased or decreased) from the levels produced in untransformed host cells.

In a further embodiment, the instant invention concerns a transformed host cell comprising in its genome a chimeric gene encoding a corn Mlo, operably linked to suitable regulatory sequences. Expression of the chimeric gene results in production of altered levels of the encoded protein in the transformed host cell. The transformed host cell can be of eukaryotic or prokaryotic origin, and include cells derived from higher plants and microorganisms. The invention also includes transformed plants that arise from transformed host cells of higher plants, and seeds derived from such transformed plants.

An additional embodiment of the instant invention concerns a method of altering the level of expression of a corn Mlo in a transformed host cell comprising: a) transforming a host cell with a chimeric gene comprising a nucleic acid fragment encoding a corn Mlo; and b) growing the transformed host cell under conditions that are suitable for expression of the chimeric gene wherein expression of the chimeric gene results in production of altered levels of corn Mlo in the transformed host cell.

An addition embodiment of the instant invention concerns a method for obtaining a nucleic acid fragment encoding all or a substantial portion of an amino acid sequence encoding a corn Mlo.

A further embodiment of the instant invention concerns a method for using the instant nucleic acid fragments and their homologs and derivatives as molecular probes to monitor inheritance of corresponding loci in genetic crosses, and thus to facilitate and accelerate plant breeding. Additionally, the instant nucleic acid fragments may be used as probes to isolate, identify and genetically map corn Mlos and other closely related disease resistance genes.

The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application.

FIG. 1 is a genetic map of chromosome 6 of corn, including the position of cDNA clone cta1.pk0005.c1. The map was constructed using Mapmaker 3.0 for the Macintosh (Lander, E. S. et al. (1987) Genomics 1:174-181) and mapping data from Burr et al. (Burr et al. (1988) Genetics 118:5)19-526).

FIGS. 2A and 2B presents an alignment of nucleotides 781 to 1390 of the contig assembled with the nucleotide sequence of cDNA insert in clone cta1.pk0005.c1 and the 5′RACE product obtained using PCR primer P3 (SEQ ID NO:9) with the sequences of the 703 bp (SEQ ID NO:17) product obtained by PCR amplification of corn genomic DNA using primers P1 and P2. The extent of the intron sequences is indicated with an arrow. Intron sequences are shown in lowercase. The gt/ag invariant intron boundary sequences are bold and underlined.

FIGS. 3 and 3B presents an alignment of nucleotides 60 to 520 from the sequence of cDNA mlo3b3 (SEQ ID NO:11) with the sequence of the 765 bp (SEQ ID NO:18) product obtained by PCR amplification of corn genomic DNA using primers P1 and P2. The extent of the intron sequences is indicated with an arrow. Intron sequences are shown in lowercase. The gt/ag invariant intron boundary sequences are bold and underlined.

FIGS. 4A and 4B presents an alignment of the amino acid sequence deduced from the contig assembled from the entire CDNA insert in clone csc1c.pk005.d8 and the 5′ RACE PCR (SEQ ID NO:23) with the amino acid sequence of the putative Mlo from Oryza sativa having NCBI gi Accession No. 6063540 (SEQ ID NO:24) and with the sequence of the Hordeum vulgare Mlo having NCBI gi Accession No.6016588 (SEQ ID NO:25). Amino acids which are conserved among all sequences are indicated by an asterisk above the alignment. Dashes are used by the program to maximize the alignment.

The following sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825.

SEQ ID NO:1 is the nucleotide sequence comprising the entire cDNA insert in clone cc3.pk0007.g3 encoding an almost entire corn Mlo.

SEQ ID NO:2 is the deduced amino acid sequence of an almost entire corn Mlo derived from the nucleotide sequence of SEQ ID NO:1.

SEQ ID NO:3 is the nucleotide sequence comprising a portion of the cDNA insert in clone cr1n.pk0154.a3 encoding the C-terminal half of a corn Mlo.

SEQ ID NO:4 is the deduced amino acid sequence of the C-terminal half of a corn Mlo derived from the nucleotide sequence of SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence comprising a portion of the cDNA insert in clone cs1.pk0071.e3 encoding the C-terminus of a corn Mlo.

SEQ ID NO:6 is the deduced amino acid sequence of the C-terminus of a corn Mlo derived from the nucleotide sequence of SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence comprising the entire cDNA insert in clone cco1.pk0052.b10 encoding an entire corn Mlo.

SEQ ID NO:8 is the deduced amino acid sequence of an entire corn Mlo derived from the nucleotide sequence of SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence comprising a contig assembled from the entire cDNA insert in clone cta1.pk0005.c1 and the 5′ RACE PCR product obtained using PCR primer P3. This sequence encodes an entire corn Mlo.

SEQ ID NO:10 is the deduced amino acid sequence of an entire corn Mlo derived from the nucleotide sequence of SEQ ID NO:9.

SEQ ID NO:11 is the nucleotide sequence comprising mlo3b3, the 3′ RACE PCR product obtained using clone cta1.pk0005.c1 and PCR primer P1, encoding a fragment of a corn Mlo.

SEQ ID NO:12 is the deduced amino acid sequence of a fragment of a corn Mlo derived from the nucleotide sequence of SEQ ID NO:11.

SEQ ID NO:13 is the nucleotide sequence comprising a portion of the cDNA insert in clone cen3n.pk0062.b7 encoding a fragment of a corn Mlo.

SEQ ID NO:14 is the deduced amino acid sequence of a fragment of a corn Mlo derived from the nucleotide sequence of SEQ ID NO:13.

SEQ ID NO:15 is the nucleotide sequence of PCR primer P1.

SEQ ID NO:16 is the nucleotide sequence of PCR primer P2.

SEQ ID NO:17 is the nucleotide sequence of the 703 bp PCR product obtained by amplification of corn genomic DNA using PCR primers P1 and P2.

SEQ ID NO:18 is the nucleotide sequence of the 765 bp PCR product obtained by amplification of corn genomic DNA using PCR primers P1 and P2.

SEQ ID NO:19 is the nucleotide sequence of PCR primer P3.

SEQ ID NO:20 is the nucleotide sequence of PCR primer P4.

SEQ ID NO:21 is the nucleotide sequence of PCR primer P5.

SEQ ID NO:22 is the nucleotide sequence comprising a contig assembled from the entire cDNA insert in clone csc1c.pk005.d8 and the 5′ RACE PCR product obtained using PCR primer P5. This sequence encodes an entire corn Mlo.

SEQ ID NO:23 is the deduced amino acid sequence of an entire corn Mlo derived from the nucleotide sequence of SEQ ID NO:22.

SEQ ID NO:24 is the amino acid sequence of the putative Mlo protein from Oryza sativa having NCBI gi Accession No. 6063540.

SEQ ID NO:25 is the amino acid sequence of the Hordeum vulgare Mlo having NCBI gi Accession No.6016588.

SEQ ID NO:26 is the nucleotide sequence comprising nucleotides 1 through 572 from SEQ ID NO:22.

SEQ ID NO:27 is the nucleotide sequence comprising nucleotides 1554 through 1802 from SEQ ID NO:22.

SEQ ID NO:28 is the amino acid sequence comprising amino acids 1 through 184 from SEQ ID NO:23.

SEQ ID NO:29 is the amino acid sequence comprising amino acids 512 through 563 from SEQ ID NO:23.

The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.

In the context of this disclosure, a number of terms shall be utilized. The terms “polynucleotide”, “polynucleotide sequence”, “nucleic acid sequence”, and “nucleic acid fragment”/“isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like. As used herein, an “isolated nucleic acid fragment” is a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. An isolated nucleic acid fragment in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA or synthetic DNA. As used herein, “contig” refers to an assemblage of overlapping nucleic acid sequences to form one contiguous nucleotide sequence. For example, several DNA sequences can be compared and aligned to identify common or overlapping regions. The individual sequences can then be assembled into a single contiguous nucleotide sequence.

The terms “Mlo” and “Mlo homolog” are used interchangeably herein. “Mlo homolog” refers to an isolated nucleic acid fragment or polypeptide with similarity to the barley Mlo gene or protein. This barley gene conveys resistance to Erysiphe graminis f sp. hordei.

As used herein, “substantially similar” refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the protein encoded by the DNA sequence. “Substantially similar” also refers to nucleic acid fragments wherein changes in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate alteration of gene expression by antisense or co-suppression technology. “Substantially similar” also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of one or more nucleotides that do not substantially affect the functional properties of the resulting transcript vis-á-vis the ability to mediate alteration of gene expression by antisense or co-suppression technology or alteration of the functional properties of the resulting protein molecule. It is therefore understood that the invention encompasses more than the specific exemplary sequences.

For example, it is well known in the art that antisense suppression and co-suppression of gene expression may be accomplished using nucleic acid fragments representing less than the entire coding region of a gene, and by nucleic acid fragments that do not share 100% sequence identity with the gene to be suppressed. Moreover, alterations in a gene which result in the production of a chemically equivalent amino acid at a given site, but do not effect the functional properties of the encoded protein, are well known in the art. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product. Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the protein molecule would also not be expected to alter the activity of the protein. Each of the proposed modifications is well within the routine skill in the art, as is determination of retention of biological activity of the encoded products. Moreover, the skilled artisan recognizes that substantially similar nucleic acid sequences encompassed by this invention are also defined by their ability to hybridize, under stringent conditions (0.1×SSC, 0.1% SDS, 65° C.), with the sequences exemplified herein. Preferred substantially similar nucleic acid fragments of the instant invention are those nucleic acid fragments whose DNA sequences are 80% identical to the coding sequence of the nucleic acid fragments reported herein. More preferred nucleic acid fragments are 90% identical to the coding sequence of the nucleic acid fragments reported herein. Most preferred are nucleic acid fragments that are 95% identical to the coding sequence of the nucleic acid fragments reported herein.

A “substantial portion” of an amino acid or nucleotide sequence comprises enough of the amino acid sequence of a polypeptide or the nucleotide sequence of a gene to afford putative identification of that polypeptide or gene, either by manual evaluation of the sequence by one skilled in the art, or by computer-automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol . 215:40-3410). In general, a sequence of ten or more contiguous amino acids or thirty or more nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene. Moreover, with respect to nucleotide sequences, gene specific oligonucleotide probes comprising 20-30 contiguous nucleotides may be used in sequence-dependent methods of gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial colonies or bacteriophage plaques). In addition, short oligonucleotides of 12-15 bases may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers. Accordingly, a “substantial portion” of a nucleotide sequence comprises enough of the sequence to afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence. The instant specification teaches partial or complete amino acid and nucleotide sequences encoding one or more particular plant proteins. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Accordingly, the instant invention comprises the complete sequences as reported in the accompanying Sequence Listing, as well as substantial portions of those sequences as defined above.

“Codon degeneracy” refers to divergence in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. Accordingly; the instant invention relates to any nucleic acid fragment that encodes all or a substantial portion of the amino acid sequence encoding the corn Mlo as set forth in SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, and 23. The skilled artisan is well aware of the “codon-bias” exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

“Synthetic genes” can be assembled from oligonucleotide building blocks that are chemically synthesized using procedures known to those skilled in the art. These building blocks are ligated and annealed to form gene segments which are then enzymatically assembled to construct the entire gene. “Chemically synthesized”, as related to a sequence of DNA, means that the component nucleotides were assembled in vitro. Manual chemical synthesis of DNA may be accomplished using well established procedures, or automated chemical synthesis can be performed using one of a number of commercially available machines. Accordingly, the genes can be tailored for optimal gene expression based on optimization of nucleotide sequence to reflect the codon bias of the host cell. The skilled artisan appreciates the likelihood of successful gene expression if codon usage is biased towards those codons favored by the host. Determination of preferred codons can be based on a survey of genes derived from the host cell where sequence information is available.

“Gene” refers to a nucleic acid fragment that expresses a specific protein, including regulatory sequences preceding (5′ non-coding sequences) and following (3′ non-coding sequences) the coding sequence. “Native gene” refers to a gene as found in nature with its own regulatory sequences. “Chimeric gene” refers any gene that is not a native gene, comprising regulatory and coding sequences that are not found together in nature. Accordingly, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. “Endogenous gene” refers to a native gene in its natural location in the genome of an organism. A “foreign” gene refers to a gene not normally found in the host organism, but that is introduced into the host organism by gene transfer. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes. A “transgene” is a gene that has been introduced into the genome by a transformation procedure.

“Coding sequence” refers to a DNA sequence that codes for a specific amino acid sequence. “Regulatory sequences” refer to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, and polyadenylation recognition sequences.

“Promoter” refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3′ to a promoter sequence. The promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an “enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as “constitutive promoters”. New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg, (1989) Biochemistry of Plants 15:1-82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

The “translation leader sequence” refers to a DNA sequence located between the promoter sequence of a gene and the coding sequence. The translation leader sequence is present in the fully processed mRNA upstream of the translation start sequence. The translation leader sequence may affect processing of the primary transcript to mRNA, mRNA stability or translation efficiency. Examples of translation leader sequences have been described (Turner, R. and Foster, G. D. (1995) Molecular Biotechnology 3:225).

The “3′ non-coding sequences” refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is usually characterized by affecting the addition of polyadenylic acid tracts to the 3′ end of the mRNA precursor. The use of different 3′ non-coding sequences is exemplified by Ingelbrecht et al., (1989) Plant Cell 1:671-680.

“RNA transcript” refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence. When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be a RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA. “Messenger RNA (mRNA)” refers to the RNA that is without introns and that can be translated into protein by the cell. “cDNA” refers to DNA that is complementary to and synthesized from a mRNA template using, for example, the enzyme reverse transcriptase. The cDNA can be single stranded or converted into the double stranded by using the Klenow fragment of DNA polymerase I. “Sense” RNA refers to RNA transcript that includes the mRNA and so can be translated into protein by the cell. “Antisense RNA” refers to a RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene (U.S. Pat. No. 5,107,065, incorporated herein by reference). The complementarity of an antisense RNA may be with any part of the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′ non-coding sequence, introns, or the coding sequence. “Functional RNA” refers to antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.

The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.

The term “expression”, as used herein, refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. “Antisense inhibition” refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein. “Overexpression” refers to the production of a gene product in transgenic organisms that exceeds levels of production in normal or non-transformed organisms. “Co-suppression” refers to the production of sense RNA transcripts capable of suppressing the expression of identical or substantially similar foreign or endogenous genes (U.S. Pat. No. 5,231,020, incorporated herein by reference).

“Altered levels” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.

“Mature” protein refers to a post-translationaliy processed polypeptide; i.e., one from which any pre- or propeptides present in the primary translation product have been removed. “Precursor” protein refers to the primary product of translation of mRNA; i.e., with pre- and propeptides still present. Pre- and propeptides may be but are not limited to intracellular localization signals.

A “chloroplast transit peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the chloroplast or other plastid types present in the cell in which the protein is made. “Chloroplast transit sequence” refers to a nucleotide sequence that encodes a chloroplast transit peptide. A “signal peptide” is an amino acid sequence which is translated in conjunction with a protein and directs the protein to the secretory system (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol . 42:21-53). If the protein is to be directed to a vacuole, a vacuolar targeting signal (supra) can further be added, or if to the endoplasmic reticulum, an endoplasmic reticulum retention signal (supra) may be added. If the protein is to be directed to the nucleus, any signal peptide present should be removed and instead a nuclear localization signal included (Raikhel (1992) Plant Phys . 100:1627-1632).

“Transformation” refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as “transgenic” organisms. Examples of methods of plant transformation include Agrobacterium-mediated transformation (De Blaere et al. (1987) Meth. Enzymol . 143:277) and particle-accelerated or “gene gun” transformation technology (Klein et al. (1987) Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference).

Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual ; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Maniatis”).

Nucleic acid fragments encoding at least a portion of several corn Mlos have been isolated and identified by comparison of random plant cDNA sequences to public databases containing nucleotide and protein sequences using the BLAST algorithms well known to those skilled in the art. These nucleic acid fragments encode plant proteins involved in the regulation of plant cell death response in corn which is associated with resistance of corn to disease. Table 1 lists the designation of the cDNA clones that comprise the nucleic acid fragments encoding corn homologs of the barley Mlo protein.

The homology with the barley Mlo protein indicated that these cDNAs corresponded to genes involved in disease resistance, but did not provide information as to the possible disease specificity in corn; Erysiphe graminis , the causative agent of powdery mildew in barley, is not a pathogen of corn. Ultimately, disease specificity information was provided by genetic mapping experiments.

PCR amplification of maize genomic DNA using two primers, P1 and P2, designed using the nucleic acid sequence of cDNA clone cta1.pk0005.c1, resulted in the amplification of two DNA segments, both homologous to cta1.pk0005.c1, but differing in the numbers of introns (see Example 4). Genetic mapping experiments (see Example 5) placed one of the two genomic segments on chromosome 6, in Bin 6.01, between markers BNL7.28 and UMC59. Three disease resistance loci map to the same bin on chromosome 6: maize dwarf mosaic virus (MDMV), wheat streak mosaic virus (WSMV) and Southern corn leaf blight (SCLB). Of the three disease resistance loci, the SCLB locus is unique in that it is the only known recessive disease resistance locus in corn. Therefore, it is highly likely that the Mlo homolog represented by the 765 bp amplification product containing three introns corresponds to the Southern corn leaf blight resistance (SCLB R ) gene. The second genomic sequence, which contains two introns, is likely to be a second, linked SCLB R gene. The existence of two linked genes has been reported by Craig and Fajeminsin (Craig, J. and Fagemisin, J. M (1969) Plant Dis. Rep . 53:742-743).

The nucleic acid fragments of the instant invention may be used to isolate cDNAs and genes encoding homologous corn Mlos from the same or other plant species. Isolation of homologous genes using sequence-dependent protocols is well known in the art. Examples of sequence-dependent protocols include, but are not limited to, methods of nucleic acid hybridization, and methods of DNA and RNA amplification as exemplified by various uses of nucleic acid amplification technologies (e.g., polymerase chain reaction, ligase chain reaction).

For example, genes encoding other Mlos, either as cDNAs or genomic DNAs, could be isolated directly by using all or a portion of the instant nucleic acid fragments as DNA hybridization probes to screen libraries from any desired plant employing methodology well known to those skilled in the art. Specific oligonucleotide probes based upon the instant nucleic acid sequences can be designed and synthesized by methods known in the art (Maniatis). Moreover, the entire sequences can be used directly to synthesize DNA probes by methods known to the skilled artisan such as random primer DNA labeling, nick translation, or end-labeling techniques, or RNA probes using available in vitro transcription systems. In addition, specific primers can be designed and used to amplify a part or all of the instant sequences. The resulting amplification products can be labeled directly during amplification reactions or labeled after amplification reactions, and used as probes to isolate full length cDNA or genomic fragments under conditions of appropriate stringency.

In addition, two short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols to amplify longer nucleic acid fragments encoding homologous genes from DNA or RNA. The polymerase chain reaction may also be performed on a library of cloned nucleic acid fragments wherein the sequence of one primer is derived from the instant nucleic acid fragments, and the sequence of the other primer takes advantage of the presence of the polyadenylic acid tracts to the 3′ end of the mRNA precursor encoding plant genes. Alternatively, the second primer sequence may be based upon sequences derived from the cloning vector. For example, the skilled artisan can follow the RACE protocol (Frohman et al., (1988) Proc. Natl. Acad. Sci. USA 85:8998) to generate cDNAs by using PCR to amplify copies of the region between a single point in the transcript and the 3′ or 5′ end. Primers oriented in the 3′ and 5′ directions can be designed from the instant sequences. Using commercially available 3′ RACE or 5′ RACE systems (BRL), specific 3′ or 5′ cDNA fragments can be isolated (Ohara et al., (1989) Proc. Natl. Acad. Sci USA 86:5673; Loh et al., (1989) Science 243:217). Products generated by the 3′and 5′ RACE procedures can be combined to generate full-length cDNAs (Frohman, M. A. and Martin, G. R., (1989) Techniques 1:165).

Availability of the instant nucleotide and deduced amino acid sequences facilitates immunological screening of cDNA expression libraries. Synthetic peptides representing portions of the instant amino acid sequences may be synthesized. These peptides can be used to immunize animals to produce polyclonal or monoclonal antibodies with specificity for peptides or proteins comprising the amino acid sequences. These antibodies can be then be used to screen cDNA expression libraries to isolate full-length cDNA clones of interest (Lerner, R. A. (1984) Adv. Immunol . 36:1; Maniatis).

The nucleic acid fragments of the instant invention may be used to create transgenic plants in which the disclosed corn Mlos are present at higher or lower levels than normal or in cell types or developmental stages in which they are not normally found. This would have the effect of altering the level of the Mlo in those cells, and thus changing the ability of corn to resist certain diseases.

Overexpression of the Mlos of the instant invention may be accomplished by first constructing a chimeric nucleic acid sequence in which the coding region is operably linked to a promoter capable of directing expression of a gene in the desired tissues at the desired stage of development. The chimeric gene may comprise promoter sequences and translation leader sequences derived from the same genes. 3′ Non-coding sequences encoding transcription termination signals may also be provided. The instant chimeric nucleic acid sequence may also comprise one or more introns in order to facilitate gene expression.

Plasmid vectors comprising the instant chimeric gene can then constructed. The choice of plasmid vector is dependent upon the method that will be used to transform host plants. The skilled artisan is well aware of the genetic elements that must be present on the plasmid vector in order to successfully transform, select and propagate host cells containing the chimeric gene. The skilled artisan will also recognize that different independent transformation events will result in different levels and patterns of expression (Jones et al., (1985) EMBO J. 4:2411-2418; De Almeida et al., (1989) Mol. Gen. Genetics 218:78-86), and thus that multiple events must be screened in order to obtain lines displaying the desired expression level and pattern. Such screening may be accomplished by Southern analysis of DNA, Northern analysis of mRNA expression, Western analysis of protein expression, or phenotypic analysis.

For some applications it may be useful to direct the instant Mlo gene product to different cellular compartments, or to facilitate its secretion from the cell. It is thus envisioned that the chimeric gene described above may be further supplemented by altering the coding sequence to encode Mlos with appropriate intracellular targeting sequences such as transit sequences (Keegstra, K. (1989) Cell 56:247-253), signal sequences or sequences encoding endoplasmic reticulum localization signals (Chrispeels, J. J., (1991) Ann. Rev. Plant Phys. Plant Mol. Biol . 42:21-53), or nuclear localization signals (Raikhel, N. (1992) Plant Phys . 100:1627-1632) added and/or with targeting sequences that are already present removed. While the references cited give examples of each of these, the list is not exhaustive and more targeting signals of utility may be discovered in the future.

It may also be desirable to reduce or eliminate expression of genes encoding Mlos in plants for some applications. In order to accomplish this, a chimeric nucleic acid sequence designed for co-suppression of the instant Mlo can be constructed by linking a nucleic acid sequence encoding a Mlo to plant promoter sequences. Alternatively, a chimeric nucleic acid sequence designed to express antisense RNA for all or part of the instant nucleic acid fragment can be constructed by linking the nucleic acid fragment in reverse orientation to plant promoter sequences. Either the co-suppression or antisense chimeric nucleic acid sequences could be introduced into plants via transformation wherein expression of the corresponding endogenous genes are reduced or eliminated.

For example, the instant nucleic acid fragments may be combined in sense or antisense orientation with a suitable plant expression vector comprising a strong plant promoter sequence and plant terminator sequences. The recombinant vector may then be introduced into plants by transformation using techniques well known to those skilled in this art. Some of the transgenic plants recovered from the transformation process will exhibit diminished expression of the gene corresponding to the instant nucleic acid fragments. These plants may be identified by Northern blot analysis. These individuals may then be tested for disease resistance, and resistant individuals recovered for future use as a source of genetically controlled resistance in plant breeding schemes. Longer cDNA clones, especially csc1c.pk005.d8, cta1.pk0005.c1, cc3.pk0007.g3 and cco1.pk0052.b10 are preferred. cDNA clones cs1.pk0071.e3, or1n.pk0154.a3, mlo3b3 and cen3n.pk0062.b7 may also be used in a similar way to inhibit expression of their corresponding genes.

The instant Mlos (or portions thereof) may be produced in heterologous host cells, particularly in the cells of microbial hosts, and can be used to prepare antibodies to the these proteins by methods well known to those skilled in the art. The antibodies are useful for detecting Mlos in situ in cells or in vitro in cell extracts. Preferred heterologous host cells for production of the instant Mlos are microbial hosts. Microbial expression systems and expression vectors containing regulators, sequences that direct high level expression of foreign proteins are well known to those skilled in the art. Any of these could be used to construct a chimeric gene for production of the instant Mlos. This chimeric gene could then be introduced into appropriate microorganisms via transformation to provide high level expression of the encoded Mlo. An example of a vector for high level expression of the Mlo in a bacterial host is provided (Example 8).

All or a substantial portion of the nucleic acid fragments of the instant invention may also be used as probes for genetically and physically mapping the genes that they are a part of, and as markers for traits linked to those genes. Such information may be useful in plant breeding in order to develop lines with desired phenotypes. For example, the instant nucleic acid fragments may be used as restriction fragment length polymorphism (RFLP) markers. Southern blots (Maniatis) of restriction-digested plant genomic DNA may be probed with the nucleic acid fragments of the instant invention. The resulting banding patterns may then be subjected to genetic analyses using computer programs such as MapMaker (Lander et at., (1987) Genomics 1:174-181) in order to construct a genetic map. In addition, the nucleic acid fragments of the instant invention may be used to probe Southern blots containing restriction endonuclease-treated genomic DNAs of a set of individuals representing parent and progeny of a defined genetic cross. Segregation of the DNA polymorphisms is noted and used to calculate the position of the instant nucleic acid sequence in the genetic map previously obtained using this population (Botstein, D. et al., (1980) Am. J. Hum. Genet . 32:314-331).

The production and use of plant gene-derived probes for use in genetic mapping is described in R. Bernatzky, R. and Tanksley, S. D. (1986) Plant Mol. Biol. Reporter 4(1):37-41. Numerous publications describe genetic mapping of specific cDNA clones using the methodology outlined above or variations thereof. For example, F2 intercross populations, backcross populations, randomly mated populations, near isogenic lines, and other sets of individuals may be used for mapping. Such methodologies are well known to those skilled in the art.

Nucleic acid probes derived from the instant nucleic acid sequences may also be used for physical mapping (i.e., placement of sequences on physical maps; see Hoheisel, J. D., et al., In: Nonmammalian Genomic Analysis: A Practical Guide , Academic press 1996, pp. 319-346, and references cited therein).

In another embodiment, nucleic acid probes derived from the instant nucleic acid sequences may be used in direct fluorescence in situ hybridization (FISH) mapping (Trask, B. J. (1991) Trends Genet . 7:149-154). Although current methods of FISH mapping favor use of large clones (several to several hundred KB; see Laan, M. et al. (1995) Genome Research 5:13-20), improvements in sensitivity may allow performance of FISH mapping using shorter probes.

A variety of nucleic acid amplification-based methods of genetic and physical mapping may be carried out using the instant nucleic acid sequences. Examples include allele-specific amplification (Kazazian, H. H. (1989) J Lab. Clin. Med . 114(2):95-96), polymorphism of PCR-amplified fragments (CAPS; Sheffield, V. C. et al. (1993) Genomics 16:325-332), allele-specific ligation (Landegren, U. et al. (1988) Science 241:1077-1080), nucleotide extension reactions (Sokolov, B. P. (1990) Nucleic Acid Res . 18:3671), Radiation Hybrid Mapping (Walter, M. A. et al. (1997) Nature Genetics 7:22-28) and Happy Mapping (Dear, P. H. and Cook, P. R. (1989) Nucleic Acid Res . 17:6795-6807). For these methods, the sequence of a nucleic acid fragment is used to design and produce primer pairs for use in the amplification reaction or in primer extension reactions. The design of such primers is well known to those skilled in the art. In methods employing PCR-based genetic mapping, it may be necessary to identify DNA sequence differences between the parents of the mapping cross in the region corresponding to the instant nucleic acid sequence. This, however, is generally not necessary for mapping methods.

For example, genetic diagnostics of the southern corn leaf blight (SCLB), maize dwarf mosaic virus (MDMV), wheat streak mosaic virus (WSMV) loci, and of other loci corresponding or tightly linked to cta1.pk0005.c1 and related cDNAs may be performed. Knowledge and availability of the nucleotide sequences corresponding to the corn Mlo homolog embodied by cDNA clone cta1.pk0005.c1 allows one skilled in the art to use one of many generally known techniques, like restriction fragment length polymorphism, allele specific PCR, single strand conformational polymorphism, allele-specific ligation, Cleaved Amplified Polymorphic Fragments (CAPS) and other similar methods discussed above and known to those skilled in the art, to follow the inheritance of a particular allele in genetic crosses during the process of plant breeding. Through this process, the plant carrying the alleles conferring disease resistance may be always recovered reliably.

For example, the 765 bp PCR fragment, which is produced only from the CM37 genotype and not from the T232 genotype using primers designed from the nucleotide sequence of cDNA clone cta1.pk0005.c1, is therefore polymorphic between these two genotypes and could be used to follow inheritance of the allele corresponding to the 765 bp PCR fragment in crosses of CM37 and T232, and in any crosses with a genotype which does not produce the 765 bp fragment in the above described PCR process.

Loss of function mutant phenotypes may be identified for the instant cDNA clones either by targeted gene disruption protocols or by identifying specific mutants for these genes contained in a maize population carrying mutations in all possible genes (Ballinger and Benzer, (1989) Proc. Natl. Acad. Sci USA 86:9402; Koes et al., (1995) Proc. Natl. Acad Sci USA 92:8149; Bensen et al., (1995) Plant Cell 7:75). The latter approach may be accomplished in two ways. First, short segments of the instant nucleic acid fragments may be used in polymerase chain reaction protocols in conjunction with a mutation tag sequence primer on DNAs prepared from a population of plants in which Mutator transposons or some other mutation-causing DNA element has been introduced (see Bensen, supra). The amplification of a specific DNA fragment with these primers indicates the insertion of the mutation tag element in or near the plant Mlo gene. Alternatively, the instant nucleic acid fragment may be used as a hybridization probe against PCR amplification products generated from the mutation population using the mutation tag sequence primer in conjunction with an arbitrary genomic site primer, such as that for a restriction enzyme site-anchored synthetic adaptor. With either method, a plant containing a mutation in the endogenous Mlo gene can be identified and obtained. This mutant plant can then be used to determine or confirm the natural function of the Mlo gene product.

The present invention is further defined in the following Examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these Examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

The disclosure of each reference sat forth herein is incorporated herein by reference in its entirety.

Zea mays

1

gcacgagggc cgagggcgag gcggcggcgc tggagttcac accgacgtgg atcgtcgcgg 60

cggtctgctc tctcatcgtg ctcctctcgc tcgtcgccga gcgatgcctc cactacctcg 120

gcaagacgct caagaggaag aaccagaagc cgctcttcga ggcgctgctc aaggtcaaag 180

aagagttgat gcttctgggg ttcatctccc tgctgctgac ggtgttccag gggatgatcc 240

ggaggacgtg catccctgaa cgctggacat tccacatgct gccatgcgag aagccagatg 300

agaaggccgg tgaggccgcc accatggagc attttgtagg gacgcttggc aggatcggta 360

ggcgtctgtt gcaggaaggc actgctgggg ctgagcaatg ccagaagaag ggaaaagttc 420

cacttttgtc ccttgaagcc atacatcagc tgcacatttt catatttgtt ctggcaatca 480

cacatgttat tttcagcgtc acaactatgc ttttaggagg tgcacagata caccaatgga 540

aacagtggga gaatggaatt aaaaaagatg ctcctggaaa tgggcctaag gtaaccaatg 600

tacatcatca tgaatttatc aagaaacgtt ttaagggtat tggcaaagat tctataatat 660

tgagttggct gcattctttt ggtaagcagt tttatagatc agtatctaaa tcagattaca 720

ccacaatgcg tcttggtttt atcatgactc actgccctgg aaatccaaaa tttgatttcc 780

atagatacat ggtaagggtt ttagaggcgg attttaagaa agtggtaggc ataagctggt 840

acttgtgggt cttcgtggtg atatttctgt tgctgaatgt taatggctgg cacacatact 900

tttggattgc tttccttccc cttattcttc tgttagccat tggcactaag ctggagcatg 960

tcatagctca gctagcccat gatgtagctg agaagcacac agcggtcgag ggcgatgtga 1020

tcgtaaaacc atcagatgaa cacttctggt tcggcaagcc tagggttatc ctttacctga 1080

tccacttcat cctctttcag aatgcgtttg agattgcgtt tttcttctgg atactgagca 1140

cttatggatt cgactcgtgc atcatgggac aagttcgttt tattgtgcca aggcttgtca 1200

tcggggtggt tattcagctt ctctgcagct acagcacctt gcctctgtat gcaattgtaa 1260

cccagatggg gagctgctac aagaaggaga tcttcaacga gcatgtgcag cagggcgtcc 1320

tgggctgggc tcagaaggtc aagatgaaaa agggactgaa gggagctgca tctgctagca 1380

aggacgaatc gattaccaat gccgattcgg caggaccttc cgttaagatt gaaatggcga 1440

aggctgggga ggatgttgag atcgttggaa acacaggttg attgggacaa tagggtgccc 1500

gtgttgtaat gatgtaacag gttaatatgc catcatcttt ttttgtagat actagatagc 1560

ttgctgtggc aataccgcaa tagcggtgaa ctagagaagg tgagtttggg cccgggagcc 1620

tcatctgtta tcggtccagt aggaagcaaa ttcttatata cgggatatcg ataagaaatg 1680

aactaagaac atgttcctgg attaaaaaaa aaaaaaaaaa aaaaa 1725

2

492

Zea mays

2

Thr Arg Ala Glu Gly Glu Ala Ala Ala Leu Glu Phe Thr Pro Thr Trp

1 5 10 15

Ile Val Ala Ala Val Cys Ser Leu Ile Val Leu Leu Ser Leu Val Ala

20 25 30

Glu Arg Cys Leu His Tyr Leu Gly Lys Thr Leu Lys Arg Lys Asn Gln

35 40 45

Lys Pro Leu Phe Glu Ala Leu Leu Lys Val Lys Glu Glu Leu Met Leu

50 55 60

Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Phe Gln Gly Met Ile Arg

65 70 75 80

Arg Thr Cys Ile Pro Glu Arg Trp Thr Phe His Met Leu Pro Cys Glu

85 90 95

Lys Pro Asp Glu Lys Ala Gly Glu Ala Ala Thr Met Glu His Phe Val

100 105 110

Gly Thr Leu Gly Arg Ile Gly Arg Arg Leu Leu Gln Glu Gly Thr Ala

115 120 125

Gly Ala Glu Gln Cys Gln Lys Lys Gly Lys Val Pro Leu Leu Ser Leu

130 135 140

Glu Ala Ile His Gln Leu His Ile Phe Ile Phe Val Leu Ala Ile Thr

145 150 155 160

His Val Ile Phe Ser Val Thr Thr Met Leu Leu Gly Gly Ala Gln Ile

165 170 175

His Gln Trp Lys Gln Trp Glu Asn Gly Ile Lys Lys Asp Ala Pro Gly

180 185 190

Asn Gly Pro Lys Val Thr Asn Val His His His Glu Phe Ile Lys Lys

195 200 205

Arg Phe Lys Gly Ile Gly Lys Asp Ser Ile Ile Leu Ser Trp Leu His

210 215 220

Ser Phe Gly Lys Gln Phe Tyr Arg Ser Val Ser Lys Ser Asp Tyr Thr

225 230 235 240

Thr Met Arg Leu Gly Phe Ile Met Thr His Cys Pro Gly Asn Pro Lys

245 250 255

Phe Asp Phe His Arg Tyr Met Val Arg Val Leu Glu Ala Asp Phe Lys

260 265 270

Lys Val Val Gly Ile Ser Trp Tyr Leu Trp Val Phe Val Val Ile Phe

275 280 285

Leu Leu Leu Asn Val Asn Gly Trp His Thr Tyr Phe Trp Ile Ala Phe

290 295 300

Leu Pro Leu Ile Leu Leu Leu Ala Ile Gly Thr Lys Leu Glu His Val

305 310 315 320

Ile Ala Gln Leu Ala His Asp Val Ala Glu Lys His Thr Ala Val Glu

325 330 335

Gly Asp Val Ile Val Lys Pro Ser Asp Glu His Phe Trp Phe Gly Lys

340 345 350

Pro Arg Val Ile Leu Tyr Leu Ile His Phe Ile Leu Phe Gln Asn Ala

355 360 365

Phe Glu Ile Ala Phe Phe Phe Trp Ile Leu Ser Thr Tyr Gly Phe Asp

370 375 380

Ser Cys Ile Met Gly Gln Val Arg Phe Ile Val Pro Arg Leu Val Ile

385 390 395 400

Gly Val Val Ile Gln Leu Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr

405 410 415

Ala Ile Val Thr Gln Met Gly Ser Cys Tyr Lys Lys Glu Ile Phe Asn

420 425 430

Glu His Val Gln Gln Gly Val Leu Gly Trp Ala Gln Lys Val Lys Met

435 440 445

Lys Lys Gly Leu Lys Gly Ala Ala Ser Ala Ser Lys Asp Glu Ser Ile

450 455 460

Thr Asn Ala Asp Ser Ala Gly Pro Ser Val Lys Ile Glu Met Ala Lys

465 470 475 480

Ala Gly Glu Asp Val Glu Ile Val Gly Asn Thr Gly

3

crnkagtgga acactatccc gagaaaccag atattgattt ccacaaatac atgactcgcg 60

ctgttgaata tgagtttaaa agagttgttg gtatcagctg gtatctgtgg ctttttgtaa 120

tcttattcct gctgctgaat ataaatggat ggcacacata cttctggttg gctttcttgc 180

ctctatttct gttacttatt gttggtgcca aactagagca cattatcact cggttggctc 240

aagaggcagc gatatcatta tcaaataata cagaggaagt tccgaaaata aagccatgca 300

aggaccattt ctggtttcac aagcctgagc tagtccttca tttgattcat ttcatcctgt 360

tccagaattc gttcgagatt agttttttcc tcctggattc tggtatcaga aggtttcggt 420

tcgtgtatga tggaacggaa gcttatgtca tttccagact tgttatcggg tgatnatcga 480

agtcacctgc agctatatca cccgccacta tacgcaacgn gaccaatatg accggcangn 540

taagctgatg gntttggcnc cgcgtgcnca natgtcaagg gcnggtttgg tttagga 597

4

80

Zea mays

4

His Tyr Pro Glu Lys Pro Asp Ile Asp Phe His Lys Tyr Met Thr Arg

1 5 10 15

Ala Val Glu Tyr Glu Phe Lys Arg Val Val Gly Ile Ser Trp Tyr Leu

20 25 30

Trp Leu Phe Val Ile Leu Phe Leu Leu Leu Asn Ile Asn Gly Trp His

35 40 45

Thr Tyr Phe Trp Leu Ala Phe Leu Pro Leu Phe Leu Leu Leu Ile Val

50 55 60

Gly Ala Lys Leu Glu His Ile Ile Thr Arg Leu Ala Gln Glu Ala Ala

65 70 75 80

5

gtgcatcatg ggacaagttc gttttattgt gccaaggctt gtcatcgggt atttaaatcg 60

ttgaaaacac atccatttca tgaggaaaag aaaaaaaaac atcaatatgt tatgttctct 120

tgacactagt atccacgctt tgtacttgca gggtggttat tcancttctc tgcagctaca 180

gcaccttgcc tctgtatgca attgtaaccc agatggggag ctgctacaag aaggagatct 240

tcaacgagca tgtgcagcag ggcgtcctgg gctgggctca gaaggtcaag atgaaaaagg 300

gactgaggga gctgcatctg ctagcaagga cgaatcgtta ccaatgccga ntcggcagga 360

ccttccgtta agattgaang gcgaagctgg ggaggatntt gagaccttgg aaanaggatg 420

attgganaaa aggtcccgnn tgaaatnagn acagttatat caacacttta aa 472

6

Val Val Ile Xaa Leu Leu Cys Ser Tyr Ser Thr Leu Pro Leu Tyr Ala

1 5 10 15

Ile Val Thr Gln Met Gly Ser Cys Tyr Lys Lys Glu Ile Phe Asn Glu

20 25 30

His Val Gln Gln Gly Val Leu Gly Trp Ala Gln Lys Val Lys Met Lys

35 40 45

Lys Gly Leu Arg

50

7

1831

Zea mays

7

ccagatatca gggcagctgg aactgaaggt ggcgggagcc gagtgatccg gcggtgagct 60

gagcggggcg ggatggcggc ggagcagggg cggtcgctgg cggagacgcc cacctggtcc 120

gtggcaaccg tcaccacgct catggtcgct gcctgcttcc tcgtcgagcg ctccctctcg 180

cgcttcgcca agtggctgcg caagaccaag cggaaggcca tgctcgccgc gctcgagaag 240

atccgcgaag agctgatgct gctcggagtc atctcgctgc tgctcagcca gacggcgcgc 300

ttcatatcgg agatctgcgt gccgtcctcg ctcttcacca gccgcttcta catctgctcc 360

gagagcgact accaggacct gctgcgcaac acggacgcca accagacggc gctcgacaag 420

aacatgttcg gtggccaacg gctgcacgtc tgtggcgagg gccatgaacc ttttgtttcg 480

tacgagggcc ttgagcagct gcaccggttt ctcttcatcc ttggtatcac tcatgtgttg 540

tacagttttg taacagtggt tctgtccatg atcaagatct atagctggag gaagtgggaa 600

accttagcag gtccaattgc tgctgaggaa ttgaaagcta ggagaaccaa ggtgatgaga 660

aggcagtcaa cctttgtttt taacaatgct tctcatccat ggagcaaaaa taaaatactt 720

atttggatgc tttgcttttt gcgtcaattc aagggctcca taataaggtc agactatttg 780

gcactgaggt tgggctttgt cacatatcac aagctaccac attcatatga cttccataaa 840

tacatggtac ggagcatgga agatgattac aatgggacta ttggtatcag ttggccactt 900

tgggcatatg cgattgtctg catattaatc aatgttcatg gtatcaatat atatttctgg 960

ttgtcctttg ttcctgttat tctggtgctt ctagtgggta ctgaacttca gcacgtcatt 1020

gctcagttgg ctttggaagt cgctgaggca acagcgcctt atgttggctc acaacttaaa 1080

ctgcgtgatg atctattttg gtttggaaag cctcgggtac tctggtggct tatacagttc 1140

atttcatttc agaatgcttt tgagctggca acattcttat ggtctctgtg ggaactcagt 1200

gcacaaacat gtttcatgaa gcactactac atggttgcca ttcggttgat ttctgggctc 1260

ttagttcagt tttggtgcag ctacagcaca ctcccgctga atgtgattat ttctcagatg 1320

ggtcccaagt tcaagaaatc actggtctcg gagaacgtga gggagtcgct gcacagctgg 1380

tgcaagaggg ttaaggacag gagccgacac aatccgctct tctcgcggaa cgggaccctc 1440

acgaccagat ccgtgtgctc cctagacacc acctacgaga cggatcacga gacgaacacg 1500

gtgtgcacgc tgtcgaggac ggcgtcggcg acgtcgctgg acgaccagtt gaccgtggtc 1560

accgtcgatg acgagccgtc ctgcattgag aaggatgtct gacgcagttg ctgattcgcc 1620

aaaactacat actgcaccga cctgtgtgtt aggaggtact ctaggactga aattgttcac 1680

ggcgtggcgc gctctgaact agtaatgtcg ccgcggagag tgcttgaccc gcgctacgtg 1740

ggagaggcat aactattgta gtgaggtaaa ttggggaggg ggtagatgaa gagtcgccgg 1800

tggatggtgt gttgcacggc aagagacgac a 1831

8

509

Zea mays

8

Met Ala Ala Glu Gln Gly Arg Ser Leu Ala Glu Thr Pro Thr Trp Ser

1 5 10 15

Val Ala Thr Val Thr Thr Leu Met Val Ala Ala Cys Phe Leu Val Glu

20 25 30

Arg Ser Leu Ser Arg Phe Ala Lys Trp Leu Arg Lys Thr Lys Arg Lys

35 40 45

Ala Met Leu Ala Ala Leu Glu Lys Ile Arg Glu Glu Leu Met Leu Leu

50 55 60

Gly Val Ile Ser Leu Leu Leu Ser Gln Thr Ala Arg Phe Ile Ser Glu

65 70 75 80

Ile Cys Val Pro Ser Ser Leu Phe Thr Ser Arg Phe Tyr Ile Cys Ser

85 90 95

Glu Ser Asp Tyr Gln Asp Leu Leu Arg Asn Thr Asp Ala Asn Gln Thr

100 105 110

Ala Leu Asp Lys Asn Met Phe Gly Gly Gln Arg Leu His Val Cys Gly

115 120 125

Glu Gly His Glu Pro Phe Val Ser Tyr Glu Gly Leu Glu Gln Leu His

130 135 140

Arg Phe Leu Phe Ile Leu Gly Ile Thr His Val Leu Tyr Ser Phe Val

145 150 155 160

Thr Val Val Leu Ser Met Ile Lys Ile Tyr Ser Trp Arg Lys Trp Glu

165 170 175

Thr Leu Ala Gly Pro Ile Ala Ala Glu Glu Leu Lys Ala Arg Arg Thr

180 185 190

Lys Val Met Arg Arg Gln Ser Thr Phe Val Phe Asn Asn Ala Ser His

195 200 205

Pro Trp Ser Lys Asn Lys Ile Leu Ile Trp Met Leu Cys Phe Leu Arg

210 215 220

Gln Phe Lys Gly Ser Ile Ile Arg Ser Asp Tyr Leu Ala Leu Arg Leu

225 230 235 240

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

245 250 255

Tyr Met Val Arg Ser Met Glu Asp Asp Tyr Asn Gly Thr Ile Gly Ile

260 265 270

Ser Trp Pro Leu Trp Ala Tyr Ala Ile Val Cys Ile Leu Ile Asn Val

275 280 285

His Gly Ile Asn Ile Tyr Phe Trp Leu Ser Phe Val Pro Val Ile Leu

290 295 300

Val Leu Leu Val Gly Thr Glu Leu Gln His Val Ile Ala Gln Leu Ala

305 310 315 320

Leu Glu Val Ala Glu Ala Thr Ala Pro Tyr Val Gly Ser Gln Leu Lys

325 330 335

Leu Arg Asp Asp Leu Phe Trp Phe Gly Lys Pro Arg Val Leu Trp Trp

340 345 350

Leu Ile Gln Phe Ile Ser Phe Gln Asn Ala Phe Glu Leu Ala Thr Phe

355 360 365

Leu Trp Ser Leu Trp Glu Leu Ser Ala Gln Thr Cys Phe Met Lys His

370 375 380

Tyr Tyr Met Val Ala Ile Arg Leu Ile Ser Gly Leu Leu Val Gln Phe

385 390 395 400

Trp Cys Ser Tyr Ser Thr Leu Pro Leu Asn Val Ile Ile Ser Gln Met

405 410 415

Gly Pro Lys Phe Lys Lys Ser Leu Val Ser Glu Asn Val Arg Glu Ser

420 425 430

Leu His Ser Trp Cys Lys Arg Val Lys Asp Arg Ser Arg His Asn Pro

435 440 445

Leu Phe Ser Arg Asn Gly Thr Leu Thr Thr Arg Ser Val Cys Ser Leu

450 455 460

Asp Thr Thr Tyr Glu Thr Asp His Glu Thr Asn Thr Val Cys Thr Leu

465 470 475 480

Ser Arg Thr Ala Ser Ala Thr Ser Leu Asp Asp Gln Leu Thr Val Val

485 490 495

Thr Val Asp Asp Glu Pro Ser Cys Ile Glu Lys Asp Val

500 505

9

1851

Zea mays

9

cccggtgatc gtatcgtcga ttggaagtga agatcaagcg atcgatagaa taaattaaag 60

gcgcgggggc aacaacaaga atggggggcg gtggcggtgg cggcaactcg cgggagcttg 120

accagacgcc gacatgggcg gtggcgtcgg tgtgcggcgt gatcgtgctc atctccatcc 180

tgctggagaa ggggctccac cacgtgggcg agttcttctc ccaccgcaag aagaaggcca 240

tggtggaggc cctggagaag gtgaaggcgg agctcatggt gctgggcttc atctcgctcc 300

tcctcgtgtt cggccagaac tacatcatca aggtctgcat cagcaaccac gccgccaaca 360

ccatgctccc ctgcaagctc gaggccgccg ccgtcgaggg caaggacggc cacggcaagg 420

aggccgccgc cgtggtcgct ggcaagaaga aggtcgccgt cgccgtccct ggaaagaaga 480

agaagaaggc cgccgccgcc gccgaccatc ttggcggtgt ggtggactgg ccgccgccct 540

actacgcgca caacgccagg atgctggcgg aggcgagcat ggcgaccaag tgccccgagg 600

ggaaagtgcc gctcatctcc atcaacgccc tgcaccagct gcacatcttc atcttcttcc 660

tcgccgtctt ccacgtctcc tacagcgcaa tcaccatggc gctcggcagg gccaagatac 720

gtgcatggaa agagtgggag aaagaagctg caggacaaga ctacgagttc tcacatgacc 780

cgacgcggtt caggttcacc cacgagactt ccttcgtgag gcagcatatg aatgtgctga 840

acaagttccc agcatcattc tacatcagca acttcttccg gcagttcttc aggtccgtga 900

ggcaggcaga ctactgcgcg ctgcgccaca gctttgtcaa cgtccatctg gcccctggca 960

gcaagtttga tttccagaag tacatcaagc ggtctctgga ggatgacttc aaggtgatcg 1020

tggggatcag tcctcctctg tgggcttctg ctctcatctt cctcttcctc aacgtcaatg 1080

gatggcacac catgctctgg atctccatca tgccggtggt gatcatcctg tcggtgggga 1140

cgaagctgca gggcatcatc tgccgcatgg cgatcgacat cacggagcgc cacgccgtca 1200

tccagggcat cccgatggtg caagtcagcg actcctactt ctggttcgca cgccccacct 1260

tcgtgctctt cctcatccac ttcaccctct tccagaatgg cttccagatc atctacttcc 1320

tctggattct gtatgagtac ggcatggact cgtgcttcaa cgactccgaa gagttcgtct 1380

ttgcacgact ctgccttggc gtggttgtcc aggtgctgtg cagctacgtg acgctcccgc 1440

tgtacgcgct cgtctcccaa atgggctcca ccatgaagca gtccatcttc gacgagcaga 1500

cctccaaggc gctcaagaac tggcgcgccg gcgccaagaa gaaggctccc accggcggct 1560

ccaagcacgg cggtggtggc tcccccaccg ccggcggcag ccccaccaag gccgacggcg 1620

acgcgtagag aggaacacgc taactttaat ttctgtgtgc ttaattgcct aggctcgtta 1680

agtcagaaca tgcatgcatg taacaccact gctggttttc atatagtgtc gacagatggt 1740

caacgtactt tttgcgatcc cacttgtatt ttttttttac aatgaagcac ccgtccgcgt 1800

ccgtggacac tgcaagtgca aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa a 1851

10

515

Zea mays

10

Met Gly Gly Gly Gly Gly Gly Gly Asn Ser Arg Glu Leu Asp Gln Thr

1 5 10 15

Pro Thr Trp Ala Val Ala Ser Val Cys Gly Val Ile Val Leu Ile Ser

20 25 30

Ile Leu Leu Glu Lys Gly Leu His His Val Gly Glu Phe Phe Ser His

35 40 45

Arg Lys Lys Lys Ala Met Val Glu Ala Leu Glu Lys Val Lys Ala Glu

50 55 60

Leu Met Val Leu Gly Phe Ile Ser Leu Leu Leu Val Phe Gly Gln Asn

65 70 75 80

Tyr Ile Ile Lys Val Cys Ile Ser Asn His Ala Ala Asn Thr Met Leu

85 90 95

Pro Cys Lys Leu Glu Ala Ala Ala Val Glu Gly Lys Asp Gly His Gly

100 105 110

Lys Glu Ala Ala Ala Val Val Ala Gly Lys Lys Lys Val Ala Val Ala

115 120 125

Val Pro Gly Lys Lys Lys Lys Lys Ala Ala Ala Ala Ala Asp His Leu

130 135 140

Gly Gly Val Val Asp Trp Pro Pro Pro Tyr Tyr Ala His Asn Ala Arg

145 150 155 160

Met Leu Ala Glu Ala Ser Met Ala Thr Lys Cys Pro Glu Gly Lys Val

165 170 175

Pro Leu Ile Ser Ile Asn Ala Leu His Gln Leu His Ile Phe Ile Phe

180 185 190

Phe Leu Ala Val Phe His Val Ser Tyr Ser Ala Ile Thr Met Ala Leu

195 200 205

Gly Arg Ala Lys Ile Arg Ala Trp Lys Glu Trp Glu Lys Glu Ala Ala

210 215 220

Gly Gln Asp Tyr Glu Phe Ser His Asp Pro Thr Arg Phe Arg Phe Thr

225 230 235 240

His Glu Thr Ser Phe Val Arg Gln His Met Asn Val Leu Asn Lys Phe

245 250 255

Pro Ala Ser Phe Tyr Ile Ser Asn Phe Phe Arg Gln Phe Phe Arg Ser

260 265 270

Val Arg Gln Ala Asp Tyr Cys Ala Leu Arg His Ser Phe Val Asn Val

275 280 285

His Leu Ala Pro Gly Ser Lys Phe Asp Phe Gln Lys Tyr Ile Lys Arg

290 295 300

Ser Leu Glu Asp Asp Phe Lys Val Ile Val Gly Ile Ser Pro Pro Leu

305 310 315 320

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

325 330 335

Thr Met Leu Trp Ile Ser Ile Met Pro Val Val Ile Ile Leu Ser Val

340 345 350

Gly Thr Lys Leu Gln Gly Ile Ile Cys Arg Met Ala Ile Asp Ile Thr

355 360 365

Glu Arg His Ala Val Ile Gln Gly Ile Pro Met Val Gln Val Ser Asp

370 375 380

Ser Tyr Phe Trp Phe Ala Arg Pro Thr Phe Val Leu Phe Leu Ile His

385 390 395 400

Phe Thr Leu Phe Gln Asn Gly Phe Gln Ile Ile Tyr Phe Leu Trp Ile

405 410 415

Leu Tyr Glu Tyr Gly Met Asp Ser Cys Phe Asn Asp Ser Glu Glu Phe

420 425 430

Val Phe Ala Arg Leu Cys Leu Gly Val Val Val Gln Val Leu Cys Ser

435 440 445

Tyr Val Thr Leu Pro Leu Tyr Ala Leu Val Ser Gln Met Gly Ser Thr

450 455 460

Met Lys Gln Ser Ile Phe Asp Glu Gln Thr Ser Lys Ala Leu Lys Asn

465 470 475 480

Trp Arg Ala Gly Ala Lys Lys Lys Ala Pro Thr Gly Gly Ser Lys His

485 490 495

Gly Gly Gly Gly Ser Pro Thr Ala Gly Gly Ser Pro Thr Lys Ala Asp

500 505 510

Gly Asp Ala

515

11

1040

Zea mays

11

aggttcaccc acgagacttc gtttgtgagg cagcatatga atgtgctcaa caagttccca 60

gcatccttct acatcagcaa cttcttccgg cagttcttca ggtccgtcag gcgggcagac 120

tactgcgcgc tgcgccacag ctttgtcaac gtccatctgg cccctggcac caagtttgat 180

ttccaaaagt acatcaagcg gtctctggag gacgacttca aggtgatcgt ggggatcagc 240

cctcctttgt gggcttctgc tctcatcttc ctattcctca atgtcaatgg atggcacacc 300

atgctctgga tctccatcat gccggtggtg atcatcctgt ccgtggggac gaagctgcag 360

ggcatcatct gccgcatggc gatcgacatc acggagcggc acgccgtgat ccagggcatc 420

ccgctggtgc aggtcagcga ctcctacttc tggttcgcac gcccaacctt cgtgctcttc 480

ctcatccact tcaccctctt ccagaatggc ttccagatca tctacttcct ctggattctg 540

tatgagtacg ggatggactc gtgcttcaac gactccgaag aattcgtctt tgcacgtctc 600

tgccttgggg tggttgttca ggtgctgtgc agctacgtga cgctccctct gtacgcgctc 660

gtctcccaga tgggctccac catgaagcag tccatcttcg acgagcagac ctccaaggcg 720

ctcaagaact ggcgcgccgg cgccaagaag aaggccccca ccggcagccc caccaaggcc 780

gacggcgacg cgtagacgta gctagcagag ggtcatcgat cgattgatcg atcgatccag 840

ctgttcgttc tacatataac ctgttactta tttgtatgta attgtaccaa tagcaatcga 900

cacgcgtgcg ccgcatgcaa tgtaccatga aactgctgat agctagagag cgacattttt 960

cttgtttggt tcgtttctgc aatgctccta ctatgtaatt gtcagttatc caaaaaaaaa 1020

aaaaaaaaaa aaaaaaaaaa 1040

12

264

Zea mays

12

Arg Phe Thr His Glu Thr Ser Phe Val Arg Gln His Met Asn Val Leu

1 5 10 15

Asn Lys Phe Pro Ala Ser Phe Tyr Ile Ser Asn Phe Phe Arg Gln Phe

20 25 30

Phe Arg Ser Val Arg Arg Ala Asp Tyr Cys Ala Leu Arg His Ser Phe

35 40 45

Val Asn Val His Leu Ala Pro Gly Thr Lys Phe Asp Phe Gln Lys Tyr

50 55 60

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

65 70 75 80

Pro Pro Leu Trp Ala Ser Ala Leu Ile Phe Leu Phe Leu Asn Val Asn

85 90 95

Gly Trp His Thr Met Leu Trp Ile Ser Ile Met Pro Val Val Ile Ile

100 105 110

Leu Ser Val Gly Thr Lys Leu Gln Gly Ile Ile Cys Arg Met Ala Ile

115 120 125

Asp Ile Thr Glu Arg His Ala Val Ile Gln Gly Ile Pro Leu Val Gln

130 135 140

Val Ser Asp Ser Tyr Phe Trp Phe Ala Arg Pro Thr Phe Val Leu Phe

145 150 155 160

Leu Ile His Phe Thr Leu Phe Gln Asn Gly Phe Gln Ile Ile Tyr Phe

165 170 175

Leu Trp Ile Leu Tyr Glu Tyr Gly Met Asp Ser Cys Phe Asn Asp Ser

180 185 190

Glu Glu Phe Val Phe Ala Arg Leu Cys Leu Gly Val Val Val Gln Val

195 200 205

Leu Cys Ser Tyr Val Thr Leu Pro Leu Tyr Ala Leu Val Ser Gln Met

210 215 220

Gly Ser Thr Met Lys Gln Ser Ile Phe Asp Glu Gln Thr Ser Lys Ala

225 230 235 240

Leu Lys Asn Trp Arg Ala Gly Ala Lys Lys Lys Ala Pro Thr Gly Ser

245 250 255

Pro Thr Lys Ala Asp Gly Asp Ala

260

13

gtggagccaa gtgacaggtt cttctggttt aaccgccctg gctgggtcct cttcctcatc 60

cacctcacgc tcttccagaa cgccttccag atggcgcatt tcgtttggac actgctcacc 120

ccagacctga agaaatgcta ccacgagagg ctgggcctga gcatcatgaa agttgcggtg 180

gggctggttc tccaggtcct ctgcagctac atcaccttcc cgctctacgc gctcgtcacg 240

cagatggggt cgcacatgaa gaagaccatc ttcgaggagc agacggccaa ggcggtgatg 300

aagtggcgca agacggccaa ggataaggtg cggcagcggg aggcggcagg cttcctcgac 360

gtgctgacga gcgccgacac cacgccgagc cacagccgcg cgacgtcgcc gagccggggc 420

aactcgccgg tgcacctgct ccacaagtac aggggcaggt cggaggaacc gcagagggng 480

ccggcgtcgc gngggcggng agcttcgggg aaatgtancc ggtggctgan cagcatng 538

14

114

Zea mays

14

Val Glu Pro Ser Asp Arg Phe Phe Trp Phe Asn Arg Pro Gly Trp Val

1 5 10 15

Leu Phe Leu Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln Met Ala

20 25 30

His Phe Val Trp Thr Leu Leu Thr Pro Asp Leu Lys Lys Cys Tyr His

35 40 45

Glu Arg Leu Gly Leu Ser Ile Met Lys Val Ala Val Gly Leu Val Leu

50 55 60

Gln Val Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu Val Thr

65 70 75 80

Gln Met Gly Ser His Met Lys Lys Thr Ile Phe Glu Glu Gln Thr Ala

85 90 95

Lys Ala Val Met Lys Trp Arg Lys Thr Ala Lys Asp Lys Val Arg Gln

100 105 110

Arg Glu

15

20

Artificial Sequence

Description of Artificial SequencePCR primer

15

caggttcacc cacgagactt 20

16

20

Artificial Sequence

Description of Artificial SequencePCR primer

16

ttgatgagga aagagcacga 20

17

705

Zea mays

17

caggttcacc cacgagactt ccttcgtgag gcagcatatg aatgtgctga acaagttccc 60

agcatcattc tacatcgtaa taagattgaa ttctaagcat cattcgatct aatatatatg 120

ctagctagct acagcaggtc gatagactga cgacgacgat catatgcaga gcaacttctt 180

ccggcagttc ttcaggtccg tgaggcaggc agactactgc gcgctgcgcc acagctttgt 240

caacgtccat ctggcccctg gcagcaagtt tgatttccag aagtacatca agcggtctct 300

ggaggatgac ttcaaggtga tcgtggggat cagtcctcct ctgtgggctt ctgctctcat 360

cttcctcttc ctcaacgtca atggtacgta cgtatacgta ggggttgttc gagatcgaga 420

tccatgcatg catcttctat ctattactat tatatgtata tacatgcatg catgcatatg 480

ctgcgtgcat gaatcatgaa tgcaggatgg cacaccatgc tctggatctc catcatgccg 540

gtggtgatca tcctgtcggt ggggacgaag ctgcagggca tcatctgccg catggcgatc 600

gacatcacgg agcgccacgc cgtcatccag ggcatcccga tggtgcaagt cagcgactcc 660

tacttctggt tcgcacgccc caccttcgtg ctctttcctc atcaa 705

18

750

Zea mays

18

caggttcacc cacgagactt cgtttgtgag gcagcatatg aatgtgctca acaagttccc 60

agcatccttc tacatcgtaa gattcatgat gcttttctac tgaattgttg tctattgcat 120

tgcatctgac gatcgatgat gctgcctgca gagcaacttc ttccggcagt tcttcaggtc 180

cgtcaggcgg gcagactact gcgcgctgcg ccacagcttt gtcaacgtat gtagggccac 240

gccagcttgt ttgttcgttc cttcttcatt ggcaatcagc agcaacaaca atgtatgtat 300

cgtatgcagg tccatctggc ccctggcacc aagtttgatt tccaaaagta catcaagcgg 360

tctctggagg acgacttcaa ggtgatcgtg gggatcagcc ctcctttgtg ggcttctgct 420

ctcatcttcc tattcctcaa tgtcaatggt aatatatatc catcttcgtc ttcctctagc 480

ttagcttagc tagggtaata atagggtcgt ccatcatgca tctgacgacg atgcatatat 540

atatatgcag gatggcacac catgctctgg atctccatca tgccggtggt gatcatcctg 600

tccgtgggga cgaagctgca gggcatcatc tgccgcatgg cgatcgacat cacggagcgg 660

cacgccgtga tccagggcat cccgctggtg caggtcagcg actcctactt ctggttcgca 720

cgcccaacct tcgtgctctt tcctcatcaa 750

19

27

Artificial Sequence

Description of Artificial SequencePCR primer

19

acattcatat gctgcctcac gaaggaa 27

20

24

Artificial Sequence

Description of Artificial SequencePCR primer

20

tagctgcaga ggacctggac aacc 24

21

24

Artificial Sequence

Description of Artificial SequencePCR primer

21

ctgcacatct tcatcttcgt gctc 24

22

cntggcaaag gacggcacat ggcggggggc gggggcggcc gggacctgcc gtcgacgccg 60

acgtgggcgg tggccctggt gtgcgccgtc atcgtgctcg tctccgtcgc catggagcat 120

ggcctccaca agctcggcca ctggttccat acgcggcaga agaaggccat gcgggaggcc 180

ctggagaaga tcaaagcaga gttgatgctg atgggcttca tctcgctgct cctcgccgtg 240

gggcagacgc ccatctccaa gatatgcatc ccggccaagg ctggcagcat catgctgccg 300

tgcaagccgc cgaaaggcgc cgccgccgcc gccgacgacg acaagagcga cggccgccgg 360

agactcctct ggtacccgcc gtaccctgga tacgatgagc ccgggcacca ccgccgtttc 420

ctcgccggcg cggctccgga cgacaactac tgcagtgacc aaggcaaggt gtccctcatc 480

tcctcggccg gcgtccacca gctgcacatc ttcatcttcg tgctcgcggt gttccatatc 540

gtctacagcg tcgccaccat ggcgctgggg cgtctcaaaa tgaggaaatg gaagaaatgg 600

gaatcggaga ccaactccct ggaataccag tacgcaaacg acccttcacg gttccggttc 660

acgcaccaga cgtcgttcgt gaagcggcac ctgggcctct cgagcacccc tggagtgaga 720

tgggtcgtgg cgttcttcag gcagttcttc gcgtccgtga ccaaggtgga ttacctgacc 780

atgcggcagg ggttcatcaa ctaccatctg tcgcccagca ccaagttcaa cttccagcag 840

tacatcaagc ggtccttgga ggacgacttc aaagtcgtcg ttggcatcag tctcccgctg 900

tggttcgtcg ccatcttcac tctcttgatc gatatcaagg gattcggcac gcttgtctgg 960

atctcttttg tcccgctcgt tatactcctg ctagttgggg ccaagctgga ggttgtcatc 1020

atggagatgg ccaaggagat acaggacaag gcgacggtca tcaagggggc gcctgtggtg 1080

gagccaagtg acaggttctt ctggtttaac cgccctggct gggtcctctt cctcatccac 1140

ctcacgctct tccagaacgc cttccagatg gcgcatttcg tttggacact gctcacccca 1200

gacctgaaga aatgctacca cgagaggctg ggcctgagca tcatgaaggt tgcggtgggg 1260

ctggttctcc aggtcctctg cagttacatc accttcccgc tctacgcgct cgtcacgcag 1320

atggggtcgc acatgaagaa gaccatcttc gaggagcaga cggccaaggc ggtgatgaag 1380

tggcgcaaga cggccaagga taaggtgcgg cagcgggagg cggcaggctt cctcgacgtg 1440

ctgacgagcg ccgacaccac gccgagccac agccgcgcga cgtcgccgag ccggggcaac 1500

tcgccggtgc acctgctcca caagtacagg ggcaggtcgg aggaaccgca gagcgggccg 1560

gcgtcgccgg ggcgggagct cggggacatg tacccggtgg ctgaccagca tcgcctgcac 1620

aggctggacc ccgagaggat gaggcccgcc tcgtccaccg ccgtcaacat tgacatcgct 1680

gatgccgatt tttcttttag catgcggtga cctgaccttg aacgaattct gtgtccttac 1740

tcttgtatag ggaagcaaaa gcatagacgg agaacataat gacacgttac gttagggaaa 1800

gt 1802

23

563

Zea mays

23

Met Ala Gly Gly Gly Gly Gly Arg Asp Leu Pro Ser Thr Pro Thr Trp

1 5 10 15

Ala Val Ala Leu Val Cys Ala Val Ile Val Leu Val Ser Val Ala Met

20 25 30

Glu His Gly Leu His Lys Leu Gly His Trp Phe His Thr Arg Gln Lys

35 40 45

Lys Ala Met Arg Glu Ala Leu Glu Lys Ile Lys Ala Glu Leu Met Leu

50 55 60

Met Gly Phe Ile Ser Leu Leu Leu Ala Val Gly Gln Thr Pro Ile Ser

65 70 75 80

Lys Ile Cys Ile Pro Ala Lys Ala Gly Ser Ile Met Leu Pro Cys Lys

85 90 95

Pro Pro Lys Gly Ala Ala Ala Ala Ala Asp Asp Asp Lys Ser Asp Gly

100 105 110

Arg Arg Arg Leu Leu Trp Tyr Pro Pro Tyr Pro Gly Tyr Asp Glu Pro

115 120 125

Gly His His Arg Arg Phe Leu Ala Gly Ala Ala Pro Asp Asp Asn Tyr

130 135 140

Cys Ser Asp Gln Gly Lys Val Ser Leu Ile Ser Ser Ala Gly Val His

145 150 155 160

Gln Leu His Ile Phe Ile Phe Val Leu Ala Val Phe His Ile Val Tyr

165 170 175

Ser Val Ala Thr Met Ala Leu Gly Arg Leu Lys Met Arg Lys Trp Lys

180 185 190

Lys Trp Glu Ser Glu Thr Asn Ser Leu Glu Tyr Gln Tyr Ala Asn Asp

195 200 205

Pro Ser Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg His

210 215 220

Leu Gly Leu Ser Ser Thr Pro Gly Val Arg Trp Val Val Ala Phe Phe

225 230 235 240

Arg Gln Phe Phe Ala Ser Val Thr Lys Val Asp Tyr Leu Thr Met Arg

245 250 255

Gln Gly Phe Ile Asn Tyr His Leu Ser Pro Ser Thr Lys Phe Asn Phe

260 265 270

Gln Gln Tyr Ile Lys Arg Ser Leu Glu Asp Asp Phe Lys Val Val Val

275 280 285

Gly Ile Ser Leu Pro Leu Trp Phe Val Ala Ile Phe Thr Leu Leu Ile

290 295 300

Asp Ile Lys Gly Phe Gly Thr Leu Val Trp Ile Ser Phe Val Pro Leu

305 310 315 320

Val Ile Leu Leu Leu Val Gly Ala Lys Leu Glu Val Val Ile Met Glu

325 330 335

Met Ala Lys Glu Ile Gln Asp Lys Ala Thr Val Ile Lys Gly Ala Pro

340 345 350

Val Val Glu Pro Ser Asp Arg Phe Phe Trp Phe Asn Arg Pro Gly Trp

355 360 365

Val Leu Phe Leu Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln Met

370 375 380

Ala His Phe Val Trp Thr Leu Leu Thr Pro Asp Leu Lys Lys Cys Tyr

385 390 395 400

His Glu Arg Leu Gly Leu Ser Ile Met Lys Val Ala Val Gly Leu Val

405 410 415

Leu Gln Val Leu Cys Ser Tyr Ile Thr Phe Pro Leu Tyr Ala Leu Val

420 425 430

Thr Gln Met Gly Ser His Met Lys Lys Thr Ile Phe Glu Glu Gln Thr

435 440 445

Ala Lys Ala Val Met Lys Trp Arg Lys Thr Ala Lys Asp Lys Val Arg

450 455 460

Gln Arg Glu Ala Ala Gly Phe Leu Asp Val Leu Thr Ser Ala Asp Thr

465 470 475 480

Thr Pro Ser His Ser Arg Ala Thr Ser Pro Ser Arg Gly Asn Ser Pro

485 490 495

Val His Leu Leu His Lys Tyr Arg Gly Arg Ser Glu Glu Pro Gln Ser

500 505 510

Gly Pro Ala Ser Pro Gly Arg Glu Leu Gly Asp Met Tyr Pro Val Ala

515 520 525

Asp Gln His Arg Leu His Arg Leu Asp Pro Glu Arg Met Arg Pro Ala

530 535 540

Ser Ser Thr Ala Val Asn Ile Asp Ile Ala Asp Ala Asp Phe Ser Phe

545 550 555 560

Ser Met Arg

24

555

Oryza sativa

24

Met Ala Gly Gly Gly Gly Gly Arg Ala Leu Pro Glu Thr Pro Thr Trp

1 5 10 15

Ala Val Ala Val Val Cys Ala Val Ile Val Leu Val Ser Val Ala Met

20 25 30

Glu His Gly Leu His Lys Leu Gly His Trp Phe His Lys Arg Glu Lys

35 40 45

Lys Ala Met Gly Glu Ala Leu Glu Lys Ile Lys Ala Glu Leu Met Leu

50 55 60

Leu Gly Phe Ile Ser Leu Leu Leu Thr Val Ala Gln Thr Pro Ile Ser

65 70 75 80

Lys Ile Cys Ile Pro Glu Ser Ala Ala Asn Ile Met Leu Pro Cys Lys

85 90 95

Ala Gly Gln Asp Ile Val Lys Gly Leu Lys Gly Lys Lys Asp His Arg

100 105 110

Arg Arg Leu Leu Trp Tyr Thr Gly Glu Glu Glu Ser His Arg Arg Ser

115 120 125

Leu Ala Gly Ala Ala Gly Glu Asp Tyr Cys Ala Gln Ser Gly Lys Val

130 135 140

Ala Leu Met Ser Ser Gly Gly Met His Gln Leu His Ile Phe Ile Phe

145 150 155 160

Val Leu Ala Val Phe His Val Thr Tyr Cys Val Ile Thr Met Ala Leu

165 170 175

Gly Arg Leu Lys Met Lys Lys Trp Lys Lys Trp Glu Leu Glu Thr Asn

180 185 190

Ser Leu Glu Tyr Gln Phe Ala Asn Asp Pro Ser Arg Phe Arg Phe Thr

195 200 205

His Gln Thr Ser Phe Val Lys Arg His Leu Gly Leu Ser Ser Thr Pro

210 215 220

Gly Leu Arg Trp Ile Val Ala Phe Phe Arg Gln Phe Phe Gly Ser Val

225 230 235 240

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

245 250 255

Leu Ser Gln Asn Ser Lys Phe Asp Phe His Lys Tyr Ile Lys Arg Ser

260 265 270

Leu Glu Asp Asp Phe Lys Val Val Val Gly Ile Ser Leu Pro Leu Trp

275 280 285

Phe Val Ala Ile Leu Val Leu Phe Leu Asp Ile Gln Gly Phe Gly Thr

290 295 300

Leu Ile Trp Ile Ser Phe Val Pro Leu Val Ile Leu Met Leu Val Gly

305 310 315 320

Thr Lys Leu Glu Met Val Ile Met Glu Met Ala Gln Glu Ile Gln Asp

325 330 335

Arg Ala Thr Val Ile Lys Gly Ala Pro Val Val Glu Pro Ser Asn Lys

340 345 350

Tyr Phe Trp Phe Asn Arg Pro Asp Trp Val Leu Phe Phe Ile His Leu

355 360 365

Ile Leu Phe Gln Asn Ala Phe Gln Met Ala His Phe Val Trp Thr Leu

370 375 380

Ala Thr Pro Gly Leu Lys Lys Cys Phe His Glu Asn Met Gly Leu Ser

385 390 395 400

Ile Met Lys Val Val Val Gly Ile Phe Ile Gln Phe Leu Cys Ser Tyr

405 410 415

Ser Thr Phe Pro Leu Tyr Ala Leu Val Thr Gln Met Gly Ser Asn Met

420 425 430

Lys Lys Thr Ile Phe Glu Glu Gln Thr Met Lys Ala Leu Met Asn Trp

435 440 445

Arg Lys Thr Ala Arg Glu Lys Lys Lys Leu Arg Asp Ala Asp Glu Phe

450 455 460

Leu Ala Gln Met Ser Gly Asp Thr Thr Pro Ser Arg Gly Ser Ser Pro

465 470 475 480

Val His Leu Leu His Lys Gln Arg Val Arg Ser Glu Asp Pro Pro Ser

485 490 495

Ala Pro Ala Ser Pro Gly Phe Ala Gly Glu Ala Arg Asp Met Tyr Pro

500 505 510

Val Pro Val Ala Pro Val Val Arg Pro His Gly Phe Asn Arg Met Asp

515 520 525

Pro Asp Lys Arg Arg Ala Ala Ser Ser Ser Ala Ile Gln Val Asp Ile

530 535 540

Ala Asp Ser Asp Phe Ser Phe Ser Val Gln Arg

545 550 555

25

533

Hordeum vulgare

25

Met Ser Asp Lys Lys Gly Val Pro Ala Arg Glu Leu Pro Glu Thr Pro

1 5 10 15

Ser Trp Ala Val Ala Val Val Phe Ala Ala Met Val Leu Val Ser Val

20 25 30

Leu Met Glu His Gly Leu His Lys Leu Gly His Trp Phe Gln His Arg

35 40 45

His Lys Lys Ala Leu Trp Glu Ala Leu Glu Lys Met Lys Ala Glu Leu

50 55 60

Met Leu Val Gly Phe Ile Ser Leu Leu Leu Ile Val Thr Gln Asp Pro

65 70 75 80

Ile Ile Ala Lys Ile Cys Ile Ser Glu Asp Ala Ala Asp Val Met Trp

85 90 95

Pro Cys Lys Arg Gly Thr Glu Gly Arg Lys Pro Ser Lys Tyr Val Asp

100 105 110

Tyr Cys Pro Glu Gly Lys Val Ala Leu Met Ser Thr Gly Ser Leu His

115 120 125

Gln Leu His Val Phe Ile Phe Val Leu Ala Val Phe His Val Thr Tyr

130 135 140

Ser Val Ile Thr Ile Ala Leu Ser Arg Leu Lys Met Arg Thr Trp Lys

145 150 155 160

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

165 170 175

Pro Ala Arg Phe Arg Phe Thr His Gln Thr Ser Phe Val Lys Arg His

180 185 190

Leu Gly Leu Ser Ser Thr Pro Gly Ile Arg Trp Val Val Ala Phe Phe

195 200 205

Arg Gln Phe Phe Arg Ser Val Thr Lys Val Asp Tyr Leu Thr Leu Arg

210 215 220

Ala Gly Phe Ile Asn Ala His Leu Ser Gln Asn Ser Lys Phe Asp Phe

225 230 235 240

His Lys Tyr Ile Lys Arg Ser Met Glu Asp Asp Phe Lys Val Val Val

245 250 255

Gly Ile Ser Leu Pro Leu Trp Gly Val Ala Ile Leu Thr Leu Phe Leu

260 265 270

Asp Ile Asn Gly Val Gly Thr Leu Ile Trp Ile Ser Phe Ile Pro Leu

275 280 285

Val Ile Leu Leu Cys Val Gly Thr Lys Leu Glu Met Ile Ile Met Glu

290 295 300

Met Ala Leu Glu Ile Gln Asp Arg Ala Ser Val Ile Lys Gly Ala Pro

305 310 315 320

Val Val Glu Pro Ser Asn Lys Phe Phe Trp Phe His Arg Pro Asp Trp

325 330 335

Val Leu Phe Phe Ile His Leu Thr Leu Phe Gln Asn Ala Phe Gln Met

340 345 350

Ala His Phe Val Trp Thr Val Ala Thr Pro Gly Leu Lys Lys Cys Tyr

355 360 365

His Thr Gln Ile Gly Leu Ser Ile Met Lys Val Val Val Gly Leu Ala

370 375 380

Leu Gln Phe Leu Cys Ser Tyr Met Thr Phe Pro Leu Tyr Ala Leu Val

385 390 395 400

Thr Gln Met Gly Ser Asn Met Lys Arg Ser Ile Phe Asp Glu Gln Thr

405 410 415

Ser Lys Ala Leu Thr Asn Trp Arg Asn Thr Ala Lys Glu Lys Lys Lys

420 425 430

Val Arg Asp Thr Asp Met Leu Met Ala Gln Met Ile Gly Asp Ala Thr

435 440 445

Pro Ser Arg Gly Ser Ser Pro Met Pro Ser Arg Gly Ser Ser Pro Val

450 455 460

His Leu Leu His Lys Gly Met Gly Arg Ser Asp Asp Pro Gln Ser Ala

465 470 475 480

Pro Thr Ser Pro Arg Thr Gln Gln Glu Ala Arg Asp Met Tyr Pro Val

485 490 495

Val Val Ala His Pro Val His Arg Leu Asn Pro Asn Asp Arg Arg Arg

500 505 510

Ser Ala Ser Ser Ser Ala Leu Glu Ala Asp Ile Pro Ser Ala Asp Phe

515 520 525

Ser Phe Ser Gln Gly

530

26

cntggcaaag gacggcacat ggcggggggc gggggcggcc gggacctgcc gtcgacgccg 60

acgtgggcgg tggccctggt gtgcgccgtc atcgtgctcg tctccgtcgc catggagcat 120

ggcctccaca agctcggcca ctggttccat acgcggcaga agaaggccat gcgggaggcc 180

ctggagaaga tcaaagcaga gttgatgctg atgggcttca tctcgctgct cctcgccgtg 240

gggcagacgc ccatctccaa gatatgcatc ccggccaagg ctggcagcat catgctgccg 300

tgcaagccgc cgaaaggcgc cgccgccgcc gccgacgacg acaagagcga cggccgccgg 360

agactcctct ggtacccgcc gtaccctgga tacgatgagc ccgggcacca ccgccgtttc 420

ctcgccggcg cggctccgga cgacaactac tgcagtgacc aaggcaaggt gtccctcatc 480

tcctcggccg gcgtccacca gctgcacatc ttcatcttcg tgctcgcggt gttccatatc 540

gtctacagcg tcgccaccat ggcgctgggg cg 572

27

249

Zea mays

27

cgggccggcg tcgccggggc gggagctcgg ggacatgtac ccggtggctg accagcatcg 60

cctgcacagg ctggaccccg agaggatgag gcccgcctcg tccaccgccg tcaacattga 120

catcgctgat gccgattttt cttttagcat gcggtgacct gaccttgaac gaattctgtg 180

tccttactct tgtataggga agcaaaagca tagacggaga acataatgac acgttacgtt 240

agggaaagt 249

28

378

Zea mays

28

Leu Lys Met Arg Lys Trp Lys Lys Trp Glu Ser Glu Thr Asn Ser Leu

1 5 10 15

Glu Tyr Gln Tyr Ala Asn Asp Pro Ser Arg Phe Arg Phe Thr His Gln

20 25 30

Thr Ser Phe Val Lys Arg His Leu Gly Leu Ser Ser Thr Pro Gly Val

35 40 45

Arg Trp Val Val Ala Phe Phe Arg Gln Phe Phe Ala Ser Val Thr Lys

50 55 60

Val Asp Tyr Leu Thr Met Arg Gln Gly Phe Ile Asn Tyr His Leu Ser

65 70 75 80

Pro Ser Thr Lys Phe Asn Phe Gln Gln Tyr Ile Lys Arg Ser Leu Glu

85 90 95

Asp Asp Phe Lys Val Val Val Gly Ile Ser Leu Pro Leu Trp Phe Val

100 105 110

Ala Ile Phe Thr Leu Leu Ile Asp Ile Lys Gly Phe Gly Thr Leu Val

115 120 125

Trp Ile Ser Phe Val Pro Leu Val Ile Leu Leu Leu Val Gly Ala Lys

130 135 140

Leu Glu Val Val Ile Met Glu Met Ala Lys Glu Ile Gln Asp Lys Ala

145 150 155 160

Thr Val Ile Lys Gly Ala Pro Val Val Glu Pro Ser Asp Arg Phe Phe

165 170 175

Trp Phe Asn Arg Pro Gly Trp Val Leu Phe Leu Ile His Leu Thr Leu

180 185 190

Phe Gln Asn Ala Phe Gln Met Ala His Phe Val Trp Thr Leu Leu Thr

195 200 205

Pro Asp Leu Lys Lys Cys Tyr His Glu Arg Leu Gly Leu Ser Ile Met

210 215 220

Lys Val Ala Val Gly Leu Val Leu Gln Val Leu Cys Ser Tyr Ile Thr

225 230 235 240

Phe Pro Leu Tyr Ala Leu Val Thr Gln Met Gly Ser His Met Lys Lys

245 250 255

Thr Ile Phe Glu Glu Gln Thr Ala Lys Ala Val Met Lys Trp Arg Lys

260 265 270

Thr Ala Lys Asp Lys Val Arg Gln Arg Glu Ala Ala Gly Phe Leu Asp

275 280 285

Val Leu Thr Ser Ala Asp Thr Thr Pro Ser His Ser Arg Ala Thr Ser

290 295 300

Pro Ser Arg Gly Asn Ser Pro Val His Leu Leu His Lys Tyr Arg Gly

305 310 315 320

Arg Ser Glu Glu Pro Gln Ser Gly Pro Ala Ser Pro Gly Arg Glu Leu

325 330 335

Gly Asp Met Tyr Pro Val Ala Asp Gln His Arg Leu His Arg Leu Asp

340 345 350

Pro Glu Arg Met Arg Pro Ala Ser Ser Thr Ala Val Asn Ile Asp Ile

355 360 365

Ala Asp Ala Asp Phe Ser Phe Ser Met Arg

370 375

29

51

Zea mays

29

Gly Pro Ala Ser Pro Gly Arg Glu Leu Gly Asp Met Tyr Pro Val Ala

1 5 10 15

Asp Gln His Arg Leu His Arg Leu Asp Pro Glu Arg Met Arg Pro Ala

20 25 30

Ser Ser Thr Ala Val Asn Ile Asp Ile Ala Asp Ala Asp Phe Ser Phe

35 40 45

Ser Met Arg

50