Polypeptides having catalytic activity for the removal of epoxide groups, nucleic acids encoding same and uses thereof

Abstract

The present application discloses a polypeptide with catalytic activity of removing epoxy group, its coding nucleic acid and use. The polypeptide of the present application can catalyze the reaction of removing epoxy group between trichothecene and glutathione under mild conditions, and produce nontoxic and harmless glutathione derivative, so as to realize the detoxification and detoxication of trichothecene. The polypeptide of the present application has wide use in the fields of agriculture, food, feed and medicine.

Description

Technical Field

[0001] This invention relates to the field of polypeptides, and more specifically to a polypeptide with deepoxidation catalytic activity, its encoded nucleic acid, and its uses. Background Technology

[0002] The basic chemical structure of trichothecene toxins is a sesquiterpene. Because they form epoxy groups at the 12th and 13th carbon positions, they are also called 12,13-epoxy trichothecene compounds. Since the 1970s, researchers have determined that the epoxy groups are the main source of toxicity in trichothecene toxins. Currently, de-epoxy trichothecene toxin derivatives can be chemically synthesized in vitro under highly alkaline conditions, but due to the harsh reaction conditions and low efficiency, industrial application is difficult. Furthermore, several anaerobic bacteria have been isolated from animal intestinal microorganisms that can detoxify trichothecene toxins, but the mechanism of action is unclear, and their dependence on anaerobic conditions greatly limits their practical industrial application. Summary of the Invention

[0003] In view of the problems existing in the prior art, the inventors provide a polypeptide with de-epoxylated catalytic activity, which can catalyze the reaction of the epoxy groups of trichothecene toxins with glutathione (GSH) under mild conditions to produce non-toxic and harmless GSH-substituted derivatives, thereby achieving detoxification of trichothecene toxins. The present invention is based at least in part on this, and specifically, the present invention includes the following:

[0004] In a first aspect, the present invention provides an isolated polypeptide with deepoxidation catalytic activity, which, at a temperature of 15-35°C, enables the catalytic reaction of trichothecene toxins with GSH in PBS buffer to remove epoxy groups and generate GSH-treated derivatives. Here, although the reaction temperature is specified as 15-35°C, this is merely to characterize or identify the polypeptide's deepoxidation catalytic activity under these conditions, and does not imply that the polypeptide of the present invention does not possess deepoxidation catalytic activity at temperatures below 15°C or above 35°C. In fact, the conditions for the catalytic reaction of the active polypeptide of the present invention are not limited to the above-mentioned temperatures.

[0005] A second aspect of the present invention provides an isolated polypeptide having de-epoxy catalytic activity, comprising an amino acid sequence selected from the group consisting of (1)-(5) below:

[0006] (1) The amino acid sequences shown in SEQ ID No.:1-35, wherein SEQ ID No.:1 represents the amino acid sequence from the decaploid of *Thymus chinensis*, SEQ ID No.:2 represents the amino acid sequence from the diploid of *Thymus chinensis*, SEQ ID No.:3-24 represents the mutant sequences of SEQ ID No.:1 that have been verified to have the original activity, and SEQ ID No.:25-35 represents the amino acid sequences from the genus *Cyclocarya*. The amino acid sequences of different species.

[0007] (2) The amino acid sequence identity with the amino acid sequence in (1) is 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and further preferably 99% or more, and originates from the same genus, preferably from the same species; further preferably, the polypeptide composed of these sequences still has protease activity. In some embodiments, the amino acid sequence of the active polypeptide has 95% or more sequence identity with the amino acid sequence in (1), and both originate from the genus *Cyclocarya*.

[0008] (3) An amino acid sequence having one or more amino acid mutations compared to the amino acid sequence of (1) or (2) and having sequence identity of 90% or more, preferably 95% or more, more preferably 97% or more, even more preferably 98% or more, and even more preferably 99% or more, wherein the amino acid mutations include the insertion, deletion or substitution of amino acids.

[0009] (4) A partial continuous sequence derived from any one of the amino acid sequences described in (1)-(3), preferably a polypeptide (or truncated polypeptide) having the partial continuous sequence still having the enzymatic catalytic activity of the original polypeptide, more preferably having a partial continuous sequence located at the N-terminus of any one of the amino acid sequences described in (1)-(3), for example, a polypeptide having the first 200 to 250 amino acid sequences from the N-terminus, for example, a polypeptide having the first 208 amino acid sequences from the N-terminus, or a polypeptide having the first 242 amino acid sequences from the N-terminus.

[0010] (5) A chimeric sequence in which other amino acid sequences are linked to the N-terminus and / or C-terminus of any of the amino acid sequences described in (1)-(4). That is, the active polypeptide of the present invention can be a chimeric polypeptide. In some embodiments, the other amino acid sequences are sequences that enhance polypeptide expression or secretion, examples of which include, but are not limited to, guide peptides, signal peptides, and rotational peptides. In some embodiments, the active polypeptide is a chimeric polypeptide of the active fragment of a full-length protein and other amino acid sequences, wherein the other amino acid sequences are sequences corresponding to other homologous proteins other than the active fragment, such as sequences of structural or functional regions. For example, when the full length of a protease from a certain species consists of two parts A+B, and the full length of another homologous protease from the same genus but a different species consists of two parts A'+B', and A and A' are homologous corresponding regions, and B and B' are homologous corresponding regions, the chimeric polypeptide can consist of A'+B or A+B'. In some embodiments, the other amino acid sequences include non-functional sequences, such as linker arms or spacer sequences. In some embodiments, the other amino acid sequences are polypeptides with independent functions, which are linked to the active polypeptide of the present invention via non-functional sequences, such as linker arms or spacer sequences.

[0011] In some embodiments, the active polypeptide of the present invention has a conserved site selected from at least one of the following: amino acid A at position 98, amino acid A at position 99.

[0012] A third aspect of the present invention provides an isolated active polypeptide having the following amino acid sequence:

[0013] V1-GDX1X2DIAAX3LQRT-V2- ADYARFNX1NVDX4AFX5AHVX1X6MX6HGLPLDPAX7X4DVX8KAEFVR-V3, where:

[0014] X1 represents G or S;

[0015] X2 represents F or L;

[0016] X3 represents Y or H;

[0017] X4 represents A or V;

[0018] X5 represents T, Q, or N;

[0019] X6 represents L or V;

[0020] X7 indicates T or S;

[0021] X8 represents T or I;

[0022] V1 is absent or represents a first variable region, the amino acid sequence of the first variable region corresponds to a series of consecutive amino acid sequences before the 92nd amino acid in SEQ.ID.No.1, and the sequence identity between the first variable region and the series of consecutive amino acid sequences is 80% or more, 85% or more, preferably 90% or more, preferably 92% or more, more preferably 95% or more, and even more preferably 98% or less, for example 99%;

[0023] V2 represents a linker arm or a second variable region, wherein the amino acid sequence of the second variable region corresponds to a series of consecutive amino acid sequences between amino acids 105-143 in SEQ.ID.No.1, and the sequence identity between the second variable region and the series of consecutive amino acid sequences is 80% or more, 85% or more, preferably 90% or more, preferably 92% or more, more preferably 95% or more, and even more preferably 98% or less, for example 99%.

[0024] V3 is absent or represents a third variable region, wherein the amino acid sequence of the third variable region corresponds to a series of consecutive amino acid sequences following the 144th amino acid in SEQ.ID.No.1, and the sequence identity between the third variable region and the series of consecutive amino acid sequences is 80% or more, 85% or more, preferably 90% or more, preferably 92% or more, more preferably 95% or more, and even more preferably 98% or less, for example 99%.

[0025] In a fourth aspect, the present invention provides an isolated nucleic acid molecule encoding the polypeptide described in the first or second aspect.

[0026] A fifth aspect of the present invention provides an isolated nucleic acid molecule having a base sequence selected from the group consisting of (a)-(e) below:

[0027] (a) Sequences shown in SEQ ID No.:36-70. Wherein, SEQ ID No.:36 represents the decyclooxygenase gene from the decaploid of *Thinopyrum longicornis*, SEQ ID No.:37 represents the decyclooxygenase gene from the diploid of *Thinopyrum longicornis*, SEQ ID No.:38-59 represent mutants of the SEQ ID No.:36 sequence, and SEQ ID No.:60-70 represent homologous gene sequences from different species of the genus *Cyclocarya*.

[0028] (b) A sequence modified based on the base sequence in (a) using host codon preference. To adapt to the needs of different hosts, the base sequence in (a) can be modified according to degenerate codons. Codon preference modification generally does not change the sequence of the product protein or polypeptide.

[0029] (c)(a) shows a conserved region sequence. Preferably, it is a conserved region sequence encoding an active polypeptide. It should be noted that a conserved region sequence does not necessarily express or encode an active polypeptide. As long as it is a conserved region, it can be used as a detection target.

[0030] (d) has a sequence identity of 95% or more with (a)-(c), preferably 97% or more, more preferably 98% or more, and most preferably 99% or more, and the sequences are from the same genus, preferably the same species;

[0031] (e) A sequence complementary to at least a portion of any of the sequences in (a)-(d). Complementary sequences include sequences that specifically hybridize to these sequences under stringent conditions. Examples include probes, primers, etc.

[0032] A sixth aspect of the present invention provides a nucleic acid construct comprising the nucleic acid described in the third and fourth aspects of the present invention and optional regulatory elements. Examples of regulatory elements include, but are not limited to, promoters, activators, enhancers, operons, ribosome binding sites, initiation signals, stop signals, cap signals, polyadenylation signals, and other signals involved in transcriptional or translational control. These regulatory elements enable the expression of the nucleic acid molecule in a intended target cell (e.g., *E. coli*, yeast cells, etc.). The nucleic acid construct includes both self-replicating and non-self-replicating constructs. Examples of self-replicating constructs include, but are not limited to, vectors, plasmids, etc.

[0033] A seventh aspect of the present invention provides a host cell comprising the nucleic acids described in the third and fourth aspects of the present invention, or the nucleic acid construct described in the fifth aspect of the present invention, introduced by genetic engineering. The host cell is not particularly limited and includes prokaryotic cells and eukaryotic cells. Examples of prokaryotic cells include, but are not limited to, *Escherichia coli*, and examples of eukaryotic cells include, but are not limited to, yeast, plant cells, or animal cells.

[0034] An eighth aspect of the present invention provides a method for producing an active polypeptide. The method comprises a genetic engineering method and a chemical synthesis method. The genetic engineering method includes the step of expressing the nucleic acid of the present invention in an intracellular (e.g., *E. coli*) or non-cellular expression system to obtain the polypeptide. The chemical synthesis method may use any method currently known.

[0035] A ninth aspect of the present invention provides a method for catalyzing the deepoxidation reaction of trichothecene compounds, comprising the step of contacting the active polypeptide described in the first and second aspects of the present invention, or the host cell described in the sixth aspect, with trichothecene compounds and GSH under suitable reaction conditions to generate GSH-treated derivatives. Suitable reaction conditions in the present invention include a reaction temperature of 1-45°C, preferably 2-40°C, more preferably 5-35°C, and even more preferably 10-30°C; a reaction time of 10 minutes to 36 hours, for example 10-60 minutes or 1.5-24 hours; and a suitable reaction solution, for example, PBS or DMSO solution, with a pH between 4.0 and 7.5, preferably between 4.5 and 7.0. Specific reaction conditions need to be adjusted by those skilled in the art according to the source of the enzyme, the enzyme activity, the substrate concentration, the reaction amount, etc., and are not particularly limited.

[0036] In a tenth aspect, the present invention provides a glutathioneized derivative having the structure shown in the following general formula (I):

[0037]

[0038] In this context, R1, R2, and R3 each independently represent a hydrogen atom, a hydroxyl group, or an ester group represented by -OCO-R', where R' is a straight-chain or branched C1-C5 alkyl group; R4 represents a hydrogen atom or a hydroxyl group; and R5 represents a hydrogen atom, =O, a hydroxyl group, or an ester group represented by -OCO-R”, where R” is a straight-chain or branched C1-C5 alkyl group. 10 alkyl.

[0039] In an eleventh aspect, the present invention provides the use of the glutathionized derivatives of the present invention as an indicator for evaluating the deepoxidation reaction of trichothecene compounds.

[0040] A twelfth aspect of the present invention provides a method for evaluating the detoxification effect of a sample contaminated with trichothecene compounds, comprising the step of using the glutathione derivative of the present invention as an evaluation index.

[0041] In a thirteenth aspect of the present invention, a method for evaluating the detoxification effect on a sample contaminated with trichothecene compounds is provided, comprising the following steps:

[0042] (1) The step of measuring the content of glutathione derivatives in the sample to obtain a measured value, or measuring the ratio of glutathione derivatives to trichothecene compounds in the sample.

[0043] (2) The steps for comparing the measured value or ratio with the reference value;

[0044] (3) Steps for evaluating the detoxification effect of the samples based on the comparison results.

[0045] In some embodiments, the reference value here is the result obtained from a control sample, or the content of glutathione derivatives in the sample before treatment, or the ratio of the content of glutathione derivatives to trichothecene compounds.

[0046] In a fourteenth aspect, the present invention provides a method for determining the catalytic activity of a polypeptide de-epoxylated compounds, comprising the steps of treating a standard sample with the polypeptide and measuring the content of the glutathione derivative of the present invention, or the content of a trichothecene compound, or the ratio of the content of the glutathione derivative to the content of the trichothecene compound in the standard sample before and after treatment. The standard sample is a standard sample of trichothecene compounds. The ratio of the content of the glutathione derivative to the content of the trichothecene compound includes both the case of glutathione derivative content: trichothecene compound content and the case of trichothecene compound content: glutathione derivative content.

[0047] A fifteenth aspect of the present invention provides a method for identifying compounds capable of affecting the catalytic activity of peptide deepoxidation, comprising the following steps:

[0048] a. The step of contacting a polypeptide with a standard sample of a trichothecene family of compounds under suitable reaction conditions to obtain a reaction system, and measuring the first formation rate of the glutathione derivative;

[0049] b. The step of applying the test compound to the same reaction system as in step a and measuring the second rate of the glutathione derivative, wherein the same reaction system as in step a includes another reaction system in which the components and their concentrations in the reaction mixture are present, as well as the case of the same reaction system at different time periods;

[0050] c. In the step of comparing the first generation rate and the second generation rate, when the second generation rate is less than the first generation rate, the test compound is identified as a peptide activity inhibitory compound; when the second generation rate is greater than the first generation rate, the test compound is identified as a peptide activity promoting compound; and when the second generation rate is equal to the first generation rate, the test compound is identified as a compound ineffective for peptide activity.

[0051] In a sixteenth aspect, the present invention provides the use of the active polypeptide of the present invention in food processing, feed processing and pharmaceuticals.

[0052] The seventeenth aspect of the present invention provides the use of the nucleic acid of the present invention in plant breeding and disease control. Attached Figure Description

[0053] Figure 1 FTCD purified SDS-PAGE analysis diagram.

[0054] Figure 2 The effect of enzyme amount on enzymatic reactions. (a) Reduction of the substrate DON in the enzymatic reaction; (b) Formation of the enzymatic reaction product DON-GSH.

[0055] Figure 3 The effect of reaction buffer pH on the enzymatic reaction. (a) Reduction of the substrate DON in the enzymatic reaction; (b) Formation of the enzymatic reaction product DON-GSH.

[0056] Figure 4 The effect of reaction temperature on the enzymatic reaction. (a) Reduction of the substrate DON in the enzymatic reaction; (b) Formation of the enzymatic reaction product DON-GSH.

[0057] Figure 5A EIC is the extraction ion current spectrum of the in vitro enzymatic reaction of DON and GSH in LC-HRMS (Method 1).

[0058] Figure 5B The mass spectrum of the daughter ions generated by the high-energy collision dissociation of DON-GSH obtained by the in vitro enzymatic reaction of DON and GSH in LC-HRMS2 (Method 2) is shown.

[0059] Figure 6A EIC is the extraction ion current spectrum of the in vitro enzymatic reaction of 3-ADON and GSH in LC-HRMS (Method 1).

[0060] Figure 6B The mass spectrum of the daughter ions generated by the high-energy collision dissociation of 3-ADON-GSH obtained by the in vitro enzymatic reaction of LC-HRMS2 (Method 2) 3-ADON and GSH.

[0061] Figure 7A EIC is the extraction ion current spectrum of 15-ADON and GSH extracted by in vitro enzymatic reaction of LC-HRMS (Method 1).

[0062] Figure 7B Mass spectra of daughter ions generated by high-energy collisional dissociation of 15-ADON-GSH obtained by in vitro enzymatic reaction of 15-ADON and GSH in LC-HRMS2 (Method 2).

[0063] Figure 8A EIC is the extraction ion current spectrum of the in vitro enzymatic reaction of LC-HRMS (Method 1) NIV and GSH.

[0064] Figure 8BThe mass spectrum of the daughter ions generated by the high-energy collision dissociation of NIV-GSH obtained by the in vitro enzymatic reaction of NIV and GSH in LC-HRMS2 (Method 2) is shown.

[0065] Figure 9A EIC is the extraction ion current spectrum of the in vitro enzymatic reaction of Fus-X and GSH by LC-HRMS (Method 1).

[0066] Figure 9B The mass spectrum of the daughter ions generated by the high-energy collisional dissociation of Fus-X-GSH obtained by the in vitro enzymatic reaction of Fus-X and GSH in LC-HRMS2 (Method 2) is shown.

[0067] Figure 10A EIC is the extraction ion current spectrum of DAS and GSH extracted by in vitro enzymatic reaction of LC-HRMS (Method 1).

[0068] Figure 10B The mass spectrum of the daughter ions generated by the high-energy collision dissociation of DAS-GSH obtained by the in vitro enzymatic reaction of DAS and GSH in LC-HRMS2 (Method 2) is shown.

[0069] Figure 11A EIC is the extraction ion current spectrum of HT-2 extracted by in vitro enzymatic reaction of LC-HRMS (Method 1) and GSH.

[0070] Figure 11B The mass spectrum of the daughter ions generated by the high-energy collisional dissociation of the HT-GSH adduct obtained by the in vitro enzymatic reaction of LC-HRMS2 (Method 2) HT-2 and GSH is shown.

[0071] Figure 12A EIC is the extraction ion current spectrum of LC-HRMS (Method 1) T-2 and GSH in vitro enzymatic reaction.

[0072] Figure 12B The mass spectrum of the daughter ions generated by the high-energy collisional dissociation of the T2-GSH adduct obtained by the in vitro enzymatic reaction of LC-HRMS2 (Method 2) T-2 and GSH is shown.

[0073] Figure 13 Effects of trichothecene compounds on the viability of human cell lines. Cells were treated with different concentration gradients of DON, 3ADON, 15ADON, FUS-X, NIV, T-2, HT-2, and DAS for 48 h, and the OD at 450 nm was measured.

[0074] Figure 14 LC-HRMS (Method 1) Ion chromatogram of toxin-treated transgenic yeast extract.

[0075] Figure 15Results of DON tolerance in transgenic FTCD Pichia pastoris.

[0076] Figure 16 FTCD and homologous sequence phylogenetic tree.

[0077] Figure 17 LC-HRMS (Method 1) ion chromatogram of DON-treated FTCD homologous sequence transgenic yeast extract. DON-GSH adduct extracted in positive ion mode, m / z 604.21730 (corresponding to [M+H)). + (Δ±5ppm). Detailed Implementation

[0078] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0079] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0080] Unless otherwise stated, 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 is described. While only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail. Unless otherwise stated, “%” means percentage based on weight.

[0081] In this document, the terms “peptide” and “protein” are used interchangeably to refer to polymers of amino acid residues and their variants, as well as synthetic and naturally occurring analogs. Both terms apply to amino acid polymers in which one or more amino acid residues are synthetic, non-naturally occurring amino acids (such as chemical analogs of the corresponding naturally occurring amino acids), and to naturally occurring amino acid polymers and their naturally occurring chemical derivatives. Such chemical derivatives include, for example, post-translational modifications and degradation products, including pyroglutamyl, isoaspartic, proteolytic, phosphorylated, glycosylated, oxidized, isomerized, and deamination variants.

[0082] In this article, the term "active polypeptide" refers to a polypeptide with deepoxidase catalytic activity, that is, an active polypeptide that converts epoxy groups into other groups or removes those groups. It is sometimes also referred to as a "protease" in this article.

[0083] In this paper, the term "sequence identity" refers to the degree to which sequences are identical on a nucleotide-by-nucleotide or amino acid-by-amino acid basis within a comparison window. Therefore, the sequence identity percentage can be calculated as follows: compare two optimally aligned sequences within the comparison window, determine the number of identical nucleic acid bases or amino acid residues in both sequences to obtain the number of matching positions, divide this number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiply the result by 100 to obtain the sequence identity percentage.

[0084] In this paper, the calculation of sequence identity or sequence similarity (which may be used interchangeably) between two sequences is performed using the following method. To determine the percentage of identity between two amino acid sequences or two nucleic acid sequences, sequences are aligned for optimal comparison purposes (e.g., vacancies may be introduced in one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, regardless of non-homologous sequences for comparison purposes). In some embodiments, the length of the reference sequence aligned for comparison purposes is at least 30% of the total length of the reference sequence, preferably at least 40%, more preferably at least 50%, and even more preferably at least 70%, 80%, 90%, or even 100%. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percentage of identity between two sequences is a function of the number of shared identical positions in the sequences, taking into account the number of vacancies and the length of each vacancy, which requires the introduction of optimal alignment for the two sequences. The sequence comparison and determination of the percentage of identity between two sequences can be achieved using mathematical algorithms. The percentage of identity between two amino acid sequences or between nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) or the ALIGN program (version 2.0).

[0085] In this document, the term "amino acid at position x" or similar expressions refer to the amino acid sequence of the decyclooxygenase from the deploid strain of *Thinopyrum longicornis*, i.e., the amino acid sequence shown in SEQ ID No.:1, unless otherwise explicitly stated. Similarly, the term "base at position y" or similar expressions refer to the base sequence of the decyclooxygenase gene from the deploid strain of *Thinopyrum longicornis*, i.e., the amino acid sequence shown in SEQ ID No.:36, unless otherwise explicitly stated.

[0086] In this document, the terms "trichophyton toxins" or "trichophyton compounds" refer to a class of compounds whose basic chemical structure is a sesquiterpene, with epoxy groups formed at the 12th and 13th carbon positions. Preferably, trichophyton toxins have the structure shown in the following general formula (II):

[0087]

[0088] In this context, R1, R2, and R3 each independently represent a hydrogen atom, a hydroxyl group, or an ester group represented by -OCO-R', where R' is a straight-chain or branched C1-C5 alkyl group, such as CH3, CH2CH3, CH2CH2CH3, or CH2(CH3)2; R4 represents a hydrogen atom or a hydroxyl group; and R5 represents a hydrogen atom, =O, a hydroxyl group, or an ester group represented by -OCO-R”, where R” is a straight-chain or branched C1-C5 alkyl group. 10 Alkyl groups, preferably CH3 or CH2CH3, and even more preferably straight-chain or branched C3-C8 alkyl groups, more preferably CH2CH(CH3)2. In some embodiments, trichothecene toxins include deoxynivalenol (DON), 15-acetyl-deoxynivalenol (15-ADON), 3-acetyl-deoxynivalenol (3-ADON), nivalenol (NIV), fusarenonX (Fus-X), diacetoxyscirpenol (DAS), T-2 toxin (T-2), and HT-2 toxin (HT-2).

[0089] In this article, the term "de-epoxy group catalytic activity" refers to the activity or function capable of removing epoxy groups (preferably those formed on the 12th or 13th carbon) from trichothecene toxins. The specific catalytic process is shown below:

[0090]

[0091] The meanings of R1-R5 are the same as those in (I) and (II).

[0092] Example

[0093] I. Preparation of FTCD-Activated Peptides

[0094] 1. Materials and Methods

[0095] The Escherichia coli DH5α strain, the expression strain BL21(DE3), the prokaryotic expression vector pET-28a(+) and the plasmid pMD19-T-FTCD were preserved in our laboratory. Among them, the plasmid pMD19-T-FTCD contains a deepoxidase gene derived from wheatgrass, and its sequence is shown in SEQ ID No. 36.

[0096] 1.2 Experimental Methods

[0097] 1.2.1 The recombinant expression vector pET28a-FTCD was constructed using the following method.

[0098] Primers with NcoI and BamHI restriction sites were designed based on the expression vector pET28a. The primer sequences are as follows (underlined sites indicate restriction sites):

[0099] Forward primer: 5'- CCATGG CTAGAAATCCACCCATCGTCATCACC-3'

[0100] Reverse primer: 5'- GGATCC TCTTCACCTCGGCATACTTGTC-3'

[0101] PCR amplification was performed using plasmid pMD19-T-FTCD as a template. The amplification products were detected by 1% agarose gel electrophoresis, and the target fragment was recovered from the gel. The target fragment and the pET28a vector were double-digested with NcoI and BamHI, respectively, and ligated using T4 ligase after gel recovery. The ligation product was transformed into *E. coli* DH5α, and colony PCR and double enzyme digestion confirmed the approximately 900 bp target gene and approximately 5000 bp pET28a vector backbone. Further sequencing verification confirmed the correct sequence and reading frame of the recombinant expression vector pET28a-FTCD.

[0102] 1.2.2 Peptide-induced expression

[0103] The recombinant expression vector plasmid pET28a-FTCD was transformed into competent E. coli strain BL21(DE3). Positive clones from the transformation plates, detected by PCR, were inoculated into test tubes containing 3 mL of LB medium containing 50 μg / mL Kana and incubated overnight at 37°C with shaking at 220 rpm. The next day, the cells were inoculated into Kana LB medium and shaken until the OD600 reached 0.6–0.8. One mL of culture was removed, centrifuged at room temperature for 2 min, the supernatant was discarded, and the bacterial pellet was resuspended in 100 μl of 1× loading buffer. IPTG was added to the remaining culture to a final concentration of 0.5 mM, and the mixture was incubated at 37°C with shaking at 220 rpm for 4 h to induce fusion protein expression. One mL of culture was removed, centrifuged at 10000 rpm for 2 min at room temperature, the supernatant was discarded, and the bacterial pellet was resuspended in 100 μl of 1× loading buffer. Centrifuge the remaining culture at 4000 r / min for 10 min, discard the supernatant, and resuspend the bacterial precipitate in PBS; after sonicating the resuspended solution, take the supernatant and precipitate respectively and add them to the loading buffer for resuspending.

[0104] 1.2.3 Purification of peptides

[0105] The protein solution was purified using a Ni column and collected using a low-pressure chromatography system. It was then added to a dialysis bag and dialyzed overnight using 50 mM Tris-HCl, 0.30 M NaCl, and pH 8.0.

[0106] Protein expression was induced by shaking with 0.5 mmol / L IPTG at 37°C for 4 h. Bacterial cells were collected and resuspended in PBS. After sonication, the supernatant was collected and purified using a Ni column and molecular sieve. SDS-PAGE electrophoresis showed that a soluble protein peptide with a molecular weight of approximately 33 kDa was obtained. The purified protein band was single, indicating good purification efficiency (see [link to SDS-PAGE]). Figure 1 ).

[0107] II. Establishment of an in vitro enzymatic reaction system for peptides

[0108] 1. Experimental Methods:

[0109] 1.1 Reagents: 0.5 mg / ml trichothecene compounds (DON, 3DON, 15ADON, FUS-X, NIV, T2, H-T2, DAS): 1 mg of trichothecene compound, add distilled water to 2 ml, filter and sterilize.

[0110] 1.2 Establishment of in vitro enzymatic reaction system

[0111] The optimal conditions for the in vitro enzymatic reaction system of FTCD peptide were established through gradient experiments on three different factors affecting the enzymatic reaction:

[0112] (1) Enzyme concentration gradient: 1 μg, 5 μg, 10 μg, 25 μg, 50 μg;

[0113] (2) Multiple buffer solutions with pH gradients: range of 3.0-10.0, disodium hydrogen phosphate-citric acid buffer (pH=3.0, 4.0, 5.0), disodium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH=6.0, 7.0), Tris-phosphate buffer (pH=8.0, 9.0, 10.0).

[0114] (3) Reaction temperature gradient: 4℃, 12℃, 15℃, 20℃, 25℃, 30℃, 37℃, 45℃, 50℃

[0115] 2. Experimental Results:

[0116] 2.1 Effect of enzyme amount on enzyme-catalyzed reaction system

[0117] Using phosphate-buffered saline (PBS) (pH = 7.0), the reaction was carried out at 25°C for 12 hours. Samples were taken at 0h, 0.5h, 1h, 3h, and 6h for LC-HRMS analysis. The changes in the concentrations of the substrate DON and the GSH adduct formed during the reaction were obtained from the area results of the first-order LC-HRMS scan, thus determining the optimal enzyme amount for the reaction. (See [link to relevant documentation]). Figure 2 .

[0118] Experiments with varying enzyme dosages showed that, within the same timeframe, the amount of DON-GSH generated was positively correlated with the amount of enzyme added, ranging from 1 to 25 μg. When the enzyme dosage exceeded 25 μg, the DON-GSH generation tended to stabilize. Therefore, 25 μg was selected as the optimal enzyme dosage for the experiment.

[0119] 2.2 Effect of pH value on enzyme-catalyzed reaction system

[0120] The results of the pH gradient experiment of the enzyme reaction buffer are as follows: Figure 3 As shown. Figure 3 This indicates that the amount of the product DON-GSH reaches its maximum at a buffer pH of 6.0, while the content of the substrate DON is the lowest. Therefore, the suitable buffer pH is between 5.0 and 7.0.

[0121] 3. Effect of reaction temperature on the enzyme-catalyzed reaction system

[0122] Based on the above experimental results, with a reaction buffer pH of 7.0 and an enzyme dosage of 25 μg, the temperature conditions were set at 4℃, 12℃, 15℃, 20℃, 25℃, 30℃, 37℃, 45℃, and 50℃, with a reaction time of 24 h. Samples were taken at 0 h, 0.5 h, 1 h, 6 h, 12 h, and 24 h for LC-HRMS analysis. The changes in the content of the reaction substrate DON and the GSH adduct formed during the reaction were obtained from the area results of the first-order LC-HRMS scan, thus determining the optimal reaction temperature.

[0123] The results of experiments conducted at different reaction temperatures are as follows: Figure 4 As shown. Figure 4 The results show that the effect of 20-25℃ on the enzymatic reaction is not significant, and the content of the products can reach the maximum value. Below 15℃, the amount of DON-GSH generated decreases with decreasing temperature. Between 30-37℃, the amount of DON-GSH generated is inversely proportional to the increase of reaction temperature. Above 37℃, the product DON-GSH cannot be detected by LC-HRMS primary scanning, indicating that the protease has basically lost its activity. Therefore, 20-25℃ is the most suitable temperature range for the enzymatic reaction.

[0124] The above experimental results indicate that the optimal conditions for in vitro enzymatic reactions of proteases are as follows: the reaction system contains 25 μg of FTCD purified protein, and after adding an appropriate amount of reaction substrate, the mixture is supplemented with buffer solution with a pH of 5.0-7.0 to a final volume of 200 μl. After mixing, the reaction is carried out at 20-25℃.

[0125] III. Active polypeptide FTCD catalyzes the deepoxidation reaction of trichothecene toxins

[0126] 1. Experimental Methods:

[0127] 1.1 In vitro enzymatic reaction:

[0128] DON, 3-DON, 15-ADON, NIV, DAS, HT-2, and T-2 toxins (1 mg) were dissolved in freshly prepared GSH (30.7 mg, 100 μmol) in PBS buffer, and protease was added. The mixtures were then incubated in a water bath at 20°C for 24 h.

[0129] 1.2 LC-HRMS ( / MS) Analysis

[0130] The in vitro reaction solution was filtered through a 0.22 μm filter membrane and then transferred to a vial for LC-HRMS detection.

[0131] Using Thermo Scientific TM Q ExactiveTM A combined quadrupole Orbitrap mass spectrometer was used. A UHPLC system (Accela, Thermo Fisher Scientific, San Jose, California, USA) was coupled with an Orbitrap equipped with an electrospray ionization (ESI) source. Chromatography was performed on a reversed-phase XBridge C18 column (150 × 2.1 mm inner diameter, 3.5 μm particle size, Waters, Dublin, Ireland) at 35 °C. The flow rate was 300 μL / min, and the injection volume was 3 μL. A U3000 liquid chromatograph was used. The mobile phase was: A: 0.1% aqueous acetic acid, B: acetonitrile; the elution gradient was: 0–0.2 min, A = 90%; 0.2–6 min, A decreasing to 10%; 6–8 min, A = 10%; 8.1 min, A increasing to 90%; 8.1–10 min, A = 90%.

[0132] (1) Full scan mode: This mode rapidly alternates between positive and negative ion scanning in the m / z range of 200-1000. The ESI interface for positive ion mode is configured as follows: sheath gas, 40; auxiliary gas, 10; capillary voltage 3.8kV; capillary temperature 350℃. The AGC target is set to 2×e5. The ESI interface for negative ion mode is set to 2.9kV; sheath gas, 4; auxiliary gas, 0. The resolution for this mode is set to 70,000.

[0133] (2) The liquid chromatography method and chromatographic conditions for Full scan + ddms (first-level full scan + automatic triggering of second-level) mode are the same as above. This method uses full scan and MS2 scan alternately, the normalized collision energy is set to 20 eV, and the resolution is set to 17500 during product ion scanning.

[0134] (3) The PRM mode can be used to quantify the relative abundance of toxins and their derivatives in a sample. After screening the precursor ion in the PRM mode, dissociation is induced at a normalized collision energy (HCID), followed by fragment detection of the daughter ion in Orbitrap with a resolution set to 17500. The normalized collision energy used depends on the specific analyte and the applied collision energy (15, 30, and 45 eV).

[0135] LC-HRMS ( / MS) analysis was performed using an Xcalibur 2.1.0 (Thermo Fisher Scientific, San Jose, California, USA). Extractable ion chromatograms (EICs) of the toxin and its derivatives were studied using peak shapes, retention times (±0.2 min), and masses (±5 ppm) of the proposed biotransformation products. Chemical structures were inferred based on secondary chromatograms and neutral loss analysis of the basic structural components.

[0136] 2. Experimental Results

[0137] 2.1 FTCD catalyzes the conversion of DON toxin into the glutathione adduct DON-GSH

[0138] Figure 5A The extraction ion current spectrum (EIC) of the in vitro enzymatic reaction of LC-HRMS1 (Method 1) DON and GSH is shown. Figure 5A As shown, the extracted ion current spectrum (EIC) of DON was obtained from LC-HRMS (Full scan mode) in negative ion mode, with a m / z of 355.13984 (corresponding to [M+CH3COO]). - Form, Δ±5ppm); positive ion mode extraction yielded the DON-GSH adduct, m / z 604.21707 (corresponding to [M+H]). + (Δ±5ppm).

[0139] Figure 5B The image shows the mass spectrum of the daughter ions generated by the high-energy collisional dissociation of DON-GSH obtained from the in vitro enzymatic reaction of DON and GSH in LC-HRMS2 (Method 2), [M+H]. + (m / z 604.21707, Δ±5ppm). This was achieved by analyzing positively charged ([M+H]) + Targeted HRMS2 analysis was performed on the ions of DON-GSH to investigate the MS fragment of the epoxy adduct. Ion fragmentation of DON-GSH produced a characteristic ion with m / z 299.0939, corresponding to C 14 H 19 O5S + This characteristic ion can be attributed to the cleavage of the side chain at C-6 and the loss of the GSH moiety except for S. This fragment can further cleave to produce an m / z of 281.08482 (C 14 H 17 O4S + ), 263.07425(C 14 H 15 O3S + ) and 231.10218(C 14 H 15 O3 + The product ion at m / z 263.07425 is the base peak of the HRMS2 mass spectrum. This product ion has lost two molecules of H2O from the base at m / z 299.0939.

[0140] Following the loss of glycine, DON-GSH yields fragment ions with m / z 529.18503 (C). 23 H 33 O 10 N2S+ Fragment ions 475.17466 (C) containing dehydrated glutamate can also be obtained. 20 H 31 O9N2S + The m / z 574.20717 (C) of the side chain at C-6 was lost. 24 H 36 O 11 N3S + The loss of dehydrated glutamate from the GSH fraction of ion fragments yields a characteristic ion (C) with m / z 445.16389. 19 H 29 O8N2S + ); it can also yield 428.13733 (C) after glutamine removal. 19 H 26 O8NS + ).

[0141] The product ion has an m / z of 308.09108 (C). 10 H 18 O6N3S + This corresponds to GSH's [M+H] + The fragment ion loss of dehydrated glutamic acid yielded m / z 179.04907 (C5H). 11 O3N2S + ); Loss of glutamine yielded m / z 162.02251 (C5H9O3NS) + In addition, m / z 130.05044 (C5H8O3N) + ), m / z 145.06077(C5H9O3N2 + The product ions of ) are all related to GSH.

[0142] 2.2 FTCD catalyzes the conversion of 3-ADON toxin into the glutathione adduct 3-ADON-GSH

[0143] Figure 6A The extraction ion current spectrum (EIC) of the in vitro enzymatic reaction of 3-ADON and GSH by LC-HRMS (Method 1). Figure 6A As shown, the extracted ion current spectrum (EIC) of 3-ADON was obtained from LC-HRMS (Full scan mode) in negative ion mode, with a m / z of 397.15041 (corresponding to [M+CH3COO]). - Form, Δ±5ppm); 3-ADON-GSH adduct extracted in positive ion mode, m / z 646.22764 (corresponding to [M+H)). + (Δ±5ppm).

[0144] Figure 6B The mass spectrum of the daughter ions generated by the high-energy collisional dissociation of 3-ADON-GSH obtained from the in vitro enzymatic reaction of LC-HRMS2 (Method 2) 3-ADON and GSH is shown. [M+H]+ (m / z 646.22764, Δ±5ppm). [M+H]+ is the positively charged daughter ion. + Targeted HRMS2 analysis of the 3-ADON-GSH epoxy adduct ions revealed that ionic fragmentation of 3-ADON-GSH produces a characteristic ion with m / z 323.09539, corresponding to C 16 H 19 O5S + This characteristic ion can be attributed to the cleavage and dehydration of the side chain attached at C-6, as well as the loss of the GSH moiety excluding S. This fragment can further cleave to produce an m / z of 263.07425 (C 14 H 15 O3S + ) and 231.10218(C 14 H 15 O3 + The daughter ion at m / z 263.07425 is the base peak of the HRMS2 mass spectrum. This product ion is formed by the removal of CH3COOH at C-3 from the ion at m / z 323.09539.

[0145] After the loss of 3-ADON-GSH glycine, fragment ions with m / z 571.19560 (C) are obtained. 25 H 35 O 11 N2S + The further breakage of the side chain at C-6 can produce m / z 541.18503 (C 24 H 33 O 10 N2S + Fragment ions of ) ; Fragment ions that have lost 1 molecule of H2O m / z 628.21707 (C 27 H 38 O 12 N3S + Losing glycine yields m / z 553.18503 (C 25 H 33 O 10 N2S + The loss of dehydrated glutamic acid yields 499.17466 (C). 22 H 31 O9N2S + ).

[0146] 2.3 FTCD catalyzes the conversion of 15-ADON toxin into the glutathione adduct 15-ADON-GSH

[0147] Figure 7A EIC is the extraction ion current spectrum (EIC) of 15-ADON extracted by in vitro enzymatic reaction of LC-HRMS (Method 1) with GSH. Figure 7A As shown, the extracted ion current spectrum (EIC) of 15-ADON obtained from LC-HRMS (Full scan mode) in negative ion mode is 397.15041 (corresponding to [M+CH3COO]). - Form, Δ±5ppm); 15-ADON-GSH adduct extracted in positive ion mode, m / z 646.22764 (corresponding to [M+H)). + (Δ±5ppm).

[0148] Figure 7B Mass spectrum of the daughter ions generated by the high-energy collisional dissociation of 15-ADON-GSH obtained from the in vitro enzymatic reaction of 15-ADON and GSH using LC-HRMS2 (Method 2), [M+H]+ (m / z 646.22764, Δ±5ppm). The positively charged ([M+H]+) + Targeted HRMS2 analysis was performed on the ions of 15-ADON-GSH to investigate the MS fragment of the epoxide adduct. Ion fragmentation of 15-ADON-GSH produced a characteristic ion with m / z 311.09475, corresponding to C 15 H 19 O5S + This characteristic ion can be attributed to the cleavage of the side chain CH3COOH attached at C-15 and the loss of the GSH moiety except for S.

[0149] Similar to 3-ADON-GSH, the loss of glycine in 15-ADON-GSH yields a daughter ion with m / z 571.1956 (C). 25 H 35 O 11 N2S + The m / z of the molecule of H2O removed is 628.21707 (C). 27 H 38 O 12 N3S + Losing glycine yields m / z 553.18503 (C 25 H 33 O 10 N2S + The loss of dehydrated glutamate yields m / z 499.17466 (C). 22 H 31 O9N2S+ ).

[0150] Characteristic ion m / z 440.13736 (C) 20 H 26 O8NS + Losing dehydrated glutamic acid yields m / z 311.09475 (C 15 H 19 O5S + Fragment ions; Characteristic ion m / z 450.15471 (C 17 H 28 O9N3S + The loss of glycine yielded m / z 375.12267 (C 15 H 23 O7N2S + ) daughter ion, loss of dehydrated glutamic acid to obtain m / z 321.1121 (C 12 H 21 O6N2S + Furthermore, this ion can also remove two molecules of H₂O to form a daughter ion with m / z 414.13295 (C). 17 H 24 O7N3S + The characteristic ion loss of glycine yields m / z 339.10091 (C). 15 H 19 O5N2S + The loss of dehydrated glutamic acid yielded m / z 285.09035 (C). 12 H 17 O4N2S + Further dehydration of this fragment can produce m / z 267.07979 (C). 12 H 15 O3N2S + ). m / z related to GSH 145.06077 (C5H9O3N2) + The characteristic ion (Δ±5ppm) is the base peak of the mass spectrum.

[0151] 2.4 FTCD catalyzes NIV toxin conversion into glutathione adduct NIV-GSH

[0152] like Figure 8A As shown, the extracted ion current spectrum (EIC) of NIV was obtained from LC-HRMS (Full scan mode) in negative ion mode, with a m / z of 371.13366 (corresponding to [M+CH3COO]). - Form, Δ±5ppm); NIV-GSH adduct extracted in positive ion mode, m / z 620.21199 (corresponding to [M+H)).+ (Δ±5ppm).

[0153] Figure 8B The image shows the mass spectrum of the daughter ions generated by the high-energy collisional dissociation of NIV-GSH obtained from the in vitro enzymatic reaction of LC-HRMS2 (Method 2) NIV and GSH, [M+H]. + (m / z 620.21199, Δ±5ppm). This was achieved by analyzing positively charged ([M+H]) + Targeted HRMS2 analysis was performed on the NIV-GSH epoxy adduct ions to investigate their MS fragmentation. Ion fragmentation of NIV-GSH produced a product ion with m / z 229.08652, corresponding to C2. 14 H 13 O3 + The product ion can be attributed to the cleavage of the side chain at C-6, the breakage of 3 H2O molecules, and the loss of the GSH moiety, while the structure retains the basic framework of NIV.

[0154] Following the loss of glycine, NIV-GSH yields a daughter ion with m / z 545.17995 (C). 23 H 33 O 11 N2S + The fragment ion 491.16938 (C) of dehydrated glutamate can also be obtained. 20 H 31 O 10 N2S + The side chain breaks at C-6, forming m / z590.20142(C). 24 H 36 O 12 N3S + The GSH portion of this ion loses dehydrated glutamate to yield a daughter ion (C) with m / z 461.15881. 19 H 29 O9N2S + ).

[0155] [M+H] + The loss of glutamine by GSH yields a daughter ion with m / z 162.02251 (C5H9O3NS). + ); Loss of dehydrated glutamic acid yields m / z 179.04907 (C5H) 11 O3N2S + This ion is the most prominent product ion in HRMS2 mass spectrometry. Furthermore, the daughter ion has a m / z of 130.05044 (C5H8O3N). + ) and daughter ion m / z 145.06077 (C5H9O3N2) + All of them are related to GSH.

[0156] 2.5FTCD catalyzes the conversion of Fus-X toxin into the glutathione adduct Fus-X-GSH.

[0157] Figure 9A The extraction ion current spectrum (EIC) of the in vitro enzymatic reaction of Fus-X and GSH by LC-HRMS (Method 1). Figure 9A As shown, the extracted ion current spectrum (EIC) of Fus-X obtained from LC-HRMS (Full scan mode) in negative ion mode is 377.12069 (corresponding to [M+Na)). + Form, Δ±5ppm); Fus-X-GSH adduct extracted in positive ion mode, m / z 662.22255 (corresponding to [M+H)). + (Δ±5ppm).

[0158] Figure 9B This is the mass spectrum of the daughter ions generated by the high-energy collisional dissociation of Fus-X-GSH obtained from the in vitro enzymatic reaction of LC-HRMS2 (Method 2) Fus-X and GSH. The mass spectra were obtained by analyzing the positively charged ([M+H)) daughter ions. + Targeted HRMS2 analysis was performed on the FusX-GSH epoxy adduct ions to investigate their MS fragmentation. Ion fragmentation of FusX-GSH produced a product ion with m / z 297.07973, corresponding to C2. 14 H 17 O5S + The product ion can be attributed to the cleavage of the side chain at C-4, the cleavage of the side chain at C-6, and the loss of the GSH moiety except for S. The structure retains only the basic framework of Fus-X.

[0159] Following the loss of glycine, FusX-GSH yields a daughter ion with m / z 587.19051 (C). 25 H 35 O 12 N2S + The side chain breaks at C-6, resulting in m / z 632.21198 (C). 26 H 38 O 13 N3S + The characteristic ion loss of dehydrated glutamic acid yields m / z 503.16937 (C). 21 H 31 O 10 N2S + The loss of glutamine yields m / z 486.14281 (C) (the daughter ion of glutamine). 21 H 28 O 10 NS +). Among them, m / z 503.16937 (C 24 H 36 O 12 N3S + The daughter ion of ) is the most prominent product ion in HRMS2 mass spectrometry.

[0160] [M+H] + The loss of glutamine by GSH yields a daughter ion with m / z 162.02251 (C5H9O3NS). + ); Loss of dehydrated glutamic acid yields m / z 179.04907 (C5H) 11 O3N2S + Furthermore, the daughter ion m / z is 130.05044 (C5H8O3N). + ) and daughter ion m / z 145.06077 (C5H9O3N2) + All of them are related to GSH.

[0161] 2.6FTCD catalyzes the conversion of DAS toxin into the glutathione adduct DAS-GSH

[0162] Figure 10A The extraction ion current (EIC) spectrum of DAS extracted by in vitro enzymatic reaction with GSH via LC-HRMS (Method 1) is shown. The EIC spectrum of DAS was obtained from LC-HRMS (Full scan mode) in positive ion mode, m / z 389.15707 (corresponding to [M+Na)). + Form, Δ±5ppm); DAS-GSH adduct, m / z 674.25894 (corresponding to [M+H]). + (Δ±5ppm).

[0163] Figure 10B The image shows the mass spectrum of the daughter ions generated by the high-energy collisional dissociation of DAS-GSH obtained from the in vitro enzymatic reaction of DAS and GSH in LC-HRMS2 (Method 2), [M+H]. + (m / z 674.25894, Δ±5ppm). This was achieved by analyzing positively charged ([M+H]) + Targeted HRMS2 analysis was performed on the ions of DAS-GSH to investigate the MS fragment of the DAS-GSH epoxy adduct. Ion fragmentation of DAS-GSH produced a product ion with m / z 229.12231, corresponding to C 15 H 17 O2 + The product ion can be attributed to the cleavage and dehydration of the side chain CH3COOH attached at C-4 and C-15, as well as the loss of the GSH moiety.

[0164] Following the loss of glycine, DAS-GSH yields a daughter ion with m / z 599.22690 (C). 27 H 39 O 11 N2S + ); Loss of glutamine yields m / z 528.18977 (C 24 H 34 O 10 NS + The daughter ion of ) can also be obtained; the daughter ion of lost dehydrated glutamic acid 545.21633 (C) can also be obtained. 24 H 37 O 10 N2S + The loss of CH3COOH yields m / z 614.23781 (C). 27 H 40 O 11 N3S + Characteristic ions of ).

[0165] m / z related to GSH: 130.05044 (C5H8O3N) + m / z 145.06077(C5H9O3N2) + ), m / z162.02251(C5H9O3NS + m / z 179.04907(C5H) 11 O3N2S + Among the daughter ions, the characteristic ion that loses dehydrated glutamate m / z is at 179.04907 (C5H). 11 O3N2S + () is the base peak of the mass spectrum.

[0166] 2.7 FTCD catalyzes the conversion of HT-2 into the glutathione adduct HT-2-GSH

[0167] Figure 11A The extraction ion current (EIC) spectra of HT-2 extracted by in vitro enzymatic reaction with GSH using LC-HRMS (Method 1) are shown. The EIC of HT-2 obtained from LC-HRMS (Full scan mode) in positive ion mode is m / z 447.19894 (corresponding to [M+Na]+, Δ±5ppm); the EIC of the HT-GSH adduct is m / z 732.30080 (corresponding to [M+H]+, Δ±5ppm).

[0168] Figure 11B The image shows the mass spectrum of the daughter ions generated by the high-energy collisional dissociation of the HT-GSH adduct obtained from the in vitro enzymatic reaction of LC-HRMS2 (Method 2) HT-2 and GSH, [M+H].+ (m / z 732.30080, Δ±5ppm). This was achieved by analyzing positively charged ([M+H]) + Targeted HRMS2 analysis was performed on the HT-GSH epoxy adduct ions to investigate their MS fragmentation. HT-GSH fragmentation produced a product ion with m / z 295.10048, corresponding to C 15 H 19 O4S + The product ion can be attributed to the cleavage of ((CH3)2CHCH2COOH) at C-8, the cleavage of CH3COOH at C-15, and the loss of the GSH moiety except for S. This structure retains the basic framework of HT-2. The m / z value of 274.10335 is [M+H]. + This is caused by the breakage of the -SH bond in the GSH form, resulting in the loss of neutral H2S.

[0169] After the side chains of HT-GSH break at C-8 and C-15, an m / z of 570.21226 (C) can be obtained. 25 H 36 O 10 N3S + The characteristic ion of ), which loses glycine to obtain m / z 495.18022 (C). 23 H 31 O8N2S + The loss of dehydrated glutamic acid yields m / z 441.16965 (C). 20 H 29 O7N2S + The loss of glutamine yields m / z 424.14309 (C). 20 H 26 O7NS + Fragment ions. The base peak of the mass spectrum is at m / z 441.16965.

[0170] In addition, m / z 130.05044 (C5H8O3N) associated with GSH was detected. + m / z 145.06077(C5H9O3N2) + m / z 162.02251(C5H9O3NS) + m / z 179.04907(C5H) 11 O3N2S + )ion.

[0171] 2.8 FTCD catalyzes the conversion of T-2 into the glutathione adduct HT-2-GSH

[0172] Figure 12AThe extraction ion current (EIC) spectrum of T-2 obtained by in vitro enzymatic reaction with GSH via LC-HRMS (Method 1) is shown. The EIC spectrum of T-2 was obtained from LC-HRMS (Full scan mode) in positive ion mode, m / z 489.20950 (corresponding to [M+Na)). + Form, Δ±5ppm); adduct of T2-GSH, m / z 774.31136 (corresponding to [M+H]). + (Δ±5ppm).

[0173] Figure 12B The image shows the mass spectrum of the daughter ions generated by the high-energy collisional dissociation of the T2-GSH adduct obtained from the in vitro enzymatic reaction of LC-HRMS2 (Method 2) T-2 with GSH, [M+H]. + (m / z 774.31136, Δ±5ppm). This was achieved by analyzing positively charged ([M+H]) + Targeted HRMS2 analysis was performed on the T2-GSH epoxy adduct ions to study their MS fragmentation. T2-GSH fragmentation produced a product ion with m / z 337.11105, corresponding to C 17 H 21 O5S + The product ion can be attributed to the breakage of the side chains attached at C-8 and C-15, and the loss of the GSH moiety except for S. The structure retains the basic framework of T-2. The m / z value of 274.10335 is [M+H]. + This is caused by the breakage of the -SH bond in the GSH form, resulting in the loss of neutral H2S.

[0174] After the side chains of T2-GSH break at C-8 and C-15, an m / z of 612.22283 (C) can be obtained. 27 H 38 O 11 N3S + The characteristic ion of is the base peak of the mass spectrum. Losing glycine from this ion yields m / z 537.19079 (C). 25 H 33 O9N2S + The loss of dehydrated glutamic acid yields m / z 483.18022 (C). 22 H 31 O8N2S + ); Loss of glutamine yielded m / z 466.15366 (C 22 H 28 O8NS + Fragment ions.

[0175] Similar to HT2-GSH, 130.05044(C5H8O3N) associated with GSH was detected.+ m / z 145.06077(C5H9O3N2) + m / z 162.02251(C5H9O3NS) + m / z 179.04907(C5H) 11 O3N2S + )ion.

[0176] 3. Experimental Conclusions

[0177] The active polypeptide of this invention can efficiently catalyze trichothecene toxins (including DON, 3DON, 15ADON, FUS-X, NIV, T2, H-T2, DAS, etc.) into glutathione adducts in vitro. Furthermore, secondary spectra show that the formation of this adduct disrupts the epoxy ring structure that plays a major role in the toxicity of trichothecene, thereby significantly reducing the toxicity of the toxins.

[0178] IV. Cytotoxicity assays of trichothecene toxin GSH derivatives

[0179] 1. Cell Culture

[0180] Using DMEM basal medium supplemented with 10% fetal bovine serum and 500 μl of penicillin-streptomycin, pancreatic cancer cell line (PATU8988), human embryonic kidney cell line 293 (293T), and normal human esophageal epithelial cells (HEEC) were cultured in a constant temperature incubator at 37°C and 5% CO2. When the cells reached 80%–90% of the flask wall height, they were passaged every 2–3 days. Cells were collected by trypsin digestion and passaged again. Based on the cell growth status, cells in the logarithmic growth phase were selected for experiments.

[0181] 2. CCK8 assay for cytotoxicity

[0182] Cell proliferation and virulence were analyzed using the Cell Counting Kit-8 (CCK-8) reagent. Three cell lines in logarithmic growth phase were seeded into 96-well plates, 100 μL per well (approximately 5 × 10⁻⁶ cells / well). 3 Cells were cultured at 37°C and 5% CO2 for 24 hours using conventional methods. The culture medium was then discarded, and the cells were divided into groups. Each group had three replicates for observation. The treatment methods for each group were as follows: blank group (zeroing well containing only culture medium), control group (DMEM medium containing 10% fetal bovine serum), and trichothecene compounds and their corresponding glutathione adducts produced after enzymatic reactions were set at low, medium, and high concentration gradients. After 48 hours of culture at 37°C, 10 μL of CCK8 solution was added to each well for further culture. After 2 hours, the culture supernatant was carefully aspirated from the wells, and the OD value of each well was measured at 450 nm using a multi-wavelength microplate reader. Cell viability was then calculated.

[0183] 3. Experimental Results

[0184] At a cell concentration of 5×10 7 L -1 Plates were seeded, and the OD450 values ​​of pancreatic cancer cell line (PATU8988), human embryonic kidney cell line 293 (293T), and normal human esophageal epithelial cells (HEEC) were detected by CCK-8 microplate reader after 48 h of treatment with trichothecene compounds and their corresponding glutathione adducts produced by enzymatic reactions. Each group was observed in triplicate. The treatment methods for each group were as follows: blank group (i.e., zeroing well containing only culture medium), control group (DMEM medium containing 10% fetal bovine serum), and the concentrations of trichothecene compounds and their corresponding glutathione adducts produced by enzymatic reactions were set according to literature results. Results are as follows: Figure 13 As shown.

[0185] like Figure 13 As shown, after treatment with corresponding concentrations of trichothecene compounds (DON, 3-ADON, 15-ADON, FUS-X, NIV, T-2, HT-2, DAS) for 48 h, the viability of PATU8988, 293T, and HEEC all decreased sharply, indicating that different trichothecene compounds have significant toxicity to cells. However, the corresponding derivatives of trichothecene compounds DON, 3-ADON, 15-ADON, FUS-X, NIV, T-2, HT-2, and DAS produced after the reaction showed that the cell viability was basically the same as that of the blank control at the same concentration, indicating that the corresponding glutathione adducts of the above eight trichothecene compounds have basically no toxic effect on cells.

[0186] The above experiments revealed that all trichothecene compounds strongly inhibited cell activity, while the corresponding glutathione adducts of most trichothecene compounds had almost no effect on cell activity at the same concentration. In conclusion, the formation of glutathione adducts from trichothecene compounds can significantly reduce the cytotoxic effects of these compounds.

[0187] V. Host cells expressing FTCD-active peptides and their functional studies

[0188] 1. Construction of yeast expression plasmid pPICZαA-FTCD

[0189] The deoxygenase gene cDNA from decaploid *Thinopyrum elongatum* is 865 bp in length (SEQ ID No.: 36) and does not contain Bsp119I and XbaI restriction sites. The designed primer sequence is as follows:

[0190] F: 5'-ATTA TTCGAA AGAAATCCACCCATCGTCATCACC-3'

[0191] R: 5'-TTGT TCTAGA CTACTTCACCTCGGCATACTTGTC-3'

[0192] The underlined portion indicates restriction endonuclease sites. The complete cDNA sequence was obtained by PCR. After purification of the PCR product, it was digested with Bsp119I and XbaI, and the expression vector pPICZαA was also digested. The large fragment of the vector and the target gene fragment were recovered separately. The recovered fragments were then ligated using T4 DNA ligase and transformed into E. coli DH5α. After colony PCR identification, positive single clones were sequenced for verification.

[0193] 2. Transformation of Pichia pastoris

[0194] First, linearize the recombinant plasmid using Sac I. Boil 1 ml of single-stranded DNA sample for 5 minutes, then rapidly cool on ice. Keep on ice. Centrifuge the competent yeast cells and remove LiCl by pipetting. Add 240 μl of 50% polyethylene glycol, 36 μl of 1M LiCl, 25 μl of 2 mg / ml single-stranded DNA, and 50 μl of plasmid DNA (5-10 μg) in sterile water in sequence. Vortex each tube vigorously until the cell pellet is completely mixed (about 1 minute). Incubate the tubes at 30°C for 30 minutes. Heat shock in a 42°C water bath for 20-25 minutes. Centrifuge the cells to obtain the pellet. Resuspend the pellet in 1 ml of YPD and incubate with shaking at 30°C. After 1 hour and 4 hours, incubate with an appropriate concentration of Zeocin. TM Inoculate 25 to 100 μl onto YPD plates. Incubate the plates at 30°C for 2-3 days.

[0195] Ten single colonies were selected for enrichment culture, yeast chromosomal DNA was extracted, and positive recombinant bacteria were detected by PCR. PCR identification is usually performed using universal primers for pPICZαA. If the yeast expression vector pPICZαA is used as a template, a target fragment of approximately 588 bp can be amplified; if pPICZαA-FTCD is used as a template, the target band plus a target fragment of 588 bp will be amplified.

[0196] 3. Enzyme Expression and Toxin Processing

[0197] The selected positive yeast colonies (X33 / pPICZαA-FTCD) and negative yeast colonies (X33 / pPICZαA) were inoculated separately into 25 ml of BMGY medium and cultured at 28℃–30℃ until the OD600 reached 2–6. After centrifugation at room temperature, the supernatant was discarded, and the cells were collected. The cells were resuspended in BMGY liquid medium until the OD600 reached approximately 1, and then transferred to 500 ml Erlenmeyer flasks and cultured at 28℃–30℃. Methanol was added every 24 h to a final concentration of 0.5% to maintain induced expression. After 48 h of induction, 5 ml of the bacterial culture was aliquoted into 15 ml centrifuge tubes, and various trichothecene compounds were added to a final concentration of 25 μg / ml. Induction was continued for another 48–72 h, and the cells were collected for LC-HRMS analysis.

[0198] Simultaneously, after inducing protein expression for 48 h in positive yeast single colonies (X33 / pPICZαA-FTCD) and negative yeast single colonies (X33 / pPICZαA), culture medium was added to dilute the cultures to 1, 1 / 5, and 1 / 20 (initial OD = 0.01). These cultures were then cultured for 5 days on solid media containing 400 μM DON and YPDA without DON, and their growth was observed. The tolerance of transgenic yeast overexpressing the active peptide and the blank vector to DON was compared.

[0199] 4. LC-HRMS

[0200] Centrifuge the dispensed samples and discard the supernatant. Quickly freeze in liquid nitrogen, add a small amount of quartz sand, grind with a plastic grinding rod, and then add 1.3 ml of pre-cooled 75% methanol-water solution (containing 0.1% formic acid). Shake for 10 seconds, sonicate at room temperature for 30 minutes, and transfer the supernatant to a new centrifuge tube. Concentrate under vacuum to a dry powder. Resuspend in 100 μL of 20% acetonitrile before injection, filter through a 0.22 μm filter membrane, and transfer to a vial for LC-HRMS detection. The detection method is the same as described above.

[0201] 5. Experimental Results

[0202] 5.1 LC-HRMS Results

[0203] LC-HRMS results as follows Figure 14 As shown. DON-GSH adducts were detected in yeast expressing active peptides treated with DON in positive ion mode using LC-HRMS (Full scan), m / z 604.21730 (corresponding to [M+H]). + 3-ADON-GSH adduct was detected in yeast expressing active peptides treated with 3-ADON, m / z 646.22764 (corresponding to [M+H, Δ±5ppm); 3-ADON-GSH adduct was detected in yeast treated with 3-ADON, m / z 646.22764 (corresponding to [M+H]). +(Δ±5ppm); 15-ADON-GSH adduct was detected in yeast treated with 15-ADON to express active peptides, m / z 646.22764 (corresponding to [M+H)). + (Δ±5ppm); NIV-GSH adducts were detected in yeast expressing active peptides treated with NIV, m / z 620.21199 (corresponding to [M+H)). + (Δ±5ppm); DAS-GSH adducts were detected in yeast expressing active peptides treated with DAS, m / z 674.25894 (corresponding to [M+H), Δ±5ppm); + (Δ±5ppm); The "HT2-2H"-GSH adduct was detected in yeast expressing active peptides treated with HT-2, m / z 730.28515 (corresponding to [M+H)). + (Δ±5ppm); The "T2-2H"-GSH adduct was detected in yeast expressing FTCD under T-2 treatment, m / z 772.29572 (corresponding to [M+H)). + (Δ±5ppm). In contrast, no GSH adduct derivatives were detected in the corresponding controls.

[0204] LC-HRMS results showed that transferring the deepoxidase gene into Pichia pastoris efficiently catalyzes trichothecene toxins (including DON, 3-DON, 15-ADON, FUS-X, NIV, T-2, HT-2, and DAS) into glutathione adducts. The transgenic yeast exhibited enhanced toxin resistance, demonstrating that FTCD can use trichothecene toxins as substrates and catalyze them into the corresponding GSH adducts, thereby playing a role in in vivo detoxification.

[0205] 5.2 Results of the DON tolerance experiment of transgenic yeast

[0206] The growth viability of transgenic yeast overexpressing FTCD and the blank vector was compared on YPDA medium with and without DON. Yeast culture medium was supplemented with a series of dilutions of 1, 1 / 5, and 1 / 20 (initial OD = 0.01), and cultured at 30°C for 5 days. Growth was then observed. Results are as follows: Figure 15 As shown, the results showed that the transgenic yeast overexpressing FTCD had significantly higher growth viability on DON-containing medium than the transgenic yeast with the blank vector.

[0207] In the DON tolerance experiment of transgenic yeast, it was found that on YPDA medium containing 400 μm DON, the growth activity of transgenic yeast containing FTCD was significantly higher than that of transgenic yeast with blank vector. This further indicates that FTCD can be expressed in yeast and can catalyze the reaction of glutathione with DON and other trichothecene compounds to detoxify, thereby improving the yeast's tolerance to DON.

[0208] VI. Functional analysis of homologous sequence genes

[0209] Based on the sequence of the deepoxidase gene (SEQ ID No.:36) obtained from *Thinopyrum longicornis*, BLASTN alignment was performed using NCBI. Under default parameters, no highly homologous annotated genes were found. However, according to... Information on homologous genes exists within this genus. The inventors, in collaboration with other laboratories, searched genomic databases and obtained 11 sequences originating from this genus, as shown in SEQ ID No.:60-70. Figure 16 As shown, these sequences share more than 90% sequence identity with the deoxygenase gene of the decaploid *Thinopyrum longicornis*. Furthermore, the inventors also isolated a gene from the diploid *Thinopyrum longicornis* with 98% sequence identity to the decaploid deoxygenase gene, the sequence of which is shown in SEQ ID No.:37.

[0210] These genes were transformed into yeast cells using the same method described above and expressed as the corresponding proteins, the amino acid sequences of which are shown in SEQ ID No.:25-35. Analysis was performed using LC-HRMS. Figure 17 As shown, the other 12 homologous sequences were transformed into Pichia pastoris and treated with DON. LC-HRMS analysis showed the formation of DON-GSH in all sequences. A distinct and specific peak was observed at RT = 1.68 min, which was the GSH adduct at C-13 (via de-epoxy group). The ion chromatogram of the DON-treated transgenic yeast extracted by LC-HRMS (Method 1) showed that the DON-GSH adduct was extracted in positive ion mode, with m / z 604.21730 (corresponding to [M+H]). + (Δ±5ppm).

[0211] Based on the above analysis, the inventors further analyzed the conservation among the proteins produced by these homologous genes, and obtained polypeptide fragments with amino acid sequences between positions 25-62, amino acid sequences between positions 92-110, and amino acid sequences between positions 144-184.

[0212] VII. FTCD Mutation Research

[0213] Using induced gene mutagenesis technology (TILLING), the decyclooxygenase gene (sequence shown in SEQ ID No.:36) derived from the decaploid variety of *Thinopyrum longicornis* was randomly mutated, resulting in mutants with altered amino acid sequences of 22 amino acids. The amino acid sequences of these mutants are shown in SEQ ID Nos:3-24, respectively. Functional analysis revealed that all 22 mutants retained the original decyclooxygenation activity to varying degrees. Two termination mutations were observed, terminating at amino acids 209 and 243, respectively; however, these two terminations did not lead to a complete loss of enzyme function. Therefore, it is indicated that the functional domain of this enzyme is primarily the N-terminus.

[0214] Sequence homology analysis revealed two relatively conserved regions: the region between positions 92-104 and the region between positions 144-184. These two conserved regions are presumed to be important areas related to catalytic activity. The region between these two conserved regions exhibits significant variation among different species. Therefore, it is speculated that the region between these two conserved regions may be a connecting region.

[0215] To verify the above hypothesis, the inventors designed a series of deletion mutants based on the mutant materials obtained from the previous screening of the Tilling population. Specifically, specific primers containing homologous sequences of the cloning vector were designed using the sequence of SEQ ID No. 36 as a template. The specific mutant types are as follows:

[0216] Table 1. Types of Deletion Mutants

[0217]

[0218] Note:

[0219] Linker1 corresponds to the amino acid sequence between 105 and 142 in SEQ ID No. 25;

[0220] Linker2 corresponds to the amino acid sequence between 103 and 141 in SEQ ID No. 26;

[0221] Linker3 corresponds to the amino acid sequence between 107 and 148 in SEQ ID No. 28;

[0222] Linker4 corresponds to the amino acid sequence between 106 and 143 in SEQ ID No. 30;

[0223] Linker5 is an artificial sequence GGGSGGSGG.

[0224] The specific experimental steps are as follows:

[0225] 1. The gene sequence corresponding to the above-mentioned deletion mutant was obtained by PCR, and the plasmid pET28a was constructed using the designed NcoI and BamHI. This plasmid was then transformed into *E. coli* DH5α, and colony PCR and sequencing verification were performed. The correct recombinant expression vector plasmid was transformed into competent *E. coli* strain BL21(DE3) cells. Protein expression was induced by shaking at 37°C for 4 hours with 0.5 mmol / L IPTG. The cells were collected, resuspended in PBS, and sonicated. The supernatant was collected and purified using a Ni column and molecular sieve. The purified protein was quantified using the BCA protein quantification method.

[0226] 2. In vitro enzymatic reaction

[0227] DON, 3-DON, and 15-ADON toxins (1 mg) were dissolved in freshly prepared GSH (30.7 mg, 100 μmol) in PBS buffer. Equal amounts of protein were added according to the in vitro purified protein concentration of several different FTCD deletion mutants, and the mixture was incubated in a water bath at 25°C for 24 h.

[0228] 3. LC-HRMS ( / MS) analysis

[0229] The in vitro reaction solution was filtered through a 0.22 μm filter and transferred to vials for LC-HRMS analysis. The relative abundance of toxins and their derivatives in the samples was quantified using the PRM mode. Enzyme activity was calculated based on substrate conversion per unit time, and the effect of different deletion mutations on protein activity was determined based on this result. DON, 3-DON, and 15-ADON toxins (1 mg) were dissolved in freshly prepared GSH (30.7 mg, 100 μmol) in PBS buffer, and an equal amount of protein was added according to the concentration of in vitro purified protein from several different FTCD deletion mutants. The mixture was incubated in a water bath at 25 °C for 24 h. Samples were taken for LC-HRMS analysis, and the effect of different deletion mutations on enzyme activity is shown in Table 2.

[0230] Table 2. Experimental results of enzyme activity of different mutants

[0231]

[0232] Table 2 shows that mutants lacking 1-90 amino acids have little impact on enzyme activity, while the loss of the first 95 amino acids has a significant impact, greatly reducing enzyme activity. Furthermore, the loss of the first 150 amino acids results in an inactive protein. On the other hand, sequences containing the conserved region predicted in this invention, i.e., mutants containing 92-184 amino acids, while experiencing activity impairment, still exhibit a high level of specific enzyme activity. Therefore, this is largely consistent with the predicted findings.

[0233] Furthermore, in the mutation results of the predicted linker region, replacing the sequence of this region in the decaploid *Leymus chinensis* with corresponding sequences from other genera showed that the activity remained essentially unchanged. Further replacement of this region with the artificially designed linker sequence GGGSGGSGG also had little impact on its activity. These results are largely consistent with the predictions.

[0234] 2. Mutation analysis of key amino acids in conserved regions

[0235] Based on the identification of the key regions of enzyme activity, the inventors further mutated the amino acids in two conserved regions in the hope of discovering key amino acids.

[0236] Specifically, gene sequences with different mutant combinations were obtained through gene synthesis. These gene sequences were expressed and purified in *E. coli*. The obtained mutant peptides were used for in vitro enzymatic reactions, and enzyme activities were analyzed using LC-HRMS / MS. The results are shown in Table 3.

[0237] Table 3. Effects of amino acid mutations in conserved regions on activity (experimental)

[0238]

[0239]

[0240]

[0241]

[0242]

[0243] Several variable sites exist within the conserved sequence of FTCD. Changes in amino acids at positions 94, 95, 100, 151, 155, 158, 162, 163, 165, 174, 175, and 178 allow FTCD to maintain a certain level of activity. Within these variable sites, amino acid changes at different sites have varying effects on FTCD activity. Changes at positions 100 and 178 have a significant impact, reducing activity by approximately 60%. Changes at other variable sites do not diminish FTCD activity by more than 50%.

[0244] Although the invention has been described with reference to exemplary embodiments, it should be understood that the invention is not limited to the disclosed exemplary embodiments. Various adjustments or changes may be made to the exemplary embodiments described in this specification without departing from the scope or spirit of the invention. The scope of the claims should be interpreted in the broadest possible sense to cover all modifications and equivalent structures and functions. sequence list <110> Shandong Nongzhian Biotechnology Co., Ltd. <120> Polypeptides with deepoxidation catalytic activity, their encoded nucleic acids, and their uses <130> BH2000025-1 <141> 2020-03-05 <160> 70 <170> SIPOSequenceListing 1.0 <210> 1 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 1 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Leu Asp Lys Tyr Ala Glu Val Lys 275,280 <210> 2 <211> 281 <212> PRT <213> Elytrigia elongatum <400> 2 Met Ala Thr Ser Thr Ser Thr Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 There is no His Asp Pro but there is no Thr Asp but there is no Gly Asp. 85 90 95 Ile Tyr Leu Gln Arg Thr Tyr Pro Only Ser Gly Only Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln 115 120 125 Gln Leu Leu Phe Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 3 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 3 Put Ala Thr Create Ala Create Thr Create Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Val Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 4 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 4 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Pro Thr Ala Ile 1 5 10 15 Ala Gly Ala Pro Pro Val Ala Gly Thr Cys Cys Ala Val Ala Pro Thr 20 25 30 Leu Ser Ala Leu Ala Leu Ala Pro Leu Ala Val Pro Thr Thr Thr Thr 35 40 45 Thr Val Leu Met Pro Ala Ile Ser Ser Val Ala Ala Ser Leu Ala Val 50 55 60 Pro Ala Cys Ala Leu Pro Ala Ala Gly Ser Ala Pro Ala Thr Leu Pro 65 70 75 80 Ile Ile His Ala Pro Ala Thr Ala Ser Leu Val Gly Ala Ser Pro Ala 85 90 95 Ile Ala Ala Thr Leu Gly Ala Thr Thr Leu Ala Ser Gly Ala Gly Ala 100 105 110 Leu Pro Pro Pro Gly Leu Leu Ala Thr Ala Val Gly Ala Ala Met Pro 115 120 125 Gly Leu Leu Ile Pro Leu Ser Gly Ile Ala Ala Ser Pro Gly Leu Ala 130 135 140 Ala Thr Ala Ala Pro Ala Ser Ala Val Ala Ala Ala Pro Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Ala Pro Ala Thr Ala Ala 165 170 175 Val Thr Leu Ala Gly Pro Val Ala Ala Ala Gly Leu Ser Ser Thr Ala 180 185 190 Ala Leu Gly Met Val Gly Gly Ala Ala Ala Leu Met Met Gly Ser Leu 195 200 205 Ala Ala Met Leu Gly Ala Leu Ala Ala Leu Pro Ala Leu Ala Ala Ser 210 215 220 Gly Pro Pro Leu Leu Gly Gly Ala Ala Thr Thr Ala Ala Met Ile Val 225 230 235 240 Gly Gly Thr Leu Ala Met Met Ala Ala Thr Leu Pro Val Ser Gly Thr 245 250 255 Gly Gly Ala Ala Ala Cys His Gly Ala Ile Pro Gly Gly Leu His Ala 260 265 270 Ala Leu Ala Leu Thr Ala Gly Val Leu 275 280 <210> 5 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 5 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Val Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 6 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 6 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Ile 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 7 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 7 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Phe Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 8 <211> 208 <212> PRT <213> Elytrigia ponticum <400> 8 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 <210> 10 <211> 242 <212> PRT <213> Elytrigia ponticum <400> 10 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly <210> 10 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 10 Put Ala Thr Create Ala Create Thr Create Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Lys Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 11 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 11 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Ser Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 12 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 12 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Lys Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 13 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 13 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Phe Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 14 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 14 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Leu Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Leu Asp Lys Tyr Ala Glu Val Lys 275,280 <210> 15 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 15 Met Ala Thr Ser Ala Ser Thr Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Tyr Leu Gln Arg Thr Tyr Pro Only Ser Gly Only Gly Asp 100 105 110 Leu Phe Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile His Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 16 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 16 Put Ala Thr Create Ala Create Thr Create Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Thr Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 17 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 17 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Phe Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 18 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 18 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Ile Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 19 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 19 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Val Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 20 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 20 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Val Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 21 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 21 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Val Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 22 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 22 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Val Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 23 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 23 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Met Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 24 <211> 281 <212> PRT <213> Elytrigia ponticum <400> 24 Met Ala Thr Ser Ala Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Pro 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg His Ala Gly Leu Ser Ser Trp Asp 180 185 190 Asp Leu Glu Met Val Gly Glu Ala Arg Asp Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Lys Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Met Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Val Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Only Leu Asp Lys Tyr Only Glu Val Lys 275,280 <210> 25 <211> 280 <212> PRT <213> Epichloe bromicola <400> 25 Met Ala Thr Ser Thr Ser Ile Ser Thr Pro Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Val Gly Asp Ser Phe Asp 85 90 95 Ile Tyr Leu Gln Arg Thr Tyr Pro Only Ser Gly Only Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln 115 120 125 Leu Leu Ile Pro Leu Ser Glu Thr Arg Ala Ser Pro Glu Leu Ala Asp 130 135 140 Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val 145 150 155 160 Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val 165 170 175 Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp 180 185 190 Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg 195 200 205 Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly 210 215 220 Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly 225 230 235 240 Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln 245 250 255 Glu Val Arg Ala Cys His Gly Ala Val Phe Gly Gln Leu His Asp Ala 260 265 270 Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 26 <211> 279 <212> PRT <213> Epichloe amarillans <400> 26 Met Ala Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile Ala Gln 1 5 10 15 Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp Lys Ser 20 25 30 Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr Trp Val 35 40 45 Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Leu Pro Ala 50 55 60 Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe Asp Thr Leu Pro Ile Ile 65 70 75 80 His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser Phe Asp Ile Ala 85 90 95 Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp Leu Phe 100 105 110 Pro Pro Gln Lys Leu Asp Tyr Ala Ala Gly Arg Asp Thr Gln Leu Leu 115 120 125 Ile Pro Leu Ser Glu Val Arg Ala Ala Ser Pro Glu Leu Ala Asp Tyr 130 135 140 Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val Gly 145 150 155 160 Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val Thr 165 170 175 Lys Ala Glu Phe Val Arg Arg Ala Ala Val Ser Ser Trp Asp Asp Leu 180 185 190 Asp Met Val Gly Asp Ala Arg Asp Lys Met Met Gln Ser Leu Arg Asn 195 200 205 Thr Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly Pro 210 215 220 Phe Leu Leu Gly Pro Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly Gly 225 230 235 240 Trp Leu Arg Met Met Arg Ala Thr Leu Pro Pro Ser Glu Trp Gln Ala 245 250 255 Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp Ala Leu 260 265 270 Asp Lys Tyr Ala Glu Val Lys 275 <210> 27 <211> 264 <212> PRT <213> Epichloe baconii <400> 27 Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile 1 5 10 15 Phe Tyr Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala 20 25 30 Val Asn Pro Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro 35 40 45 Tyr Thr Thr Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala 50 55 60 Ser Leu Asn Leu Pro Ala Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe 65 70 75 80 Asn Thr Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Val Gly 85 90 95 Asp Ser Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Asp Thr Gln Leu 100 105 110 Leu Ile Pro Leu Ser Glu Val Arg Ala Ala Ser Ser Asp Leu Ala Asp 115 120 125 Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val 130 135 140 Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val 145 150 155 160 Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Asp Asp 165 170 175 Phe Glu Met Ala Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg 180 185 190 Asn Thr Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly 195 200 205 Pro Phe Leu Leu Gly Arg Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly 210 215 220 Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln 225 230 235 240 Ala Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp Ala 245 250 255 Leu Asp Lys Tyr Ala Glu Val Lys 260 <210> 28 <211> 286 <212> PRT <213> Epichloe festucae <400> 28 Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr 1 5 10 15 Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn 20 25 30 Pro Trp Lys Thr Arg Leu Ala Leu Asn Phe Lys Ala Val Thr Tyr Thr 35 40 45 Thr Thr Trp Val Lys Met Pro Asp Ile Ser Gly Val Arg Ala Ser Leu 50 55 60 Asn Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Thr Asp Phe Asn Thr 65 70 75 80 Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser 85 90 95 Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala 100 105 110 Gly His Leu Phe Pro Pro Leu Pro Pro Pro Gln Lys Leu Asp Tyr Ala 115 120 125 Val Gly Arg Asp Met Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala 130 135 140 Ser Ser Glu Leu Ala Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala 145 150 155 160 Ala Phe Thr Ala His Val Gly Val Met Val His Gly Leu Pro Leu Asp 165 170 175 Pro Ala Thr Ala Asp Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly 180 185 190 Val Ser Ser Trp Glu Asp Phe Glu Met Val Gly Glu Ala Arg Glu Lys 195 200 205 Met Met Gln Ser Leu Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe 210 215 220 Arg Arg Asp Ala Ser Gly Pro Phe Leu Leu Gly Gln Gln Ala Thr Tyr 225 230 235 240 Ala Asp Leu Ile Val Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu 245 250 255 Pro Ala Ser Glu Trp Gln Glu Val Arg Ala Trp His Gly Ala Val Phe 260 265 270 Gly Arg Leu His Asp Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 285 <210> 29 <211> 281 <212> PRT <213> Epichloe gansuensis <400> 29 Met Ala Thr Ser Thr Ser Thr Ser Ala Ser Thr Pro Ile Ile Phe Tyr 1 5 10 15 Asp Ile Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn 20 25 30 Pro Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr 35 40 45 Thr Thr Trp Val Glu Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu 50 55 60 Asn Leu Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr 65 70 75 80 Leu Pro Ile Ile His Asp Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser 85 90 95 Phe Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala 100 105 110 Asp Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Val Val Gly Ser His 115 120 125 Val Gln Pro Phe Ile Pro Leu Ser Asp Ile Arg Ala Ser Glu Phe Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Leu His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu 180 185 190 Asp Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Phe 195 200 205 Arg Thr Met Leu Glu Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Thr 210 215 220 Gly Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Trp His Gly Ala Val Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 30 <211> 281 <212> PRT <213> Epichloe typhina <400> 30 Met Ala Thr Ser Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp 1 5 10 15 Ile Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro 20 25 30 Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr 35 40 45 Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn 50 55 60 Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu 65 70 75 80 Pro Ile Met His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe 85 90 95 Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly 100 105 110 Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu 180 185 190 Asp Phe Glu Met Val Gly Glu Val Arg Glu Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 31 <211> 281 <212> PRT <213> Epichloe uncinata <400> 31 Met Ala Thr Ser Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp 1 5 10 15 Ile Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro 20 25 30 Trp Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr 35 40 45 Thr Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn 50 55 60 Val Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu 65 70 75 80 Pro Ile Met His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe 85 90 95 Asp Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly 100 105 110 Asp Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met 115 120 125 Gln Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala 130 135 140 Asp Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His 145 150 155 160 Val Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp 165 170 175 Val Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu 180 185 190 Asp Phe Glu Met Val Gly Glu Val Arg Glu Lys Met Met Gln Ser Leu 195 200 205 Arg Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser 210 215 220 Gly Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val 225 230 235 240 Gly Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp 245 250 255 Gln Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp 260 265 270 Ala Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 32 <211> 280 <212> PRT <213> Epichloe sylvatica <400> 32 Met Thr Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Lys Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Lys Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Met His Asp Pro Ala Thr Asp Ser Leu Leu Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Gly Arg Asp Met Gln 115 120 125 Leu Leu Ile Pro Leu Ser Glu Val Arg Ala Ser Pro Glu Leu Ala Asp 130 135 140 Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val 145 150 155 160 Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val 165 170 175 Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp 180 185 190 Leu Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg 195 200 205 Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly 210 215 220 Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val Gly 225 230 235 240 Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln 245 250 255 Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp Ala 260 265 270 Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 33 <211> 280 <212> PRT <213> Epichloe aotearoae <400> 33 Met Ala Thr Pro Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Met His Asp Pro Ala Thr Ser Ser Leu Ile Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Ser Gln Lys Leu Asp Tyr Ala Val Ala Arg Asp Thr Gln 115 120 125 Leu Leu Ile Pro Leu Ser Glu Ile Arg Ala Ser Ser Glu Leu Ala Asp 130 135 140 Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val 145 150 155 160 Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val 165 170 175 Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp 180 185 190 Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg 195 200 205 Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly 210 215 220 Pro Phe Leu Leu Gly Gln Arg Ala Thr Tyr Ala Asp Leu Ile Val Gly 225 230 235 240 Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln 245 250 255 Glu Ala Arg Ala Cys His Gly Ala Ile Phe Gly Gln Leu His Asp Ala 260 265 270 Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 34 <211> 275 <212> PRT <213> Epichloe glyceriae <400> 34 Met Ala Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile Ala Gln Arg Pro 1 5 10 15 Pro Val Ala Glu Thr Cys Cys Ala Val Asn Pro Trp Lys Ser Arg Leu 20 25 30 Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr Trp Val Ser Met 35 40 45 Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val Pro Ala Cys Arg 50 55 60 Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro Ile Ile His Asp 65 70 75 80 Pro Ala Thr Gly Ser Leu Ile Gly Asp Ser Phe Asp Ile Ala Ala His 85 90 95 Leu Gln Arg Ala Tyr Pro Ala Ser Gly Ala Gly Asp Leu Phe Pro Pro 100 105 110 Gln Glu Leu Asp Tyr Val Val Ala Arg Asp Thr Arg Leu Leu Val Pro 115 120 125 Leu Ser Glu Thr Arg Ala Ser Glu Phe Ala Asp Tyr Ala Arg Phe Asn 130 135 140 Ser Asn Val Asp Ala Ala Phe Thr Ala His Val Gly Leu Met Val His 145 150 155 160 Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val Thr Lys Ala Glu Phe 165 170 175 Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp Phe Glu Leu Val Gly 180 185 190 Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg Asn Val Leu Gly Asp 195 200 205 Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly Pro Phe Leu Leu Gly 210 215 220 Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly Gly Trp Leu Arg Met 225 230 235 240 Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln Glu Ala Arg Ala Trp 245 250 255 His Gly Ala Val Phe Gly Gln Leu His Asp Ala Leu Asp Lys Tyr Ala 260 265 270 Glu Val Lys 275 <210> 35 <211> 280 <212> PRT <213> Epichloe brachyelytri <400> 35 Met Ala Thr Ser Thr Ser Thr Ser Thr Ser Thr Pro Ile Ile Phe Tyr Asp Ile 1 5 10 15 Ala Gln Arg Pro Pro Val Thr Glu Thr Cys Cys Ala Val Asn Pro Trp 20 25 30 Lys Ser Arg Leu Ala Leu Asn Phe Lys Ala Val Pro Tyr Thr Thr Thr 35 40 45 Trp Val Lys Met Pro Asp Ile Ser Ser Val Arg Ala Ser Leu Asn Val 50 55 60 Pro Ala Cys Arg Lys Phe Ala Asp Gly Ser Asp Phe Asn Thr Leu Pro 65 70 75 80 Ile Ile His Asp Pro Ala Thr Asp Ser Leu Ile Gly Asp Ser Phe Asp 85 90 95 Ile Ala Ala Tyr Leu Gln Arg Thr Tyr Pro Ala Ser Gly Ala Gly Asp 100 105 110 Leu Phe Pro Pro Gln Lys Leu Asp Tyr Ala Val Ser Arg Asp Met Gln 115 120 125 Leu Leu Ile Pro Leu Ser Glu Met Arg Ala Ser Ser Glu Leu Ala Asp 130 135 140 Tyr Ala Arg Phe Asn Ser Asn Val Asp Ala Ala Phe Thr Ala His Val 145 150 155 160 Gly Leu Met Val His Gly Leu Pro Leu Asp Pro Ala Thr Ala Asp Val 165 170 175 Thr Lys Ala Glu Phe Val Arg Arg Ala Gly Val Ser Ser Trp Glu Asp 180 185 190 Phe Glu Met Val Gly Glu Ala Arg Glu Lys Met Met Gln Ser Leu Arg 195 200 205 Asn Met Leu Gly Asp Leu Ala Ala Leu Phe Arg Arg Asp Ala Ser Gly 210 215 220 Pro Phe Leu Leu Gly Gln Lys Ala Thr Tyr Ala Asp Leu Ile Val Gly 225 230 235 240 Gly Trp Leu Arg Met Met Arg Ala Thr Leu Pro Ala Ser Glu Trp Gln 245 250 255 Glu Val Arg Ala Trp His Gly Ala Ile Phe Gly Gln Leu His Asp Ala 260 265 270 Leu Asp Lys Tyr Ala Glu Val Lys 275 280 <210> 36 <211> 846 <212> DNA <213> Tinopyrum_ponticum_(wild_type_for_mutants) <400> 36 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 37 <211> 846 <212> DNA <213> Tinopyrum_elongatum <400> 37 atggccacct ccacctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtc aagatgccag atatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc atcggcgact ccttggacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gcagcagctg ctcttcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc tttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gcgagtggca ggaggcgaga 780 gcctgccacg gggctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 38 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 38 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgtctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 39 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 39 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatctcgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 40 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 40 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgtcgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 41 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 41 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac catctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 42 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 42 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat tcagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 43 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 43 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctctgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 44 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 44 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctgat tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 45 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 45 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcaa aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 46 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 46 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatctcgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 47 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 47 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccaaactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 48 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 48 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccttc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 49 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 49 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ctagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 51 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 51 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcacgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 51 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 51 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacacagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 52 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 52 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcttcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 53 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 53 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gatcaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 54 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 54 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgtcgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 55 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 55 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgt ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 56 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 56 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgtagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 57 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 57 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgtc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 58 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 58 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctgggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgatg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 59 <211> 846 <212> DNA <213> Tinopyrum_ponticum <400> 59 atggccacct ccgcctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtg aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact cctttgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gccgcagctg ctcatcccgc tgtccgagat tcgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg acttcccttg gatcctgcca ccgccgacgt gaccaaggcc 540 gagtttgtgc ggcacgcggg gctctcatcg tgggacgact tggaaatggt tggcgaggcg 600 cgcgacaaga tgatgcagtc cctccgaaac atgctggggg acctggctgc cttgtttcgg 660 aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720 ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780 gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 60 <211> 843 <212> DNA <213> Epichloe_bromicola <400> 60 atggccacct ccacctccat ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtgacag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtc aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaaattc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc gtcggcgact ccttcgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctccggcgcc ggcgacctct ttccccccca gaagctcgac 360 tacgcagtcg gcagggacat gcagctgctc atcccgctgt ccgagactcg ggcatcccca 420 gagcttgcag actacgcccg cttcaacagc aacgttgacg cagcctttac cgcacacgtg 480 ggcctcatgg tccacgggct tcccttggat cctgccaccg cggacgtgac caaggccgag 540 tttgtgcggc gcgcgggggt ctcgtcgtgg gaggacttcg aaatggttgg tgaggcgcgc 600 gagaagatga tgcagtccct ccggaacatg ctgggggacc tggctgcctt gttccggaga 660 gatgcgagcg ggccgttctt gctgggacag aaggccacct atgcggatct gattgtcggt 720 ggctggctgc ggatgatgcg ggcgacgttg ccggcgagtg agtggcagga ggtgagagcc 780 tgccacgggg ctgtcttcgg gcaactgcat gatgcgctgg acaagtatgc cgaggtgaag 840 tag 843 <210> 61 <211> 840 <212> DNA <213> Epichloe_amarillans <400> 61 atggccacct ccacctccac cccaatcatc ttctacgaca tagcccagcg gccccccgtc 60 acagaaacgt gctgcgccgt caacccttgg aaatccagac tggccctcaa cttcaaggcc 120 gtcccctaca caaccacctg ggtcaagatg ccagacatca gcagcgtccg cgccagcctc 180 aacctgccag cgtgtcgcaa gttcgccgac ggcaccgact tcgacaccct gcccatcatc 240 cacgaccccg cgaccggctc cctcatcggc gactccttcg acatcgccgc ctacctgcag 300 cgcacgtatc ccgcctccgg cgccggcgac ctcttccccc cccagaagct cgactacgcc 360 gccggcaggg acacgcagct gctcatcccg ctgtccgagg ttcgcgccgc atccccggag 420 ctcgcagact acgcccgctt caacagcaac gttgacgcag ccttcaccgc gcacgtgggc 480 ctcatggtcc acgggcttcc cttggaccct gccaccgcgg acgtgaccaa ggccgagttt 540 gtgcggcgcg cggccgtctc atcgtgggac gacctcgaca tggttggcga cgcgcgcgac 600 aagatgatgc agtccctccg gaacacgctg ggggacctgg ccgccttgtt tcggagagat 660 gcgagcgggc cgttcttgct gggacccaag gccacgtacg cggatctgat tgtcggtggc 720 tggttgcgca tgatgcgggc gacgttgccg ccgagtgagt ggcaggcggc gagagcttgg 780 cacggggctg tcttcgggca gctgcatgat gcgctggaca agtacgccga ggtgaagtag 840 <210> 62 <211> 795 <212> DNA <213> Epichloe_baconii <400> 62 atggccacct ccacctccac ctccacctcc acctccaccc caatcatctt ctacgacata 60 gcccagcggc cccccgtcac agaaacatgc tgcgccgtca acccttggaa atccagactg 120 gccctcaact tcaaggccgt cccctacaca accacctggg tcaagatgcc cgacatcagc 180 agcgtccgcg ccagcctcaa cctgccagcg tgtcgcaagt tcgccgacgg caccgacttc 240 aacaccctgc ccatcatcca cgaccccgcg accggctccc tcgtcggcga ctccttcgac 300 atcgccgcct acctgcagcg cacggacacg cagctgctca tcccgctgtc cgaggttcgc 360 gccgcatcct cggacctcgc agactacgcc cgcttcaaca gcaacgttga cgcagccttt 420 accgcgcacg tgggcctcat ggtccacggg cttcccttgg accctgccac cgcggacgtg 480 accaaggccg agtttgtgcg gcgcgcgggg gtctcatcgt gggacgactt cgagatggca 540 ggcgaggcgc gcgagaagat gatgcagtcc ctccggaaca cgctggggga cctggccgcc 600 ttgtttcgga gagatgcgag cgggccgttc ttgctgggac gcaaggccac gtacgcggat 660 ctgattgtcg gtggctggtt gcgcatgatg cgggcgacgt tgccggcgag tgagtggcag 720 gcggcgagag cttggcacgg ggctgtcttc gggcagctgc atgatgcgct ggacaagtat 780 gccgaggtga agtag 795 <210> 63 <211> 861 <212> DNA <213> Epichloe_festucae <400> 63 atggccacct ccacctccac ctccacctcc accccaatca tcttctacga catagcccag 60 cggccccccg tcacagaaac atgctgcgcc gtcaaccctt ggaaaaccag actggccctc 120 aacttcaagg ccgtcaccta cacaaccacc tgggtcaaga tgccagacat cagcggcgtc 180 cgcgccagcc tcaacgtgcc agcgtgtcgc aaattcgccg acggcaccga cttcaacacc 240 ctgcccatca tccacgaccc cgcgaccggc tccctcatcg gcgactcctt cgacatcgcc 300 gcctacctgc agcgcaccta tcccgcctcc ggcgctggcc acctcttccc cccccttccc 360 ccccctcaga agctcgacta cgccgtcggc agggacatgc agctgctcat cccgctgtcc 420 gaggttcgcg catcctcgga gctcgcagac tacgcccgct tcaacagcaa cgttgacgca 480 gcctttaccg cgcacgtcgg cgtcatggtc cacgggcttc ccttggatcc tgccaccgcg 540 gacgtgacca aggccgagtt cgtgcggcgc gcgggggtct catcgtggga ggacttcgaa 600 atggtcggtg aggcgcgcga gaagatgatg cagtccctcc ggaacatgct gggggacctg 660 gccgccttgt ttcggagaga tgcgagcggg ccgttcttgc tgggacagca ggccacgtac 720 gcggatctga ttgtcggtgg ctggttgcgg atgatgcggg cgacgttgcc ggccagtgag 780 tggcaggagg tgagagcttg gcacggggct gtttcgggc ggctgcatga tgcgctggac 840 aagtatgccg aggtgaagta g 861 <210> 64 <211> 846 <212> DNA <213> Epichloe_gansuensis <400> 64 atggccacct ccacctccac ttccgcctcc accccaatca tcttctacga catagcccag 60 cgccccccccg tcacagaaac atgctgcgcc gtcaaccctt ggaaatccag actggccctc 120 aatttcaagg ccgtccccta cacaaccacc tgggtcgaga tgccagacat cagcagcgtc 180 cgcgccagtc tcaacctgcc agcgtgtcgc aaattcgccg acggctccga cttcaatacc 240 ctgcccatca tccacgaccc cgcgaccggc tccctcatcg gcgactcctt cgacatcgcc 300 gcctacctgc agcgcacgta tcccgctcg ggcgccgacg acctcttccc cccccagaag 360 ctggactacg tagtcggcag ccatgtccag ccgttcatcc cgctgtctga cattcgcgca 420 tcagagtttg cagattacgc ccgcttcaac agcaacgttg acgcagcctt taccgcacac 480 gtgggcctca tgctccacgg acttcccttg gatcctgcca ccgcggacgt gaccaaggca 540 gaattcgtgc gacgcgccgg ggtctcgtcg tgggaggatt ttgaaatggt tggtgaggcg 600 cgggagaaga tgatgcagtc ctttcggact atgctggagg acctggctgc cttgttccgg 660 agagatgcga ccgggccgtt cttgctggga cagaaggcta cgtatgcgga tctgattgtc 720 ggcgggtggt tgcggatgat gcgcgcgacg ttgccggcga gtgagtggca ggaggcgaga 780 gcttggcatg gggccgtctt cggacaactg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 65 <211> 846 <212> DNA <213> Epichloe_typhina <400> 65 atggccacct cctccacctc cacctccacc ccaatcatct tctacgacat agcccagcgg 60 ccccccgtcg cagaaacatg ctgcgccgtc aacccttgga aatccagact ggccctcaac 120 ttcaaggccg tcccctacac aaccacctgg gtcaagatgc cagacatcag cagcgtccgc 180 gccagcctca acgtgccagc gtgtcgtaaa ttcgccgacg gctccgactt caacaccctg 240 cccatcatgc acgaccccgc gaccgactcc ctcatcggcg actccttcga tatcgccgcc 300 tacctgcagc gcacgtatcc cgcctccggc gccggcgacc tcttcccccc ccagaagctc 360 gactacgcag tcggcaggga catgcagctg ctcatcccgc tgtccgaggt ccgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg gcttcccttg gatcctgcca ccgcagacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg ggtctcgtcg tgggaggact tcgaaatggt tggcgaggtg 600 cgcgagaaga tgatgcagtc cctccggaac atgctcgggg acctggctgc cttgtttcgg 660 agagatgcga gcgggccgtt cctgctgggg cagagggcca cgtatgcgga cctgattgtc 720 ggtggctggt tgcgcatgat gcgcgcgacg ttgccggcga gtgagtggca ggaggcgaga 780 gcctgccacg gggccatctt cgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 66 <211> 846 <212> DNA <213> Epichloe_uncinata <400> 66 atggccacct cctccacctc cacctccacc ccaatcatct tctacgacat agcccagcgg 60 ccccccgtcg cagaaacatg ctgcgccgtc aacccttgga aatccagact ggccctcaac 120 ttcaaggccg tcccctacac aaccacctgg gtcaagatgc cagacatcag cagcgtccgc 180 gccagcctca acgtgccagc gtgtcgtaaa ttcgccgacg gctccgactt caacaccctg 240 cccatcatgc acgaccccgc gaccgactcc ctcatcggcg actccttcga catcgccgcc 300 tacctgcagc gcacgtatcc cgcctccggc gccggcgacc tcttcccccc ccagaagctc 360 gactacgcag tcggcaggga catgcagctg ctcatcccgc tgtccgaggt ccgcgcatca 420 ccagagctcg cagactacgc ccgcttcaac agcaacgttg acgcagcctt taccgcgcac 480 gtgggcctca tggtccacgg gcttcccttg gatcctgcca ccgcggacgt gaccaaggcc 540 gagtttgtgc ggcgcgcggg ggtctcgtcg tgggaggact tcgaaatggt tggcgaggtg 600 cgcgagaaga tgatgcagtc cctccggaac atgctcgggg acctggctgc cttgtttcgg 660 agagatgcga gcgggccgtt cctgctgggg cagagggcca cgtatgcgga cctgattgtc 720 ggtggctggt tgcgcatgat gcgcgcgacg ttgccggcga gtgagtggca ggaggcgaga 780 gcctgccacg gggccatctt cgggcagctg catgatgcgc tggacaagta tgccgaggtg 840 aagtag 846 <210> 67 <211> 843 <212> DNA <213> Epichloe_sylvatica <400> 67 atgaccacct ccacctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacaaaac cacctgggtc aagatgccag acatcagcag cgtccgcgcc 180 agcctcaagg tgccagcgtg tcgtaaattc gccgacggct ccgacttcaa caccctgccc 240 atcatgcacg accccgcgac cgactccctc ctcggcgact ccttcgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctccggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtcg gcagggacat gcagctgctc atcccgctgt ccgaggtccg cgcgtcacca 420 gagctcgcag actacgcccg cttcaacagc aacgttgacg cagcctttac cgcgcacgtg 480 ggcctcatgg tccacgggct tcccttggat cctgccaccg cggacgtgac caaggccgag 540 tttgtgcggc gcgcgggggt ctcgtcgtgg gaggaccttg aaatggttgg cgaggcgcgc 600 gagaagatga tgcagtccct ccggaacatg ctcggggacc tggctgcctt gtttcggaga 660 gatgcgagcg ggccgttcct gctggggcag agggccacgt atgcggacct gattgtcggt 720 ggctggttgc gcatgatgcg cgcgacgttg ccggcgagtg agtggcagga ggcgagagcc 780 tgccacgggg ccatcttcgg gcagctgcat gatgcgctgg acaagtatgc cgaggtgaag 840 tag 843 <210> 68 <211> 843 <212> DNA <213> Epichloe_aotearoae <400> 68 atggccaccc ccacctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcgcag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtc aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgccagcgtg tcgcaagttc gccgacggct ccgacttcaa caccctgccc 240 atcatgcacg accccgcgac ctcttccctc atcggcgact ccttcgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctcgggcgcc ggcgacctct tcccctccca gaagctcgac 360 tacgcagtcg ccagggacac gcagctgctc atcccgctgt ccgagattcg cgcatcatca 420 gagctcgcag actacgcccg cttcaacagc aacgttgacg cagcctttac cgcgcacgtg 480 ggcctcatgg tccacgggct tcccttggat cctgccaccg ccgacgtgac caaggccgag 540 tttgtgcggc gcgcgggcgt ctcatcgtgg gaggacttcg aaatggttgg cgaggcgcgc 600 gagaagatga tgcagtccct ccggaacatg ctgggggacc tggctgcctt gtttcggaga 660 gatgcgagcg ggccgttcct gctggggcag agggccacgt atgcggacct gattgtcggt 720 ggctggttgc gcatgatgcg ggcgacgttg ccggcgagtg agtggcagga ggcgagagcc 780 tgccacgggg ctatcttcgg gcagctgcat gatgcgctgg acaagtatgc cgaggtgaag 840 tag 843 <210> 69 <211> 828 <212> DNA <213> Epichloe_glyceriae <400> 69 atggccacct ccaccccaat catcttctac gacatagccc agcggccccc cgtcgcagaa 60 acatgctgcg ccgtcaaccc ttggaatcc agactggccc tcaacttcaa ggccgtcccc 180. tacacaacca cctgggtcag catgccagac atcagcagcg tccgcgccag cctcaacgtg ccggcgtgtc gcaaattcgc cgacggctcc gacttcaaca ccctgcccat catccacgac 240 cccgcgaccg gctccctcat cggcgactcc ttcgacatcg ccgcccacct gcagcgcgcc 300 tatcccgcct ccggcgccgg cgacctcttc cccccccagg agctggacta cgtggtcgcc 360 agggacacgc ggctgctcgt cccgctgtcc gagactcgcg catcagagtt cgcggactac 420 gcccgcttca acagcaacgt tgacgcagcc tttaccgcac acgtgggcct catggtccac 480 gggcttccct tggaccctgc caccgcggac gtgaccaagg cggagtttgt gcggcgcgcg 540 ggagtctcgt cgtgggagga tttcgaattg gttggtgagg cgcgcgagaa gatgatgcag 600. tccctccgga acgtgctggg ggacctggct gccttgtttc ggagagatgc gagcggggccg 660 ttcttgctgg gacagaaggc cacgtatgcg gatctgattg tcggtggctg gttgcggatg 720 atgcgggcga cgttgcccgc gagtgagtgg caggaggcga gagcctggca tggcgctgtc 780 ttcgggcagc tgcatgatgc gctggacaag tatgccgagg tgaagtag 828 <210> 70 <211> 843 <212> DNA <213> Epichloe_brachyelytri <400> 70 atggccacct ccacctccac ctccacccca atcatcttct acgacatagc ccagcggccc 60 cccgtcacag aaacatgctg cgccgtcaac ccttggaaat ccagactggc cctcaacttc 120 aaggccgtcc cctacacaac cacctgggtc aagatgccag acatcagcag cgtccgcgcc 180 agcctcaacg tgcctgcgtg tcgcaaattc gccgacggct ccgacttcaa caccctgccc 240 atcatccacg accccgcgac cgactccctc atcggcgact ccttcgacat cgccgcctac 300 ctgcagcgca cgtatcccgc ctccggcgcc ggcgacctct tcccccccca gaagctcgac 360 tacgcagtca gcagggacat gcagctgctc atcccgctgt ccgagatgcg cgcatcatca 420 gagctcgcag actacgcccg cttcaacagc aacgttgacg cagcctttac cgcgcacgtg 480 ggcctcatgg tccacgggct tcccttggat cctgccaccg cggacgtgac caaggccgag 540 tttgtgcggc gcgcgggggt ctcatcgtgg gaggatttcg aaatggttgg tgaggcgcgc 600 gagaagatga tgcagtccct ccggaacatg ctgggggacc tggctgcctt gtttcggaga 660 gatgcgagcg ggccgttctt gctggggcag aaggccacgt atgcggatct gattgtcggt 720 ggctggttgc gcatgatgcg ggcgacgttg ccggcgagtg agtggcagga ggtgagagcc 780 tggcacgggg ctatcttcgg gcagctgcat gatgcgctgg acaagtatgc cgaggtgaag 840 tag 843

Claims

1. An isolated polypeptide with deepoxidation catalytic activity, characterized in that, Its amino acid sequence is selected from one of the groups (1)-(2) and (4)-(5) below: (1) The amino acid sequences shown in SEQ ID No:1-35; (2) The amino acid sequence of SEQ ID No:1 or 2 is more than 99% identical to the amino acid sequence of the same species as the corresponding species of the genus *Cypripedium*. (4) Based on SEQ ID No:1, the consecutive sequences shown in Table 2, numbers 3-8; (5) A chimeric sequence in which other amino acid sequences are attached to the N-terminus and / or C-terminus of any of the amino acid sequences described in SEQ ID No:1-2 or 25-35 and still have de-epoxy catalytic activity, wherein the other amino acid sequences are sequences that enhance peptide expression or secretion.

2. An isolated active polypeptide, characterized in that, Its amino acid sequence is shown in any one of (A)-(C) below: (A) The amino acid sequence corresponding to positions 92-184 of SEQ ID No:1; (B) A sequence corresponding to amino acid sequences 92-104 and 144-184 of SEQ ID No:1, with a linker sequence between them, wherein the linker sequence does not substantially affect the peptide activity, and is selected from at least one of the following: amino acid sequences corresponding to amino acid sequences between 105-142 of SEQ ID No:25, amino acid sequences between 103-141 of SEQ ID No:26, amino acid sequences between 107-148 of SEQ ID No:28, amino acid sequences between 106-143 of SEQ ID No:30, and the artificial sequence GGGSGGSGG; or (C) Based on (A), and simultaneously possessing the following characteristics as listed in Table 3: numbers 2-3, 6, 11, 14, 17-18, 22-23, 25, 29-30, 32, 35-46, 49-51, 53, 55-56, 58-71, 73-75, 78-82, 84, 86, 87, 89, 91-95, 98-99, 102-106, 109-111. Mutation types shown in 114-115, 118-121, 125, 129-134, 144-145, 150-151, 155-156, 159-160, 162, 165-167, 169, 171, 173-174, 185-187, 189-191, 195, 198-200, 202, 212 or 214.

3. An isolated nucleic acid molecule, characterized in that, It encodes the polypeptide according to claim 1 or 2.

4. An isolated nucleic acid molecule, characterized in that, Its base sequence is selected from one of the following groups (a)-(e): (a) The sequences shown in SEQ ID No:36-70; (b) A sequence modified by host codon preference based on the base sequence in (a); (c) The conserved region sequence of the sequence shown in (a) encodes a polypeptide sequence between positions 92-104 and 144-184 of SEQ ID No:1, and further comprises a linker between the two, the linker being selected from at least one of the following: amino acid sequence corresponding to positions 105-142 of SEQ ID No:25, amino acid sequence corresponding to positions 103-141 of SEQ ID No:26, amino acid sequence corresponding to positions 107-148 of SEQ ID No:28, amino acid sequence corresponding to positions 106-143 of SEQ ID No:30, and artificial sequence GGGSGGSGG; (d) A sequence that is 99% or more identical to the sequence shown in SEQ ID No:36 or 37, is derived from *Thinopyrum longicornis* and encodes a polypeptide with de-epoxylated catalytic activity, or a sequence that is 99% or more identical to the sequence shown in SEQ ID No:60-70, is derived from the same species of the *Cypripedium* genus and encodes a polypeptide with de-epoxylated catalytic activity.

5. A nucleic acid construct, characterized in that, It comprises the nucleic acid as described in claim 3 or 4 and optional regulatory elements.

6. A host cell, characterized in that, It comprises the nucleic acid as described in claim 3 or 4, or the nucleic acid construct as described in claim 5, introduced by genetic engineering.

7. A method for producing a polypeptide according to claim 1 or 2, characterized in that, This includes the step of expressing the nucleic acid according to claim 3 or 4 through genetic engineering to obtain a polypeptide, or the step of obtaining a polypeptide through chemical synthesis.

8. A method for catalyzing the deepoxidation reaction of trichothecene compounds, characterized in that, It includes the step of contacting the polypeptide according to claim 1 or 2 or the host cell according to claim 6 with a trichothecene compound and glutathione to generate a glutathione derivative.

9. The method for catalyzing the deepoxidation reaction of trichothecene compounds according to claim 8, wherein, The trichothecene compounds include deoxynivalenol, 15-acetyldeoxynivalenol, 3-acetyldeoxynivalenol, nivalenol, fusarenone-X, diacetoxyfusarenol, T-2 toxin, and HT-2 toxin.

10. Use of the polypeptide according to claim 1 or 2 in food processing, feed processing and pharmaceuticals.

11. The use of nucleic acids according to claim 3 or 4 in plant breeding and disease control.

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