CN111549042B - Application of nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker - Google Patents
Application of nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker Download PDFInfo
- Publication number
- CN111549042B CN111549042B CN202010146400.9A CN202010146400A CN111549042B CN 111549042 B CN111549042 B CN 111549042B CN 202010146400 A CN202010146400 A CN 202010146400A CN 111549042 B CN111549042 B CN 111549042B
- Authority
- CN
- China
- Prior art keywords
- sequence
- plant
- seq
- nucleic acid
- wheat
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
- C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Cell Biology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses application of nucleic acid molecules in plant transgenosis, molecular breeding, disease control and molecular markers. The invention expresses the nucleic acid molecule for coding the de-epoxidase in the plant through gene engineering, thereby efficiently removing the epoxy group of the trichothecene toxins, directly reducing the endotoxin content in the plant, simultaneously improving the disease resistance of the crops and obviously improving the quality of the crops. The invention makes up the blank that no definite gene can detoxify through catalyzing trichothecene toxin by removing epoxy group at present, and has wide application prospect in the aspects of plant transgenosis, molecular breeding and disease control.
Description
Technical Field
The invention relates to the field of molecular botany, in particular to application of a nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular markers.
Background
Fusarium sp is a kind of worldwide distributed fungus, which can not only live through winter and summer in soil, but also infect various plants (grain crops, economic crops, medicinal plants and ornamental plants), causing various diseases of root rot, stem-base rot, flower rot, ear rot and the like of the plants, wherein more than 100 host plants infect host plant vascular bundle systems, destroy the vascular bundles of the transport and guide tissues of the plants, generate toxins in the processes of growth, development and metabolism to harm the crops, cause crop wilting and death, influence yield and quality, and is one of the most difficult important diseases for production control. Fusarium infected plants or seeds contain various mycotoxins, mainly including trichothecenes (CTCs), zearalenone (zearalenone), butenolide (butenolide), fumonisin (fumonisins fb) and other toxins.
Diseases caused by infection of cereals by fusarium graminearum (mainly including f.asiticum, f.graminearum Schwabe and f.pseudograminearum) mainly include scab and stem rot of wheat, barley, oats, corn and millet. Currently, wheat, barley and corn gibberella are serious fungal diseases which are difficult to solve worldwide. For example, the crop yield reduction and mycotoxin contamination of grains caused by wheat scab become one of the most urgent food safety problems to be solved in China and worldwide. Fusarium graminearum infects wheat spikes in the populus florescence of wheat and secretes a large amount of trichothecene toxins, so that the pathogenicity of pathogenic bacteria is obviously increased, and the destructive damage to the yield is caused. Meanwhile, after eating wheat grains polluted by the toxins, the appetite loss or abolition, gastrointestinal inflammation and bleeding, vomiting, diarrhea, necrotizing dermatitis, dyskinesia, poor blood coagulation, anemia, reduction of the number of white blood cells, reduction of immune functions, abortion and the like can be caused, and the health of people and livestock is seriously threatened.
Disclosure of Invention
In view of the problems of the prior art, the inventors provide a nucleic acid molecule encoding a de-epoxidase, by which the epoxide group in the toxin can be effectively removed, thereby achieving detoxification. The present invention has been accomplished based on this. Specifically, the present invention includes the following.
In a first aspect of the present invention, there is provided use of a nucleic acid molecule having a base sequence selected from the group consisting of the following (a) to (e):
(a) a sequence shown as SEQ ID No. 1-35;
(b) a sequence modified by host codon bias based on the base sequence in (a);
(c) a conserved region sequence of the sequence set forth in (a);
(d) a sequence having 95% or more sequence identity to (a) to (c) and derived from the same genus;
(e) a sequence complementary to at least a portion of any one of (a) - (d).
In the sequence of (a), SEQ ID No.1 represents a deoxylase gene derived from a decaploid of Elytrigia elongata, and SEQ ID No.2 represents a deoxylase gene derived from a diploid of Elytrigia elongata. SEQ ID No. 3-24 show mutants of the sequence SEQ ID No. 1. SEQ ID Nos. 25 to 35 show the amino acid sequence derived from the genus Methylopsis (A. bergamot)
Genus) from different species.
In the present invention, in the sequence of (b), the modification of the codon bias of the host means that the base sequence of (a) is subjected to base substitution according to degenerate codons in order to adapt to the requirements of different hosts for expression. Codon bias engineering does not generally alter the sequence of the product protein or polypeptide.
In the sequence of (c), the conserved region sequence is a region corresponding to 98% or more, preferably 99% or more, more preferably 100% identity in different species of the genus Elytrigia elongata and the genus Leptospira. The conserved region sequence may also refer to a partially contiguous region of 100% sequence identity for different species in the genus elytrigia elongata, or to a corresponding partially contiguous region of 100% sequence identity for different species in the genus xylaria. The sequence of the conserved region may correspond to the amino acid sequence of the conserved region of the active polypeptide. It is noted that the conserved sequence of bases does not necessarily express or encode an active polypeptide. So long as it is a conserved region, can be used as a detection target. In certain embodiments, when the base sequence shown in SEQ ID No.1 is used as a positional reference, the nucleic acid molecule comprises at least one base sequence selected from the group consisting of: the nucleotide sequence between positions 436-470, 430-476 and 808-846.
In the sequence (d), the sequence identity with the base sequence in (a) to (c) is generally 90% or more, preferably 95% or more, more preferably 97% or more, still more preferably 98% or more, and further preferably 99% or more. In general, the sequences in (d) are derived from the same genus, preferably from the same species, on the basis of sequence identity. In certain embodiments, the nucleic acid molecule has a base sequence that has greater than 95% sequence identity to the amino acid sequence of (a), (b), or (c) and is derived from the genus Elytrigia elongata or the genus Leptospira.
A sequence complementary to at least a portion of any one of (a) - (d) in the sequence of (e), wherein the complementary sequence comprises a sequence that specifically hybridizes to such sequence under stringent conditions. For example, probes, primers, and the like. The length of the nucleic acid molecule or oligonucleotide molecule having these sequences is not particularly limited, and may be 15 to 200bp, for example, 15 to 40bp, 150 to 180bp, etc.
In certain embodiments, the nucleic acid molecule is capable of encoding a protease with catalytic activity for the removal of epoxide.
The plant in the present invention is not particularly limited, and may be a food crop, a commercial crop, a medicinal plant, or the like, or may be a herb plant or a woody plant. Examples of grain crops include, but are not limited to, for example, rice, wheat, barley, rice, highland barley, millet, soybean, and the like. Examples of woody plants include fruit trees such as woody evergreen or rosaceous fruit trees. Examples of plants also include alfalfa, elytrigia repens, lolium perenne, maize mexican grass, pennisetum hydridum, pennisetum purpureum, barley, peanut, cotton, and the like. The plant of the present invention may also be a hybrid plant. For example, a plant obtained by crossing the above-mentioned plants.
In certain embodiments, the disease comprises a plant disease caused by a bacterium of the genus fusarium, cephalosporium, fischerella or trichoderma. Such plant diseases may be exemplified by root rot, stem base rot, flower rot and ear rot.
In a second aspect of the present invention, there is provided a plant cell comprising an exogenous nucleic acid molecule having one base sequence selected from the group consisting of (a) to (e) introduced by genetic engineering means.
In a third aspect of the present invention, there is provided a transgenic plant obtained by introducing an exogenous nucleic acid into a host plant by genetic engineering means.
The nucleic acid molecule can encode the de-epoxidase, so that the transgenic plant has the epoxy group removing capability aiming at the trichothecene compounds, and the content of the toxins in the plant is further reduced. Examples of such toxins include, but are not limited to, Deoxynivalenol (DON), 15-acetyldeoxynivalenol (15-ADON), 3-acetyldeoxynivalenol (3-ADON), Nivalenol (NIV), fusanone-X (Fus-X), Diacetyloxynivalenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2).
Drawings
FIG. 1 is an ion chromatogram extracted from transgenic wheat infected by Fusarium graminearum (F.g) under LC-HRMS full scan mode.
FIG. 2 is an ion chromatogram extracted from F.g infected transgenic wheat in LC-HRMS (/ MS) PRM mode.
FIG. 3 shows the results of the quantitative detection of toxins and their derivatives in LC-HRMS (/ MS) PRM mode. Wherein (a) represents the quantitative result of DON-GSH; (b) representing DON quantitative results; (c) represents the quantitative result of 3-ADON-GSH; (d) shows the quantitative results for 3-ADON.
FIG. 4 shows the results of phenotypic identification experiments for transgenic plants against gibberellic disease. Wherein, the picture (a) shows the disease condition of the inoculated 21d (in the picture, A represents a transgenic positive plant, and B represents transgenic receptor material); (b) counting the number of the outbreak spikelets of 3 strains which are overexpressed for the ThFhb 7; (c) statistics of the number of the outbreak spikelets of 3 strains originally expressed for the ThFhb7 transformation.
FIG. 5 shows the results of extensive resistance of the ThFhb7 transgenic line to Fusarium species. Wherein (a) represents the result of resistance of the ThFhb7 transgenic line to Fusarium graminearum; (b) represents the results of resistance of the ThFhb7 transgenic line to Fusarium asianum; (c) shows the results of resistance of the ThFhb7 transgenic line to Fusarium pseudograminearum.
FIG. 6 shows the results of experiments on resistance of ThFhb7 to wheat stalk rot. Wherein (a) shows the pathogenesis of ThFhb7 to wheat stem basal rot; (b) shows the statistical result of the death rate of ThFhb7 transgenic plants in the stem-base rot resistance experiment.
FIG. 7 shows the identification result of resistance to Gibberella zeae to ThFhb7 gene.
FIG. 8 shows an alignment of partial mutant sequences of ThFhb 7.
FIG. 9 shows the phenotypic identification of mutants against gibberellic disease.
FIG. 10 is the NCBI alignment of ThFhb 7.
FIG. 11 shows ThFhb7 and a homologous sequence evolutionary tree.
FIG. 12 is an ion chromatogram of LC-HRMSDON treatment of transgenic yeast.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. Further, for numerical ranges in this disclosure, it is understood that the upper and lower limits of the range, and each intervening value therebetween, is specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control. Unless otherwise indicated, "%" is percent by weight.
As used herein, the term "base at position y" or the like is referenced to the base sequence of the deoxyribose enzyme gene derived from a decaploid of Elytrigia elongata, i.e., to the amino acid sequence shown in SEQ ID No.1, unless otherwise specifically indicated.
Example 1
Transgenic wheat scab resistance experiment
1. Overexpression vector construction
The CDS region 847bp of the Thinopyrum elongatum ThFhb7 gene is amplified by taking the genomic DNA of the tetraploid Thinopyrum elongatum as a template, and the designed primer sequence is as follows:
a forward primer: 5' -TGCAGCCCGGGGATCCAGAAATCCACCCATCGTCATCACC-3'; reverse primer: 5' -ACCTGTAATTCACACGTGCTACTTCACCTCGGCATACTTGTC-3'。
The underlined section is the extension sequence complementary to the end of the linearized vector. The ThFhb7 cDNA complete gene sequence is obtained by PCR, after the carrier pCAMBIA3300 is treated by endonuclease BamHI, the PCR product and linearized plasmid are purified and recovered, the PCR product and linearized plasmid are inserted into MCS at the downstream of pCAMBIA3300 strong promoter by using In-Fusion HD cloning kit and are transformed into Escherichia coli DH5 alpha, after colony PCR identification, the positive monoclonal bacteria liquid is sequenced and verified, and the sequence is shown as SEQ ID No. 1. The plasmid with correct sequence verification is sent to a wheat transformation platform of national key laboratory of Shandong agricultural university for wheat transgenosis, and the receptor is wheat variety fielder.
2. Original expression vector construction
The genomic DNA of the tetraploid thinopyrum elongatum is taken as a template to amplify 847bp of a promoter region and a CDS region of a gibberellic disease resistant gene ThFhb71308bp of the thinopyrum elongatum, and the designed primer sequence is as follows:
a forward primer: 5' -ACATGATTACGAATTCTTCTACTAGTGCCCCACCtACG-3'
Reverse primer:
5′-ACCTGTAATTCACACGTGCGACCAGCCAGGAAACACCACTG-3′.a
the underlined section is the extension sequence complementary to the end of the linearized vector. Obtaining a sequence containing a ThFhb7 promoter and an open reading frame through PCR, treating a vector pCAMBIA3300 with an endonuclease EcoRI, purifying and recovering a PCR product and a linearized plasmid, inserting the fragment into the pCAMBIA3300 vector without the ubi promoter by using an In-Fusion HD cloning kit, transforming the vector into escherichia coli DH5 alpha, and sequencing a positive monoclonal bacterium solution for verification after colony PCR identification. The plasmid with correct sequence verification is sent to a wheat transformation platform of national key laboratory of Shandong agriculture university for wheat transgenosis, and the receptor is wheat variety Kenong 199.
3. PCR detection of ThFhb7 transgenic plant
Taking young leaves of the transgenic plants, and extracting the wheat genome DNA by a CTAB method. And respectively developing cross-vector primers of an over-expression vector and an original expression vector by using the sequence information of the pCAMBIA3300 expression vector, the ThFhb7 promoter region and the CDS region, and carrying out PCR on the transgenic plant. The amplification products were detected by electrophoresis on a 1% agarose gel. Wherein, the cross-vector primer of the over-expression vector is F: 5'-TGCAGTCCCTCCGAAACATG-3'; 5'-CAAATGGACGAACGGATAAACC-3' is added. The original expression cross-vector primer is F: 5'-AGCGGAAACACGCATCTGACCT-3'; 5'-TTACCCGCCAATATATCCTGTC-3' is added.
4. RT-PCR detection of ThFhb7 transgenic plant
Grinding wheat seedling leaves in liquid nitrogen into powder, and utilizing
Reagent, extract according to instructions. Genomic DNA was removed using a reverse transcription kit and RNA was reverse transcribed into cDNA. Designing a fluorescent quantitative primer F according to the sequence of the ThFhb7 gene: 5'-TGATTCTTCTTCCGTTTCTAAGGA-3', respectively; r: 5'-ATGTCAAAGGAGTCGCCGACGA-3' are provided. Using Roche
480 RT-PCR analysis was performed. Housekeeping gene beta-actin is used as an internal standard. Calculating the relative expression quantity of ThFhb7 by utilizing CT value, selecting high-expression wheat transgenic line, strictly bagging and continuously selfing to obtain T3And (5) further carrying out phenotype identification on the fusarium head blight resistance.
5. Phenotype identification of gibberellic disease resistance of ThFhb7 transgenic plants
Taking out Fusarium graminearum strain, inoculating and activatingThen, the activated hyphae are picked and inoculated in a CMC spore production culture medium, and conidia are generated through culture and induction. Filtering to remove mycelium with gauze, and collecting bacterial liquid in an aseptic conical flask; centrifuging to remove the supernatant, adding appropriate amount of ddH2O, regulating the spore concentration to 2 x 105seed/mL, used for single flower inoculation; subpackaging into centrifuge tubes, and storing at-20 deg.C for use. At the initial stage of wheat flowering, sucking 10 mul of spore suspension liquid by a liquid-moving machine, and injecting the spore suspension liquid between the palea and the palea at the middle upper part of the spikelet; covering a plastic bag, removing the plastic bag after preserving moisture for 72h, inoculating about 30 spikes to each transgenic line, and inoculating one spikelet to each single plant. After inoculation for 21d, the number of small diseased ears (NDS) was counted.
6. Fusarium graminearum strain activation
Scraping hypha on the potato culture medium with toothpick, placing in mung bean culture medium, and shaking at 28 deg.C and 200rpm for 3 days; after fungus shaking is finished, filtering the bacterial liquid by using filter cloth, subpackaging the bacterial liquid into 50ml centrifuge tubes, centrifuging the centrifuge tubes to remove supernatant, adding 30ml of sterilized water, and shaking and uniformly mixing the sterilized water; centrifuging at 4000rpm for 20min, and removing supernatant; the precipitate was resuspended in a small amount of sterile water, the bacterial load was examined microscopically and diluted to 1X 10 according to the spore load5Concentration per ml.
7. Inoculating strain of wheat material by single flower drip method
At wheat flowering stage, 10uL fusarium graminearum conidia (concentration of 1X 10 liters per mm) were inoculated to two spikelets at the base of the same spike (usually two flowers at the left and right bases of the third spike at the top of the spike) in the same ear position5One). Marking the inoculation part, bagging and moisturizing. During the whole inoculation process, the spikelets are prevented from being directly exposed to the air to avoid drying. Sampling the scions 72h after inoculation, quickly freezing the collected samples with liquid nitrogen, and transferring to an ultra-low temperature refrigerator of-80 ℃ for storage and standby.
8. Toxin derivative extraction
After grinding the wheat samples, pre-cooled 1.5ml 75% methanol in water (containing 0.1% formic acid) was added. Shaking for 10s, performing ultrasonic treatment at room temperature for 30min, collecting supernatant, and transferring to a new centrifuge tube. Vacuum concentrating to dry powder. Before injection, 100 μ L20% acetonitrile was used for resuspension, and after filtration through a 0.22 μm filter, the sample was transferred to a sample vial for LC-HRMS detection.
9. The experimental results are as follows:
to identify the biochemical and biological effects of ThFhb7 in wheat, T was first overexpressed in wheat driven by the maize ubiquitin promoter3The generation homozygous transgenic wheat head is inoculated with fusarium graminearum. In the wheat flowering period, fusarium graminearum is inoculated for 72 hours, and then sampling is carried out for detection. As shown in FIG. 1, DON-GSH adduct, M/z 604.21730 (corresponding to [ M + H ] 604.21730, was detected from Fg-infected ThFhb7 wheat in positive ion mode of LC-HRMS (full scan)]+Δ ± 5 ppm); NIV-GSH adduct, M/z 620.21199 (corresponding to [ M + H ] was detected in Fg-infected ThFhb7 wheat]+, Δ. + -. 5 ppm). Whereas in the corresponding control (fusarium graminearum infected wheat transgenic recipient material) no corresponding GSH adduct production was detected.
As 3-ADON and 15-ADON are isomers with the same molecular weight, and a Full scan mode first-order spectrum cannot be distinguished, the LC-HRMS (/ MS) positive ion PRM mode is selected for detection, and the detection result is shown in figure 2. With the parent ion M/z 646.22764 (corresponding to [ M + H ]]+Δ ± 5ppm), daughter ion m/z 321.11210 detected 3-ADON-GSH adduct from Fg infected ThFhb7 wheat; with the parent ion M/z 646.22764 (corresponding to [ M + H ]]+Δ ± 5ppm), the daughter ion m/z 450.15471 detected 15-ADON-GSH adduct from Fg infected ThFhb7 wheat. Whereas no corresponding GSH adduct production was detected in the corresponding control wheat transgene receptor fielder.
And (4) conclusion: single fusarium graminearum strains can generate trichothecene toxins when infecting wheat ears in the flowering period within 72 hours, DON, 3-ADON, 15-ADON and a small amount of NIV are definitely detected by LC-HRMS in the experiment, but other toxins such as T2, HT-2 and the like are not detected due to extremely low content. The experimental results show that: when fusarium graminearum infects transgenic wheat over-expressing ThFhb7, DON, 3-ADON, 15-ADON and NIV can be efficiently catalyzed into glutathione adduct. The transgenic ThFhb7 wheat improves the toxin resistance, proves that ThFhb7 can take trichothecene toxins as substrates and catalyze the trichothecene toxins into corresponding GSH adducts, and can improve the gibberellic disease resistance of wheat.
Second, quantitative detection of toxin in transgenic wheat and its derivative
In view of the above, it was demonstrated that ThFhb7 can catalyze toxins such as DON, 3ADON, 15ADON, NIV, etc. to GSH form derivatives in wheat in the case of fusarium graminearum infection. To further demonstrate the content variation of these toxins, the toxins were further quantified using a high-resolution liquid LC-HRMS (/ MS) PRM profile. Because the PRM mode has higher requirements on the toxin content, the experiment only carries out related quantitative detection on DON and 3ADON in transgenic wheat which excessively expresses ThFhb7, and the specific result is shown in figure 3.
Inoculating fusarium graminearum to the ThFhb7 wheat head, taking materials at different time points, and then measuring the contents of DON, DON-GSH, 3-ADON and 3-DON-GSH. FIG. 3(a) shows that transgenic wheat containing ThFhb7 specifically catalyzes the formation of DON-GSH, whereas the control material JW1 without ThFhb7 does not detect accumulation of DON-GSH. The results in FIG. 3(b) show that after inoculation with Fg, substrate DON is significantly reduced relative to wheat without ThFhb7 (about 1/3) due to the fact that DON-GSH is synthesized in large quantities in the presence of ThFhb 7. Similarly, FIG. 3(c) transgenic wheat containing ThFhb7 can synthesize 3-ADON-GSH, while control material JW without ThFhb713-ADON-GSH (green) is not detected. Figure 3(d) wheat with ThFhb7 was significantly less (about 1/3) than control recipient material 3-ADON without ThFhb7 after spike inoculation with Fg. The experiment proves that ThFhb7 can efficiently derive DON and 3-ADON to generate DON-GSH after wheat is infected by fusarium graminearum, so that accumulation of DON toxin in wheat bodies is reduced, and the detoxification effect is achieved.
The quantitative detection results of the toxin and the derivatives thereof show that the fusarium graminearum infected wheat can generate a large amount of DON and 3-ADON. The results of quantification of PRM show that: when F.g infects wheat with trans-gibberellic disease resistance gene ThFhb7, the content of DON and 3-ADON is reduced by about 2/3 compared with the control, and the DON-GSH and 3-ADON-GSH are correspondingly increased along with the reduction of DON and 3-ADON. The ThFhb7 transgenic wheat improves the toxin resistance, and proves that ThFhb7 can catalyze the wheat into a corresponding GSH adduct by using DON and 3-ADON as substrates and can play a role in vivo detoxification.
Third, transgenic wheat scab resistant phenotype and statistical data
On the basis of toxin analysis after the transgenic wheat is infected by fusarium graminearum, disease resistance investigation is carried out on the transgenic wheat. Comprises an overexpression transgenic line forcibly expressed under a ubiquitin promoter, wherein the receptor material is wheat variety fielder; and the original expression transgenic line generated by transforming the original promoter of ThFhb7 donor material in wheat variety Kenong 199, and performing related disease resistance investigation.
The fusarium graminearum spore suspension is prepared by the method, and the concentration is adjusted to 2 multiplied by 105one/mL, 10. mu.l of spore suspension was inoculated onto the upper middle part of the ear while bagging and moisturizing. When the spores germinate on the spikelets and obvious hyphae can be seen, the bags are removed. Inoculating about 30 spikes to each strain, observing the disease condition, counting the number of small pathogenic spikes after 21d of inoculation, and performing differential analysis. The results are shown in fig. 4, where both the overexpressed and originally expressed transgenic lines were found to have significantly smaller numbers of diseased spikelets than the control recipient material.
And (4) conclusion: the overexpression vector and the original expression vector of the ThFhb7 gene are constructed. Transferring the two vectors into corresponding wheat receptors to obtain T0Strictly self-pollinating the ThFhb7 high-expression wheat line to T by PCR and RT-PCR screening3. The scab resistant spike of the positive transgenic line is identified by using a single flower instillation method, and the morbidity spikelet number of the over-expression transgenic line and the morbidity spikelet number of the original expression transgenic line are obviously lower than that of a control receptor material. The experimental results show that the expression of ThFhb7 can obviously improve the gibberellic disease resistance of wheat.
Example 2
This example is a test of transgenic wheat for resistance to various fusarium and stalk rot.
1. Experimental materials:
plant materials of agricultural 199 and ThFhb7 original expression nuclear transgenic strains and strain materials of wheat scab dominant species fusarium graminearum (F.graminearum, or F.g), fusarium asiaticum (F.asiticum) and wheat stem-rot dominant species pseudofusarium graminearum (F.pseudograminearum) are stored in the laboratory.
2. Experimental methods
The Fusarium strain was activated in a manner similar to that described above and stored for use. Selecting full transgenic receptor material Kenong 199 and transgenic line seed, culturing to two leaves and one heart to three leaves and one heart, selecting flat leaf part with consistent width, dividing into 3-3.5cm leaf segments, requiring neat fracture of leaf segment without causing damage to mesophyll tissue, making a circular wound at the middle position of upper surface of leaf segment, inoculating 1-2.5 μ L of 2 × 105Each/mL of different Fusarium spore suspensions, 24 replicates per strain, were cultured at 25 ℃ under humidified conditions for 3 days and the spread of lesions observed.
Selecting JM22 and RJM22 seedlings with consistent growth vigor, culturing to 4 leaves and one heart, making a regular round wound at the stem base part by using a pipette tip, and preparing 2.5 mu L of pseudofusarium graminearum conidium suspension at the wound part; and (5) moisturizing and attacking for 15d at the temperature of 28 ℃, observing the attack condition of the plants, and counting the mortality.
3. Results of the experiment
3.1 in vitro leaf Multi-Strain resistance identification
Identifying the extensive resistance of Fusarium strain by using in-vitro leaf Fhb transgenic strain, culturing seedlings to two leaves and one heart to three leaves and one heart, selecting the same leaf position, and dividing the leaf into 3-3.5cm leaf segments, wherein the fracture of the leaf segment is required to be neat without causing the damage of mesophyll tissues, a round wound is made at the middle position of the upper surface of the leaf segment by using a liquid transfer device wall head, and the round wound is inoculated with 2.5 mu L of 2 multiplied by 10 mu L of liquid transfer agent with the concentration of 2 multiplied by 105Each/mL of different Fusarium spore suspensions, 24 replicates per strain, were cultured at 25 ℃ under humidified conditions for 3 days and the spread of lesions observed. Fusarium graminearum and Fusarium asia are the two major strains responsible for head blight. Fusarium pseudograminearum is the predominant strain responsible for the stalk rot of wheat. First, a round wound was made in the center of the excised leaf of a two-leaf one-heart seedling by using a pipette tip, and 2. mu.L of a Fusarium conidia suspension was inoculated at the wound site and moisturized at 25 ℃ for 3 days. Then theThe area of necrotic spots was measured for fusarium resistance assessment. As a result, as shown in FIGS. 5(a), 5(b) and 5(c), the ThFhb7 transgenic plant showed a significant reduction in the lesion area of the leaf compared to the susceptible control family of agricultural 199 inoculated with spore suspensions of three species, Fusarium graminearum, Fusarium asia and Fusarium pseudograminearum.
3.2 Stem root rot resistance results
The results of inoculating the recipient material and the ThFhb7 transgenic material with a Fusarium graminearum conidium suspension 15d using the method of shoot base wound inoculation are shown in FIG. 6(a), and indicate that the incidence of transgenic wheat shoot base is reduced compared to the control. The results in FIG. 6(b), (c) show that the mortality rate of the plants with the ThFhb7 gene is significantly lower than that of the wild type. Pseudograminearum is a dominant species responsible for the stem rot of wheat. In FIG. 6(a), the stalk base rot disease was observed by making a round wound at the base of the stalk of a four-leaf single-heart wheat seedling with a pipette tip, inoculating 2. mu.L of a Fusarium conidium suspension at the wound site, and moisturizing the wound for 15 days at 25 ℃. FIG. 6(b) is a graph showing the statistical analysis of mortality and the differential analysis after 15 days of illness at 25 ℃.
And (4) conclusion: the extensive resistance of the ThFhb7 transgenic strain to Fusarium species is identified by utilizing the ThFhb7 transgenic strain and the excised leaf of the recipient material farm 199, and the result shows that the scab areas of the three strains, namely the Fusarium graminearum, the Fusarium asianum and the Fusarium pseudograminearum, on the leaf are all obviously smaller than those of an infectious control, which indicates that the ThFhb7 has a relatively broad-spectrum resistance to the Fusarium; among them, Fusarium pseudograminearum is the dominant species responsible for the stem rot of wheat. In addition, the experiment proves that the short-fragment translocation line material R-JM22 containing ThFhb7 and the ThFhb7 gene strain which are taken as the background by JM22 are inoculated with Fusarium graminearum to identify the wheat stem basal rot resistance of the two materials, the result is consistent with the leaf phenotype, and the two materials show good wheat stem basal rot resistance, which indicates that ThFhb7 can improve the resistance of wheat to the stem basal rot.
Example 3
This example is transgenic maize T0Preliminary resistance experiments on the generation plants.
1. Experimental Material
The tetraploid elytrigia repens is used for amplifying a target gene ThFhb7 sequence fragment, and the overexpression vector is used for transforming a receptor material into KN 5585. The expression vector pCAMBIA3300 was supplied by the Chinese academy of agricultural sciences, and the E.coli (Escherichia coli) DH 5. alpha. strain was kept in the laboratory.
2. Experimental methods
Maize transgenesis was performed by the national focus laboratory of Shandong university of agriculture, using a method similar to that of example 1. Meanwhile, PCR detection, RT-PCR detection and plant scab resistance phenotype identification of the ThFhb7 transgenic maize plant are carried out by adopting the method.
T to be converted to ThFhb73Selecting positive transgenic seedling from plant, culturing to two leaves and one heart, selecting the part with flat leaves and consistent width, dividing into 3-3.5cm leaf segments, requiring the leaf segment fracture to be neat without causing mesophyll tissue damage, making a round wound at the middle position of the upper surface of the leaf segment, inoculating 1-2.5 μ L of the round wound with concentration of 2 × 105Each mutant was replicated 10 times in each of the fusarium graminearum spore suspensions at 25 ℃ under humid conditions for 3 days, and the spread of lesions was observed.
3. Results of the experiment
3.1 identification of resistance to gibberellic disease of plants transformed with ThFhb7 gene
Using three T3The in vitro leaf phenotype of the transgenic positive lines (W1, W2, W3) identifies gibberellic disease resistance in transgenic ThFhb7 maize plants. A round wound is made in the center of the in vitro leaf, and fusarium graminearum spore suspension is inoculated on the wound; the cells were incubated for 3 days under humid conditions, and the lesion size was counted and analyzed differently from the control.
Selecting three highly expressed T3Transgenic lines, with excised leaves for gibberellic disease resistance evaluation, showed a significant reduction in lesion area compared to the recipient material susceptible control. The area of necrotic spots 3 days after inoculation was counted and at least 10 leaves ex vivo were used for each sample. The experimental results are shown in FIG. 7, and show that the over-expression vector of ThFhb7 gene is obtained by constructing the over-expression vector and transferring the over-expression vector into cornT0And (3) carrying out in vitro leaf disease resistance identification on a ThFhb7 high-expression corn strain through PCR and RT-PCR screening, and carrying out gibberellic disease resistance identification on a positive transgenic strain, wherein the lesion area of the in vitro leaf of the transgenic corn is obviously smaller than that of a susceptible control. The experiment preliminarily shows that the expression of ThFhb7 can also obviously improve the resistance of corn to fusarium graminearum infection.
Example 4
This example is directed to mutations in nucleic acid molecules and functional studies thereof.
1. Experimental Material
Wheat 22 was used as background and was cross-bred into ThFhb7 wheat material, Nos. A052-2 and A079-3, created and stored earlier in the laboratory.
2. Construction of mutant populations
In the early stage of a laboratory, according to EMS pretreatment experiment results, 0.8% EMS solution is selected to be processed in a dark place, is vibrated for 10 hours at the speed of 150rpm under the condition of 25 ℃, is continuously washed for 3 to 4 hours by tap water, processes 2500 seeds each time, processes 10000 seeds in 4 batches, and is all planted in a field. M1 plants are strictly bagged for selfing, and 1 seed is taken from each plant after maturation to form M2 single seed-borne population. Each M2 individual plant was labeled and the leaf was taken for subsequent DNA extraction and harvested as individual plants after maturation.
TILLING assay
3.1PCR amplification
Since the full length of the gene was 864bp, we sequenced the full length of the gene and screened for mutants using a traditional Sanger sequencing platform. Full-length primer of ThFhb7
F：5'-TTCATCATCCTGCTAGGCGATAAGA-3'
R：5'-CTACTTCACCTCGGGGCATACTTGTC-3'
We used UNG enzyme treatment in combination with targeted preamplification using dUTP, in which deoxythymidine triphosphate (dTTP) was replaced with deoxyuridine triphosphate (dUTP). Prior to initiation of PCR, any uracil-containing PCR products will be degraded, i.e., residual contaminants will be eliminated, using uracil DNA N-glycosylase (UNG). PCR amplification was performed in a total volume reaction of 10. mu.l, containing 1 × Multiplex PCR Mastermix (UNG) (CWBIO Bio, China), 0.7. mu.M of each primer and 100ng of template DNA. The amplification curve included 1 cycle at 50 ℃ for 2-8 minutes and 5 minutes at 95 ℃ followed by 35 cycles of 30s at 94 ℃, 30s at 60 ℃ and 50s at 72 ℃ and finally 10 minutes at 72 ℃. The homozygous and heterozygous status of each point mutation was verified by manual inspection of the signal peak maps by DNAMAN. Homozygous mutants were screened for further phenotypic characterization of FHB resistance.
3.2 phenotypic identification of resistance to gibberellic disease
Culturing the selected homozygous mutant plant until two leaves and one heart to three leaves and one heart, selecting the part with flat leaves and consistent width, dividing the part into 3-3.5cm leaf segments, requiring the leaf segment to have regular fracture without causing mesophyll tissue damage, making a round wound at the middle position of the upper surface of the leaf segment, inoculating 1-2.5 μ L of the round wound with concentration of 2 × 105Each mutant was replicated 10 times in each of the fusarium graminearum spore suspensions at 25 ℃ under humid conditions for 3 days, and the spread of lesions was observed.
4. Results of the experiment
4.1 mutant screening
The about 4000M2 mutant was screened for mutations by direct sequencing (Sanger) of the full-length PCR product of ThFhb7 (primers as before). To avoid possible template contamination during PCR, deoxyuridine triphosphate (dUTP) was used in place of deoxythymidine triphosphate (dTTP) in PCR, and uracil DNA N-glycosylase (UNG) treatment was performed to degrade any uracil-containing PCR product in the template. The final screen yielded 24 amino acid variants with partial alignment shown in FIG. 8.
4.2 phenotypic identification of mutant scab resistance
The phenotypic identification of mutants against gibberellic disease is shown in FIG. 9, where the wheat lines containing ThFhb7 were subjected to EMS mutagenesis. By using excised leaves for scab resistance assessment, a total of 7 mutants with amino acid changes in the CDS region were significantly different from the disease-resistant control. The area of necrotic spots 3 days after inoculation was counted and at least 10 leaves ex vivo were used for each sample.
Through the combination of UNG enzyme treatment and targeted pre-amplification using dUTP, residual contamination and false positive methods are eliminated, and finally 24 mutants are obtained through screening. The resistance to gibberellic disease was assessed by using leaves ex vivo, 5 missense mutations, 2 stop mutations out of 7 significantly different mutants of the ThFhb7 gene from the disease-resistant control; although the disease susceptibility of the 7 mutants is different, the lesion areas are all significantly larger than those of the disease-resistant control (statistics of the area of the necrotic spots of the mutants in FIG. 9). Functional analysis showed that the proto-decyloxy activity was retained to varying degrees in each of the 24 mutants. Wherein the stop mutation is located at the C-terminus of ThFhb7, and ends at amino acids 209 and 243, respectively, but both ends do not result in complete loss of function of the enzyme. Thus, it was shown that the functional domain of the enzyme is mainly N-terminal. In addition, the 5 missense mutations at 34, 48, 98, 99 and 106 in the mutant had a greater effect on the activity of the de-epoxy group. Therefore, it can be regarded as a key amino acid.
Example 5
This example is a functional analysis of homologous sequence genes.
1. Sequence comparison
On the basis of the sequence of the de-epoxidase gene of elytrigia elongata (SEQ ID No.1), blastn alignment was performed by NCBI without searching for any annotated highly homologous genes under default parameters. The results of the NCBI alignment are shown in FIG. 10. But according to
The genus has information of homologous genes, and the inventor jointly searches genome databases of other laboratories to obtain 11 sequences derived from the genus, wherein the sequences are respectively shown as SEQ ID No. 25-35. As shown in FIG. 11, these sequences have more than 90% sequence identity with the de-epoxidase gene of the decaploid Elytrigia elongata. In addition, the inventor also separates a gene with 98 percent of sequence identity with the decaploid desacycloxygenase gene from the elytrigia elongata diploid, and the sequence is shown as SEQ ID No. 2.
Experiments on toxins transferred into pichia pastoris from sequences homologous to ThFhb7 gene and derivatives thereof
From
11 homologous genes are obtained from the genus, the sequence is verified to be correct by sequencing, and a yeast expression vector of the homologous sequence is constructed by using pPICZ alpha A-ThFhb 7. The recombinant plasmid was then linearized with Sac I and a 1ml single stranded DNA sample was boiled for 5 minutes and then rapidly cooled on ice. Kept on ice. The yeast competed and LiCl removed with a pipette. For each transformation, the following reagents were added in the order given to the cells. PEG protects cells from the deleterious effects of high LiCl concentrations. Each tube was vortexed vigorously until the cell pellet was completely mixed (about 1 minute). The tubes were incubated at 30 ℃ for 30 minutes. The thermal shock was carried out in a water bath at 42 ℃ for 20-25 minutes. The cells were centrifuged at 6000 to 8000rpm to pellet. The pellet was resuspended in 1ml YPD and incubated with shaking at 30 ℃. After 1 and 4 hours, 25 to 100. mu.l were plated on YPD plates containing appropriate concentrations of Zeocin. The plates were incubated at 30 ℃ for 2-3 days. And selecting 10 single colonies for enrichment culture, extracting yeast chromosome DNA, and detecting positive recombinant bacteria by PCR. Generally, pPICZ alpha A universal primers are adopted for PCR identification, and if a yeast expression vector pPICZ alpha A is taken as a template, a target fragment can be amplified; if pPICZ alpha A-ThFhb7 homologous sequence is used as template, the target fragment with the size of +588bp of the target band is amplified.
3. Expression of homologous sequences and toxin manipulation
The screened positive yeast single colony (X33/pPICZ alpha A-ThFhb7 homologous sequence) and negative yeast single colony (X33/pPICZ alpha A) were inoculated to 25ml of BMGY medium, and cultured at 28 ℃ -30 ℃ until OD600 was 2-6. Centrifuging at 4000rpm at room temperature for 5min, discarding the supernatant, collecting the cells, resuspending the cells with 50ml-100ml (0.5% -1% methanol) BMMY liquid medium to OD600 ═ 1, transferring to a 500ml conical flask, culturing at 28 ℃ -30 ℃, and adding methanol to the final concentration of 0.5% every 24h to maintain inducible expression. After the induction for 48h, subpackaging the bacterial liquid with 5ml into 15ml centrifuge tubes, adding various trichothecene compounds into the centrifuge tubes until the final concentration is 25 mu g/ml, continuing the induction for 48h-72h, and collecting thalli for LC-HRMS analysis.
LC-HRMS analysis
The dispensed samples were centrifuged at 4 ℃ to remove the supernatant. Quick freezing in liquid nitrogen, adding a little quartz sand, grinding with plastic grinding rod, and adding pre-cooled 1.3ml 75% methanol water (containing 0.1% formic acid). Shaking for 10s, performing ultrasonic treatment at room temperature for 30min, collecting supernatant, and transferring to a new centrifuge tube. Vacuum concentrating to dry powder. Before injection, 100uL 20% acetonitrile heavy suspension, through 0.22 u m filter membrane filtration, transfer to the injection vial for LC-HRMS detection.
LC-HRMS (/ MS) analysis data were analyzed using Xcaliibur 2.1.0. Extracted Ion Chromatograms (EICs) of toxins and derivatives thereof were studied using the proposed chromatographic peak shape, retention time (+ -0.2 minutes) and mass (+ -5 ppm) of the bioconversion products. And (4) analyzing neutral loss according to a secondary map and a substance basic structure to conjecture a chemical structure.
The genes are respectively transferred into yeast cells to be expressed into corresponding proteins, and the proteins are analyzed by LC-HRMS. The results of the experiment are shown in FIG. 12. The other 12 homologous sequences were transferred into Pichia pastoris and treated with DON. LC-HRMS detection shows that DON-GSH is generated. Ion chromatograms from transgenic yeast processed by LC-HRMSDON extraction in positive ion mode to give DON-GSH adduct, M/z 604.21730 (corresponding to [ M + H ]]+，Δ±5ppm)。
While the present invention has been described with reference to exemplary embodiments, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. Many modifications and variations may be made to the exemplary embodiments of the present description without departing from the scope or spirit of the present invention. The scope of the claims is to be accorded the broadest interpretation so as to encompass all modifications and equivalent structures and functions.
Sequence listing
<110> Shandong university of agriculture
Application of <120> nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker
<130> BH2000026-1
<141> 2020-03-05
<160> 35
<170> SIPOSequenceListing 1.0
<210> 1
<211> 846
<212> DNA
<213> Tinopyrum_ponticum_(wild_type_for_mutants)
<400> 1
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
<210> 2
<211> 846
<212> DNA
<213> Tinopyrum_elongatum
<400> 2
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 atgctggggg acctggctgc tttgtttcgg 660
aaagatgcga gcgggccgtt cctgttgggg cagagggcca cgtatgcgga catgattgtc 720
ggtggctggt tgcgcatgat gcgggcgacg ttgccggtga gcgagtggca ggaggcgaga 780
gcctgccacg gggctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840
<210> 3
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 3
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 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
<210> 4
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 4
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
<210> 5
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 5
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 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
<210> 6
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 6
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 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
<210> 7
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 7
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
<210> 8
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 8
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 cctctgaaac 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
<210> 9
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 9
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
ggtggctgat tgcgcatgat gcgggcgacg ttgccggtga gtgagtggca ggaggcgaga 780
gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840
<210> 10
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 10
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
<210> 11
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 11
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 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 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
<210> 12
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 12
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 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
<210> 13
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 13
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
<210> 14
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 14
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
ctagagctcg 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
<210> 15
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 15
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 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 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
<210> 16
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 16
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
<210> 17
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 17
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 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
<210> 18
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 18
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 gatcaaggcc 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
<210> 19
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 19
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
<210> 20
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 20
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 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
<210> 21
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 21
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 acgtagcctt 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
<210> 22
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 22
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
<210> 23
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 23
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 gcgggcgatg ttgccggtga gtgagtggca ggaggcgaga 780
gcctgccacg gagctatctt tgggcagctg catgatgcgc tggacaagta tgccgaggtg 840
<210> 24
<211> 846
<212> DNA
<213> Tinopyrum_ponticum
<400> 24
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
<210> 25
<211> 843
<212> DNA
<213> Epichloe_bromicola
<400> 25
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> 26
<211> 840
<212> DNA
<213> Epichloe_amarillans
<400> 26
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> 27
<211> 795
<212> DNA
<213> Epichloe_baconii
<400> 27
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> 28
<211> 861
<212> DNA
<213> Epichloe_festucae
<400> 28
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 gtcttcgggc ggctgcatga tgcgctggac 840
aagtatgccg aggtgaagta g 861
<210> 29
<211> 846
<212> DNA
<213> Epichloe_gansuensis
<400> 29
atggccacct ccacctccac ttccgcctcc accccaatca tcttctacga catagcccag 60
cgcccccccg 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 tcccgcctcg 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
<210> 30
<211> 846
<212> DNA
<213> Epichloe_typhina
<400> 30
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
<210> 31
<211> 846
<212> DNA
<213> Epichloe_uncinata
<400> 31
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
<210> 32
<211> 843
<212> DNA
<213> Epichloe_sylvatica
<400> 32
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> 33
<211> 843
<212> DNA
<213> Epichloe_aotearoae
<400> 33
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> 34
<211> 828
<212> DNA
<213> Epichloe_glyceriae
<400> 34
atggccacct ccaccccaat catcttctac gacatagccc agcggccccc cgtcgcagaa 60
acatgctgcg ccgtcaaccc ttggaaatcc agactggccc tcaacttcaa ggccgtcccc 120
tacacaacca cctgggtcag catgccagac atcagcagcg tccgcgccag cctcaacgtg 180
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 gagcgggccg 660
ttcttgctgg gacagaaggc cacgtatgcg gatctgattg tcggtggctg gttgcggatg 720
atgcgggcga cgttgcccgc gagtgagtgg caggaggcga gagcctggca tggcgctgtc 780
ttcgggcagc tgcatgatgc gctggacaag tatgccgagg tgaagtag 828
<210> 35
<211> 843
<212> DNA
<213> Epichloe_brachyelytri
<400> 35
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 (6)
1. Use of a nucleic acid molecule for removing a monoterminal mycotoxin in a plant, wherein the sequence of the nucleic acid molecule is selected from one of the group consisting of (a), (b), and (d):
(a) the sequence shown as SEQ ID No. 1-35;
(b) a sequence which is modified by host codon bias according to degenerate codons on the basis of the sequence of (a) and encodes an amino acid sequence which is identical to the encoded amino acid sequence of the corresponding sequence in (a);
(d) a sequence which has more than 99 percent of sequence identity with SEQ ID No.1 or 2 and is derived from elytrigia elongata and encodes an enzyme with the catalytic activity of the dehydroepoxidation, or a sequence which has more than 99 percent of sequence identity with SEQ ID No. 25-35 and is derived from a corresponding species of the genus corynebacterium.
2. Use according to claim 1, wherein the plant is a plant capable of infecting fusarium.
3. The use according to claim 1, wherein the plant is a food crop, a fruit tree, a flower or tobacco.
4. Use according to claim 1, wherein the plant is selected from the group consisting of corn, wheat, barley, rice, oat, millet, highland barley, alfalfa, elytrigia, ryegrass, maize mexican grass, pennisetum pratense, pennisetum purpureum, peanut, cotton, sunflower, sweet potato, soybean, tomato, watermelon, cantaloupe, grape, hevea brasiliensis, banana, agaricus bisporus, carrot, bok choy, cabbage, cucumber, lily, cymbidium faberi, butterfly orchid, cyclamen, poinsettia, gerbera, chrysanthemum, platycodon grandiflorum and carnation, or a plant obtained by crossing these plants.
5. Use of a nucleic acid molecule for disease control, wherein the sequence of the nucleic acid molecule is selected from the group consisting of (a), (b), and (d):
(a) the sequence shown as SEQ ID No. 1-35;
(b) a sequence which is modified by host codon bias according to degenerate codons on the basis of the sequence of (a) and encodes an amino acid sequence which is identical to the encoded amino acid sequence of the corresponding sequence in (a);
(d) a sequence which has more than 99 percent of sequence identity with SEQ ID No.1 or 2, is derived from elytrigia elongata and codes an enzyme with the catalytic activity of the dehydroepoxidation, or a sequence which has more than 99 percent of sequence identity with SEQ ID No. 25-35, is derived from a corresponding species of the genus corynebacterium;
the disease is a plant disease related to trichothecene toxins caused by fusarium, cephalosporium, fistular or trichoderma fungi.
6. A plant cell comprising an exogenous nucleic acid molecule introduced by genetic engineering means, wherein said nucleic acid molecule has a base sequence selected from the group consisting of (a), (b), and (d):
(a) the sequence shown as SEQ ID No. 1-35;
(b) a sequence which is modified by host codon bias according to degenerate codons on the basis of the base sequence in (a) and encodes an amino acid sequence identical to the amino acid sequence encoded by the corresponding sequence in (a);
(d) a sequence which has more than 99 percent of sequence identity with SEQ ID No.1 or 2 and is derived from elytrigia elongata and encodes an enzyme with the catalytic activity of the dehydroepoxidation, or a sequence which has more than 99 percent of sequence identity with SEQ ID No. 25-35 and is derived from a corresponding species of the genus corynebacterium.
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010146400.9A CN111549042B (en) | 2020-03-05 | 2020-03-05 | Application of nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker |
| CA3174549A CA3174549A1 (en) | 2020-03-05 | 2020-12-11 | Nucleic acid molecule, and polypeptide having epoxy group-removal catalytic activity and use thereof |
| AU2020433667A AU2020433667A1 (en) | 2020-03-05 | 2020-12-11 | Nucleic acid molecule, and polypeptide having epoxy group-removal catalytic activity and use thereof |
| PCT/CN2020/135821 WO2021174949A1 (en) | 2020-03-05 | 2020-12-11 | Nucleic acid molecule, and polypeptide having epoxy group-removal catalytic activity and use thereof |
| CA3176479A CA3176479A1 (en) | 2020-03-05 | 2020-12-11 | Nucleic acid molecule, and polypeptide having epoxy group-removal catalytic activity and use thereof |
| US17/905,591 US20230063483A1 (en) | 2020-03-05 | 2020-12-11 | Nucleic acid molecules, polypeptides having epoxy group-removing catalytic activity and use thereof |
| BR112022017582A BR112022017582A2 (en) | 2020-03-05 | 2020-12-11 | NUCLEIC ACID AND POLYPEPTIDE MOLECULE THAT HAS CATALYTIC ACTIVITY TO REMOVE THE EPOXY GROUP AND USE THEM |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010146400.9A CN111549042B (en) | 2020-03-05 | 2020-03-05 | Application of nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN111549042A CN111549042A (en) | 2020-08-18 |
| CN111549042B true CN111549042B (en) | 2021-09-21 |
Family
ID=71998077
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010146400.9A Active CN111549042B (en) | 2020-03-05 | 2020-03-05 | Application of nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111549042B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2020433667A1 (en) * | 2020-03-05 | 2022-09-29 | Shandong Agricultural University | Nucleic acid molecule, and polypeptide having epoxy group-removal catalytic activity and use thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544398A (en) * | 2016-11-09 | 2017-03-29 | 山东农业大学 | A kind of wheat scab resistance authentication method |
| CN107630106A (en) * | 2017-11-21 | 2018-01-26 | 山东农业大学 | A kind of molecular labeling of quick detection E. elongata anti gibberellic disease gene |
-
2020
- 2020-03-05 CN CN202010146400.9A patent/CN111549042B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106544398A (en) * | 2016-11-09 | 2017-03-29 | 山东农业大学 | A kind of wheat scab resistance authentication method |
| CN107630106A (en) * | 2017-11-21 | 2018-01-26 | 山东农业大学 | A kind of molecular labeling of quick detection E. elongata anti gibberellic disease gene |
Non-Patent Citations (3)
| Title |
|---|
| Epichloe festucae Fl1 chromosome 3, complete sequence;GenBank登录号：CP031387.1;《GenBank》;20181123;参见序列及相关信息 * |
| High-density mapping of the major FHB resistance gene Fhb7 derived from Thinopyrum ponticum and its pyramiding with Fhb1 by marker-assisted selection;Jun Guo等;《Theoretical and Applied Genetics》;20150729;第128卷(第11期);第2301-2316页，参见全文 * |
| 普通小麦-十倍体长穗偃麦草衍生新品种抗赤霉病基因的分子鉴别;刘新伦等;《中国农业科学》;20171031;第50卷(第20期);第3908-3917页，参见全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN111549042A (en) | 2020-08-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP2854514B1 (en) | Selection of symbiota by screening multiple host-symbiont associations | |
| CN106148353B (en) | Brown planthopper resistant gene in rice Bph6 and its close linkage molecular labeling | |
| CN112626040B (en) | ZmRBOHB gene and application of encoding protein thereof in resistance to maize ear rot | |
| CN107937416A (en) | Improve gene and its application of nitrogen fertilizer for paddy rice utilization ratio and yield | |
| CN107034223A (en) | The anti-plant hopper gene BGIOSGA015651 of adjusting and controlling rice and its application | |
| CN108588041B (en) | Gossypium barbadense cytochrome P450 gene, and coding protein and application thereof | |
| CN111549042B (en) | Application of nucleic acid molecule in plant transgenosis, molecular breeding, disease control and molecular marker | |
| CN111876514B (en) | Rapid detection method for gibberellin miniascape generated in bakanae disease germ of rice | |
| CN117082972A (en) | Plant resistance genes and means for their identification | |
| CN113573576B (en) | Downy mildew resistance in spinach | |
| CN110004155B (en) | Disease-resistant gene and protein for controlling plant aversion and aphid resistance characters and application thereof | |
| CN109694876A (en) | Cultivate the method for low Cd accumulation rice and its purposes of associated materials | |
| CN109706154B (en) | CsPR3 gene and application thereof in cucumber fusarium wilt resistance | |
| CN112662804B (en) | Primer group, kit and method for detecting pathogenic variation of avirulence gene AvrPi9 of rice blast | |
| CN113637678A (en) | Application of gene GhSWEET42 in prevention and treatment of cotton verticillium wilt | |
| CN106834337A (en) | The DON tolerances and FHB resistances of arabidopsis are improved using wheat cdna | |
| CN111635906A (en) | Gossypium barbadense GbCYP72A2 gene, and coding protein and application thereof | |
| Kareem et al. | Comparison of Rhizoctonia solani Isolated from Soil in Baghdad–Iraq Genetically with World Isolates | |
| WO2021174949A1 (en) | Nucleic acid molecule, and polypeptide having epoxy group-removal catalytic activity and use thereof | |
| Stahr et al. | Long-read sequencing genome assembly of Ceratocystis fimbriata enables development of molecular diagnostics for sweetpotato black rot | |
| Manjunatha et al. | Morphological and molecular identification of stem rot pathogen in berseem (Trifolium alexandrinum L.) | |
| CN115209725B (en) | Downy mildew resistance of spinach | |
| CN114591984B (en) | Application of OsAP79 gene in inducing rice to resist brown planthoppers | |
| CN117447567B (en) | OsNramp5 mutant and related products and application thereof | |
| CN116716190B (en) | Chaetomium spirochaeta 201 and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |