CN118109482A - Rye stripe rust resistance gene and application thereof - Google Patents
Rye stripe rust resistance gene and application thereof Download PDFInfo
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Abstract
The invention provides a stripe rust resistance gene and application thereof, wherein the nucleotide sequence of the stripe rust resistance gene is shown as SEQ ID NO. 2 or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with the nucleotide sequence shown as SEQ ID NO. 2. The stripe rust resistance gene of the invention is overexpressed in stripe rust hypersensitive material tobacco grower 19, and transgenic positive plants show near immunity.
Description
Technical Field
The invention belongs to the field of plant genetic engineering, and relates to a rye SECCE Rv1G0452990 stripe rust resistance gene and application thereof.
Background
Wheat (Triticum aestivum L.) is one of the important grain crops in China, and the maintenance and improvement of the yield are important guarantees of the national grain safety. Wheat stripe rust (WHEAT STRIPE rust) is a common disease of wheat production and is a foliar disease caused by Puccinia striiformis westend.f. sp. The most pronounced feature of stripe rust is the production of pronounced yellow or orange spores in the leaves, generally aligned along the veins. 88% of the wheat production area of the world is easy to be infected by stripe rust, the wheat yield is reduced by 500 ten thousand tons due to the stripe rust each year, the lost economic value is as high as 10 hundred million dollars (see reference ：Colin,R,Wellings.Global status of stripe rust:a review of historical and current threats.Euphytica.2011,179:129-141). China is one of the largest stripe rust epidemic areas in the world, four pandemics have occurred since the country was built, and the yield loss is about 130 hundred million kilograms respectively in 1950, 1964, 1990 and 2002 (see reference ：Wan A,Zhao Z,Chen X,et al.Wheat stripe rust epidemic and virulence of Puccinia striiformis f.sp.triticiin China in 2002.Plant Disease,2004,88:896-904).. Therefore, the breeding of the stripe rust resistant wheat variety and the excavation and utilization of disease resistant genes have important significance for guaranteeing the national grain safety).
Modern breeding aimed at high yield greatly weakens the genetic diversity of crops, and loss of genetic diversity not only limits further improvements in yield and quality, but also makes it difficult for crops to cope with global climate change and pest epidemic. The related species of wheat contain a plurality of excellent genes and are important genetic resources for genetic improvement of common wheat. Therefore, the introduction of a useful gene of a wheat closely related species into a conventional cultivated wheat is one of important and effective ways of improving wheat varieties.
Rye (SECALE CEREALE l.), a annual or perennial herbaceous crop of the genus lolium of the family gramineae, is one of the wild kindred species of wheat. Rye is a diploid species whose genome is RR. Rye has excellent characters lacking in common cultivated wheat and is an important wild germplasm resource for genetic improvement of common wheat. Triticale is a new species artificially synthesized by wheat (Triticum) and rye (Secale) species via intergeneric sexual crosses and doubling of hybrid chromosome numbers. The wheat hybrid rice combines the characteristics of high yield and quality of wheat, disease resistance and cold resistance of rye, high lysine content and the like, has huge hybrid growth advantages, and can be used as feed, brewing, biological energy and grain. The triticale obtained by doubling diploid rye by crossing with wheat of different ploidy has mainly six ploidy (AABBRR) and eight ploidy (AABBDDRR) 2 types.
There is no literature report on the research content of the stripe rust resistance gene sequence and molecular markers in triticale Rozovskaya-7.
Disclosure of Invention
According to the invention, the two triticale parents Rozovskaya-7 and 4100 are respectively expressed as near immunity and high sense by inoculating and identifying the physiological race CYR32-34 of the stripe rust. The F1 is obtained after crossing two triticale parents, and the F2 group is obtained after selfing. 200 single plants in the F2 group are inoculated and identified, 144 plants are found to be resistant to diseases and 56 plants are found to be high in sense. And respectively selecting 40 extreme disease-resistant and 40 extreme disease-sensitive materials from the F2 population to form an anti-pool and a sensitive pool, taking leaves to extract total RNA, sequencing, and finding that the stripe rust resistance gene is positioned at the tail end of 6RL of rye through BSR (Bulk SEGREGANT RNA sequencing) analysis. 11 co-dominant Indel markers were developed near 6RL using two parental resequencing data, and the stripe rust resistance gene was further mapped to the 1.25Mb interval (corresponding to Lo7 reference genome 6r:878.556-879.809 Mb) using fine mapping with F2 consisting of 1246 individuals. Subsequently 24 genes in the interval were analyzed in combination with anti-pool sensory pool transcriptome data, and 19 genes were found to have FPKM <1 in both anti-pool and sensory pool, possibly pseudogenes. The other 5 genes only had SECCE expression levels of the Rv1G0452990 in the anti-pool higher than in the sensory pool, thus SECCE Rv1G0452990 was identified as a candidate gene. Using the rye reference genome sequence (https:// ftp.ebi.ac.uk/ensemblgenomes/pub/release-56/plant/fasta/secale _ cere ale/dna /), the SECCE6Rv1G0452990 sequence in Rozovskaya-7 was cloned and a specific molecular marker 6R2990-1 was developed. To verify SECCE6Rv1G0452990 function, genetic transformation with stripe rust-sensitive wheat as a receptor material is performed by using an overexpression vector of SECCE6Rv1G0452990, and a transgenic result shows that the over-expression of SECCE6Rv1G0452990 can improve the resistance of wheat to stripe rust, and the result shows that SECCE6Rv1G0452990 is a stripe rust resistance gene.
Specifically, the invention provides the following technical scheme:
in one aspect, the invention provides a stripe rust resistance gene characterized in that the nucleotide sequence of the stripe rust resistance gene is as shown in SEQ ID NO.2 or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity with the nucleotide sequence shown in SEQ ID NO. 2.
In another aspect, the invention provides an expression vector, characterized in that the expression vector comprises a stripe rust resistance gene as described above.
In another aspect, the invention provides a host cell, characterized in that the host cell comprises a stripe rust resistance gene or expression vector as described above.
In another aspect, the present invention provides a method for detecting the presence or absence of a stripe rust resistance gene in a plant to be tested, characterized in that the method comprises detecting the presence of a stripe rust resistance gene according to claim 1 or a specific molecular marker thereof, preferably the sequence of said specific molecular marker is as shown in SEQ ID NO. 3, preferably the sequence of SEQ ID NO. 3 is detected using the primers shown in SEQ ID NO. 4 and SEQ ID NO. 5.
In another aspect, the invention provides a method of increasing the resistance of a plant to stripe rust, the method comprising introducing into the plant or a cell of the plant a stripe rust resistance gene or expression vector or host cell as described above.
In another aspect, the invention provides a method of growing a stripe rust resistant plant, the method comprising introducing a stripe rust resistant gene or expression vector or host cell as described above into the plant or cells of the plant.
In another aspect, the invention provides the use of a stripe rust resistance gene or expression vector or host cell as described above for increasing the stripe rust resistance of a plant or for growing a stripe rust resistant plant.
In another aspect, the present invention provides a method for obtaining a plant cell or plant carrying a stripe rust resistance gene, characterized in that the method comprises introducing a stripe rust resistance gene or expression vector or host cell as described above into the plant cell or the plant.
In another aspect, the invention provides a molecular marker for detecting the existence of a stripe rust resistance gene of a plant to be detected, which is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 3.
In some embodiments, the plant is wheat or rye.
In some embodiments, the stripe rust is caused by physiological races CYR32, CYR33 and/or CYR 34.
Definition of the definition
GISH: genomic in situ hybridization techniques (genomic in situ hybridization) developed based on fluorescence in situ hybridization techniques. The technique employs total genomic DNA from one species as a label probe, and is blocked with total genomic DNA from another species at an appropriate concentration, and in situ hybridization is performed on the target chromosome. Between the blocking DNA and the labeled DNA probes, the blocking DNA preferentially hybridizes with non-specific sequences, the remaining specific sequences are hybridized mainly by the labeled probes, and finally, the chromosome sets, chromosomes or chromosome fragments of different sources are detected.
Inoculation identification: healthy triticale seedlings are cultivated in a greenhouse, inoculated by a method of manually scattering stripe rust spores, and wheat plants are subjected to moisturizing, illumination cultivation and the like to attack diseases so as to identify stripe rust resistance of the material.
Near immunity and high feel: according to the resistance degree of wheat varieties to stripe rust, the disease resistance of wheat varieties can be classified into six grades of immunity, high resistance, medium feeling and high feeling. Immunization did not produce any visible spore pile on the leaves. Near immunity is the appearance of small dead spots on the leaves, which do not produce a spore stack. The high sense is that the spores on the leaves are large and much, and the periphery is not green.
Extreme disease resistance and extreme disease susceptibility: the research of disease resistance inheritance mainly adopts a Mendelian analysis method, namely, hybridization of disease resistance and disease-sensitive plants is carried out, and the disease resistance separation condition of offspring is analyzed. In the F2 separation group, the most disease-resistant material is the extremely disease-resistant material, and the most disease-resistant material is the extremely disease-resistant material.
BSR: the mixed population separation analysis method (Bulked SEGREGANT ANALYSIS, BSA) is a rapid method for positioning the target trait genes by using an extreme trait individual mixed pool. In the segregating population, offspring DNA of extreme characters are selected and mixed in equal quantity, library construction and sequencing are respectively carried out on a mixed gene pool (GenePool) and parents, the Frequency (AF) of sequencing data of different libraries in polymorphic Sites (SNP) is calculated, and correlation is carried out on the sequencing data and a target phenotype, so that gene positioning or linkage molecular marker development related to the target characters is realized.
Co-dominant markers: meanwhile, dominant and recessive alleles can be detected, and the genetic markers of homozygous and heterozygous genotypes can be distinguished.
Indel label: insertion-deletion (InDel) refers to the insertion or deletion of nucleotide fragments of different sizes in the sequence at the same genomic locus between closely related species or different individuals of the same species, i.e., one or more bases in one sequence at a position that is homologous to another sequence. The InDel polymorphism molecular marker is a marker for PCR amplification by designing specific primers based on sequences at two sides of an insertion/deletion site, and the marker belongs to a length polymorphism marker in nature, and can be typed by using a convenient electrophoresis platform.
Ubiqutin promoter: ubiquitin (UBQ or Ubi) gene promoters, which are constitutive promoters, show significantly higher levels of gene expression in most tissues and many cell types of plants, especially in monocotyledonous plants (e.g. maize), and are often used to drive the overexpression of a gene.
Specific molecular markers: the specific molecular marker is developed based on functional Single Nucleotide Polymorphism (SNP) or insertion and deletion (InDels) loci in a gene sequence, and has the advantages of high selection efficiency, co-dominance, co-separation with a target gene, no influence of genetic background and the like.
FPKM: FPKM (FRAGMENTS PER Kilobase of exon model per Million FRAGMENTS MAPPED) is a commonly used method of quantifying gene expression, and is used to measure the level of expression of a gene (or transcript). FPKM is a formula for measuring the relative expression of genes. FPKM is the number of fragments aligned to a gene divided by the number of all sequences aligned to the genome (in Million) and the length of RNA (in KB).
Genome re-sequencing: methods for sequencing the genomes of different individuals from species whose genomic sequences are known. For analysis of differences between the genomes of different individuals, such as the discovery of single nucleotide polymorphism sites, insertion deletion sites, structural variation sites, copy number variation sites, and the like.
1BL/1RS translocation line: refers to wheat-rye translocation chromosomes formed by translocation of the short arm of the 1R chromosome of rye to the long arm of the wheat 1B chromosome. As the short arm of 1R is provided with genes for resisting wheat leaf rust, stem rust, stripe rust and powdery mildew, the germplasm containing 1BL/1RS is widely applied to the breeding of wheat in the world.
Drawings
FIG. 1 shows the chromosomal composition of hexaploid triticale Rozovskaya-7 stripe rust resistance identification and GISH identification. Panel a is hexaploid triticale Rozovskaya-7 and 4100 stripe rust identification, mingxian (Mingxian 169) was used as a disease control. Panel b and c are GISH assays of triticale Rozovskaya-7 and 4100, respectively, probed with the rye genome (green). The results of the inoculation of the stripe rust indicated Rozovskaya-7 immunization against stripe rust. GISH results showed that Rozovskaya-7 and 4100 were hexaploid triticale.
FIG. 2 shows the identification of hexaploid triticale Rozovskaya-7 and 4100F2 stripe rust. F2 plants were inoculated with a mixture of three physiological micro-species CYR32, CYR33, CYR34 in a leaf-heart period, and the statistical phenotype was counted after 14d inoculation. 1,4100; 2, rozovskaya-7;3-5, F2 disease-resistant plants; 6-8, F2 infected plants. The result shows that the separation of disease resistance occurs in the F2 group, and the difference between the F2 disease-resistant single plant and the F2 disease-sensitive single plant is obvious.
FIG. 3 shows the amplification result of SECCE Rv1G0452990 specific molecular marker 6R2990-1 in triticale and common wheat and transgenic seedlings. From left to right, the primers for the Marker,6 common wheat (Nicotiana 19, chinese spring, fielder, shannon 19, shannon 20 and Shannon 122), 31 BL/1RS translocation lines (dwarf 58, kennon 199 and Nongda 399) and 18 hexaploid triticale (4100、Rozovskaya-7、L20191212、PI 542533、PI 542560、PI 552974、PI 559373、PI 590946、PI 610226、CIXT33、PI 511870、PI 587384、PI542563、PI 610224、PI 610227、PI 628658、PI 610225、CIXT101).Tubilin have the amplification results that the DNA used by the invention has reliable quality, and the 6R2990-1 amplification results indicate that SECCE Rv1G0452990 can not be detected in the 6 common wheat and 31 BL/1RS translocation lines, and SECCE6Rv1G0452990 can not be detected in the 4 triticale and 14 triticale. The results indicate that SECCE Rv1G0452990 is not present in common wheat and 1BL/1RS translocation lines, and is present in triticale.
Figure 4 shows the stripe rust resistance results of transgenic positive plants. A mixture of three physiological micro-seeds CYR32, CYR33, CYR34 was inoculated in one-leaf one-heart period, and the statistical phenotype was counted after 14d inoculation. The inoculation result shows that SECCE Rv1G0452990 transgenic positive material is near immune to stripe rust, while SECCE Rv1G0452990 transgenic negative material is hypersensitive to stripe rust, and SECCE Rv1G0452990 is proved to be a stripe rust resistance gene.
Detailed Description
The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.
Example 1 identification of rye stripe rust resistance genes
The inventor uses the stripe rust physiological race CYR32, CYR33 and CYR34 (inoculated on a stripe rust identification platform of the national academy of sciences of China and development biology) to carry out mixed inoculation (the inoculation method refers to a powder spraying method in 3.3 wheat stripe rust propagation and preservation section in national standard NY/T1443.1-2007. In actual operation, the inoculation effect is judged by the disease condition of a disease control material), and three mixed physiological races of CYR32, CYR33 and CYR34 are found to be near-immune and high-sense respectively by two triticale parents Rozovskaya-7 (saved by Han Fangpu laboratories of the national academy of sciences of China and development biology) and 4100 (provided by Ni Zhongfu professor of national academy of agriculture). To clone the stripe rust resistance gene in Rozovskaya-7, two triticale parents were crossed to give F1 and selfed to give F2 populations. 200 single plants in the F2 group are inoculated and identified, 144 plants are found to be resistant to diseases and 56 plants are found to be high in sense. And respectively selecting 40 extreme disease-resistant and 40 extreme disease-sensitive materials from the F2 population to form an anti-pool and a sensitive pool, taking total RNA extracted from leaves for sequencing (carrying out transcriptome sequencing at An Nuo reach gene technology company), and finding that the stripe rust resistance gene is positioned at the tail end of 6RL of rye through BSR (Bulk SEGREGANT RNA sequencing) analysis. 11 co-dominant Indel markers were developed near 6RL using genome re-sequencing data of ten times the sequencing depth of the two parents, and the stripe rust resistance gene was further mapped to the 1.25Mb interval (corresponding to Lo7 reference genome 6r:878.556-879.809 Mb) using F2 progeny obtained after crossing of the two parents, which is a population for gene mapping, with disease resistance: 3:1, for fine mapping. Subsequently 24 genes in the interval were analyzed in combination with anti-pool sensory pool transcriptome data, and 19 genes were found to be FPKM <1 in anti-pool and sensory pool, possibly pseudogenes. The other 5 genes only had SECCE.sup.6Rv1G 0452990 expression levels in the anti-pool higher than in the sense pool and higher (Table 2), and therefore SECCE.sup.6Rv1G 0452990 was determined as candidate genes.
Table 1 shows the statistics for the group Rozovskaya-7 and 4100F2 stripe rust identification. The total inoculation of 200 strains, 144 disease-resistant strains and 56 high-sensitivity strains basically accords with the separation ratio of 3:1.
TABLE 2 screening of disease resistance genes
Example 2 detection of the Secale stripe rust resistance Gene
The genomic DNA of the plant material to be detected is amplified by PCR by using a molecular marker 6R2990-1 primer, and the 1776bp of material can be amplified to be SECCE Rv1G 0452990.
6R2990-1 primer F:5'-CCAGAGAGCGTGTTATGGCT-3' (SEQ ID NO: 4);
6R2990-1 primer R:5'-AGACGGAGATAGATTGTTCACTT-3' (SEQ ID NO: 5).
Wherein the F primer is positioned in the promoter region of the gene, and the R primer is positioned at the front end of the CDS region.
TABLE 3PCR System
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94℃for 30 seconds, annealing at 60℃for 30 seconds, extension at 72℃for 1 minute, and running for 35 cycles; finally, the extension is carried out at 72 ℃ for 10 minutes. The PCR amplification product may be stored at 4 ℃.
PCR product electrophoresis detection system: 1.2% agarose gel electrophoresis, voltage: 120V, time: 25 minutes, buffer: 1 xTAE.
Example 3 functional identification of rye stripe rust resistance genes
An over-expression vector of SECCE6Rv1G0452990 was constructed using Ubiqutin promoter. Wheat transgene vector pWMB (supplied by the institute of crop She Xingguo teacher, national institute of sciences) was first linearized using restriction enzymes kpnl and SacI, SECCE Rv1G0452990 CDS was amplified using primers with recombinant linkers, the above PCR products and linearized vector were recovered using a universal DNA purification recovery kit of the root, and the linearized vector was ligated with the amplified gene fragment using a recombinant cloning kit of the root EasyGeno. And genetic transformation is carried out on the wheat variety tobacco grower 19 with high stripe rust (the transformation method refers to Chinese patent ZL 201710422896.6), and the transgenic positive plants have improved stripe rust resistance compared with the control, which shows that SECCE Rv1G0452990 genes from rye can endow wheat with stripe rust resistance.
TABLE 4 pWMB110 vector linearization System
| Medicine | Volume (mul) | Manufacturer' s | Specification of specification |
| CutSmart Buffer | 5 | NEW ENGLAND | 1.25ml |
| kpnI | 1 | NEW ENGLAND | 0.4ml |
| SacI | 1 | NEW ENGLAND | 0.1ml |
| PWMB110 plasmid 110 | 10 | 250ng/ul | |
| ddH2O | 33 | ||
| Together, a total of | 50 |
Incubate at 37℃for 3 hours.
Amplification of Gene CDS Using recombinant primers the primers used were as described above:
2990-110-F:GGTCGACTCTAGAGGATCCCCGGGTACCGAATGGCGATGGTGCTGGATGC(SEQ ID NO:6)
2990-110-R:TGAACGATCGGGGAAATTCGAGCTCCTAGCTATCATGCACTTTCT(SEQ ID NO:7)
The PCR product and the linearized vector were purified and recovered according to the procedure of the Tiangen general-purpose DNA purification and recovery kit (TIANGEN, cat# DP 214-03).
The gene fragment was ligated into the linearized pWMB110,110 vector by recombinant means, and the specific procedure was carried out according to the Tiangen EasyGeno recombinant cloning kit (TIANGEN cat# VI 201-02).
TABLE 5 statistical results of stripe rust inoculation of transgenic plants
The disease resistant triticale parent Rozovskaya-7 was near immunized against stripe rust, while the disease resistant parent 4100 was hypersensitive to stripe rust. Transgenic acceptor material Nicotine 19 and SECCE Rv1G0452990 transgenic material negative materials are all sensitive to stripe rust, while SECCE Rv1G0452990 transgenic positive materials are sensitive to stripe rust, indicating SECCE Rv1G0452990 is a stripe rust resistant gene.
Sequence(s)
1 Rye Lo7 reference genome Gene SECCE CDS sequence of Rv1G0452990 (4266 bp):
ATGGCGATGGTGCTGGATGCGTTTGCACACTACGTGGGCCACATGCTCGCGCAGCTAGCAGCAGATGAGGTGGGGACCATGCTGGGCGTCTCCGGCGAGATCAAAAAAATGGGCGACAAGCTTCGGGATCTCAACAACTTCTTGGCTGATGCCGATAGGAGGAACATAACTGACGTGACCATCCAAGAGTGGGTGGGGCAGCTCAAGCGTGCCATGTACGAAGCTACCGACATCCTTGACCTCTGTCAGCTCAAGGCCATGGAGCGTGGATCATCTACTACTCTAGACGCAGGGTGTTTCAACCCCTTGCTCTTCTGCATGCGCAATCCCTCCCATGCTCATGAGATCGGCACCCGCATCAAAAATCTCAACAAGAGGCTCGACTCCATCAAACAACGGAGTGCTCCTTTCAACTTCGTCAATCTCGGGTCTTATGAGGATCATAACAACAACGCCCATGGCTTTCGCCATGGTAATCCAAGTCGGGAGACGGTAGGGGACTTTGACCGATCAGCTGTTGTTGGGGACAAGATTGAAGAAGATACAAGAGCACTGG
TGGCCCAAATCATGCAGACGGGAAAGGATGTCAACAATGGCATCATT
GTGGTCGCTATCGTAGGTGTTGGTGGGATCGGCAAGACCACCCTCGC
CCAGAAGGTCTTCAATGATGAGGCAATCCAAGGTGAATTCAGCAAA
AAGATATGGTTGAGTGTCAACCAAAACTTCAGTGATGTTGATCTGCT
GAGAAGGGCCATCATCGAAGCCGGAGGAGATGCCCAACCACCTGAA
AGTGCAAAGACCAGCCTTCATGAAACCCTCAAGAACATATTGATTGA
CCACAAGACCTTTCTGGTAATGGATGATGTGTGGAACCATAGAGCAT
GGGATGACGTGCTGAAAATACCCTTAGTCAATGTTGCTGGTTCAGGC
AGCCGAGTCCTCGTTACTACCAGAGAAGAAGGTGTTGCCCGAGGGG
TGAAAGCCATTTGGCCGTACCACCATATCGATACATTACTGCCTGAAG
ATGCCTGGTCATTGCTCAAGAAACAGGTATGCTCAAGGGAGTTAGAT
GAAGACCACATCAATACGCTAAAGGATATAGGACTGAAAATTATACA
GAAATGTGGTTGTTTACCAATTGCTGTTAAAGTGATGGGAGGACTCT
TGCGTGAAAGAGGGGGGCTACGTCGTGACTGGCAGCAGGTTTTGGA
TGATTCTAAATGGTCAACAACTAAAATGCCTGATGATCTCAACCACA
CAGTATACTTAAGCTATGAATATATGCCTTCTTACCTGAAGCAGTGCTT
TCTGTACTACTCTTTTCTTCCTAAAAGTAGACGTTTTCATATGGAGCA
AGTCGTGGCAATGTGGATAAGCGAAGGATTTATTCATGGAAACTCTG
GTGATTTAGAAGAATTGGGGAGAAATTACTACAAGGAGTTGGTATCT
AGGAACCTTATAGAGCCAGATAAATCATATGCTGATATATGGTTTTGC
AGCATGCATGATGTTGTTCGCTCATTTGCTCGGTATATGACTAAAGAT
GAAGCACTTGTTACTCAAGACGGAGACACTGATATGCTTGCTAAACT
TGCTTCACAAAAGTTTCTTCGGTTGTCCATAGAAACTAGCCGATCAC
AATCAGGTGAACTTGATTGGAAATCCCTACAGGCGCAGCAATCAGTG
AGAACACTGATCTCAACCATCCAGATTAAGATGATGCCTGGTGATTC
ATTGGTTACCTTTTCTAGTTTGCGGACTCTGCATATAGAATCTGCTGAT
ATGGCTGTATTGCTTGAATTGTTGCATCAACTCAAGCATGTGAGGTAT
CTAGCACTAGTAAATGCTGGTATATCTGTACTTCCAGGGAACATTGGC
AAGATGAAACTATTGCAATTCCTTGACCTTGGTGGATGTACAAAATT
GGTTAACCTTCCAGACAACATTGTGAATCTTGGCCAGCTGAGGTTAT
TTGCACTTCCCAGAGCAAGTACGGTACCTAGAGGGTTTAGTGGCCTG
ACAAATATGAGGATATTACGTATGTTTCGAGCCCACATGGATAATGAT
TGGTGCAGTTTGGACGAGTTGGGGCCTCTTTCACAGCTCAGATTTCT
TGGATTAAATGAATTAGAGAATGTATCTGCTGCCTCGTTTGCTTCTAA
TGCTAGGCTCGGCGAGAAGATGCATCTTATCACTCTACTCCTGGGTT
GCACTAGTAAGCTGGGAGATGATGGGTTCGTCAAAGAGAAGGAAGG
TGTCTCTGAGGAAGAGCAGCAACGAATTGAGAAGGTTCTTGATAAG
CTCTACCCTCCACCTGGTGTAGAAGATCTTCAAATTAGTGGGTATTTT
GGCCGGCAACTCCCGAGCTGGATAATGTCCACATCAACGGTGCCCCT
CAACAACTTGAAGACTATATTTTTTGTTGATCTGGCTTGTTGCACACA
ACTTCCCAATGGGCTGTGCCACCTCCCCAATCTGCAGTTTCTACAGG
TCTCTCGTGCTCCATGCATCAAACATGTTGGGGCTGGATTCTTGCAG
GCGGCAGCAGCTTCATTTCCAAGGTTAAACAAATTGATCTTGTTAGG
CATGGTGGAATGGGAGGAGTGGGAGTGGGAGGAGCAAGTACAAGC
CATGCCCCGTTTGGAGGAGCTTGTGCTCAATAAATGCAGACTAAGGC
ATGTTCCTCCAGGCCTTGCCTCCAATGCAAGCTCTTTGAAAATATTTG
TTCTAGTGCATGTCAAGCAACTTAGCTACATTGAGAGCTTTCCTTCTG
TTGTTGAGCTTATAGTGACTGGATGCCCAGACCTGGAGAGGATCAGC
AATCTCCCTAATCTGCAGAAGCTTACCATCCAAAACTGCCCAAAGTT
GAAGGTGCTGGAGCGTATCACTTCACTCGAGAGGCTGATCCTGAAG
GATTATACCATGGAAAAACTCCCAGAATACATGCGAGACTTAAAGCC
AAGGCATTTGCAGTTATTCTGCAGGCTATGGTTGCTCTATGCGGTAGC
CGCGGGACAATCTGGCACTGAGTGGGACAAGTTCAACCAAGTGGAG
CATGTCAAGGCATATGCACGTGATGGAGACAACCAAAGAAAATGGT
ACGTTTTGTACACGGGAGGAGACAACTACAAGCTGGATTCAAATATT
AGCAGCTCTACCATAGTTGAAGAACCCTTATCATCTTGTATGGTGGAC
CGACAAGGATTTGAGTCTCTGTACAGAATGAGAAGAAGCACCTTCA
GTTATGTTTGCAGCTTGGTGAGGATCCCATTTTTTGAAGATATGATGG
GAAGGGAGCCCACCTTTATCGATGGGAGAGTGTTGTCTTTACAAGAC
AGAGTGGCTGTCGCTCTGAGAATGCTGAATTCTGGTGACTCTCCGGT
TAACGTAGGATCCTCCCTTGGTGTGAGCGAGTCAACTTCCTTGCTGG
TAACTAAGGTGTTTGTCAAGGCCATGCTGGAGCCATTATCGCACCAC
TTCGATTGGCCAGGCTCTGCTAAAATGCAGAAGATCAAGTGCAAGTT
TGACAAGATACATGGCCTACCAAACTGTTACGGTGCTGTACATACAA
CCCAAATCACATTTGGATCACAAGTTCATGACCGTGAGGAGAATGAT
TCTGTGCTAATGCGAGTAATTGTTGATCCTGATATGAGGTTCACACAG
GTTTGGTTGGCCTCTGATCTCTTGGAGTTGGACTCTGATCTCTTGAAG
TACTATGAGGAGGATGCTGTGGTGAATGGCAGTAAGATGAAGTTATC
AGATGGCTCAGAAGATGAACAATACATAATTGGTGATGCAGGATACC
CTCTTCGTCCCTGGATCCTCACACCTTACCTGTTGGAGGATGGCCTCT
CACTCTCAGACGCCAAAGTTGAGTTCAACAGGAGACATTCTGCAGT
GACAGCTTTTGCGATAAGGGCGCTAGCAAAGTTAAAAGACACATGG
AAGTGCCTCCAGGGAGAAGGGTGGCATCCAGATAATAATGACATACT
AGATTTGACAATCTGGGTATGCTGCATGTTGCATAACATAGTGATAGA
CATGGAGAAGGAGAAGGACGAGGATCAGGAGGAAGGTGAGTATGA
GGATGAGGGCCAGGGGGAGTTGCAGCAGGTAGCAGCTGTCGGGGT
GAGGGGTGCACTGTCCCAGCACTTGATAAAGTCTGTAGAAGAGGAA
CAAGGAGCAGAAGATAAAAACAAGGAAGAAGAAGCACAACAACGG
AAGGCAACATCTCGAGGGAAGGAGAAAGTGCATGATAGCTAG
CDS sequence of SECCE Rv1G0452990 in SEQ ID NO. 2 hexaploid triticale Rozovskaya-7 (4248 bp):
ATGGCGATGGTGCTGGATGCGTTTGCACACTACGTGGGCCACATGCTCGCTCAGCTAGCAGCAGATGGTGTGGGGACGATGCTGGGCGTCTCCGGCGAGATCGACAAGATGGCCGACAAGCTCCGGGACCTCAAAAACTTCTTGGCTGATGCTGATAGGAGGAACATCACCGATGAGACCGTTCAAGAGTGGGTGGGGCAGCTCAAGCGTGCCATGTATGAAGCTACCGACATCCTCGACCTCTGTCAGCTCAAGGCCATGGAGCGTGGATCATCTACTACTCTAGATGCAGGGTGTTTCAACCCCTTGCTCTTCTGCATGCGGAATCCTTCCCATGCTCATGAGATCGGCACCCGCATCAAAAAGCTCAA
CATGAGGCTCGACTCCATCAAAGAACGGAGTGCTGCTTTCAACTTCA
TCAATCTCGGGTCTTATGAGGATCATAACAACAACGCCCATGGCTTTC
GCCATGGTAATCCAAGTCGGGAGACGGTAGGGGACTTTGACCGATC
AGCTGTTGTTGGGGACAAGATTGAAGAAGACACAAGAGCACTGGTG
GCCCAAATCATGCAGACGGAAAAGGATGTCAACAATGGCATCATTGT
GGTCGCTATCGTAGGTGTTGGTGGGATCGGCAAGACCACCCTCGCCC
AGAAGGTCTTCAATGATGAGGCAATCCAAGGTGAATTCAGCAAAAA
GATATGGTTGAGCGTCAACCAAAACTTCAGTGATGTTGATCTACTGA
GAAGGGCCATCATCGAAGCCGGAGGAGATGCCCAACCACCTGAAAG
TGCAAAGACCAGCCTTCATGAAACCCTCAAGAACACATTGATTGACC
ACAAGACTTTTCTGGTAATGGATGATGTGTGGAACCATAGAGCATGG
GATGACGTGCTGAAAATACCCTTAGTCAATGTTGCTGGTTCAGGCAG
CCGAGTCCTTGTTACTACCAGAGAAGAAGGTGTTGCCCGAGGGGTG
AAAGCTATCTGGCCGTACCACCATATCGATACATTACTGCCTGAAGAT
GCCTGGTCATTGCTCAAGAAACAGGTATGCTCAAGGGAGTTAGATGA
AGAGCACATCAATACGCTAAAGGATATAGGACTGAAAATTATACAGA
AATGTGGTTGTTTACCAATTGCTGTTAAAGTGATGGGAGGACTCTTG
CATGAAAGAGGGGGGCTACGTCGTGACTGGCAGCAGGTTTTGGATG
ATTCTAAATGGTCAACAACTAAAATGCCTGATGATCTCAACCACACA
GTATACTTAAGCTATGAATATATGCCTTCTTACCTGAAGCAGTGCTTTC
TGTACTACTCTTTTCTTCCTAAAAGTAGACGTTTTCATATGGAGCAAG
TCGTGGCAATGTGGATAAGCGAAGGATTTATTCATGGAAACTCTGGT
GATTTAGAAGAATTGGGGAGAAATTACTACAAGGAGTTGGTATCTAG
GAACCTTATAGAGCCAGATAAATCATATGTTGATATATGGTTTTGCAG
CATGCATGATGTTGTTCGCTCATTTGCTCGGTATATGACTAAAGATGA
AGCACTTGTTACTCAAGATGGAGACACTGATATGCTTGCTAAACTTG
CTTCACAAAAGTTTCTTCGGTTGTCCATAGAAACTAGCCGATCACAA
TCAGGTGAACTTGATTGGAAATCCCTACAGGCGCAGCAATCAGTGA
GAACACTGATCTCAACCATCCAGATTAAGATGATGCCTGGTGATTCAT
TGGTTACCTTTTCTAGTTTGCGGACTCTGCATATAGAATCTGCTGATAT
GGCTGTATTGCTTGAATTGTTGCATCAACTCAAGCATGTGAGGTATCT
AGCACTAGTAAATGCTGGTATATCTGTACTTCCAGGGAACATTGGCA
AGATGAAACTATTGCAATTCCTTGACCTTGGTGGATGTACAAAATTG
GTTAACCTTCCAGACAACATTGTGAATCTTGGCCAGCTGAGGTTATTT
GCACTTCCCAGAGCAAGTACGGTACCTAGAGGGTTTAGTGGCCTGAC
AAATATGAGGATACTACGTATGTTTCGAGCCCACATGGATAATGATTG
GTGCAGTTTGGACGAGTTGGGGCCTCTTTCACAGCTCAGATTTCTTG
GATTAAATGAATTAGAGAATGTATCTGCTGCCTCGTTTGCTTCTAATG
CTAGGCTCGGCGAGAAGATGCATCTTATCACTCTACTCCTGGGTTGC
ACTAGTAAGCTGGGAGATGATGGGTTCGTCAAAGAGAAGGAAGGTG
TCTCTGAGGAAGAGCAGCAACGAATTGAGAAGGTTCTTGATAAGCT
CTACCCTCCACCTGGTGTAGAAGATCTTCAAATTAGTGGGTATTTTGG
CCGGCAACTCCCGAGCTGGATAATGTCCACATCAACGGTGCCCCTCA
ACAACTTGAAGACTATATTTTTTCTTGATCTGGCTTGTTGCACACAAC
TTCCCAATGGGCTGTGCCACCTCCCCAATCTGCAGTTTCTACAGGTC
TCTCGTGCTCCATGCATCAAACATGTTGGGGCTGGATTCTTGCAGGC
GGCAGCAGCTTCATTTCCAAGGTTAAACAAATTGATCTTGTACGGCA
TGGTGGAATGGGAGGAGTGGGAGTGGGAGGAGCAAGTACAAGCCA
TGCCCCGTTTGGAGGAGCTTGTGCTCAATAAATGCAGACTAAGGCAT
GTTCCTCCAGGCCTTGCCTCCAATGCAAGCTCTTTGAAAATATTTGTT
CTAGTGCATGTCAAGCAACTTAGCTACATTGAGAGCTTTCCTTCTGTT
GTTGAGCTTATAGTGACTGGATGCCCTGACCTGGAGAGGATCACCAA
TCTCCCCAATCTACAGAAGCTTGACATCCAGAACTGCCCAAAGTTGA
AGGTGCTGGAGCATATCACTTCACTCGAGCGGCTAGTCCTGGAGGAT
TACACCATGGAAAGACTCCCAGAATACATGCGAGACATAAAGGCAA
GGCATTTGCAGTTATTTTGCAGGCTATGGTTGCTCTATGCGGTAGCCG
CGGGACAATCTGGCACTGAGTGGGACAAGTTCAGCCAAGTGGAGCA
TGTCAAGGCATATGCACGTGATGGAGACAACCAAAGAAAATGGTAC
GTTTCGTACACGGGAGGAGATAACTACAAGCTGGATTCAAATATTAG
CAGCTCTGCCATATTTGAAGAAACCTTATCATCTTGTATGGTGGATGC
ACAAGGATTTGACTCTCTGTACAAAATGAGAAGAAGTACCTTCAGTT
ATGTCTGCAGCTTGGTGAGGATCCCATTTTTCGAAGATATGATGGCAA
GGGAACATACCTTTGTTGATGGGAGATTGTTGTCTTTGCAAGACGGA
GTAGCTGTCGCTCTGAGGGTACTGAACTCTGGTGACTCTCCGGTTAC
CGTAGGATCCTCTCTTGGTGTGAACGAGTCAACTGTCTCCCTGGTAA
CTCAGGTGTTTGTTGAGGCCATGAAAAATCCAATAGTGCACCACTTC
AAATGGCCAACCTCTGCTAAAATGGGGAAGATCAAGCGCAAGTTTG
ACAAGATACACGGCCTGCCCAACTGCTGCGGTGTTGTACATACAACC
CAAATCACGTTTGGATCACAAGAGCGTGGCAGTGAGGAGAATGAGT
CTGTGCTAATGCGAGCCGTCGTTGATTCAGATATGAGGTTCACACAG
GTTTGGTTGGCCTCTGATCTCTTGGAGTTGGACTCTGATCTCTTGAAT
TCCTTTGAGGAGGGTGCTGTGGTGAATGGCAGTAAGCTGAAGTTATC
AAATGGCTCGGAAGTCGGAGATTACATAATTGGTGATGCAGGATACC
CTATTCGTCCCTGGATCCTCACACCTTACCTGTTAGAGGATGGCCTCC
CACTCTCAGAGGCCAAAGTTGAGTTCAACAGGAGATATTCTGCAGT
GACAGCTGTTGCGCAAAGGGCGCTAGCAAAGTTAAAAGACACATGG
AAGTGCCTCCAGGGAGAAGGGTGGCATCCAGATAATGACATGTTGA
GATGGACAATCTGGGTATGCTGCATGTTGCATAACATAGTGATAGACA
TGGAGGAGAAGGAGAAAGATGAGGAAGGTGAGGATGAGGGTCAGG
AGGAATTGCGGCAGGTAGCAGACGGGGTGAGGGGTGAACTCTCCCA
ACACTTGATAAAGTCTGTAGAGGAGGAACAAGGAGCAGAAGATAAA
AACAAGGAAGAAGAAGCACAGCAACGGAAGGCAACATCTCGAGGA
AAGGAGAAAGTGCATGATAGCTAG
SEQ ID NO. 3R 2990-1 amplification sequence:
CCAGAGAGCGTGTTATGGCTGCATTCATCGATTGATGCAGAGGCCGGGGGTATTCCTCCTTTTCAAAAAAAAAAAAAATAAGTACCGCGTCACCTGTATATATCTAGCTCATGCTCGTCCACGAACCGGAGATTGGGTTCATTGAGCACTAAAAGGTTGAAGGTTCACATCAATTGCCATCTTATACGGAAGAGCATATGTAGACGTGGTCCGAAATAAACTGAGAATAGTCAA
GGGTTAACCGTGTCCTCTCCCATCTTATCCTCATCTATCTTGTGCCATT
AGTATGCGTCTTGTGGCTCTCTCTCGTCTCTCCAGCCATTATTATCCTG
CTAGCTTCAGGGACAACCAGGAGTTGCTTTGAGGAGCTTAACGAGC
AGAACATCCATCCATCCCAAAGGTTAGTTACACCTAGCTAATTAAGCT
TCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTCCCTCTCGCATG
CTTAACTTTCACCTTACCTTTATTATTACAGTATAAGTAGCCAAGTACA
ACTAATGGCGATGGTGCTGGATGCGTTTGCACACTACGTGGGCCACA
TGCTCGCTCAGCTAGCAGCAGATGGTGTGGGGACGATGCTGGGCGT
CTCCGGCGAGATCGACAAGATGGCCGACAAGCTCCGGGACCTCAAA
AACTTCTTGGCTGATGCTGATAGGAGGAACATCACCGATGAGACCGT
TCAAGAGTGGGTGGGGCAGCTCAAGCGTGCCATGTATGAAGCTACC
GACATCCTCGACCTCTGTCAGCTCAAGGCCATGGAGCGTGGATCATC
TACTACTCTAGATGCAGGGTGTTTCAACCCCTTGCTCTTCTGCATGCG
GAATCCTTCCCATGCTCATGAGATCGGCACCCGCATCAAAAAGCTCA
ACATGAGGCTCGACTCCATCAAAGAACGGAGTGCTGCTTTCAACTTC
ATCAATCTCGGGTCTTATGAGGATCATAACAACAACGCCCATGGCTTT
CGCCATGGTAATCCAAGTCGGGAGACGGTAGGGGACTTTGACCGATC
AGCTGTTGTTGGGGACAAGATTGAAGAAGACACAAGAGCACTGGTG
GCCCAAATCATGCAGACGGAAAAGGATGTCAACAATGGCATCATTGT
GGTCGCTATCGTAGGTGTTGGTGGGATCGGCAAGACCACCCTCGCCC
AGAAGGTCTTCAATGATGAGGCAATCCAAGGTGAATTCAGCAAAAA
GATATGGTTGAGCGTCAACCAAAACTTCAGTGATGTTGATCTACTGA
GAAGGGCCATCATCGAAGCCGGAGGAGATGCCCAACCACCTGAAAG
TGCAAAGACCAGCCTTCATGAAACCCTCAAGAACACATTGATTGACC
ACAAGACTTTTCTGGTAATGGATGATGTGTGGAACCATAGAGCATGG
GATGACGTGCTGAAAATACCCTTAGTCAATGTTGCTGGTTCAGGCAG
CCGAGTCCTTGTTACTACCAGAGAAGAAGGTGTTGCCCGAGGGGTG
AAAGCTATCTGGCCGTACCACCATATCGATACATTACTGCCTGAAGAT
GCCTGGTCATTGCTCAAGAAACAGGTTTGTACATACTATGTTTGCGTC
AATTAATATGAGTAGAAGGGAGTACTAATCTCTCAATGAAGTGAACA
ATCTATCTCCATCT
The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.
Claims (10)
1. The stripe rust resistance gene is characterized in that the nucleotide sequence of the stripe rust resistance gene is shown as SEQ ID NO. 2 or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% identity with the nucleotide sequence shown as SEQ ID NO. 2.
2. An expression vector comprising the stripe rust resistance gene according to claim 1.
3. A host cell comprising the stripe rust resistance gene according to claim 1 or the expression vector according to claim 2.
4. Method for detecting the presence or absence of a stripe rust resistance gene in a plant to be tested, characterized in that the method comprises detecting the presence of a stripe rust resistance gene according to claim 1 or a specific molecular marker thereof, preferably the sequence of said specific molecular marker is shown in SEQ ID NO. 3, preferably the sequence of SEQ ID NO. 3 is detected using the primers shown in SEQ ID NO. 4 and SEQ ID NO. 5.
5. A method of increasing the resistance of a plant to stripe rust or a method of growing a stripe rust resistant plant, the method comprising introducing the stripe rust resistance gene of claim 1 or the expression vector of claim 2 into the plant or a cell of the plant.
6. A method of obtaining a plant cell or plant carrying a stripe rust resistance gene, characterized in that the method comprises introducing the stripe rust resistance gene according to claim 1 or the expression vector according to claim 2 into the plant cell or the plant.
7. Use of a stripe rust resistance gene according to claim 1 or an expression vector according to claim 2 or a host cell according to claim 3 for increasing the stripe rust resistance of a plant or for growing a stripe rust resistant plant.
8. The molecular marker for detecting the existence of the stripe rust resistance gene of the plant to be detected is characterized in that the nucleotide sequence of the molecular marker is shown as SEQ ID NO. 3.
9. The method according to any one of claims 4,5 or 6 or the use according to claim 7 or the molecular marker according to claim 8, wherein the plant is wheat or rye.
10. The stripe rust resistance gene according to claim 1 or the method according to any one of claims 4,5 or 6 or the use according to claim 7 or the molecular marker according to claim 8, characterized in that the stripe rust is caused by physiological micro-species CYR32, CYR33 and/or CYR 34.
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| Country | Link |
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