CN116640768B - Wheat powdery mildew resistance gene PmTR1 and application thereof - Google Patents
Wheat powdery mildew resistance gene PmTR1 and application thereof Download PDFInfo
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Abstract
The invention provides a wheat powdery mildew resistance gene PmTR1 and application thereof. The cDNA sequence of the PmtR1 is shown as SEQ ID NO.1, the amino acid sequence of the encoded protein is shown as SEQ ID NO.2, and the whole genome DNA sequence is shown as SEQ ID NO. 3. The invention provides cloning of powdery mildew resistance genes on rye 6RS chromosomes for the first time, and the PmTR1 genes are overexpressed in wheat for the first time. Silencing PmtR1 can transform disease-resistant wheat into disease, and excessive expression of the gene can make the disease-resistant wheat variety Fielder obtain resistance. The wheat powdery mildew resistance gene PmTR1 provided by the invention provides new gene resources and ideas for cultivating powdery mildew resistance wheat varieties, and has important application value for widening the genetic basis of wheat and genetically improving the powdery mildew resistance of wheat.
Description
Technical Field
The invention relates to the technical field of molecular breeding, in particular to a wheat powdery mildew resistance gene and application thereof.
Background
Wheat powdery mildew caused by powdery mildew (blumeriagraminif. Sp. Tricicic) is one of the main diseases that causes a decrease in wheat yield. The severe areas of wheat powdery mildew may even lead to 40% reduction in yield (Sun HG, et al, 2018). Although chemical control of wheat diseases can achieve a certain effect, manpower, material resources and financial resources are wasted, and particularly environmental pollution is caused. Therefore, the breeding method is the most economical and effective method for breeding disease-resistant varieties in preventing and controlling wheat diseases.
Currently, there are more than 80 wheat powdery mildew resistance genes formally named internationally, and more than 40 wheat powdery mildew resistance genes provisionally named internationally (McIntosh RA, et al, 2021). However, with rapid variation of wheat powdery mildew strains and widespread use of single disease resistance genes, many disease resistance genes are successively lost. Therefore, there is a need to develop new disease-resistant gene resources to cope with the constantly mutated powdery mildew.
Rye is a kindred genus of wheat, is naturally cross-pollinated, contains abundant genes such as disease resistance, yield and universality, and is one of powdery mildew resistance gene resource libraries of wheat (An DG, et al, 2019). Triticale is an important bridge material utilizing beneficial genes of rye as a new species for wheat and rye developed by distant hybridization. The development and utilization of excellent disease-resistant genes in wheat and triticale have important practical significance for widening the genetic basis of wheat, increasing the diversity of resistance sources and cultivating new disease-resistant varieties.
Cloning of disease resistance genes, the descriptionThe structure and the functions of the plant disease resistance agent are obvious, the disease resistance agent is helpful for analyzing the disease resistance essence of plants, and a breeding idea which is more beneficial to the development of the resistance is provided for the disease resistance breeding of crops. However, many wheat closely related species lack high quality whole genome sequence information, and the exogenous chromosome is difficult to recombine and exchange after being introduced into the wheat genetic background, so that the cloning of disease-resistant genes derived from the wheat closely related species is difficult and serious. At present, only powdery mildew resistance genes cloned in rye arePm8(humni S, et al, 2013) andPm17(Singh SP, et al, 2018), but they all have lost resistance in production.
Disclosure of Invention
The invention aims to provide a wheat powdery mildew resistance gene and application thereof, and provides a new gene resource and thought for cultivating powdery mildew resistance wheat varieties. The PmTR1 gene is excessively expressed in the infected wheat, and a method is provided for cultivating the wheat powdery mildew resistant variety.
The invention is realized by the following steps:
the wheat powdery mildew resistance gene provided by the invention is named as wheat powdery mildew resistance gene PmTR1, the cDNA sequence of which is shown as SEQ ID NO.1, and the genomic DNA sequence of which is shown as SEQ ID NO. 3.
The protein coded by the wheat powdery mildew resistance gene PmTR1 has an amino acid sequence shown in SEQ ID NO. 2.
The wheat powdery mildew resistance gene PmTR1 is applied to the improvement of wheat varieties.
The wheat powdery mildew resistance gene PmTR1 is applied to improvement of wheat powdery mildew resistance.
The wheat powdery mildew resistance gene PmTR1 provided by the invention is obtained by taking wheat-rye derived germplasm TR1 as a material through powdery mildew strain identification, powdery mildew resistance genetic analysis, disease resistance gene physical positioning and transcriptome analysis processes. The excessive expression of the PmtR1 gene can obviously improve powdery mildew resistance of infected wheat.
The TR1 is derived germplasm with genetic stability obtained by hybridization by taking the octaploid triticale as a female parent and the common wheat as a male parent, and is named as TR1. The field disease resistance identification and agronomic character evaluation for many years show that TR1 has the characteristic of high wheat powdery mildew resistance and the comprehensive agronomic character is excellent. Further, by genome in situ hybridization, fluorescence in situ hybridization and rye chromosome specific marker identification, the TR1 is confirmed to be a wheat-rye T1RS.1BL translocation line and a 6R diabody additional line.
The invention provides cloning of powdery mildew resistance genes on rye 6RS chromosomes for the first time, and the PmTR1 genes are overexpressed in wheat for the first time. The wheat powdery mildew resistance gene PmTR1 provided by the invention provides new gene resources and ideas for cultivating powdery mildew resistance wheat varieties, and has important application value for widening the genetic basis of wheat and genetically improving the powdery mildew resistance of wheat.
Drawings
FIG. 1 is a diagram of the identification of the genomic in situ hybridization (FIG. 1 a) and the fluorescent in situ hybridization (FIG. 1 b) of wheat-rye-derived germplasm T1 RS.1BL translocation and 6R diabody addition line TR1. Wherein FIG. 1a probed with "empire" rye genomic DNA (green); FIG. 1b probed with pSc119.2 (green) and pAs1 (red); the scale bar indicates 10 μm.
FIG. 2 is a graph showing the observation of hyphal infection of TR1 after inoculation of powdery mildew strain at different leaf phases for 4 days. Coomassie brilliant blue staining was used with a scale of 1mm.
FIG. 3 is a schematic structural diagram of powdery mildew resistance gene PmTR1. Wherein, FIG. 3a is a schematic diagram of the whole length structure of genomic DNA of PmtR1, wherein, the upper number indicates the base position, and the lower number indicates the length of the intron region; FIG. 3b is a schematic representation of the predicted protein domain of PmtR1, with the lower numbers indicating amino acid positions.
FIG. 4 is a graph showing the change of PmTR1 expression level in leaves of TR1 plants with different growth periods by fluorescent quantitative PCR.
FIG. 5 is a graph of the phenotype of onset of the TR1 plants after silencing of PmTR1 using the barley streak mosaic virus-mediated gene silencing technique (BSMV-VIGS) (FIG. 5 a) and the amount of PmTR1 expression in leaves (FIG. 5 b). Wherein TR1 represents a representative strain not inoculated with any viral plants; BSMV 00 represents a representative strain of the inoculated control virus BSMV 00 plant; BSMV: pmTR1 represents a representative strain of a plant inoculated with the recombinant virus BSMV: pmTR1.
FIG. 6 is a graph showing the phenotype of positive transgenic plants (FIGS. 6a and 6 b) and the expression level of PmTR1 in leaves (FIG. 6 c). Wherein, FIG. 6a is a four-leaf stage disease phenotype diagram, FIG. 6b is a plant stage disease phenotype diagram, and FIG. 6c is a PmTR1 expression level diagram in leaves. WT stands for wild-type Fielder; l3 represents a disease-susceptible transgenic positive line; r1, R2, R3, R4 and R5 respectively represent different disease-resistant transgenic positive strains.
Detailed Description
The present invention will be described in detail with reference to specific examples. The experimental methods in the examples are all conventional methods unless otherwise specified. The experimental materials, reagents and the like used in the examples are all commercially available unless otherwise specified.
Example 1 screening of the Gene Pmtr1
The experiment uses wheat-rye derived germplasm TR1 as a material to screen a gene PmTR1.
1. Breeding of TR1
(1) Parent breeding: in 2009, in the test station of the unit of the present application, octaploid triticale (2n=2x=56, AABBDDRR) 09R1-38 with excellent comprehensive properties such as a large number of ears, high yield per plant and the like were selected as female parent and common wheat (2n=2x=42, AABBDD) breed balance 5471 as male parent.
(2) Hybridization: the hybridization combination is prepared by taking triticale 09R1-38 as a female parent and common wheat scale 5471 as a male parent. Manually emasculating the female parent triticale 09R1-38 in the next day after heading, and bagging and isolating; pollination with male parent at 9-11 am and 3-4 pm the next day after female parent flowering, repeating for 3 times (days); continuously bagging after pollination, and harvesting a large amount of F in the year 6 of 2010 1 And (5) generation of hybrid seeds.
(3) Identification and backcross: will F in 10 months of the same year 1 The hybrid seeds are planted in a test station, multi-row single-seed dibbling (same as below) is carried out according to the plant spacing of 7.5cm, the row spacing of 25cm and the row length of 1.5m, the resistance to powdery mildew is identified by artificial inoculation, and disease-resistant plants are reserved. 5 months 2010, harvest F 1 The plants are evaluated and selected by agronomic characters to select 20 plants to resist powdery mildew F 1 Backcrossing with the wheat parent scale 5471 in the step (2) as a male parent, and harvesting for 6 monthsCross BC 1 F 1 Seed.
The resistance to powdery mildew was identified by artificial inoculation: generally, at the beginning of 4 months of spring year, the bacterial source fully covered with the mixed bacterial strain spores of powdery mildew is used for artificial inoculation induction (Mingxian 169), and watering is carried out for 3-4 times within 45 days from the jointing period to the grouting period so as to maintain a high-humidity environment, so that powdery mildew is fully induced. After 3-4 weeks, disease resistance is investigated when the disease-sensitive control Mingxian 169 is fully developed, and a 0-9-level grading method is adopted: 0-immunity, 1-2 level-high resistance, 3-4 level-medium resistance, 5-6 level-medium feeling, 7-9 level-high feeling.
Accurate identification of agronomic characters: the detailed investigation of agronomic characters of each generation of wheat-rye materials is carried out, wherein the detailed investigation comprises plant height, spike number, sterile spike number, thousand grain weight and the like, and the wheat district test control variety scale 4399 and parent scale 5471 are used as controls to directionally select the offspring materials with excellent high yield characters.
(4) Identification and selfing: and (3) planting the backcrossed seeds obtained in the step (3) in 10 months in the same year, performing cytological identification on backcrossed offspring plants, and manually inoculating and identifying the resistance to powdery mildew, thereby accurately identifying the comprehensive agronomic characters. Harvesting and threshing all powdery mildew-resistant plants in 6 months 2011, directionally selecting the wheat with morphological characteristics towards common wheat, carrying out selfing on the high powdery mildew-resistant plants with normal fertility and excellent comprehensive agronomic characters, and carrying out continuous selfing breeding for 10 generations so as to finally obtain the wheat-rye material named as TR1.
2. Identification of TR1
Through genome in situ hybridization and fluorescence in situ hybridization and rye chromosome specific marker identification, TR1 is confirmed to be a wheat-rye T1RS.1BL translocation and 6R two-body addition line (figure 1). The specific identification steps are as follows:
(1) Root tip treatment and preparation of split phase
Placing TR1 seeds in a culture dish, adding distilled water to submerge the seeds, covering the culture dish, and placing the culture dish in a constant-temperature incubator at 23 ℃; cutting 1-1.5. 1.5 cm root tip when the length of seed root is about 2cm, placing on the side wall of 0.5. 0.5 mL centrifuge tube (cover punching) with water sprayed on the tube wall, and N 2 O is treated for 2 hours, and then 90 percent glacial acetic acid is added into the centrifuge tubePassing root tip, and fixing on ice for 8 min; washing root tip with distilled water for three times, cutting off white growth point part of root tip with blade, adding into enzyme solution (1% pectase and 2% cellulase dissolved in 1×citric acid buffer solution), and water-bathing at 38deg.C for 30-40 min; washing with 70% ethanol for 3 times, mashing root tip with dissecting needle, and keeping the crushed root tip in 70% ethanol; 6000 Centrifuging at rpm for 3 min, pouring out ethanol, adding 20 μl glacial acetic acid, and placing on ice; the slide glass is placed in a wet paper box, the centrifuge tube 20 and s are slightly vibrated, the cell suspension is slightly blown for a plurality of times by a liquid-transferring gun, about 8 mu L of the cell suspension is sucked and dropped on the slide glass, and after the slide glass is covered with a cover for moisturizing for 8 min, the cell suspension can be detected under an optical microscope.
(2) Genomic in situ hybridization/fluorescence in situ hybridization
Labeling the whole genome DNA of the "empire" rye as a rye genome probe, labeling plasmids pSc119.2 and pAs1 as fluorescent in-situ hybridization probes, and diluting the probes to 10 ng/mu L by using 2 XSSC.1 XSTE; the slide with the complete TR1 split phase after the microscopy was crosslinked (1250X 100. Mu.J/cm) 2 ) Placing the glass slide on a flat table twice, sucking 10 mu L of the oscillated probe to drop on the cell area of the glass slide, and covering with a plastic cover glass; putting the glass slide into a hybridization aluminum tray, and carrying out denaturation hybridization in a boiling water bath for 5 min; taking out, putting the preheated aluminum box into a 55 ℃ oven for overnight; eluting the glass slide in 2 XSSC for 1 min, washing the cover glass off, and slightly airing; a small amount of 4', 6-diamidino-2-phenylindole (DAPI) is dripped on a cell area of a glass slide, a rectangular cover glass is covered, and microscopic examination and photographing are carried out under a microscope. After genome in situ hybridization identification and photographing, the same slide glass is placed in absolute ethyl alcohol 1h which is placed in advance at minus 20 ℃ for 30 min in 70% ethyl alcohol which is pre-cooled in 2 XSSC for 20 min and minus 20 ℃, hybridization signals are eluted, and fluorescent groups are quenched; sequential fluorescence in situ hybridization of the same split phase was then performed using plasmid probes pSc119.2 and pAs 1.
(3) Rye chromosome specific marker analysis
Target bands can be amplified in TR1 by using the rye 1R chromosome short arm specific marker SW5284, the 6R chromosome short arm specific marker SW22057 and the 6R chromosome long arm specific marker SW22063 (publicly known, han GH, et al, 2020), indicating that TR1 does carry rye 1RS and 6R chromosomes.
In order to screen the gene for controlling the powdery mildew resistance of TR1, powdery mildew resistance genetic analysis, resistance identification and spectrum resistance analysis are respectively carried out on the gene. The method comprises the following steps:
(4) Powdery mildew resistance genetic analysis
Selection of a disease-susceptible main cultivar, shixin 733, which does not contain T1 RS.1 BL translocation, and hybridization with TR1 to construct F 2 Populations to determine powdery mildew resistance gene sources. Inoculation of TR1, shixin 733 and F thereof with powdery mildew epidemic strain E09 2 The identification result of the seedling disease resistance of the population shows that TR1, shixin 733 and all F 2 The plants all showed a sense of high. Then transplanting the plant to a field, wherein the colony shows resistance separation on powdery mildew in the adult stage, and the colony is analyzed by using rye 6R chromosome and 1RS chromosome specific markers, and all F containing 6R chromosome 2 Plants all showed high resistance, but all did not contain 6R chromosome F 2 Plants all showed high sense, whereas the 1RS chromosome was not related to the anti-sense phenotype. Thus, the powdery mildew resistance gene of TR1 is localized to its 6R chromosome.
(5) Anti-spectrum analysis of multiple strains
TR1 was inoculated with 23 powdery mildew strains (E01, E02, E05, E06, E09, E11, E13, E15, E16, E17, E18, E20, E21, E23-1, E23-2, E26, E30-1, E30-2, E31, E32, E49, E60 and E69) at seedling stage, and YT1 all showed high sensation. Wheat powdery mildew mixed strains (comprising E09, E18 and E20) are inoculated in a field for three years continuously to carry out disease resistance identification in the adult stage, and when the disease control Mingxian 169 is fully developed, TR1 shows high resistance. Thus, YT1 may carry adult powdery mildew resistance genes.
(6) Identification of resistance to powdery mildew Strain E09 at different growth stages
To clarify the timing of the onset of resistance to expression of TR1, the different leaf stages of TR1 plants were inoculated with powdery mildew strain E09, and hyphal staining observation experiments were performed after 96 h with the infected strain TR1-1RS containing only T1RS.1BL translocation without 6R as a control. The results show that the TR1-1RS is inoculated with powdery mildew in the first, second, third and fourth leaf phases, and a large amount of hypha development can be observed under a microscope; in contrast, when powdery mildew is inoculated in the first and second leaf stages of TR1, the hyphae with obvious growth are observed, and after powdery mildew is inoculated in the three leaf stage, no hyphae with obvious growth are observed, and after powdery mildew is inoculated in the four leaf stage, no hyphae are observed (FIG. 2).
The identification result shows that the 6RS chromosome of the derived germplasm TR1 carries a new gene related to the growth period which shows the resistance to wheat powdery mildew from the trefoil period, and the gene is different from other reported powdery mildew resistance genes and is temporarily named as PmTR1.
Furthermore, the inventors utilized Co 60 -gamma radiation, creating a large number of offspring materials carrying TR1 with different 6R chromosome size fragments, carrying out resistance identification by using powdery mildew strain E09, and positioning PmtR1 in a physical interval corresponding to 6R chromosome short arm 34.2 Mb of rye Lo7 genome according to different resistance performances of each material to powdery mildew strains and combining genome in situ hybridization and molecular marker analysis results.
Example 2 acquisition of sequence of powdery mildew resistance Gene PmtR1
The differentially expressed genes of different leaf stages in the above physical interval of rye "Lo7" were analyzed in combination with transcriptome sequencing to obtain 5 candidate genes. Designing primers for disease-resistant genes with 1 nucleotide binding site rich in leucine repetitive sequence (NBS-LRR), and amplifying in cDNA sample induced by powdery mildew of TR1 to obtain the full-length sequence of PmtR1.
The cDNA sample preparation and amplification procedure was as follows:
under the condition of room temperature, placing the TR1 seeds in a culture dish, adding clear water for germination, and transplanting the seeds into a pot after the seeds are exposed to the white. When TR1 was grown to the trefoil phase, inoculation was performed with bacterial sources full of powdery mildew spores, sampling was performed 0.5h after inoculation, RNA was extracted with Trizol reagent (TAKARA Co.), and reverse transcription was performed with FastQuant RT Kit (Tiangen Co.). Then, using P1 (5'-CCTCCCTTGGAATCACGCTCAC-3') and P2 (5'-CCCACTAGTGCAGAACAACAAAGGACGATTTATGTTACTC-3') as primers, using cNDA of TR1 as a template, and performing PCR amplification to obtain a full-length cDNA fragment of PmtR1, wherein the specific sequence is shown as SEQ ID NO.1, and the open reading frame ORF is 2739bp, and 912 amino acids can be encoded; pmtR1 codes typical CC-NBS-LRR protein, and the amino acid sequence of the coded protein is shown as SEQ ID NO. 2; sequencing results show that the genome DNA of the PmTR1 has the total length of 5567bp and contains 3 exons and 2 introns (figure 3) by taking the DNA of the TR1 as a template, and the specific sequence of the genome DNA is shown as SEQ ID NO. 3.
EXAMPLE 3 analysis of expression Pattern of powdery mildew resistance Gene PmtR1
During the trefoil phase of the seedling of the TR1 plant, powdery mildew strain E09 is inoculated, and the expression pattern of the response of PmtR1 to powdery mildew infection is analyzed. Total RNA was extracted from leaves 1, 2 and 3 of seedlings taken at 12 time points of 0h, 0.5h, 1h, 1.5h, 2h, 4h, 8h, 12h, 24h, 36h, 48h and 72h, respectively. To be used forTaActinThe gene is internal reference, and the primers P3 (5'-AGGTGCCCTGAGGTGCTGTT-3') and P4 (5'-GCCAAAATAGAGCCACCGAT-3') are used for PCR amplificationTaActin. qRT-PCR analysis was performed using P5 (5'-CTCGTCTGATCCTTCAGTGTCTCAG-3') and P6 (5'-CCGCCACCACTTTAGTAGAAAGTG-3') as qPCR primers for PmtR1.
The results show that compared to seedlings 1 and 2 leaves, the 3 rd leaf PmTR1 appears to be rapidly up-regulated at 0.5h inoculation with powdery mildew and significantly higher than the 1 st and 2 nd leaf PmTR1 expression levels, followed by a gradual decrease, peaking again at 48h (fig. 4).
EXAMPLE 4 silencing analysis of powdery mildew resistance Gene PmtR1
Specific primers P7 (5'-CTTGAATTCTTGTCAATTGTTCTCATGGGTCA-3') and P8 (5'-CTTGTCGACGCCACGTCTTCACAATTATATA-3') are designed according to the structural characteristics of the PmtR1 gene to construct the BSMV-VIGS vector. After sequencing, the recombinant gamma vector gamma-PmtR 1, alpha vector, beta vector and gamma vector (known and commonly used) are respectively subjected to the following steps ofBssHII、MluI、SpeI、MluI is linearized. After in vitro transcription, 10 mu L of gamma-PmtR 1, alpha vector and beta vector are respectively mixed in equal amounts to form the recombinant virus BSMV: pmtR1. And mixing the alpha vector, the beta vector and the gamma vector with 10 mu L of the alpha vector, the beta vector and the gamma vector to form the control virus BSMV of 00.
In the three-leaf-one-heart period, 10 TR1 plants are rubbed and inoculated with recombinant virus BSMV: pmTR1 and control virus BSMV:00 respectively, and 10 TR1 plants are not inoculated with any virus and serve as disease resistance phenotype control. On day 4 of the tribological inoculation of the virus, powdery mildew strain E09 was inoculated separately to each plant. On day 14 of inoculation, hyphal staining observation experiments were started. On the 17 th day of inoculation, obvious powdery mildew-sensing symptoms can be observed on the leaves of TR1 plants inoculated with recombinant virus BSMV: pmtR1, and powdery mildew hyphae on the 4 th or 5 th leaves of the plants normally develop; in contrast, no apparent powdery mildew symptoms were observed on leaves of TR1 plants vaccinated with BSMV 00 and without virus.
Further qRT-PCR analysis is carried out on each TR1 plant, and the result shows that the PmTR1 gene silencing mediated by the barley streak mosaic virus can lead the TR1 plant to be infected after being inoculated with powdery mildew strains, and the expression level of the PmTR1 in the TR1 plant infected after being inoculated with the BSMV: pmTR1 virus is obviously reduced (figure 5); while TR1 plants inoculated with BSMV 00 and without virus showed high resistance to wheat powdery mildew. Thus, pmTR1 is an essential gene in TR1 against wheat powdery mildew.
Example 5 application of PmtR1 in wheat resistance breeding
The full-length cds of PmtR1 was amplified using primers P9 (5'-ATTCCCGGGATGTCTGCACACATCGTCAGCG-3') and P10 (5'-ATTACTAGT TCAAAGTTTGACCACATTTATAGAA-3') using the plasmid of the full-length coding region of PmtR1 as a template. Product warpSmaI andSpeafter the enzyme digestion, the enzyme digestion is connected to an expression vector pLGY02 to obtain an over-expression vector pLGY02-PmtR1. The corn Ubiquitin promoter is utilized for driving, the constructed pLGY02-PmtR1 vector is transformed into the callus of the wheat variety Fielder with high powdery mildew infection through agrobacterium mediation, and the PmtR1 over-expression transgenic wheat is obtained.
After transformation is completed, transferring differentiated seedlings to rooting and seedling strengthening culture medium, hardening seedlings and transplanting when the seedlings grow to about 10cm, and using transgenic positive identification primers P11 (5'-CCTGCCTTCATACGCTAT-3') and P12 (5'-AACGCACTCATGGTCATT-3') to test T 0 The generation is subjected to primary screening of transgenic positive plants, and the PCR identification is carried out, so that the positive line of the line with the size of 1058 and bp meshes can be obtained. Planting the screened positive plants singly, and harvesting T 1 Seeds were grown and planted while wild type Fielder was planted as a control. Propagation of spores of powdery mildew strain E09 is carried out by using infectious wheat Mingxian 169, and further using Blumeria graminisNap xian 169 versus leaf-heart period T of son 1 The generation plants are inoculated with powdery mildew strain E09 by a sweeping method, then the disease conditions of leaf powdery mildew are observed and counted in different leaf periods, meanwhile, positive transgenic plants are confirmed by using a transgenic positive identification primer, leaf RNA is extracted, and the expression conditions of the positive transgenic plants PmTR1 are analyzed by qRT-PCR. Will T 1 Transplanting the disease-resistant single plant of the generation into a greenhouse, and harvesting T 2 Seed generation, for T 2 The generation seeds were continued to be identified for powdery mildew resistance for phenotypic validation.
The results show that all plants with 1 st and 2 nd leaves are severely ill, and from the trefoil stage, 5 transgenic positive lines R1, R2, R3, R4 and R5 show the same resistance response as the TR1 plants, and 3 rd leaf leaves have no obvious spores or only a small amount of spores at the tip of the leaf. In the four-leaf stage, as shown in FIG. 6, there were no obvious spores on leaf 4 of all disease-resistant transgenic positive lines, whereas disease-resistant transgenic positive line L3 and each leaf of wild-type Fielder were severe. In the adult stage, each plant was again inoculated with powdery mildew strain E09, and both transgenic positive plants exhibited high resistance, while both L3 and wild-type field exhibited significant symptoms of disease. Meanwhile, qRT-PCR analysis shows that the expression quantity of PmtR1 in 5 transgenic positive lines is obviously higher than that in a susceptible positive line L3, and the expression quantity is not expressed in a wild type field.
The result shows that the PmtR1 has resistance to powdery mildew strains, plays an important role in the powdery mildew resistance process of wheat, and the over-expression of the PmtR1 can enable the receptor wheat variety Fielder with high powdery mildew resistance. In the future field breeding process, the gene can be utilized to carry out genetic improvement on the wheat variety through technologies such as variety hybridization, transgene, gene editing and the like, so that the resistance of the wheat variety to powdery mildew is improved.
Claims (3)
1. The wheat powdery mildew resistance gene PmtR1 is characterized in that the cDNA sequence is shown as SEQ ID NO.1, and the genomic DNA sequence is shown as SEQ ID NO. 3.
2. The protein encoded by the wheat powdery mildew resistance gene PmtR1 as set forth in claim 1, wherein the amino acid sequence is shown in SEQ ID No. 2.
3. The application of the wheat powdery mildew resistance gene PmTR1 as claimed in claim 1 in improving wheat powdery mildew resistance.
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