CN114621975A - Application of rice blast resistance related gene OsWRKY5 - Google Patents
Application of rice blast resistance related gene OsWRKY5 Download PDFInfo
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- 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
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Abstract
The invention discloses an application of a rice blast resistance related gene OsWRKY5, belonging to the technical field of plant genetic engineering. The amino acid sequence of the rice blast resistance related gene OsWRKY5 is shown as SEQ ID NO. 33. The inventor identifies and confirms that OsWRKY5 participates in the defense reaction of rice against rice blast germs, and is an important positive regulation gene participating in rice disease resistance. The invention is helpful for better understanding the action mechanism of OsWRKY5, and the cloning of OsWRKY5 lays a foundation for further understanding rice-pathogenic bacteria interaction and disease-resistant signal conduction pathways, and has great application value in breeding.
Description
Technical Field
The invention belongs to the technical field of plant genetic engineering, relates to application of a rice disease resistance related gene, and particularly relates to application of a rice blast resistance related gene OsWRKY 5.
Background
Rice is one of the most important foods for humans, and lives more than half of the world's population (Khush, 2005). The rice blast is caused by filamentous fungus magnaporthe oryzae (Magnaporthe oryzae), is one of the biggest diseases damaging rice, and often causes serious yield reduction of rice. Crop losses due to rice blast are estimated to be $ 660 million worldwide each year, sufficient to live 6000 million people.
The occurrence of rice blast is closely related to host defense mechanisms. During the evolution of interactions with pathogens, plants have correspondingly evolved a complex series of immune responses to combat the pathogens (Korean juan et al, 2018). Except that small molecular substances such as chitin, flagellin flg22 and PAMPs (polysaccharide associated molecules, PAMPs) such as lipopolysaccharide are recognized by Pattern Recognition Receptors (PRRs) on the cell surface to trigger PTI (PAMP-triggered immunity) immune response, a series of secreted proteins (i.e. "elicitors" encoded by pathogenic bacteria can also trigger PTI immune response. Allergic reactions are triggered when the avirulence (Avr) gene of pathogenic bacteria is recognized by a matching host resistance (R) gene. However, in the sexual propagation process of the rice blast fungus, rapid genetic change can occur through the active transposon, so that the expression of the avirulence (Avr) gene is deleted, host defense is avoided, and the occurrence of the rice blast is caused. Up to now, over 100 major rice blast R genes have been identified, 30 of which have been molecularly cloned, and the successfully cloned pairs of resistance genes and avirulence genes are Pik/Avr-Pik, Pib/Avr-Pib, Pi-ta/Avr-Pita, Piz-t/Avr-Pizt, Pi9/Avr-Pi9, Pia/Avr-Pia, Pi-CO39/AvrPi-CO39, Pii/Avr-Pii and Pi54/Avr-Pi54 (Caoni et al, 2019).
Transcription factors are a class of DNA binding proteins that specifically bind to homeopathic elements in the downstream gene promoter region to activate or inhibit plant growth, development and various biotic and abiotic stress responses in order to achieve plant stress tolerance (Nakashimaet et al, 2009). Currently, 6 major families of transcription factors have been demonstrated by transgenic or multiple molecular testing methods to be associated with biotic and abiotic stresses in plants, which are: AP2/ERF (APETALA2ethylene responsive factor); bHLH (basic helix-loop-helix); NAC (NAM), Arabidopsis transcription activity factor (ATAF1/2) and cup-shaped mutant gene (CUC2), WRKY and bZIP (basic lead kappa) (Singh et al, 2002; Seoet al, 2015), wherein the WRKY family transcription factor is one of the largest class of transcription factors in plants, and the transcription factor of the family usually contains one or two WRKY conserved domains (Seoet., 2015) composed of 60 amino acids at the N-terminal of the WRKY domain, which is a DNA binding domain (DNA binding domain) with conserved amino acid sequence at the N-terminal, KYGQK, zinc finger structural protein (C2H2 or C2HC) which plays a very important role in plant evolution (Rop. 2007), and plays a role in guiding the plant reaction of multiple protein kinases (MAP), and plays a role in the downstream reaction of the protein cascade, MAP kinase, and plays a role in guiding the plant reaction (MAP). Particularly in the perception of PAMPs or pathogen effector proteins and downstream signaling (Meng et al, 2013).
The identification of the disease-resistant related gene is helpful for deeply disclosing the disease-resistant mechanism of the rice and the specific interaction mechanism of the rice and pathogenic bacteria, further breeding or cultivating related disease-resistant varieties, better controlling and reducing the damage of the rice blast germs to the rice and enhancing the disease resistance of plants. The researches have important application values for the research of the gene function of the rice and the breeding of disease-resistant rice.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide the application of the rice blast resistance related gene OsWRKY 5. OsWRKY5 belongs to WRKY family transcription factors, the inventor of the invention discovers for the first time that after being induced by rice blast bacteria, OsWRKY5 can be infected and induced by the rice blast bacteria to be activated, over-expression OsWRKY5 can enhance rice blast resistance, a knockout OsWRKY5 plant shows susceptible disease, the expression quantity of disease process related genes PR1b, PR1a and PR10 in the over-expression OsWRKY5 plant is obviously higher than that of a wild type, and the gene positively regulates the rice blast resistance.
The purpose of the invention is realized by the following technical scheme:
the invention provides application of a rice blast resistance related gene OsWRKY5 in improving rice blast resistance of rice.
The application is preferably rice blast resistance breeding or transgenic rice cultivation.
The amino acid sequence of the rice blast resistance related gene OsWRKY5 is shown in SEQ ID NO. 33.
The cDNA full-length sequence of the rice blast resistance related gene OsWRKY5 is shown as SEQ ID NO. 34.
The full-length genome sequence of the rice blast resistance related gene OsWRKY5 is shown as SEQ ID NO. 35.
The application is to up-regulate the expression of the rice blast resistance related gene OsWRKY5 in rice.
The rice blast fungus is rice blast fungus (M.oryzae) GDYJ 7.
An application of an overexpression vector aiming at the rice blast resistance related gene OsWRKY5 in improving the resistance of rice to rice blast, wherein the vector is used for overexpressing an OsWRKY5 gene.
The host bacterium containing the overexpression vector is applied to improving the rice blast resistance of rice.
The application is rice blast resistance breeding or transgenic rice cultivation.
A method for cultivating transgenic rice specifically comprises the following steps: constructing an overexpression vector containing the rice blast resistance related gene OsWRKY5 by using a plant expression vector; then transforming rice tissues by using the overexpression vector; the transformed rice tissue is cultivated into rice plants.
The transformation mode is an agrobacterium transformation method.
The agrobacterium used in the agrobacterium transformation method is EHA 105.
The invention is identified by Co-immunoprecipitation (Co-IP) technology in the earlier stage to the disease-resistant protein Pik1-H4The interactive transcription factor OsWRKY 5. OsWRKY5 belongs to the second major group a in the WRKY family: C-Xm-C-Xn-HXC/H, OsWRKY5 has transcription activation characteristics, but WRKY domain is only responsible for binding with genomic DNA and has no transcription activation characteristics.
The invention relates to plant gene cloning and function analysis, and provides a transcription factor OsWRKY5 for improving rice blast resistance. The total length of the OsWRKY5 genome sequence is 5,421bp, the total length of CDS is 1,425bp, and the CDS comprises 5 exons and 4 introns and encodes 474 aa. The OsWRKY5 protein structure contains a conserved WRKYGQK structural domain, and a C is arranged behind the WRKYGQK conserved structural domain2H2Type of zinc finger C-X5-C-X23HXH, a subgroup a (C-Xm-C-X n-HXC/H) protein belonging to the second major class under the WRKY family.
The invention also provides an expression vector and a host bacterium containing the gene and a primer for amplifying any segment of the gene.
The invention relates to application of a rice blast resistance related gene OsWRKY 5. Expression pattern analysis shows that the expression of OsWRK Y5 is induced by rice blast fungus. The function of OsWRKY5 is lost to reduce rice blast resistance, and OsWRKY5 positively regulates the rice blast resistance of rice blast resistance. The expression level of disease course related genes PR1b, PR1a and PR10 in an OsWRKY5 overexpression plant is obviously higher than that of a wild type, and the expression level in an OsWRKY5 gene editing plant is obviously lower than that of the wild type.
OsWRKY5 positively regulates rice blast resistance. The corresponding vector is constructed to transform rice, the disease-resistant pathways involved in the vector are researched, and the vector is integrated into a complex disease-resistant regulation and control network, so that a certain theoretical guidance is provided for rice blast resistance breeding of the rice.
Compared with the prior art, the invention has the following advantages and effects:
the method provided by the invention clones a gene OsWRKY5 of WRKY family protein from rice by using a reverse transcription PCR technology, and proves that OsWRKY5 participates in the defense reaction of rice to rice blast germs, and is an important positive regulation gene participating in rice disease resistance. The invention is helpful for better understanding the action mechanism of OsWRKY5, and the cloning of OsWRKY5 lays a foundation for further understanding rice-pathogenic bacteria interaction and disease-resistant signal conduction pathways, and has great application value in breeding.
Drawings
FIG. 1 is a schematic diagram of the structure of the OsWRKY5 full-length gene; wherein, the black area is an exon, and the blank checkered area is a 5 'untranslated area and a 3' untranslated area.
FIG. 2 is a graph showing the result of comparing the amino acid sequence similarity of OsWRKY5 with other WRKY family proteins.
FIG. 3 is a graph showing the results of induction of OsWRKY5 in disease-resistant materials by Magnaporthe oryzae.
FIG. 4 is a graph showing the results of measurement of OsWRKY5 transcription activation characteristics.
FIG. 5 is a graph showing the results of analysis of the relative expression levels of genes (PR10, PR1b, PR1a) involved in disease course investigation of transgenic plants inoculated with Pyricularia oryzae 8 d; wherein, the graph A is a relative expression analysis result, and the graph B is a plant shoot graph; WT is a wild type plant, OX-OsWRKY5 is an OsWRKY5 overexpression transformation strain, and Cas-OsWRKY5 is a gene editing plant.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The raw materials and equipment used in the invention are all conventional commercial products and can be directly obtained by market purchase if no special description is made.
The primer sequences used in the examples of the present invention were synthesized by Suzhou Jinzhi Biotechnology GmbH and Shanghai Bioengineering bioengineering GmbH.
The rice blast resistant strain H4 and the two soft occupation (ZE) in susceptible varieties of the rice have been disclosed in the literature "rice blast resistant protein Pik2Cloning of the-H4 Gene and screening of the interacting protein [ J]Guangdong agricultural science, 2014, (04): 156-.
The magnaporthe oryzae (m.oryzae) GDYJ7 is disclosed in the literature "wupeng, identification of MicroRNA of rice responding to magnaporthe oryzae infection and preliminary functional research (D), 2019".
The overexpression vector pOX has been disclosed in the literature "cloning and functional analysis of rice SDG711 and SDG723 [ D ]. southern China university of agriculture, 2012".
The pGTR plasmid and pRGEB32 vector and Agrobacterium EHA105 have been described in "Xie, K, Min kenberg B., Yang Y., Boosting CRISPR/Cas9 m multiplex editing capability with the endogeneous tRNA-processing system. proceedings of the National Academy of Sciences 2015,112 (11)".
Example 1 sequence analysis and cloning of OsWRKY5
1) Extraction of total RNA of rice leaf and cloning of OsWRKY5
Taking the rice seedlings in the medium-two soft leaf stage and the four leaf stage, grinding the rice seedlings into powder by using liquid nitrogen in a mortar, transferring the powder into a 1.5mL centrifuge tube, adding 1mL Trizol reagent (Invitrogen company) into each 100mg material, and uniformly mixing; adding chloroform at a ratio of 200 μ L per 100mg material, mixing well at 10,000g, centrifuging at 4 deg.C for 15min, discarding the lower organic phase, collecting the water phase, and transferring into a centrifuge tube; adding 600 μ L isopropanol, mixing, standing at room temperature for 20min, centrifuging at 10,000g and 4 deg.C for 15min, collecting precipitate, and dissolving in ultrapure water without RNase after isopropanol volatilizes. Total RNA from rice leaves stored at-80 ℃ was removed, and 2. mu.L of oligo (dT) was added thereto16(10mM), and placing the mixture in a water bath at 70 ℃ for 5min after uniformly mixing; after a water bath for 5min, dNTP mix (10mM) 2. mu.L, 5 XT Buffer 4. mu. L, RNase inhibitor 1. mu.L (10U/. mu.L), RNase-free ddH were added to the EP tube on ice2O8. mu. L, ReverTraAce 1. mu.L; placing the EP tube in a PCR instrument, reacting at 30 deg.C for 10min, 42 deg.C for 60min, 99 deg.C for 5min, and 40 deg.C for 5min to obtain the firstSingle stranded cDNA of strand, using primers: OsWRKY 5-F: 5'-ATGGAGAT GATGGTGCAGAAGC-3' (SEQ ID NO:1), OsWRKY 5-R: 5'-TCAGGTGGGA GACGTGCCG-3' (SEQ ID NO: 2). The PCR reaction system is as follows: mu.L of cDNA template, 5. mu.L of upstream and downstream primers (10. mu.M) each 1.5. mu. L, dNTP (2mM), and 10 XKOD PCR buffer 5. mu. L, MgSO 42. mu.L (25mM), 1. mu.L of KOD Plus, Plus ddH2O to 50. mu.L. The amplification conditions were: denaturation at 94 ℃ for 3min, denaturation at 55 ℃ for 30s, denaturation at 68 ℃ for 2min for 32 cycles, and extension at 72 ℃ for 10min to obtain full-length OsWRKY5cDNA, and then sending the full-length OsWRKY5cDNA to Shanghai Invitrogen company for sequencing analysis.
2) OsWRKY5 sequence analysis and homology alignment analysis
The OsWRKY5 genome sequence has the full length of 5,421bp, the CDS has the full length of 1,425bp, and comprises 5 exons and 4 introns, and codes 474aa (figure 1). The NCBI Blast Protein function is utilized to detect that the OsWRKY5 Protein structure respectively contains conserved WRKYGQK structural domains. Through CLC sequence comparison of the OsWRKY5 gene sequence and the homologous family gene sequence, the conserved domain of OsWRKY5 protein WRKYGQK is followed by C2H2Type of zinc finger C-X5-C-X23HXH, subgroup a belonging to the second main class under the WRKY family (C-X)M-C-XN-HXC/H) protein (FIG. 2).
Example 2 analysis of expression Pattern of OsWRKY5
Will resist the disease variety H4And the susceptible variety ZE is respectively inoculated with rice blast fungus GDYJ7 (ddH)2O as control) and samples were taken at different time points after inoculation and changes in expression of OsWRKY5 were quantified using real-time fluorescence. The primer sequences used were as follows:
OsWRKY5-qPCR-F：5'-GGCTCCAATGATCAGTGATGGA-3'(SEQ ID NO:3)
OsWRKY5-qPCR-R：5'-AGCCATTGTGCATCGGTAGT-3'(SEQ ID NO:4)
Actin-F：5'-GAAGATCACTGCCTTGCTCC-3'(SEQ ID NO:5)
Actin-R：5'-CGATAACAGCTCCTCTTGGC-3'(SEQ ID NO:6)
the result shows (figure 3) that after the rice blast fungus is infected, the expression level of OsWRKY5 shows a trend that the expression level firstly rises, then falls and then rises in the disease-resistant material H4, and reaches a peak value 72H after inoculation, while the expression level of OsWRKY5 is always lower in a susceptible variety ZE, and the change trend is not obvious, and the result shows that after the disease-resistant reaction occurs, OsWRKY5 can be infected and induced by the rice blast fungus to be activated.
Example 3 analysis of transcriptional activation Activity of OsWRKY5
The OsWRKY5 protein is divided into three segments according to the structure domain, namely an N end (1-200aa), a WRKY (201-300aa) and a C end (301-474 aa). These fragments and the full-length sequence were ligated with pGBKT7 plasmid (Clontech) to construct recombinant plasmids, which were transferred into yeast strain Y2HGold (Clontech), respectively, and the specific transcriptional activation region of transcription factor OsWRKY5 was detected in the yeast expression system. The primer sequences used were as follows:
BD-OsWRKY5-F：
5'-atggccatggaggccgaattcATGGAGATGATGGTGCAGAAGC-3'(SEQ ID NO:7)
BD-OsWRKY5-R：
5'-ccgctgcaggtcgacggatccTCAGGTGGGAGACGTGCCG-3'(SEQ ID NO:8)
BD-OsWRKY5-N-F：
5'-atggccatggaggccgaattcATGGAGATGATGGTGCAGAAGC-3'(SEQ ID NO:9)
BD-OsWRKY5-N-R：
5'-ccgctgcaggtcgacggatccCTCCGCCGCCGGCGCCTG-3'(SEQ ID NO:10)
BD-OsWRKY5-WRKY-F：
5'-atggccatggaggccgaattcATGGCGCCGTGCCGGAAG-3'(SEQ ID NO:11)
BD-OsWRKY5-WRKY-R：
5'-ccgctgcaggtcgacggatccGGAGAGGAGCATGGCGGC-3'(SEQ ID NO:12)
BD-OsWRKY5-C-F：
5'-atggccatggaggccgaattcGGCCCCGCCGTCAGCCGC-3'(SEQ ID NO:13)
BD-OsWRKY5-C-R：
5'-ccgctgcaggtcgacggatccGGTGGGAGACGTGCCGCA-3'(SEQ ID NO:14)
the results show (FIG. 4) that only the full-length OsWRKY5 protein and the segmented OsWRKY5-N have transcription activation characteristics. Shows that the WRKY structural domain of the OsWRKY5 protein is only responsible for andPik1-H4the helical coil domain (CC domain) of (a) interacts or binds to downstream genomic DNA sequences without being responsible for the activation properties of the transcription factor itself.
Example 6 identification of blast resistance of transgenic plants OsWRKY5
1) Construction of OsWRKY5 overexpressed transgenic rice
OsWRKY5-pOX-F and OsWRKY5-pOX-R are used for amplifying an OsWRKY5cDNA fragment, and the cDNA is connected to a binary vector pOX vector by a homologous recombination method. Obtaining an OX-OsWRKY5 recombinant vector, transforming Escherichia coli DH5 alpha, and identifying a positive transformant for a subsequent agrobacterium transformation experiment by sequencing, wherein the agrobacterium is EHA105, and the transformation material is H4. The primer sequences used were as follows:
OsWRKY5-pOX-F：
5'-gggtaccggcgcgccaagcttATGGAGATGATGGTGCAGAAGC-3'(SEQ ID NO:15)
OsWRKY5-pOX-R：
5'-caattcacacttgtaggatccTCAGGTGGGAGACGTGCCG-3'(SEQ ID NO:16)
2) construction of OsWRKY5 knockout transgenic rice
Construction of the OsWRKY5 knock-out vector is described in professor sieka 2015 published as an article (xiet al, 2015). Two suitable targets are searched for in a CRISPR/Cas9 online target design website (http:// criprp. hzau. edu. cn/CRISPR2/) by utilizing a CDS sequence of OsWRKY5, and a gRNA-target sequence-tRNA-gRNA-target sequence-tRNA fragment is constructed by amplification with pGTR plasmid as a template according to target design primers. Cas9 protein expression vector pRGEB32 is subjected to single enzyme digestion by BsaI, the obtained fragment with the target spot is subjected to recombinant connection with the vector, the recombinant vector is transformed into escherichia coli DH5 alpha and then sequencing identification is carried out, a positive transformant is used for a subsequent agrobacterium transformation experiment, the used agrobacterium is EHA105, and the transformation material is H4. The primer sequences used were as follows:
OsWRKY5-pOX-F：
5'-gggtaccggcgcgccaagcttATGGAGATGATGGTGCAGAAGC-3'(SEQ ID NO:17)
OsWRKY5-pOX-R：
5'-caattcacacttgtaggatccTCAGGTGGGAGACGTGCCG-3'(SEQ ID NO:18)
OsWRKY5-bd1-F：
5'-taGGTCTCCATCCCATCCGCAgttttagagctagaa-3'(SEQ ID NO:19)
OsWRKY5-bd1-R：
5'-cgGGTCTCAGGATCAGCAGCAtgcaccagccgggaa-3'(SEQ ID NO:20)
OsWRKY5-bd2-F：
5'-taGGTCTCCTGATGGTGGCGAgttttagagctagaa-3'(SEQ ID NO:21)
OsWRKY5-bd2-R：
5'-cgGGTCTCAATCAGAGTCAGCtgcaccagccgggaa-3'(SEQ ID NO:22)
Recom-F：
5'-GTGCAGATGATCCGTGGCAACAAAGCACCAGTGGT-3'(SEQ ID NO:23)
Recom-R：
5'-CTATTTCTAGCTCTAAAACAAAAAAAAAAGCACCGACTCGGTG-3'(SEQ ID NO:24)
3) identification of resistance of rice blast of transgenic plants
After the transgenic plants are screened and identified by hygromycin and identified by fluorescent quantitative PCR, homozygous plants (3 plants each) with overexpression and complete function deletion and gene editing are selected for the next experiment. The rice blast germ GDYJ7 (ddH) is inoculated to the over-expressed and gene-edited plants by a spraying method and a punching method respectively2O as control), and the disease was investigated after 8 d. And further detecting the expression levels of the disease course related genes PR1b, PR1a and PR10 by fluorescence quantification. The primer sequences used were as follows:
PR10-F：5'-AAGTCGGATGTGCTCGAGG-3'(SEQ ID NO:25)
PR10-R：5'-GATGTCCTTCTCCTTCTCC-3'(SEQ ID NO:26)
PR1a-F：5'-AAGCTGGAGCACTCGGACT-3'(SEQ ID NO:27)
PR1a-R：5'-ACACCACCTGCGTGTAGTG-3'(SEQ ID NO:28)
PR1b-F：5'-ACTGGACGGCGGCGAGCGCG-3'(SEQ ID NO:29)
PR1b-R：5'-CTTATAGTTGCATGTGATG-3'(SEQ ID NO:30)
Actin-F：5'-GAAGATCACTGCCTTGCTCC-3'(SEQ ID NO:31)
Actin-R：5'-CGATAACAGCTCCTCTTGGC-3'(SEQ ID NO:32)
the results show (fig. 5): under the disease-resistant background (WT), an OsWRKY5 gene editing plant shows infection, an OsWRKY5 overexpression plant has no obvious resistance change, the expression quantity of genes PR1b, PR1a and PR10 related to the disease course in the OsWRKY5 overexpression plant is obviously higher than that of a wild type, the expression quantity in the OsWRKY5 gene editing plant is obviously lower than that of the wild type, the OsWRKY5 is presumed to positively control rice blast resistance, and the rice blast resistance can be improved by overexpressing the OsWRKY5 in rice germplasm.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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<223> OsWRKY5-pOX-R
<400> 18
caattcacac ttgtaggatc ctcaggtggg agacgtgccg 40
<210> 19
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> OsWRKY5-bd1-F
<400> 19
taggtctcca tcccatccgc agttttagag ctagaa 36
<210> 20
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> OsWRKY5-bd1-R
<400> 20
cgggtctcag gatcagcagc atgcaccagc cgggaa 36
<210> 21
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> OsWRKY5-bd2-F
<400> 21
taggtctcct gatggtggcg agttttagag ctagaa 36
<210> 22
<211> 36
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> OsWRKY5-bd2-R
<400> 22
cgggtctcaa tcagagtcag ctgcaccagc cgggaa 36
<210> 23
<211> 35
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Recom-F
<400> 23
gtgcagatga tccgtggcaa caaagcacca gtggt 35
<210> 24
<211> 43
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Recom-R
<400> 24
ctatttctag ctctaaaaca aaaaaaaaag caccgactcg gtg 43
<210> 25
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PR10-F
<400> 25
aagtcggatg tgctcgagg 19
<210> 26
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PR10-R
<400> 26
gatgtccttc tccttctcc 19
<210> 27
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PR1a-F
<400> 27
aagctggagc actcggact 19
<210> 28
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PR1a-R
<400> 28
acaccacctg cgtgtagtg 19
<210> 29
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PR1b-F
<400> 29
<210> 30
<211> 19
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> PR1b-R
<400> 30
cttatagttg catgtgatg 19
<210> 31
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Actin-F
<400> 31
<210> 32
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Actin-R
<400> 32
<210> 33
<211> 502
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> amino acid sequence of OsWRKY5
<400> 33
Met Glu Met Met Val Gln Lys Gln Arg His Glu Glu Gly Glu Glu Glu
1 5 10 15
Arg Gly Gly Leu Cys Ala Arg Glu Ile Lys Glu Leu Asp Phe Phe Ser
20 25 30
Ala Ala Gly Ala Gly Ala Gly Arg Arg Asp Asp Asp Asp Val Leu Arg
35 40 45
Ala Asp Gly Ile Ser Ser Ser His Ala Gly Phe Met Val Ser Thr Ala
50 55 60
Leu Asp Leu Leu Thr Ala Val Asn Asp Gly Asp His His Glu Glu Lys
65 70 75 80
Lys Gly Gln Ser Asn Ile His Gln Ser Lys Gln Met Asp Ala Ala Ala
85 90 95
Thr Thr Val Glu Gly Glu Leu Arg Gln Ala Gly Glu Glu Asn Arg Arg
100 105 110
Leu Arg Arg Arg Leu Glu Glu Leu Thr Ser Ser Tyr Gly Ala Leu Tyr
115 120 125
His Gln Leu Val Gln Ala Gln Gln Leu His Thr Lys His Gln Gln Gln
130 135 140
Ala Pro Ile Ala Gly Val Gln Leu Leu Asp Ala Leu Ala Ala Ala Ser
145 150 155 160
Pro Ala Ser His Arg Arg Arg Ala Ala Ala Ala Val Asp Gly Asp Arg
165 170 175
Thr Ala Asp Ser Asp Gly Gly Glu Gly Asp Glu Asn Val Ser Pro Ser
180 185 190
Leu Gly Ser Lys Arg Pro Ala Ala Ala Ala Thr Leu Thr Arg Leu Thr
195 200 205
Pro Glu Ser Gly Ser Gly Gly Glu Asn Asn Gly Gly Gly Glu Gln Ala
210 215 220
Pro Ala Ala Glu Met Ala Pro Cys Arg Lys Ala Arg Val Ser Val Arg
225 230 235 240
Ala Arg Ser Glu Ala Pro Met Ile Ser Asp Gly Cys Gln Trp Arg Lys
245 250 255
Tyr Gly Gln Lys Met Ala Lys Gly Asn Pro Cys Pro Arg Ala Tyr Tyr
260 265 270
Arg Cys Thr Met Ala Ser Gln Cys Pro Val Arg Lys Gln Val Gln Arg
275 280 285
Cys Ala Glu Asp Lys Ser Ile Leu Ile Thr Thr Tyr Glu Gly Thr His
290 295 300
Ser His Pro Leu Pro Pro Ala Ala Ala Ala Met Ala Lys Thr Thr Ser
305 310 315 320
Ala Ala Ala Ala Met Leu Leu Ser Gly Pro Ala Val Ser Arg Asp Ala
325 330 335
Leu Phe Ala Ala His His His Val Val Ala Pro Pro Pro Phe Phe His
340 345 350
His Pro Tyr Ala Gly Ser Thr Met Ala Thr Leu Ser Ala Ser Ala Pro
355 360 365
Phe Pro Thr Ile Thr Leu Asp Leu Thr Gln Pro Pro Pro Thr Thr Thr
370 375 380
Thr Thr Ala Ala Ala Ala Met Leu Gln Leu His Arg Pro Tyr Ala Phe
385 390 395 400
Ser Ser Leu Pro Phe Ser Met Tyr Gly Ala Gly Gly Gly Ser His Arg
405 410 415
Pro Pro Val Val Leu Pro Pro Pro Ser Ser Val Val Glu Thr Met Thr
420 425 430
Ala Ala Ile Thr Arg Asp Pro Asn Phe Thr Thr Ala Val Ala Ala Ala
435 440 445
Leu Ser Ser Ile Met Ala Gly Gly Gly Ala Gln Ala Arg Thr Pro Pro
450 455 460
Arg Gly Gly Ser Asp Ala Ala Gly Asp Ile Asn Gly Gly Gly Gly Ala
465 470 475 480
Asp His Ala Thr Ala Gly Ala Arg Ala Ala Ala Ala Ala Thr Gln Pro
485 490 495
Cys Gly Thr Ser Pro Thr
500
<210> 34
<211> 1509
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> full-length cDNA sequence of OsWRKY5
<400> 34
atggagatga tggtgcagaa gcaacgacac gaggaggggg aagaggagcg aggaggcttg 60
tgcgcccgcg agatcaagga gctcgacttc ttctccgccg ccggtgccgg tgccggccgg 120
agagacgacg acgacgtcct ccgtgcggat gggatcagca gcagccacgc cggatttatg 180
gtcagcactg cgctggactt gctgacagcg gtcaacgacg gtgatcatca tgaggagaag 240
aaagggcagt caaatattca ccaaagcaag cagatggatg cggcggcgac gacggtggag 300
ggggagctcc ggcaagccgg cgaagagaac cggcggctgc ggcggaggct ggaggagctc 360
accagcagct atggcgccct ctaccaccag ctcgtccagg cgcagcagct gcacaccaag 420
catcagcagc aggctccgat cgccggcgtg cagttgctgg acgcgctcgc ggcggcgtct 480
ccggcgagcc accggcgacg agcggcggcg gcggtggacg gcgatagaac agctgactct 540
gatggtggcg agggcgacga gaacgtgtcg ccttctcttg gtagcaagag gccggcggcg 600
gcagcgacgt taacgcggct gacgccggag agcggcagcg gcggggagaa taatggcggc 660
ggcgagcagg cgccggcggc ggagatggcg ccgtgccgga aggccagggt gtcggtgcga 720
gcacgatccg aggctccaat gatcagtgat ggatgccaat ggaggaagta cgggcagaag 780
atggccaagg gtaacccttg ccctagagcc tactaccgat gcacaatggc ttcgcaatgc 840
cccgtcagaa aacaggtgca acgatgcgcg gaggacaaga gcatcctcat caccacctac 900
gagggcaccc acagccaccc gctgccgccg gccgccgccg ccatggccaa gaccacctcc 960
gccgccgccg ccatgctcct ctccggcccc gccgtcagcc gcgacgcgct gttcgccgcc 1020
caccaccacg tcgtcgcgcc gccgcccttc ttccaccacc cctacgccgg gtccaccatg 1080
gccacgctct ccgcctccgc cccgttcccg accatcacgc tcgacctcac ccagccgccg 1140
ccgacgacga cgaccaccgc cgccgccgcc atgctccagc tccaccgccc ttatgccttc 1200
tcctcattgc cgttctcgat gtacggcgcc ggcggcggct cgcaccggcc gcccgtcgtc 1260
ctgcccccgc cgtcgtcggt ggtggagacg atgaccgccg cgatcaccag ggaccccaac 1320
ttcaccacgg cggtggcggc cgcgctctcc tcgatcatgg cgggaggcgg cgcccaagct 1380
cgaacacctc cacgcggcgg gagtgacgcc gccggagaca tcaacggagg cggcggcgcc 1440
gaccacgcca ctgccggagc acgcgccgcg gcggcggcga cgcagccttg cggcacgtct 1500
cccacctga 1509
<210> 35
<211> 5421
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> full-length genome sequence of OsWRKY5
<400> 35
ctctccttct cactctactg atcgatcgat cgagctctat ccatggagat gatggtgcag 60
aagcaacgac acgaggaggg ggaagaggag cgaggaggct tgtgcgcccg cgagatcaag 120
gagctcgact tcttctccgc cgccggtgcc ggtgccggcc ggagagacga cgacgacgtc 180
ctccgtgcgg atgggatcag cagcagccac gccggattta tggtcagcgt aagtcgtcgt 240
cgtcgtcgtc aagatctagc taatagcttg cttgacgata catccatgct tgcatatata 300
tgtttcatga attatgggat ttaggttgca agatgcatgt gatctcgatc gatggcttga 360
tcgatggatc ttgattagta tttctgattg tgtttgatcg atcgatcgag cagactgcgc 420
tggacttgct gacagcggtc aacgacggtg atcatcatga ggagaagaaa gggcagtcaa 480
atattcacca aagcaaggta attaagcatc ttacttactt tactcatctt gatctattat 540
gggggatcaa ttcacacaca tatatatata tacttttcat gaccttaagg gagtacgtac 600
gtcggaatta attcataaca ttatatatat atatgcagca gatggatgcg gcggcgacga 660
cggtggaggg ggagctccgg caagccggcg aagagaaccg gcggctgcgg cggaggctgg 720
aggagctcac cagcagctat ggcgccctct accaccagct cgtccaggcg cagcagctgc 780
acaccaagca tcagcagcag gtatgtaatt aatactaatt tttgcaattc gtgacaataa 840
tctcaattaa cccgatcaaa tcaaatatgt tttccggcct gctgctgcta attgatcgat 900
caaccaaaaa attgatacac gtacgactta tttgattctc tcacactaac ttatagtgat 960
ctctagtcaa cgctgtactt tcatgtgcag caaattatat actacatggt actatttgtt 1020
tttctttcaa tagaagaaac cgatgatatg ttacgcagca actagtaatt aaattatgtc 1080
tcacatgaaa ctgctcctat aaatccgcat agctgattgc aacctggctt cccattttgt 1140
gtttcaaatt aacgatcgac ggcttattct gctcctgatt tcttgttcta tatatacata 1200
tgcacaatta actcatcgat gaggattaag ggaaaaaatt gatttttttc gacgaacagt 1260
acacggtcat gcagatgcta gacgccacta attaacacaa atcgtgcaag cacgcattgc 1320
cgcggttcaa atatgtcgtt agttcagatc ataatcaaac taatcaagta aattaagtct 1380
gttaccaact actattcaat tcagaatgta gaccgtgatg tcaatgatgt atatattttt 1440
tcctaaaaca tttggatata gacctatatg gatgaaccta accaatattg attggcactc 1500
tcgattatca cagtgacgtg cggtttagtt tgtgcaagat ttcttgctaa atattcatga 1560
ttagctagtt tgtcaaacct ctgcactaat ttgtcaatta ttcaaaaatt atatttatac 1620
atatgtttat tttctaggtg catcatggac atgcccatgc atattgtaac taaaacatag 1680
aattatatat gtacacatat ataattcgat aaatttgcta tttttgatgc atatattcct 1740
ttatatatct aataactcca acattacatg tatattgccc aatacgtttt tggctcataa 1800
ttggcacata tttttgcatg ataaattcga tagtatactt acctagatct gtagtcaaga 1860
gaaatacatt atttggcaca tttttttgca tgtatatatc gatctcttct catcttctgt 1920
gcatccattt tccaattaat taattaaggt gtgtgcctca agcatcaccc tttgaatttt 1980
gtgatcgtcg aagggagacc gtgcatgcat accgtacgtg tcagcataaa ttcaacacca 2040
tgatttattt aggccggtga tcaaatccat cattctttta taacaaatta attgcaagag 2100
atcgacgtgt gtcgatcgag atacaaatgc acaagtgtgc agtgtgtttc tagccacaga 2160
atcaaacaag agagaatcca tgctgcaaga gaccaaacac aaaagaactt gaaactgaag 2220
ctaagctgca tttacttagt agtagttggc acatgctaat ccttgatttt ttcgatcact 2280
ttagtcttta tcagtaatag caattgacgt acgtgctact gttcgatcga ttggtgtagg 2340
ctccgatcgc cggcgtgcag ttgctggacg cgctcgcggc ggcgtctccg gcgagccacc 2400
ggcgacgagc ggcggcggcg gtggacggcg atagaacagc tgactctgat ggtggcgagg 2460
gcgacgagaa cgtgtcgcct tctcttggta gcaagaggcc ggcggcggca gcgacgttaa 2520
cgcggctgac gccggagagc ggcagcggcg gggagaataa tggcggcggc gagcaggcgc 2580
cggcggcgga gatggcgccg tgccggaagg ccagggtgtc ggtgcgagca cgatccgagg 2640
ctccaatggt aagtaattaa ttttattcgt agtgtattat acttcctccg tttcatacta 2700
taagactttc tagtattgcc catatttata tatattaatg aatctaaata tatatgtgta 2760
cttagattta ttagcattta tataaatatg aacaatgtta gaaagtctta taattaacct 2820
gaaacggatg gagtacttgt gaacttcatt gcaaattaaa ccggctatta cacatgcacc 2880
atatagctga tgaaaaaact gaaagttata tatatgcttt ataatcagta attaaattaa 2940
ctttaagcat atatacatgt ttatatatgt agatcagtga tggatgccaa tggaggaagt 3000
acgggcagaa gatggccaag ggtaaccctt gccctagagc ctactaccga tgcacaatgg 3060
cttcgcaatg ccccgtcaga aaacaggtac atcatctaca ctagtctttt gtatattttt 3120
tagaagatta ttactccaat attactgatt ccgcgtttgt agcacacgta tgttttatta 3180
gttttctctc acacaatgtc tgttaattag agcaactatt taattaacct gagtatgtgt 3240
ctaaactttt ctagaatttt ctgtgacatt tgctagttgt gtatacaaaa tattcacggg 3300
aaacttatgt gcacggatat gttccaaaca tgatatatat gtaaaagatt atataagcaa 3360
caaccatcta agtcattatt cgtaaaattg ttttgggagc tgaatgtcct gatctattgg 3420
gtgttgtcat aattgttgag aacaggaata aattggggat attttctata tgatggacca 3480
gcataaaatg tcgagggaaa ccgattaaat tagtgccggc ctcgtgccaa aagtacactt 3540
tgttcatttc aatatgaatc ggtagcaata tttacatata gataggacaa catattattc 3600
ctgggcaccc tgcaatcgat caaggaatat atgacaattt gcttattgca attcttaaat 3660
agaaaacaat agaacatgta ctatcaaatt aattaattag tagtactacg tactaccgct 3720
acctcggaat agatatatag taccatggaa atttattatt tgctatttgt acctcatttt 3780
tatatatatt gaaagtatat gaacaactcc aagaaaacat actacaaatg tgtacaattt 3840
tttaaagggc acgtaggcac taatgtgaac atatatagta tatattagta caaggtcatt 3900
ggcttgcagg atacaccctt tgcacacgat ataaatgaat aataattaat taacaacatg 3960
gctgcaaatc tcttctgtat cttacttgac cctactggtc catatatata tatatatata 4020
tataaattaa cagcatgatg gattagctgc actaatttat tccaagcaca aatcatgacg 4080
tcccaccaaa accctgacac atattgtcca agttgaaata ttagtcaatc atcgaataaa 4140
aatatctcaa tctgcaactt gtttggctta tacgagcatc caagaaaaga aaataaacac 4200
tctagcaaat atatatgcta tgtttggaat aagattttat cattttcttt taaaatctat 4260
ataattactt cggtactttt aaattgttaa tctatactcc ctcgtaacaa agtataagca 4320
tttctaaaac tcgaattttg caccagatta tcctgcattt ctgatacact gattctaatt 4380
cacgaaaatc cctacatttt tagcccacta agagagtacg tagtatgttc ttgagaggca 4440
cataaacaat tcaaaatttt aaattttgtt actgggagga aatttttttc cctcgggacg 4500
tattttggct atttgctaaa taatctaaac atgcttatat tttaacagag aagtacatat 4560
taatttttag aaaagtaatt aagaagctag ctacggatag tcgttgaagt tttaaaaatt 4620
ttgatcaaat tttatcttaa acatcaattt aattagctat ggctagatcg agggattaac 4680
taattaatac agtaatttgc gcgaaaagag atgaaaatta agtaattaac ggtgactaaa 4740
aaactgaaat aatttgtgta cgtgcaggtg caacgatgcg cggaggacaa gagcatcctc 4800
atcaccacct acgagggcac ccacagccac ccgctgccgc cggccgccgc cgccatggcc 4860
aagaccacct ccgccgccgc cgccatgctc ctctccggcc ccgccgtcag ccgcgacgcg 4920
ctgttcgccg cccaccacca cgtcgtcgcg ccgccgccct tcttccacca cccctacgcc 4980
gggtccacca tggccacgct ctccgcctcc gccccgttcc cgaccatcac gctcgacctc 5040
acccagccgc cgccgacgac gacgaccacc gccgccgccg ccatgctcca gctccaccgc 5100
ccttatgcct tctcctcatt gccgttctcg atgtacggcg ccggcggcgg ctcgcaccgg 5160
ccgcccgtcg tcctgccccc gccgtcgtcg gtggtggaga cgatgaccgc cgcgatcacc 5220
agggacccca acttcaccac ggcggtggcg gccgcgctct cctcgatcat ggcgggaggc 5280
ggcgcccaag ctcgaacacc tccacgcggc gggagtgacg ccgccggaga catcaacgga 5340
ggcggcggcg ccgaccacgc cactgccgga gcacgcgccg cggcggcggc gacgcagcct 5400
tgcggcacgt ctcccacctg a 5421
Claims (10)
1. The application of the rice blast resistance related gene OsWRKY5 in improving the resistance of rice to rice blast is characterized in that:
the amino acid sequence of the rice blast resistance related gene OsWRKY5 is shown in SEQ ID NO. 33.
2. The application of the rice blast resistance related gene OsWRKY5 in improving the resistance of rice to rice blast as claimed in claim 1, is characterized in that:
the application is rice blast resistance breeding or transgenic rice cultivation.
3. The use of the rice blast resistance-related gene OsWRKY5 in improving resistance of rice to rice blast as claimed in claim 1 or 2, wherein:
the cDNA full-length sequence of the rice blast resistance related gene OsWRKY5 is shown as SEQ ID NO. 34.
4. The use of the rice blast resistance-related gene OsWRKY5 in improving resistance of rice to rice blast as claimed in claim 1 or 2, wherein:
the full-length genome sequence of the rice blast resistance related gene OsWRKY5 is shown as SEQ ID NO. 35.
5. The use of the rice blast resistance-related gene OsWRKY5 in improving resistance of rice to rice blast as claimed in claim 1 or 2, wherein:
up-regulating the expression of the rice blast resistance related gene OsWRKY5 in rice.
6. The use of the rice blast resistance-related gene OsWRKY5 in improving resistance of rice to rice blast as claimed in claim 1 or 2, wherein:
the rice blast fungus is rice blast fungus (M.oryzae) GDYJ 7.
7. Use of an overexpression vector for the rice blast resistance-related gene OsWRKY5 as claimed in claim 1, 3 or 4 for improving resistance of rice to rice blast, characterized in that:
the overexpression vector is used for overexpression of OsWRKY5 gene.
8. Use of a host bacterium comprising the overexpression vector of claim 7 for increasing rice blast resistance.
9. Use according to claim 7 or 8, characterized in that:
the application is rice blast resistance breeding or transgenic rice cultivation.
10. A method for breeding transgenic rice, which is characterized in that:
constructing an overexpression vector containing the rice blast resistance-related gene OsWRKY5 described in claim 1, 3 or 4 using a plant expression vector; then transforming rice tissues by using the overexpression vector; the transformed rice tissue is cultivated into rice plants.
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| CN202011436740.1A CN114621975B (en) | 2020-12-11 | 2020-12-11 | Application of rice blast resistance related gene OsWRKY5 |
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| CN114621975B (en) | 2023-05-02 |
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