Rice blast resistance gene pi57bas and its encoding protein and application

Abstract

The present invention discloses a rice blast resistance gene Pi57bas and its encoding protein, preparation method and application. The sequence of the rice blast resistance gene Pi57bas and its encoding protein are disclosed. The gene Pi57bas can be obtained by designing primers, taking rice genomic DNA containing the gene Pi57bas as template and through PCR amplification method; it can also be obtained by chemical synthesis method. The gene Pi57bas is stably inherited to offspring through analysis of disease-resistant plants obtained by genetic transformation of the gene and their offspring. Pi57bas and its alleles Pi57 have different resistance spectrum, are excellent resistance source for disease-resistant breeding and can be used for breeding rice varieties resistant to rice blast.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering technology, specifically to rice blast resistance genes and their encoded proteins and applications. Background Technology

[0002] Rice ( Oryza sativa ) is one of the world's most important food crops, produced by fungi ( Magnaporthe oryzae Rice blast, caused by [unspecified disease], is one of the most serious diseases affecting rice production areas. It can typically cause yield losses of 10%-30%, and in severe cases, even total crop failure, significantly impacting both rice yield and quality. [1-2] Practice has proven that breeding and promoting disease-resistant rice varieties is the most economical, effective, and environmentally friendly measure to control this disease, and the discovery, research, and utilization of disease-resistant genes are the core of disease-resistant breeding. [1,3] .

[0003] However, the high genetic instability and diversity of field pathogens can easily lead to the rapid loss of resistance in rice varieties due to the emergence of new physiological races. Therefore, it is of great significance to discover and enrich resistance sources, especially new genes with broad-spectrum disease resistance characteristics, for disease resistance breeding.

[0004] The genus *Oryza* includes cultivated rice in Asia (… Oryza sativa L.), African cultivated rice ( O. glaberrima Steud.) and 22 wild species, of which 6 wild species belong to the same AA genome as cultivated rice in Asia, including common wild rice (Stud.) O. rufipogon ), Niwara wild rice ( O. nivara ), Changxiong wild rice ( O. longistaminata ), short-tongued wild rice ( O. barthii ), Zhanying wild rice ( O. glumaepatula ) and Southern Wild Rice ( O. meridionalis They are an excellent gene pool for improving Asian cultivated rice. Asian cultivated rice can be further subdivided into tropical japonica rice, temperate japonica rice, indica rice, and so on. aus and basmati 5 subgroups [4-5] .

[0005] In the identification and discovery of novel disease resistance genes, allele mining not only reveals the genetic variations and evolution of target genes and analyzes key amino acid sites that interact with the avirulence genes of pathogens, but also uncovers superior alleles with different resistance spectra. [6-8] .

[0006] Our research group has completed the development of a broad-spectrum rice blast resistance gene. Pi57The cloning of the gene has been granted a national patent (CN109280082 B, patent number ZL201811221584.X). Based on previous research, this study was conducted on six wild species (common wild rice, Niwara wild rice, long-male wild rice, short-tongued wild rice, awn-bearing wild rice, and southern wild rice) and two cultivated species (Asian cultivated rice and African cultivated rice) that share the same AA genome as Asian cultivated rice. Pi57 Allele detection and identification analysis of a gene. A novel allele for rice blast resistance was discovered in the Asian cultivated rice variety DOM ZARD. The gene's resistance to rice blast was confirmed using a transgenic function verification strategy, and it could be distinguished from other alleles through resistance spectrum analysis. This gene originates from Asian cultivated rice. basmati Varieties of the (bas) subgroup, hence the allele was named Pi57bas . Summary of the Invention

[0007] To address the problems of gene linkage redundancy, narrow resistance spectrum of existing rice blast resistance genes, and easy loss of resistance in conventional hybridization breeding, this invention provides a rice blast resistance gene. Pi57bas Its encoded proteins and applications.

[0008] The technical solution of the present invention is as follows: 1. A rice blast resistance gene Pi57bas Its nucleotide sequence is: (a) A sequence as shown in SEQ ID NO:1; or (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:2.

[0009] 2. The rice blast resistance gene described in Scheme 1 Pi57bas The encoded protein has the amino acid sequence shown in SEQ ID NO:2.

[0010] 3. A gene containing rice blast resistance Pi57bas The complete coding region of the cDNA sequence is shown in SEQ ID NO:3.

[0011] 4. Used to amplify the rice blast resistance gene described in Scheme 1 Pi57bas The primer combination consists of the following four primers: the primer shown in SEQ ID NO:4 and the primer shown in SEQ ID NO:5 for amplifying the 5' end fragment, and the primer shown in SEQ ID NO:6 and the primer shown in SEQ ID NO:7 for amplifying the 3' end fragment.

[0012] 5. A recombinant expression vector comprising the rice blast resistance gene described in Scheme 1. Pi57bas .

[0013] 6. A host cell comprising the recombinant expression vector described in Scheme 5.

[0014] 7. The host cell described in Scheme 6 is Agrobacterium, Escherichia coli, or rice cells.

[0015] 8. A rice blast resistance gene as described in preparation method 1 Pi57bas The method, containing the gene Pi57bas Using rice genomic DNA as a template or the genomic DNA of the rice variety DOM ZARD as a template, PCR amplification was performed using the primer combination described in Scheme 4 to obtain two DNA fragments: a 5' end fragment and a 3' end fragment. These fragments were then digested with enzymes and ligated to obtain the rice blast resistance gene described in Scheme 1. Pi57bas Alternatively, the rice blast resistance gene described in Scheme 1 can be obtained through chemical synthesis. Pi57bas .

[0016] 9. The rice blast resistance gene described in Scheme 1 Pi57bas Application in rice blast resistance breeding. Furthermore, the rice blast resistance gene... Pi57bas Application in rice blast resistance breeding: The rice blast resistance gene described in Scheme 1 Pi57bas By transferring the rice varieties susceptible to rice blast, rice varieties with resistance to rice blast were bred.

[0017] 10. A method for detecting or amplifying the rice blast resistance gene described in Scheme 1. Pi57bas The kit contains the primer combination described in Scheme 4.

[0018] Furthermore, the kit includes at least one of reagents for PCR amplification, reagents for DNA extraction, and reagents for PCR product analysis.

[0019] This invention also provides the rice blast resistance gene. Pi57bas Applications in the preparation of recombinant expression vectors.

[0020] The present invention also provides the application of the prepared recombinant expression vector in the preparation of host cells.

[0021] Compared with the prior art, the innovative points and beneficial effects of this invention are as follows: This invention gene Pi57bas Alleles at this locus Pi57 It exhibits different resistance spectrum characteristics against rice blast fungus, representing a novel broad-spectrum resistance gene and an excellent resistance source for rice blast resistance breeding.

[0022] Using the gene of this invention Pi57basTransformation technology can be used to transfer these genes into rice varieties susceptible to rice blast, resulting in new rice varieties resistant to rice blast. This avoids the gene linkage burden problem of traditional breeding and greatly shortens the breeding cycle.

[0023] Seeds of rice blast-resistant plants obtained through gene conversion and their offspring can be used to transmit the gene of this invention through conventional sexual hybridization. Pi57bas Stably inherited by offspring. Due to genes. Pi57bas It has broad-spectrum resistance to rice blast, and new rice blast-resistant varieties bred using this gene can be widely promoted and applied.

[0024] The sequence number SEQ ID NO:1 in the sequence listing represents the gene. Pi57bas The nucleotide sequence.

[0025] The sequence number SEQ ID NO:2 in the sequence listing represents the gene. Pi57bas The encoded protein amino acid sequence.

[0026] The sequence shown in SEQ ID NO:3 contains the gene for rice blast resistance. Pi57bas The complete coding region of the cDNA sequence.

[0027] The sequence number SEQ ID NO:4 in the sequence listing represents the amplified gene. Pi57bas The base sequence of the forward primer FULL57-5F-SalI-Fw for the 5' end fragment (abbreviated as: primer shown in SEQ ID NO:4).

[0028] The sequence number SEQ ID NO:5 in the sequence listing represents the amplified gene. Pi57bas The base sequence of the reverse primer 57-Mid-Rv for the 5' end fragment (abbreviated as: primer shown in SEQ ID NO:5).

[0029] The sequence number SEQ ID NO:6 in the sequence listing represents the amplified gene. Pi57bas The base sequence of the forward primer 57-Mid-Fw for the 3' end fragment (abbreviated as: primer shown in SEQ ID NO:6).

[0030] The sequence number SEQ ID NO:7 in the sequence listing represents the amplified gene. Pi57bas The base sequence of the reverse primer FULL57-3R-AscI-Rv for the 3' end fragment (abbreviated as: primer shown in SEQ ID NO:7).

[0031] The sequence number SEQ ID NO:8 in the sequence listing represents the amplified gene. Pi57bas Forward primer Pi57bas for the cDNA fragment - cDNA - The base sequence of Fw.

[0032] The sequence number SEQ ID NO:9 in the sequence listing represents the amplified gene. Pi57bas The reverse primer Pi57bas for the cDNA fragment - cDNA - The base sequence of Rv.

[0033] The sequence number SEQ ID NO:10 in the sequence listing represents the amplified hygromycin B phosphotransferase gene. hpt The base sequence of the forward primer Hpt-Fw.

[0034] The sequence number SEQ ID NO:11 in the sequence listing represents the amplified hygromycin B phosphotransferase gene. hpt The base sequence of the reverse primer Hpt-Rv.

[0035] The sequence number SEQ ID NO:12 in the sequence listing represents the amplified gene. Pi57bas The base sequence of the forward primer Pi57bas-SP-Fw for molecular identification.

[0036] The sequence number SEQ ID NO:13 in the sequence listing represents the amplified gene. Pi57bas The base sequence of the reverse primer Pi57bas-SP-Rv for molecular identification.

[0037] In this article, rice blast resistance genes Pi57bas Also known as genes Pi57ba s or Pi57bas Gene 。 Hygromycin B phosphotransferase gene ( hpt Also known as the hygromycin B phosphotransferase gene hpt , hpt or hpt Genes. In this article, all terms related to resistance, disease resistance, or susceptibility refer to resistance to rice blast or susceptibility to rice blast. Attached Figure Description

[0038] Figure 1 Hygromycin B phosphotransferase gene in T0 generation transgenic plants hpt Identify the electrophoresis image.

[0039] M: DL2000 molecular weight marker; Numbers 1-15: Contains recombinant plasmid pCAMBIA1300AscI- Pi57bas -2-14 PCR amplification products of genomic DNA from different T0 generation transgenic plants; No. 16: PCR amplification product of water-free blank control; No. 17: PCR amplification product of genomic DNA from the recipient rice resistance single-gene line IRBLks-F5 (negative control); No. 18: containing... Pi57bas The recombinant plasmid pCAMBIA1300AscI- of the gene Pi57bas -2-14 is the PCR amplification product of the template (positive control). (The rice resistance single-gene line IRBLks-F5 contains the rice blast resistance gene.) Pik-s However, it was susceptible to the inoculated rice blast fungus 09BSH-10-5A, so it was used as a negative control and also as a transgenic recipient variety.

[0040] Figure 2 Phenotypic response of T0 generation transgenic plants inoculated with rice blast fungus 09BSH-10-5A.

[0041] Figure 2 In the middle, number 1-15: with Figure 1 Leaves of T0 generation transgenic plants corresponding to the specified numbers; Leaf of IRBLks-F5: Leaf of the susceptible recipient rice resistance single-gene line IRBLks-F5 used for genetic transformation (susceptible control); Leaf of DOM ZARD: Contains the rice blast resistance gene. Pi57bas The donor material was a leaf from the Asian cultivated rice variety DOM ZARD (disease-resistant control).

[0042] Figure 3 Co-segregation analysis diagram of T1 generation plants inoculated with rice blast fungus 09BSH-10-5A.

[0043] Figure 3 Upper middle picture ( Figure 3 a) is a phenotypic response diagram of T1 generation plants inoculated with rice blast fungus 09BSH-10-5A; Figure 3 lower middle image ( Figure 3 b) is a molecular identification diagram of T1 generation plants; M: 20 bp DNA molecular weight marker (100 ng / 1 μL). Figure 3 Number 1: Donor material rice variety DOM ZARD; Number 2: Disease-susceptible recipient rice resistance single-gene line IRBLks-F5; Numbers 3 to 12: T1 generation plants. Detailed Implementation

[0044] The present invention will be further described in detail below with reference to the accompanying drawings and implementation process. It should be understood that the specific embodiments described herein are only for aiding understanding of this statement and do not limit the present invention. All equivalent substitutions or improvements that do not depart from the spirit of the present invention are within the protection scope of the present invention.

[0045] Unless otherwise specified in the embodiments, all methods employed are conventional techniques well-known to those skilled in the art.

[0046] Example 1 Alleles Pi57bas Amplification and Analysis I. Genes Pi57bas Full-length genomic DNA amplification and sequencing analysis (1) Primer design (genomic DNA) According to known Pi57 Genomic DNA and cDNA sequences of genes [9] Two pairs of segmented amplification primers were designed to amplify alleles. Pi57bas The full-length genome DNA sequence contains 1 gene sequence. Pst I. Restriction site: Both amplified sequences contain this restriction site, which is used for the ligation of the digested fragments during subsequent cloning.

[0047] Amplification of the 5' end fragment: Forward primer FULL57-5F-SalI-Fw, the base sequence of which is shown in SEQ ID NO:4, with the 5' end introduced. Sal The restriction enzyme site and reverse primer 57-Mid-Rv have the base sequence shown in SEQ ID NO:5. The 5' end fragment contains the gene. Pi57bas Its own promoter.

[0048] Amplification of the 3' end fragment: forward primer 57-Mid-Fw, its base sequence is shown in SEQ ID NO:6; reverse primer FULL57-3R-AscI-Rv, its base sequence is shown in SEQ ID NO:7, with the 5' end introduced. Asc I. Restriction site. The 3' end fragment contains the gene. Pi57bas The terminator of itself.

[0049] DOM ZARD, a rice variety belonging to the basmati subgroup of Asian cultivated rice, was extracted using the CTAB method.

[10] DNA was collected and diluted to a concentration of 50 ng / μL for use as a template for PCR amplification. PCR amplification was performed using the long-chain high-fidelity enzyme PrimeSTAR® GXL DNAPolymerase.

[0050] (2) PCR amplification PCR reaction system (50 μL): 10 μL 5× PrimeSTAR GXL Buffer, 1 μL PrimeSTAR GXL DNA Polymerase, 4 μL 2.5 mM each dNTP Mixture, 2 μL 10 μM forward primer, 2 μL 10 μM reverse primer, 1 μL 50 ng / μL DNA template, and finally added to 50 μL with MillQ sterile ultrapure water. PCR reaction conditions: 95℃ pre-denaturation for 3 min; 98℃ denaturation for 10 s, 68℃ sterilization and extension for 6 min, for a total of 34 cycles.

[0051] Both the 5' end fragment and the 3' end fragment were amplified using the PCR reaction system and PCR reaction conditions described in (2) of this embodiment.

[0052] (3) Electrophoresis detection and sequencing analysis After the PCR reaction, the PCR amplification products of the 5' end fragment and the amplified 3' end fragment were detected by 1% agarose gel electrophoresis. The results showed that the amplified 5' end fragment showed a single target band of the expected size at approximately 6.7 kb, and the amplified 3' end fragment showed a single target band of the expected size at approximately 3.2 kb, indicating that the 5' end fragment and 3' end fragment were successfully amplified. The two amplified fragments were sequenced and assembled to obtain... Pi57bas The full-length genome sequence of the gene is 9807 bp, and the DNA sequence is shown in SEQ ID NO:1.

[0053] II. Contains rice blast resistance genes Pi57bas Obtaining the complete coding region cDNA sequence (1) Primer design Design a pair located in the gene Pi57bas Primers flanking the coding region: forward primer Pi57bas - cDNA - Fw, whose base sequence is shown in SEQ ID NO:8, and whose reverse primer is Pi57bas - cDNA - Rv, whose base sequence is shown in SEQ ID NO:9, is used to amplify genes containing rice blast resistance. Pi57bas The complete coding region of the cDNA sequence.

[0054] (2) Reverse transcription and PCR amplification Total RNA was extracted from young leaves of the Asian cultivated rice variety DOM ZARD using an RNA extraction kit (Ominiplant RNA Kit (DNase I), Kangwei Century Biotechnology Co., Ltd.). First-strand cDNA was synthesized using PowerScript II Reversetranscriptase (Takara). Using the first-strand cDNA as a template, forward primer Pi57bas was used… - cDNA - Fw (SEQ ID NO:8) and reverse primer Pi57bas - cDNA -PCR amplification was performed using Rv (shown in SEQ ID NO:9). The PCR reaction system (50 μL) consisted of: 10 μL of 5× PrimeSTAR GXL Buffer, 1 μL of PrimeSTAR GXL DNA Polymerase, 4 μL of 2.5 mM each dNTP Mixture, 2 μL of 10 μM forward primer, 2 μL of 10 μM reverse primer, 1 μL of cDNA template, and finally, MillQ sterile ultrapure water to a final volume of 50 μL. PCR reaction conditions: 95℃ pre-denaturation for 3 min, 98℃ denaturation for 10 s, 62℃ annealing for 30 s, and 68℃ extension for 4 min, for a total of 34 cycles.

[0055] (3) Electrophoresis detection and sequencing After the PCR reaction was completed, the PCR product was detected by 1% agarose gel electrophoresis to show a single target band. The product was then sent to a sequencing company for sequencing, and sequence alignment confirmed its correctness, yielding the gene containing rice blast resistance. Pi57bas The complete coding region cDNA sequence is shown in SEQ ID NO:3, 3728 bp.

[0056] III. Genes Pi57bas Sequence analysis (1) Exon / intron structure analysis Using the obtained genes Pi57bas The complete coding region cDNA sequence (SEQ ID NO:3) and Pi57bas Comparative analysis of the gene's genomic DNA sequence (SEQ ID NO:1) showed that: Pi57bas The gene's genomic sequence contains 3 introns and 4 exons. In SEQ ID NO:1, intron 1 is located at positions 3085-3412, intron 2 at positions 3674-6340, and intron 3 at positions 8268-8584; exon 1 is located at positions 1950-3084, exon 2 at positions 3413-3673, exon 3 at positions 6341-8267, and exon 4 at positions 8585-8989.

[0057] (2) Analysis of coding region and amino acid sequence The sequence shown in SEQ ID NO:3 contains a 5' uncoding region (5'UTR) and a complete coding region (CDS), wherein the coding region (CDS) is located at positions 1158 to 3728, the coding region sequence is 2571 bp in length, and encodes a protein composed of 856 amino acids, the amino acid sequence of which is shown in SEQ ID NO:2.

[0058] Example 2: Genetic transformation and complementation verification experiment of the rice blast resistance gene Pi57bas I. Construction and Genetic Transformation of Recombinant Expression Vectors (1) PCR amplification and enzyme digestion Following the method in Example 1, using the genomic DNA of the resistant donor rice variety DOM ZARD as a template, the 5' end fragment (approximately 6.7 kb) of the gene was amplified using the forward primer FULL57-5F-SalI-Fw (SEQ ID NO:4) and the reverse primer 57-Mid-Rv (SEQ ID NO:5), and the 3' end fragment of the gene was amplified using the forward primer 57-Mid-Fw (SEQ ID NO:6) and the reverse primer FULL57-3R-AscI-Rv (SEQ ID NO:7).

[0059] Using restriction endonucleases from NEB Sal I and Pst Digest the 5' end fragment containing the promoter (approximately 6.7 kb) with two restriction enzymes; then digest with NEB restriction endonucleases. Pst I and Asc Digest the 3' end fragment (approximately 3.2 kb) of the above gene with I double enzyme digestion; use Sal I and Asc Plant expression pCAMBIA1300 AscI was double-digested with enzymes. The digestion system was 50 μL: 10×rCutSmart TM 5 μL buffer, 20 U / μL Sal I and 20 U / μL Pst Digest the 5' end fragment with 1 μL of each of the enzymes (digest the 3' end fragment with a combination of 20 U / μL Pst I and 10 U / μL Asc I, 1 μL of each enzyme), and 30-43 μL of PCR product. Finally, add MillQ water to 50 μL, and then double digest at 37℃ for 1.5 h. After separation by agarose gel electrophoresis, the digested products are recovered, purified, and quantified using a gel extraction kit.

[0060] (2) Ligation reaction and cloning The above-mentioned gene-containing samples, which underwent double enzyme digestion, purification, and quantification, were... Pi57basThe 5' and 3' fragments of the self-promoter and the plant expression pCAMBIA1300AscI were mixed at a molar ratio of 5' fragment: 3' fragment: plant expression pCAMBIA1300AscI of 3:3:1 and ligated using NEB's T4 ligase. The ligation system of the target fragment and vector using T4 ligase was as follows: 2 μL of ligation buffer (10×T4), 1 μL (10 ng) of double-digested pCAMBIA1300AscI vector, 1 μL (22.4 ng) of double-digested 5' fragment containing the promoter, 1 μL (11 ng) of double-digested 3' fragment, 0.5 μL of T4 ligase, and double-digested water to a final volume of 20 μL; ligation was performed overnight at 10°C; the ligation product was precipitated with anhydrous ethanol and transformed into E. coli DH10B competent cells by electroporation to obtain positive clones, which were then sequenced. Sequencing results showed that the inserted fragment sequence was consistent with the expected sequence. Pi57bas The recombinant expression vector pCAMBIA1300AscI- Pi57bas -2-14 is used for subsequent genetic transformation.

[0061] (3) Genetic transformation of rice callus and acquisition of transgenic plants Agrobacterium-mediated genetic transformation (Hiei Y, Ohta S, Komari T, et al. Efficient transformation of rice ( Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. The Plant Journal, 1994, 6:271-282) Conduct genetic transformation experiments on the gene: the pCAMBIA1300AscI- obtained above Pi57bas The plasmid of -2-14 was transformed into Agrobacterium strain EHA105, and Agrobacterium-mediated genetic transformation was used to transform the plasmid. Pi57bas The gene was transferred into the rice resistance single-gene line IRBLks-F5 to obtain T0 generation transgenic plants. (The rice resistance single-gene line IRBLks-F5 (see reference

[11] ) contains the rice blast resistance gene P. ik-s However, it is susceptible to rice blast fungus 09BSH-10-5A (when inoculated).

[0062] II. Molecular identification and disease resistance phenotype identification of T0 generation transgenic plants (1) Molecular identification of T0 generation transgenic plants The obtained T0 generation transgenic plants were transformed using the genetic transformation vector pCAMBIA1300AscI- Pi57bas Hygromycin B phosphotransferase gene carried on -2-14 ( hpt Molecular identification was performed through PCR amplification.

[0063] Genomic DNA was extracted from the T0 generation transgenic plants and the recipient rice resistance single gene line IRBLks-F5 (negative control) obtained in Example 1 (3) using the CTAB method. PCR amplification was performed using forward primer Hpt-Fw (SEQ ID NO:10) and reverse primer Hpt-Rv (SEQ ID NO:11) to detect the hygromycin B phosphotransferase gene (hpt).

[0064] PCR amplification of T0 generation transgenic plants: Genomic DNA of T0 generation transgenic plants was used as a template.

[0065] Negative control: Genomic DNA of the recipient rice resistance single-gene line IRBLks-F5 was used as a template.

[0066] Blank control: Sterile water was used instead of template.

[0067] Positive control: recombinant plasmid pCAMBIA1300AscI- Pi57bas -2-14 is the template.

[0068] The PCR amplification reaction system and conditions are as follows: The total PCR reaction volume is 20 µL, containing 10 µL of 2× Es TaqMaster Mix, 1 µL each of 10 µM forward primer Hpt-Fw and 10 µM reverse primer Hpt-Rv, 1 µL (10 ng) of genomic DNA, and double-distilled sterile water to a final volume of 20 µL. The PCR amplification conditions are: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and a final extension at 72℃ for 7 min. After PCR amplification, the amplified products were detected by 1% agarose gel electrophoresis, and the results are as follows. Figure 1 .

[0069] The results show (see) Figure 1 Numbered 1-15: 15 T0 generation transgenic plants were hpt Positive (expected band of 557 bp was amplified), number 18: recombinant plasmid pCAMBIA1300AscI- Pi57bas -2-14 has a stripe hpt Positive (positive control), No. 17: The recipient rice resistance single-gene line IRBLks-F5 and the blank control (No. 16) both showed no bands. hptNegative. This indicates that the gene of this invention... Pi57bas The genetic transformation has been successfully completed by transferring the transgenic rice into the recipient rice variety, resulting in 15 T0 generation transgenic positive plants.

[0070] (2) Identification of disease resistance phenotype in T0 generation transgenic plants Using the rice blast fungus 09BSH-10-5A (see reference

[12] ), which is pathogenic to the recipient rice resistance single-gene line IRBLks-F5, 15 T0 generation transgenic positive plants corresponding to the T0 generation transgenic plants identified in the second (1) T0 generation in this example were inoculated using the spray inoculation method. The inoculation results are as follows: Figure 2 As shown. Figure 2 Samples numbered 1-15 were leaves from the aforementioned T0 generation transgenic positive plants; sample number IRBLks-F5 was a leaf from the rice resistance single-gene line IRBLks-F5 (susceptible control); and sample number DOM ZARD was a leaf from the donor Asian cultivated rice variety DOM ZARD (resistant control). Disease incidence was assessed 7-10 days after inoculation (see...). Figure 2 The grading standards for rice blast are as follows: Rice blast disease is classified into 6 levels (refer to Dong Liying et al., Pathogenicity analysis of single-gene lines of rice blast resistance in Yunnan Province, Southwest China Journal of Agricultural Sciences, 2012, 25(2): 467-473). Level 0: No lesions; Level 1: Brown spot lesions, diameter ≤1 mm, no sporulation ability; Level 2: Elliptical lesions, 1 mm < diameter ≤2 mm, with brown edges around the lesions and grayish-white center, with sporulation ability; Level 3: Elliptical lesions, 2 mm < diameter ≤3 mm, with brown edges around the lesions and grayish-white center, with sporulation ability; Level 4: Typical spindle-shaped or fusiform lesions, diameter >3 mm, with or without fusion of lesions; Level 5: Lesion type is the same as Level 4, but due to fusion between lesions, the upper half of the leaf dies. Among them, Levels 0 to 2 are classified as resistance reaction (R), and Levels 3 to 5 are classified as susceptibility reaction (S).

[0071] result Figure 2 As shown: Phenotypic identification of T0 generation transgenic plants revealed that among 15 T0 generation transgenic plants, 11 showed disease resistance, while only 4 plants (plants 4, 7, 13, and 14) showed susceptibility. The rice resistance single-gene line IRBLks-F5 (susceptible control) showed susceptibility, while the donor Asian cultivated rice variety DOM ZARD (resistant control) showed resistance. This indicates that the gene... Pi57bas The transgenic plants of the T0 generation have been successfully expressed, conferring resistance to rice blast.

[0072] III. Molecular identification and disease resistance phenotype identification of T1 generation plants (1) Molecular identification of T1 generation plants After self-pollination of T0 generation plants, T1 generation seeds were obtained. Genomic DNA was extracted from T1 generation plants using the CTAB method as a template, and amplified using [a method for gene amplification]. Pi57bas PCR amplification was performed using the specific primers: forward primer Pi57bas-SP-Fw (SEQ ID NO:12) and reverse primer Pi57bas-SP-Rv (SEQ ID NO:13).

[0073] Set the following comparison: Positive control: Genomic DNA from the Asian cultivated rice variety DOM ZARD was used as a template; Negative control: Genomic DNA of the recipient rice resistance single-gene line IRBLks-F5 was used as a template.

[0074] The PCR amplification reaction system and conditions are as follows: The total PCR reaction volume is 20 µL, containing 10 µL of 2× Es TaqMaster Mix, 1 µL each of 10 µM forward primer Pi57bas-SP-Fw and 10 µM reverse primer Pi57bas-SP-Rv, 1 µL (10 ng) of genomic DNA, and double-distilled sterile water to a final volume of 20 µL. The PCR amplification conditions are: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and a final extension at 72℃ for 7 min. After PCR amplification, the amplified products were detected by 8% polyacrylamide gel electrophoresis, and the results are as follows. Figure 3 lower middle image ( Figure 3 (b) No. 1 is the donor material, the Asian cultivated rice variety DOM ZARD; No. 2 is the susceptible recipient rice resistance single gene line IRBLks-F5; No. 3-12 are T1 generation plants; No. 1 and No. 7-12 were amplified positively, while No. 2-6 were amplified negatively.

[0075] (2) Phenotypic identification of T1 generation plants Rice blast fungus 09BSH-10-5A was used to inoculate the donor Asian cultivated rice variety DOMZARD, the recipient rice resistance single-gene line IRBLks-F5, and 10 T1 generation plants corresponding to the molecularly identified T1 generation plants using a spray inoculation method. Simultaneously, the recipient rice resistance single-gene line IRBLks-F5 was set as a susceptible control, and the donor Asian cultivated rice variety DOMZARD was used as a resistant control. Disease incidence was investigated 7-10 days after inoculation (see...). Figure 3 The image above, i.e. Figure 3 a). The grading standard for rice blast is as described in Example 2(2) of this document.

[0076] The results show that ( Figure 3 a) The donor Asian cultivated rice variety DOM ZARD (number 1) and the T1 generation plants (numbers 7-12) that were positive for PCR amplification all showed disease resistance, while the recipient rice resistance single-gene line IRBLks-F5 (number 2) and the T1 generation negative plants (numbers 3-6) all showed disease susceptibility. Figure 3 Upper middle picture ( Figure 3 a) and the results corresponding to the figure below (Figure 3 b) indicate that the gene of this invention Pi57bas It has been stably integrated into the rice genome and can be stably inherited by offspring, and the disease resistance phenotype is consistent with the gene. Pi57bas The gene exhibits co-segregation characteristics, indicating that the gene of this invention... Pi57bas Materials that acquire resistance can be transferred into susceptible varieties through conversion methods.

[0077] Example 3: Rice blast resistance allele Pita-2 , Pi57 and Pi57bas Anti-spectral difference analysis I. Experimental Materials (1) Pi57bas T2 generation transgenic homozygous plants ( Pi57bas T2-6) acquisition The T1 generation disease-resistant plants (phenotypically disease-resistant and molecularly positive) obtained in Example 2 were planted and self-pollinated individually in bags during the flowering period to harvest T2 generation seeds. Each T2 generation plant constitutes a line, and 17 plants were planted in each line. Molecular and phenotypic identification were performed according to methods (1) and (2) in Example 2. If all plants in a line exhibit a disease-resistant phenotype and are molecularly positive, then the line is a homozygous line, and all its plants are homozygous. Pi57bas Transgenic homozygous individuals. The sixth homozygous line obtained through screening and identification was named Pi. 57bas T2-6 is used for subsequent anti-spectral analysis experiments.

[0078] (2) Other rice materials Asian cultivated rice variety DOM ZARD (see reference

[10] ): contains Pi57bas Allele (donor material of this invention, disease-resistant control, abbreviated as DOM ZARD).

[0079] Rice infiltration line IL-E1454 ( Pi57 ): Contains known Pi57 Allele (abbreviated as IL-E1454, see reference

[13] ).

[0080] Rice resistance single-gene line IRBLta2-Pi: contains Pita-2 The gene (abbreviated as IRBLta2-Pi, see reference

[14] ).

[0081] Rice resistance single-gene line IRBLks-F5: contains Pik-s The gene, the receptor material of the present invention (abbreviated as IRBLks-F5, disease control, see reference

[11] ).

[0082] The susceptible control variety is Lijiangxintuanheigu: the susceptible variety (abbreviated as LTH, see reference

[15] ).

[0083] (3) Tested rice blast strains Eight single-spore strains of rice blast fungus with different pathogenicities were selected: 09BSH-10-5A, CH1419, CH0999, 2011-1-1-11-2, HN09-1C-7, 08-9-3a-1, CH1019, and Y363. These strains are referenced in [12, 16-18].

[0084] II. Inoculation and Resistance Identification The above eight strains were inoculated into six rice materials (DOM ZARD, ) using a spray inoculation method. Pi57bas T2-6, IL-E1454, IRBLta2-Pi, IRBLks-F5, LTH). Each strain should be inoculated with at least 10 plants per material, and the experiment should be repeated in 3 replicates. Disease incidence should be investigated 7-10 days after inoculation. The rice blast resistance grading criteria are the same as in Example 2, section 2(2).

[0085] III. Results and Analysis The results of the anti-antigen spectrum determination are shown in Table 1. The anti-antigen spectrum determination shows that: The susceptible control rice resistance single-gene line IRBLks-F5 and the susceptible control variety LTH both showed susceptibility to all eight tested strains.

[0086] DOM ZARD, a resistance donor rice variety for Asian cultivated rice ( Pi57bas )and Pi57bas T2 generation transgenic homozygous strain Pi57bas T2-6 ( Pi57bas The strain showed resistance to 7 out of 8 tested strains, but was infected with rice blast strain CH1019.

[0087] Rice infiltration line IL-E1454 ( Pi57 It was susceptible to two strains, CH1019 and Y363, and resistant to the remaining six strains.

[0088] Rice resistance single-gene line IRBLta2-Pi ( Pita-2It was susceptible to three strains: 09BSH-10-5A, 2011-1-1-11-2, and CH1019, and resistant to the remaining five strains.

[0089] This shows that the rice blast resistance allele located on chromosome 12 of rice Pita-2 , Pi57 and Pi57bas The eight inoculated strains exhibited different resistance spectra, indicating the presence of a rice blast resistance gene. Pi57 Alleles cloned at the locus Pi57bas (The gene of this invention) is a novel functional allele with resistance to rice blast.

[0090] IV. Conclusion A gene for rice blast resistance cloned on rice chromosome 12. Pi57bas It is an allele with a new disease resistance function, possessing broad-spectrum disease resistance. Homozygous disease-resistant plants can be obtained through breeding selection, and it can be applied to rice disease resistance breeding to select rice varieties resistant to rice blast, thereby solving the problem of rice blast damage in rice production.

[0091]

[0092] References [1] Khush GS, Jena KK. Current status and future prospects forresearch on blast resistance in rice ( Oryza sativa L.). In: Wang GL, Valent B(eds) Advances in genetics, genomics and control of rice blast disease. Springer, 2009, Dordrencht, p1–10 [2] Nie Yanfang, Leng Meiqin, Li Guanjun, Li Jieling, Li Huaping, Li Yunfeng. Functional analysis of the dipeptidyl peptidase V gene DppV of *Magnaporthe oryzae*. *Chinese Journal of Plant Protection*, 2021, 48(4): 712-722. [3] Deng Y, Zhai K, Xie Z, Yang D, Zhu X, Liu J, Wang X, Qin P, YangY, Zhang G, Li Q, Zhang J, Wu S, Milazzo J, Mao B, Wang E, Xie H, Tharreau D,He Z. Epigenetic regulation of antagonistic receptors confers rice blastresistance with yield balance. Science, 2017, 355: 962-965. [4] Garris AJ, Tai TH, Coburn J, Kresovich S, McCouch S. Geneticstructure and diversity in Oryza sativa L. Genetics, 2005, 169: 1631-1638. [5] Zhou J, Yang Y, Lv Y, Pu Q, Li J, Zhang Y, Deng X, Wang M, WangJ, Tao D. Interspecific hybridization is an important driving force fororigin and diversification of Asian cultivated rice Oryza sativa L. Frontiersin Plant Science, 2022, 13:932737. [6] Kumar GR, Sakthivel K, Sundaram RM, Neeraja CN, Balachandran SM,Rani NS, Viraktamath BC, Madhav MS. Allele mining in crops: Prospects andpotentials. Biotechnology Advances, 2010, 28: 451-461 [7] Ashkani S, Yusop MR, Shabanimofrad M, Azadi A, Ghasemzadeh A,Azizi P, Latif MA. Allele mining strategies: Principles and utilization for blast resistance genes in rice ( Oryza sativa L.). Current Issues in MolecularBiology, 2015, 17: 57-74 [8] Lv Q, Huang Z, Xu X, Tang L, Liu H, Wang C, Zhou Z, Xin Y, Xing J, Peng Z, Li X, Zheng T, Zhu L. Allelic variation of the rice blastresistance gene Pid3 in cultivated rice worldwide. Scientific Reports, 2017, 7:10362; doi: 10.1038 / s41598-017-10617-2. [9] Dong Liying, Yang Qinzhong, Liu Shufang, Xu Peng, Li Xundong, Li Jing, Zhou Jiawu, Tao Dayun. A rice blast resistance gene Pi57 Its encoded proteins and applications, 2019-06-11, China, ZL201811221584.X.

[10] For the rice variety DOM ZARD, see the literature "Wang et al. Genomic variation in 3,010 diverse accessions of Asian cultivated rice. Nature, 2018, 557: 43–49 (Supplementary Data 1)".

[11] For the rice resistance monogenic line IRBLks-F5, see the literature "Tsunematsu et al. Development of monogenic lines of rice for blast resistance. Breeding Science, 2000, 50:229-234".

[12] Rice blast strain 09BSH-10-5A, see literature "Dong et al.Identification and finemapping of Pi69 (t), a new gene conferring broad-spectrum resistanceagainst Magnaporthe oryzae from Rice Steud. Front. Plant Sci. 2020,11:1190.”

[13] Rice infiltration line IL-E1454 (see reference "Dong") and others Fine mapping of Pi57 (t)conferring broad spectrum resistance against Magnaporthe rice inintrogression line IL-E1454 derived from Long-stemmed rice . PLOS ONE, 2017,12(10): e0186201; Xu and others . Identification and mapping of a novel blastresistance gene Pi57 (t) in Long-stemmed rice . Euphytica, 2015, 205(1): 95-102.”

[14] For the rice resistance monogenic line IRBLta2-Pi, see the literature "Tsunematsu et al. Development of monogenic lines of rice for blast resistance. Breeding Science, 2000, 50:229-234".

[15] Lijiang Xintuan Heigu LTH (See reference "Tsunematsu et al. Development of monogenic lines of rice for blast resistance. Breeding Science, 2000, 50:229-234")

[16] Rice blast strains CH0999, CH1019, CH1419, 2011-1-11-2, 08-9-3a-1 are referred to the literature “Pathogenicity analysis and mating type distribution of rice blast fungus population in Yunnan Province, Journal of Plant Protection, 2023, 50(2): 316-324”.

[17] For strain HN09-1C-7, see the literature "Xu and others .Identification and mapping of anovel blast resistance gene Pi57 (t) in Long-stemmed rice . Euphytica, 2015,205(1): 95-102.”

[18] Rice blast fungus Y363, see reference "Liu Shufang et al., holding Pi9 Differences in resistance to rice blast fungus at seedling and adult stages in the single-gene rice line IRBL9-W. Chinese Journal of Rice Science, 2021, 35(3): 303-310”

Claims

1. A rice blast resistance gene Pi57bas Its nucleotide sequence is: (a) A sequence as shown in SEQ ID NO:1; or (b) A nucleotide sequence encoding the amino acid sequence shown in SEQ ID NO:

2.

2. The rice blast resistance gene as described in claim 1 Pi57ba The protein encoded by s has the amino acid sequence shown in SEQ ID NO:

2.

3. A gene containing rice blast resistance Pi57bas The complete coding region cDNA sequence, the nucleotide sequence of which is shown in SEQ ID NO:

3.

4. A primer combination for amplifying the rice blast resistance gene Pi57bas as described in claim 1, characterized in that, The primer combination consists of the following four primers: the primer shown in SEQ ID NO:4 and the primer shown in SEQ ID NO:5 for amplifying the 5' end fragment, and the primer shown in SEQ ID NO:6 and the primer shown in SEQ ID NO:7 for amplifying the 3' end fragment.

5. A recombinant expression vector comprising the rice blast resistance gene as described in claim 1. Pi57bas .

6. A host cell comprising the recombinant expression vector of claim 5.

7. The host cell according to claim 6, wherein the host cell is Agrobacterium, Escherichia coli or rice cell.

8. A method for preparing the rice blast resistance gene of claim 1 Pi57bas The method is characterized by, With the rice blast resistance gene as described in claim 1 Pi57bas Using rice genomic DNA as a template, PCR amplification was performed using the primer combination described in claim 4 to obtain two DNA fragments, which were then digested and ligated to obtain the rice blast resistance gene described in claim 1. Pi57bas Alternatively, the rice blast resistance gene described in claim 1 can be obtained through chemical synthesis. Pi57bas .

9. The application of the rice blast resistance gene Pi57bas as described in claim 1 in rice blast resistance breeding, characterized in that, The rice blast resistance gene described in claim 1 Pi57bas By transferring the rice varieties susceptible to rice blast, rice varieties with resistance to rice blast were bred.

10. A kit for detecting or amplifying the rice blast resistance gene Pi57bas as described in claim 1, comprising the primer combination as described in claim 4.

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