Application of FAH1 gene in improving resistance of rice to rice blast

Abstract

The invention discloses application of an FAH1 gene in improving resistance of rice to rice blast. The nucleotide sequence of the FAH1 gene is as shown in SEQ ID NO.1, and the amino acid sequence coded by the FAH1 gene is as shown in SEQ ID NO.2. The FAH1 gene is knocked out in a rice plant, and the resistance of rice to pyricularia grisea can be remarkably improved. The invention provides a basis for the key molecular genetic mechanism of rice blast resistance, and has important guiding significance for cultivating resistant rice varieties and improving the rice quality.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, and more specifically to rice. FAH1 Application of genes in improving rice resistance to rice blast. Background Technology

[0002] Rice ( Oryza sativa Rice is my country's most important food crop, and one of the most actively promoted, strongly supported, and widely planted and produced grain crops, holding a dominant position in my country's grain production. The healthy, stable, and sustainable development of the rice industry is of paramount importance to ensuring my country's food security and the development of the rice processing industry. However, its production is frequently threatened by various pathogens, often leading to large-scale yield reductions and quality degradation, seriously threatening global food security. Rice blast fungus (… Magnaporthe oryzae ), rice aspergillus ( Ustilaginoidea virens ) and white leaf blight ( Xanthomonas oryzae pv. oryzae Diseases such as rice blast and rice blight often cause reduced rice yields and lower quality. Rice blast alone causes enough damage to rice worldwide each year to feed 60 million people. Current disease control methods mainly involve the use of chemical pesticides and planting resistant varieties. However, long-term reliance on large-scale pesticide application to maintain crop yields has caused serious environmental damage. Furthermore, breeding high-yielding, resistant rice varieties through traditional methods is time-consuming, and due to species differences, many broad-spectrum disease-resistant genes are difficult to promote and apply in different crop production. In addition, with continuous environmental degradation and the accelerated evolution of pathogen races, long-term planting of single resistant varieties has led to the gradual loss of resistance in many varieties, resulting in unsatisfactory disease control effects in actual production.

[0003] With continuous technological advancements, scientists have developed new gene-editing technologies that enable precise and predictable modification of endogenous genes in crops, safely and controllably obtaining desired traits. Among these, emerging genome editing technologies have gradually become a primary tool in plant science research, widely applied in global plant biology research and crop genetic improvement. Crop genetic improvement breeding strategies relying on genome editing technology can overcome current breeding bottlenecks, reduce breeding time, and improve crop disease resistance, yield, and quality, thus playing a significant role in ensuring global food security.

[0004] Sphingolipids are a collective term for a class of lipids with complex structures and diverse functions. They are ubiquitous in the membrane components of eukaryotic cells and some bacteria, maintaining the integrity of membrane structures. In plant cells, sphingolipids account for approximately 40% of plasma membrane lipids. The diversity of sphingolipid structures leads to the diversity of their functions. Sphingolipids participate in various biological processes, such as acting as signaling molecules in intercellular and intracellular signal transduction, programmed cell death, and abscisic acid-dependent stomatal closure in guard cells. Sphingolipid homeostasis is crucial for plant growth and development; alterations in the synthesis, transport, and degradation of sphingolipids can induce corresponding phenotypes in plants, thereby affecting their survival and reproduction. Ceramides are key intermediates in sphingolipid metabolism, typically consisting of a long-chain base (LCB) linked to a fatty acid (FA) via an amide bond. Loss of function of the ceramide kinase ACD5 leads to activation of the jasmonic acid pathway, regulating sphingolipid metabolism and increasing ceramide accumulation levels, thus accelerating spontaneous cell death.

[0005] FAH, known as sphingolipid fatty acid 2-hydroxylase, can produce 2-HFA in Saccharomyces cerevisiae and mammals. In mammals, 2-hydroxysphingolipids are particularly abundant in epidermal tissues and the nervous system, as they are considered essential for the permeability barrier of the epidermis and the formation of rigid myelin. On the other hand, 2-HFAs are also mainly contained in the sphingolipids of higher plants: saturated and monounsaturated 2-hydroxy fatty acids with carbon chain lengths between 16 and 26 carbon atoms, accounting for more than 90% of the total fatty acid content of glucosylceramide, ceramide, and glycosylinositol phosphatidylceramide—these substances are the dominant sphingolipids in plant tissues. Summary of the Invention

[0006] The main technical problem solved by this invention is to overcome the current lack of rice blast resistance gene resources, and to provide... FAH1 The application of genes in improving rice resistance to rice blast provides a basis for breeding new disease-resistant rice lines through molecular breeding methods.

[0007] The first objective of this invention is to provide the application of the FAH1 gene in improving rice resistance to rice blast, wherein... FAH1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and its gene symbol in NCBI is LOC4352850.

[0008] Specifically, the aforementioned FAH1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.2.

[0009] Specifically, the improvement of rice's resistance to rice blast is achieved by knocking out [a specific pathogen] in rice. FAH1Genetic mutant strains were obtained to improve rice resistance to rice blast.

[0010] Preferably, it involves knocking out the CRISPR-Cas9 knockout in rice. FAH1 Gene.

[0011] Preferably, it involves knocking out the CRISPR-Cas9 knockout in rice. FAH1 Genes, Acquisition FAH1 A mutant strain with a 3bp deletion of the TAA protein in the second exon of the gene enhances rice resistance to rice blast. This mutant strain mutates the OsFAH1 protein (SEQ ID NO. 2) to the protein shown in SEQ ID NO. 3.

[0012] The second objective of this invention is to provide a method for improving rice resistance to rice blast, characterized by knocking out [a specific pathogen] in rice. FAH1 Genetic mutant strains were obtained to improve rice resistance to rice blast.

[0013] Preferably, it involves knocking out the CRISPR-Cas9 knockout in rice. FAH1 Gene.

[0014] Preferably, it involves knocking out the CRISPR-Cas9 knockout in rice. FAH1 Genes, Acquisition FAH1 Mutants that delete 3bp-TAA in the second exon of the gene enhance rice resistance to rice blast.

[0015] Preferably, the rice can be any variety of rice, such as the japonica rice variety Kitaake.

[0016] Preferably, the knockout vector is pRGEB32.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] This invention constructs FAH1 The gene was placed into a CRISPR vector and transformed into the japonica rice variety Kitaake, resulting in a transgenic line. FAH1 A 3bp deletion (TAA) in the second exon of the gene significantly enhances its resistance to rice blast fungus compared to wild-type materials. This invention provides a basis for the key molecular genetic mechanism of rice resistance to rice blast, and has important guiding significance for breeding resistant rice varieties and improving rice quality. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the OsFAH1 gRNA editing site and the CRISPR vector. In diagram a, we see the vector assembly diagram; in diagram b, we see the OsFAH1 gene structure, with the gRNA design site marked in red.

[0020] Figure 2 yes fah1 Phenotypic results of rice before and after treatment with rice blast fungus, relative to wild-type rice; where Figures a and b represent wild-type Kitaake and mutant materials, respectively. fah1 Phenotypic analysis (a) and diseased area percentage (b) of T0 generation materials after 14 days of treatment with rice blast fungus; Figures c and d show the results of wild-type Kitaake and mutant materials, respectively. fah1 Phenotypic analysis (c) and diseased area percentage (d) of T1 generation materials 14 days after treatment with rice blast fungus. Detailed Implementation

[0021] The present invention will be further illustrated below with reference to specific embodiments, but these embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise specified, the reagents and materials used in the following embodiments are commercially available.

[0022] Example 1: Rice FAH1 Phenotypic Analysis of Gene Mutant Materials Against Rice Blast Fungus

[0023] one, FAH1 The construction of CRISPR-Cas9 knockout vectors and the identification of related genetic transformation materials are detailed below, including the experimental methods and results:

[0024] 1. Experimental Methods

[0025] 1) The applicant constructed a CRISPR knockout vector for the OsFAH1 gene:

[0026] The CRISPR vector construction method is derived from the article "Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system." (Xie K, Minkenberg B, Yang Y. Boosting CRISPR / Cas9 multiplex editing capability with the endogenous tRNA-processing system. Proc Natl Acad Sci US A. 2015 Mar 17;112(11):3570-5.)

[0027] Unless otherwise specified, the primer sequences used in the following steps are the same as those in the published article:

[0028] (1) Primer design:

[0029] With the assistance of the online website CRISPR-P 2.0, a guide RNA for genome editing was selected, and relevant primers were designed. Finally, a CRISPR-Cas9 targeting sequence was designed on the second exon of the FAH1 gene: GGCCCCTCCTTGCTAACAATTGG, with a low off-target rate. The gRNA scaffold sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC was then appended after the target site.

[0030] Primers designed with the target as a reference are shown below:

[0031] FAH1-F: 5'-TAGGTCTCCCTTGCTAACAATgttttagagctagaa-3'

[0032] FAH1-R: 5'-TCGGGTCTCACAAGGAGGGGCCtgcaccagccggg-3'

[0033] The targeting sequence was cloned into the pRGEB32 CRISPR / Cas9 vector (provided by Huazhong Agricultural University, commercially available, product name is pRGEB32), and the targeting design was as follows. Figure 1 As shown in a.

[0034] (2) One-round PCR: Primer pairing was L5AD5-F and FAH1-R, and FAH1-F and L3AD5-R (two PCRs were performed respectively, yielding two products). PCR amplification was performed using gRNA (GGCCCCTCCTTGCTAACAATTGG GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC) as a template. Reaction conditions were: 95℃ pre-denaturation for 3 min; 35 cycles of 95℃ for 30 sec, 55℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0035] L5AD5-F: CGGGTCTCAGGCA GGATGGGCAGTCTGGGCA ACAAAGCACCAGTGG

[0036] L3AD5-R: TAGGTCTCCAAAC GGATGAGCGACAGCAAAC AAAAAAAAAAGCACCGACTCG

[0037] (3) GG reaction: After purifying the PCR product obtained by amplification, a GG reaction was performed (3ul of product (12.5-25ng); 5ul of 2*T7 ligase buffer (NEB); 1ul of BSA; 0.25ul of BsaI (10U / ul, NEB); 0.25ul of T7 DNA ligase). The reaction conditions were: 37℃ for 5min, 25℃ for 10min, 35 cycles; 20℃ for 1h.

[0038] (4) GG PCR: The reaction product was diluted with 190ul of double-distilled water and then the target fragment was amplified using S5AD5-F and S3AD3-R. The PCR product was then purified.

[0039] S5AD5-F:CGGGTCTCAGGCA GGATGGGCAGTCTGGGCA

[0040] S3AD3-R: TAGGTCTCCAAAC GGATGAGCGACAGCAAAC

[0041] (5) Enzyme digestion and ligation: The fragment (PCR product from step (4)) and pRGEB32 vector were digested with FOKI and BSAI, respectively, and reacted at 37°C for 1 h (fragment) and 37°C for 5 h (vector). After digestion, the digested fragment (digested PCR product) containing the target site and gRNAscaffold was ligated with the digested pRGEB32 vector, and then transformed into E. coli DH5α. Positive clones were screened by enzyme digestion, and the obtained recombinant CRISPR vector was named KOFAH1 ( Figure 1 a).

[0042] 2) Transformation of plasmid vectors and genotyping of transgenic plants

[0043] (1) The obtained vector was subsequently transformed into Kitaake rice using Agrobacterium-mediated transformation. The transformation was carried out at Wuhan Aidijing Biotechnology Co., Ltd., and stable plants were obtained through genetic transformation, thus obtaining the knockout mutant material. fah1 (2) Extraction of knockout mutant material using the CTAB method. fah1 Total DNA from plant leaves was detected by PCR using specific primers (detection primers). The target fragment size was 444 bp. The PCR product was then subjected to Sanger sequencing (by Wuhan Tianyi Huayu Gene Technology Co., Ltd.) to identify the editing sites in positive transgenic plants.

[0044] The PCR detection primers are as follows:

[0045] SeqFAH1F:CGTTTCACCGCTGTATAATGA

[0046] SeqFAH1R:CTTCTGAAAGTGTGTGTCCC.

[0047] The extracted leaf DNA was subjected to PCR (50 μL system: 1 μL DNA; 2 μL SeqFAH1F; 2 μL SeqFAH1R; 2×green Taq mix (Vazyme); 30 μL double-distilled water). The reaction conditions were: 95℃ pre-denaturation for 3 min; 30 cycles of 95℃ for 30 sec, 54℃ for 30 sec, and 72℃ for 30 sec; extension at 72℃ for 5 min.

[0048] 2. Experimental Results

[0049] like Figure 1 As shown in b, CRISPR-Cas9 technology was used to obtain [the desired result]. FAH1 The gene (with a 3bp deletion (TAA) in the second exon) has a knockout effect. fah1 Plant.

[0050] two, fah1 The phenotypic analysis of plant resistance to rice blast fungus, including specific experimental methods and results, is as follows:

[0051] 1. Experimental Methods

[0052] (1) Put rice seeds ( fah1 The plants (including Kitaake wild-type rice) were soaked in water until they germinated, about 2-3 days. Then they were transplanted into plastic pots (14 cm in diameter and 15 cm in height), with 15 plants per pot. They were placed in a greenhouse with 14 hours of light / 10 hours of darkness, 80% relative humidity, and a temperature of 28°C for about 10 days. Then they were transplanted into blue pots and subjected to rice blast stress treatment when they reached the tillering stage.

[0053] (2) The rice blast fungus race is EA18, and there are relevant literature reports on it: Genome Sequence of Magnaporthe oryzae EA18 Virulent to Multiple Widely Used Rice Varieties (WangY, Yang L, Ma C, et al. Genome Sequence of Magnaporthe oryzae EA18 Virulentto Multiple Widely Used Rice Varieties. Mol Plant Microbe Interact. 2022 Aug;35(8):727-730.). EA18 strains were picked and inoculated onto tomato oat medium (OTA) plates and cultured at 28℃. After the mycelium on the plate had grown to a full extent (about 7 days), puncture inoculation treatment could be performed. Select healthy rice plants at the tillering stage. Use a punch to make a ring around the outermost edge of an EA18 agar plate. Create a potential circular wound on a leaf about 7-8 cm from the leaf tip using a mouse-ear punch. Use tweezers to pick up the mycelium cake and place it against the wound, sealing it with transparent tape. Transfer the inoculated plants to a greenhouse and continue growing under high humidity conditions (28°C). Investigate disease incidence 14 days later. Photograph diseased leaves, calculate lesion area using ImageJ, and compare with the control group to identify the resistance of the edited strains. Visualize the inoculation results using GraphPad software.

[0054] 2. Experimental Results

[0055] fah1 Phenotypic results of rice plants compared to wild-type rice Kitaake after treatment with rice blast fungus EA18 are as follows: Figure 2 As shown, it can be seen that compared to wild-type rice plants, fah1 The T0 generation material showed significantly enhanced resistance to rice blast fungus. Figure 2 a), while the percentage of rice blast diseased area measured by ImageJ software ( Figure 2 b) Statistical data also support this phenotypic result. Similarly, the results for the T1 generation mutant materials are consistent with those for the T0 generation materials. Figure 2 (c, d). These results indicate that FAH1 negatively regulates the rice response to blast fungus stress.

[0056] The above-described embodiments are preferred embodiments for ease of understanding of the present invention. However, the present invention is not limited to the above embodiments, that is, it does not mean that the present invention must rely on the above embodiments to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions of the raw materials used in the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0057] SEQ ID NO.1 ( FAH1 (nucleotide sequence)

[0058] ATGGTTGCAGAGGCCTTTACAGTAGATTTGAATGAACCTCTTGTATTTCAGGTTGGACAT

[0059] TTAGGTGAGCAGTATCAGGAATGGGTTCACCAACCAATTGTTAGCAAGGAGGGGCCAAGACTTTTTGCCAACGATGTCTTAGAGTTCTTAACACGCACGGAATGGTGGGCAATTCCTCTTATCTGGTTGCCTGTTGTATGTTGGTGCCTGACTAAATCAGTCGA AATGGGACACACACTTTCAGAAGTAGCTCTGATGGTTGTGTTTGGAATATGCCTATGGACATTGATCGAGTATATAATGCATCGATTCCTGTTCCACATAAATACTAAAAGTTACTGGACAAACACAGCACATTACCTTCTTCATGGAATTCATCACAAGCACC CGACTGACGGCCTTCGACTTGTCTTTCACCAGCAGCTGCAGCTATCTTGTGCTTTCCGTTCTGGAATCTAATCAGGCTAATTACTACTCCGACTACAACTCACGGCGTTGTTTGGAGGCGGCCTGTTGGGTTATGTGATGTATGACTGCACACACTACTATCTG CATCATGGGCAGCCGTCATCTGATCCAGGAAAACACCTCAAGAAATACCATCTGAACCATCACTTCAGAATCCAAAACAAGGGGTTCGGAATAACATCGACGCTGTGGGATCATGTGTTTGGTACATTGCCCTCGACGAAAACCATCGACAAGAAGAGCTCTTGA

[0060] SEQ ID NO.2 ( FAH1 amino acid sequence)

[0061] MVAEAFTVDLNEPLVFQVGHLGEQYQEWVHQPIVSKEGPRLFANDVLEFLTRTEWWAIPLIWLPVVCWCLTKSVEMGHTLSEVALMVVFGICLWTLIEYIMHRFLFHINTKSYWTNTAHYLLHGIHHKHPTDGLRLVFPPAAAAILCFPFWNLIRLITTPTTTHGVFGGGLLGYVMYDCTHYYLHHGQPSSDPGKHLKKYHLNHHFRIQNKGFGITSTLWDHVFGTLPSTKTIDKKSS

[0062] SEQ ID NO.3 ( fah1 amino acid sequence)

[0063] MVAEAFTVDLNEPLVFQVGHLGEQYQEWVHQPIGKEGPRLFANDVLEFLTRTEWWAIPLIWLPVVCWCLTKSVEMGHTLSEVALMVVFGICLWTLIEYIMHRFLFHINTKSYWTNTAHYLLHGIHHKHPTDGLRLVFPPAAAAILCFPFWNLIRLITTPTTTHGVFGGGLLGYVMYDCTHYYLHHGQPSSDPGKHLKKYHLNHHFRIQNKGFGITSTLWDHVFGTLPSTKTIDKKSS

Claims

1. FAH1 The use of a gene in improving rice resistance to rice blast, characterized in that, The FAH1 The nucleotide sequence of the gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, characterized in that, The FAH1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.

2.

3. Use according to claim 1, characterized in that, The method for improving the resistance of rice to rice blast is by knocking out the gene in rice to improve the resistance of rice to rice blast. FAH1 The method for improving the resistance of rice to rice blast is by knocking out the gene in rice to improve the resistance of rice to rice blast.

4. Use according to claim 3, characterized in that, FAH1 5. Use according to claim 4, characterized in that, By knocking out rice with CRISPR-Cas9 FAH1 Genes, Acquisition FAH1 Mutants that delete 3bp-TAA in the second exon of the gene enhance rice resistance to rice blast.

6. A method for improving resistance to rice blast in rice, the method comprising introducing into a rice plant a nucleic acid molecule encoding a protein having the amino acid sequence of SEQ ID NO:

2. is a knock-out of the rice FAH1 gene to increase rice resistance to rice blast.

7. The method of claim 6, wherein, are genes knocked out in rice by CRISPR-cas9 FAH1 .

8. The method of claim 7, wherein, By knocking out rice with CRISPR-Cas9 FAH1 Genes, Acquisition FAH1 Mutants that delete 3bp-TAA in the second exon of the gene enhance rice resistance to rice blast.

9. The method of claim 6, wherein, FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1 FAH1

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