Rice defense gene LOCOs02g41680 and application thereof in bacterial blight resistance

Abstract

The invention provides a rice defense gene LOCOs02g41680 and application of the rice defense gene LOCOs02g41680 in bacterial leaf blight resistance, and relates to the technical field of modern biotechnology gene engineering. The rice defense gene LOCOs02g41680 disclosed by the invention has a nucleotide sequence as shown in SEQ ID NO.1, and consists of 2643 nucleotides; 2, the sequence length of the coding region sequence is 2142 nucleotides, and the coding region sequence is shown in SEQ ID NO. A protein with 713 amino acids is coded, and the amino acid sequence is shown in SEQ ID NO.3. The rice defense gene LOCOs02g41680 disclosed by the invention can be used for creating a rice disease-resistant material for resisting bacterial blight, the disease resistance of the rice can be improved when the rice defense gene LOCOs02g41680 is applied to the rice, and valuable gene resources can be provided for breeding new varieties of the rice for resisting the bacterial blight.

Description

Technical Field

[0001] The invention belongs to the field of genetic engineering, and specifically relates to a rice defense gene LOC_Os02g41680 and an application thereof in bacterial blight resistance. Background Art

[0002] Rice is the staple food for more than half of the world's population, but rice yields often decline or even fail due to infection by many pathogens. Rice bacterial blight is one of the most common diseases in rice production worldwide. It is a bacterial disease caused by Xanthomonas oryzae pv.oryzae (Xoo), which seriously affects the growth and yield of rice.

[0003] Cultivating and applying new broad-spectrum disease-resistant rice varieties is the most economical and effective means of prevention and control. The premise of cultivating new disease-resistant rice varieties is to dig out more genes related to rice disease resistance. Therefore, continuously digging out new genes related to broad-spectrum disease resistance of rice, revealing the regulatory mechanism of rice disease resistance from the molecular level, and creating related mutants will provide useful resources for rice disease resistance research and even breeding work. The present invention is based on the inventor's research and discovery that the rice LOC_Os02g41680 gene strongly responds to the infection of the rice pathogen Xoo and upregulates expression. At present, there is no research report on the relationship between the LOC_Os02g41680 gene and rice disease resistance. If used improperly, the rice bacterial blight resistance gene is easily overcome by the bacterial blight fungus, resulting in serious yield reduction, which requires finding more new bacterial blight resistance genes. Therefore, the present invention will provide new effective bacterial blight resistance genes for rice. Summary of the invention

[0004] Aiming at providing new gene resources for disease-resistant and high-yield rice breeding, the first object of the present invention is to provide a rice defense gene LOC_Os02g41680, which can enhance the ability of rice to resist bacterial blight. The second object of the present invention is to provide an application of the rice defense gene LOC_Os02g41680.

[0005] To achieve the above object, the technical solution of the present invention is as follows:

[0006] The rice defense gene LOC_Os02g41680 of the present invention has a nucleotide sequence as shown in SEQ ID NO.1; the rice defense gene LOC_Os02g41680 has a coding region sequence as shown in SEQ ID NO.2; and the encoded protein of the rice defense gene LOC_Os02g41680 has an amino acid sequence as shown in SEQ ID NO.3.

[0007] The second object of the present invention is to provide application of rice defense gene LOC_Os02g41680 in disease resistance.

[0008] Preferably, the disease resistance described in the present invention is resistance of rice to bacterial blight.

[0009] The application includes a recombinant expression vector, an expression box, a transgenic cell line or a genetically engineered bacterium of the rice LOC_Os02g41680 gene and a protein encoded by the gene.

[0010] Preferably, the rice LOC_Os02g41680 gene, and its recombinant expression vector, expression cassette, transgenic cell line or genetically engineered bacteria and gene-encoded protein are used in any one or more of the following (A1)-(A5):

[0011] (A1) Application in regulating rice resistance to bacterial blight;

[0012] (A2) Use in the preparation of a product for regulating resistance of rice to bacterial blight;

[0013] (A3) Application in breeding rice with high resistance to bacterial blight;

[0014] (A4) Use in the preparation of a product having high resistance to bacterial blight;

[0015] (A5) Application in breeding of rice with high resistance to bacterial blight;

[0016] Wherein, the regulation comprises the following (B1) or (B2):

[0017] (B1) improving the resistance of rice to bacterial blight by up-regulating the expression level of the LOC_Os02g41680 gene, or up-regulating the content or activity of the protein encoded by the LOC_Os02g41680 gene;

[0018] (B2) Reducing the resistance of rice to bacterial blight pathogen by inhibiting the expression level of the LOC_Os02g41680 gene, or inhibiting the content or activity of the protein encoded by the LOC_Os02g41680 gene.

[0019] Preferably, in the application of breeding rice with high resistance to bacterial blight, rice strains resistant to bacterial blight are obtained by screening rice plants that efficiently express the rice LOC_Os02g41680 gene.

[0020] The present invention provides a method for regulating rice resistance to bacterial blight. Knocking out the LOC_Os02g41680 gene in the rice can reduce the resistance of the rice to bacterial blight; overexpressing the LOC_Os02g41680 gene in the rice can increase the resistance of the rice to bacterial blight.

[0021] The invention provides a method for creating transgenic rice resistant to bacterial blight, wherein the rice LOC_Os02g41680 gene is transferred into rice plants to obtain transgenic rice that efficiently expresses the rice LOC_Os02g41680 gene.

[0022] Preferably, the coding region sequence of the rice LOC_Os02g41680 gene is cloned into a plant expression vector, first transferred into Agrobacterium, and then transferred into rice cells through rice callus transformation to obtain transgenic rice that efficiently expresses the rice LOC_Os02g41680 gene.

[0023] Beneficial Effects of the Invention

[0024] The present invention found that the rice LOC_Os02g41680 gene plays an important role in the resistance of rice to bacterial blight. After knocking out the LOC_Os02g41680 gene, the rice reduced its disease resistance to bacterial blight; while overexpressing the LOC_Os02g41680 gene increased the disease resistance of rice to bacterial blight. Overexpression of the LOC_Os02g41680 gene can improve the disease resistance of rice to bacterial blight, so overexpression of the rice LOC_Os02g41680 gene can be used to improve the resistance of rice to bacterial blight. In terms of agronomic traits, the rice strains overexpressing the LOC_Os02g41680 gene are taller and have fewer tillers than the wild type, and there are no significant differences in seed size and thousand-grain weight, so they can be used as germplasm resources and intermediate materials for disease-resistant breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The figures show the resistance phenotypes of rice LOC_Os02g41680 gene knockout mutants (including three strains KO 27-1, KO 27-2 and KO 27-3) and overexpression plants (including three strains OX 2-7, OX 2-8 and OX 2-9) to bacterial blight; A: lesion images of leaf clippings of transgenic materials grown for two weeks 12 days after inoculation, with a scale of 1 cm; B: statistics of lesion lengths of wild-type materials and mutant materials.

[0026] Figure 2 These are pictures of rice LOC_Os02g41680 gene knockout mutants, overexpression plants and wild-type Nipponbare plants at the mature stage; A: wild-type Nipponbare; B: rice LOC_Os02g41680 gene knockout mutant; C: rice LOC_Os02g41680 gene overexpression plant.

[0027] Figure 3Agronomic traits of rice LOC_Os02g41680 gene knockout mutant, overexpression plants and wild-type Nipponbare plants; A: seed size; B: seed length, seed width and seed thickness (n=10); C: plant height (cm); D: number of tillers; E: 1000-grain weight (g). DETAILED DESCRIPTION

[0028] In order to illustrate the present invention in detail, the following examples will help those skilled in the art to further understand the present invention, but are not intended to limit the present invention in any form. It should be noted that the embodiments are preferred embodiments of the present invention and are intended to illustrate the implementation conditions that can be used to implement the present invention, rather than to limit the experimental conditions.

[0029] The technical solution in the embodiment of the present invention is described below in conjunction with the drawings of the specification. Here, the coding region sequence of rice LOC_Os02g41680 gene (SEQ ID NO.2) is used as an example to illustrate the method for creating transgenic rice, which is applicable to creating transgenic rice using the nucleotide sequence of rice LOC_Os02g41680 gene (SEQ ID NO.1).

[0030] The present invention provides a rice defense gene LOC_Os02g41680, the coding region sequence of the rice defense gene LOC_Os02g41680 is shown in SEQ ID NO. 2. The sequencing results show that the CDS sequence contains a stop codon of 2142 bp in length, encoding a protein of 713 amino acids. Studies have found that overexpression of the LOC_Os02g41680 gene can improve the ability of rice to resist bacterial blight ( Figure 1 A).

[0031] The present invention provides a recombinant expression vector pRHVcHA-LOC_Os02g41680 containing the rice defense gene.

[0032] The present invention provides a recombinant engineering bacterium containing the rice defense gene LOC_Os02g41680.

[0033] The present invention also proposes the use of the rice defense gene LOC_Os02g41680 in improving the resistance of rice to bacterial blight.

[0034] Further, in a preferred embodiment of the present invention, the application as described above comprises the following steps:

[0035] The coding region sequence of the rice LOC_Os02g41680 gene was cloned into the pRHVcHA vector to obtain a recombinant expression vector;

[0036] After the recombinant expression vector is transformed into Agrobacterium, the transformed Agrobacterium is used to infect and transform rice callus tissue to further obtain transgenic rice plants.

[0037] Furthermore, in a preferred embodiment of the present invention, the variety of rice is Nipponbare.

[0038] The LOC_Os02g41680 gene of the present invention can also be used to transform other varieties of rice to obtain transgenic rice plants with resistance to bacterial blight, which is not specifically limited in the present invention.

[0039] The present invention uses Agrobacterium transformation to transform Nipponbare to obtain transgenic rice plants. By studying the disease resistance of transgenic rice plants and wild-type Nipponbare rice, it can be seen that the LOC_Os02g41680 gene can improve the disease resistance of rice, especially the resistance to bacterial blight, and its application in the field of genetic engineering has great potential economic value and application prospects.

[0040] The features and performance of the present invention are further described in detail below in conjunction with specific embodiments.

[0041] Example 1: Obtaining rice LOC_Os02g41680 gene knockout mutant material.

[0042] The amino acid sequence of rice LOC_Os02g41680 protein is shown in SEQ ID NO. 3, and the coding sequence (CDS sequence) of rice LOC_Os02g41680 gene is shown in SEQ ID NO. 2. The present invention uses CRISPR / Cas9 technology to knock out the target gene in a targeted manner, thereby obtaining a knockout rice mutant of the LOC_Os02g41680 gene.

[0043] The steps for constructing the knockout vector are as follows:

[0044] (1) First, based on the sequence of the LOC_Os02g41680 gene, determine the target site and design the adapter primer LOC_Os02g41680-U6F / R. The primer sequence is as follows:

[0045] LOC_Os02g41680-U6F:

[0046] TCCATTTCAGTACCAGAAGAgttttagagctagaaatagcaagtta

[0047] LOC_Os02g41680-U6R:

[0048] TCTTCTGGTACTGAAATGGAaacctgagcctcagcgcagc

[0049] OsU6P-F:GTCGTTTCCCGCCTTCAGTTtatgtacagcattacgtagg

[0050] OsU6T-R:CTGTCAAACACTGATAGTTTAAACgatggtgcttactgtttag

[0051] (2) Vector plasmid extraction and enzyme digestion

[0052] The plasmid was extracted using a kit, and the TKC-Ruby vector was digested with restriction endonuclease Pme I. The digestion system was as follows:

[0053]

[0054] (3) gRNA expression cassette amplification (two rounds of nested PCR amplification):

[0055] The first round of PCR was performed in two reactions, both using the constructed vector with U6 promoter and terminator as template, and the primers used were combined as follows.

[0056] PCR1:OsU6P-F+LOC_Os02g41680-U6R

[0057] PCR2: OsU6T-R+LOC_Os02g41680-U6F

[0058] The PCR reaction system is as follows:

[0059]

[0060] The PCR reaction conditions were:

[0061]

[0062] In the second round of PCR, the products of PCR1 and PCR2 were recovered by gel recovery, and then 0.5 μL of each was taken as a template, and OsU6P-F+OsU6T-R was used as primers for amplification. The second round of PCR amplification reaction system was as follows:

[0063]

[0064] The second round of PCR amplification reaction conditions are as follows:

[0065]

[0066] (4) Construction of recombinant plasmid

[0067] The recovered gRNA expression cassette was connected to the TKC-Ruby vector that had been completely digested by Pme I using Gibson Assembly, and the reagent used was a single fragment homologous recombination enzyme from Novozymes. The reaction system was as follows:

[0068]

[0069] The reaction conditions were: 37°C for 30 min.

[0070] The ligated plasmid was transformed into E. coli using the heat shock method, and the positive clones were selected for detection. After the sequencing was correct, the plasmid was used to transform rice callus tissue, and the rice LOC_Os02g41680 gene knockout mutant was obtained using the Nipponbare strain as the background.

[0071] Example 2: Obtaining rice LOC_Os02g41680 gene overexpression material.

[0072] (1) Design a pair of specific primers based on the sequence information of the LOC_Os02g41680 gene:

[0073] LOC_Os02g41680-F tccccgggtgagctcggtaccATGCAGCAGCAGCAGAAGAAG

[0074] LOC_Os02g41680-R cgcactagtaagcttggtaccTTTGCACCATCCAAAGCAGG

[0075] (2) extracting total DNA from rice Nipponbare leaves as a PCR reaction template, performing PCR amplification with the above primers, and cloning and sequencing the PCR products, specifically comprising the following steps:

[0076] The plant genomic DNA was extracted using the CATB (cetyltrimethylammonium bromide) method. PCR was performed using LOC_Os02g41680-F and LOC_Os02g41680-R as primers, and the PCR products were sequenced. The PCR reaction system was as follows:

[0077]

[0078] The PCR reaction conditions were:

[0079]

[0080] The sequencing results showed that the CDS of rice LOC_Os02g41680 gene was 2142bp long and encoded a protein of 713 amino acids.

[0081] (3) Connecting the obtained LOC_Os02g41680 gene to the pRHVcHA vector comprises the following steps:

[0082] The sequence amplified by LOC_Os02g41680-F and LOC_Os02g41680-R primers was connected to the pRHVcHA vector backbone after double digestion with NotI and KpnI using the Novozyme recombination kit, and after screening on kanamycin plates, colony PCR identification and sequence determination were performed. The recombinant plasmid with the correct sequence was used for rice callus transformation, and rice LOC_Os02g41680 gene overexpression plants were obtained with the Nipponbare strain as the background.

[0083] Example 3 Identification of resistance to bacterial blight in transgenic plants

[0084] (1) Rice seedling cultivation: Soak rice seeds in prochloraz for 24 hours and then rinse with clean water. Soak the seeds in clean water for 2-3 days, changing the clean water every 12 hours. Plant the rice seedlings in the rice seedling cultivation medium after the rice turns white. After two weeks, move the rice to flooded soil that has been fertilized with basal fertilizer. Fertilizers are urea and potassium dihydrogen phosphate, 0.5 grams each per pot.

[0085] (2) Pathogen infection: For seedlings at the three- to four-leaf stage, the leaf clipping inoculation method was used to suspend Xoo in NA medium in 10 mM sterilized MgCl2 solution (pH = 7.0) until the OD 600 = 0.5, dipped in Xoo suspension with sterilized scissors, cut the leaf tip (about 2-3 cm) from the leaf. After shading for 24 hours, grow under the conditions of 28-32℃ (light, 12 hours), 28-32℃ (dark, 12 hours), and 90% relative humidity, and measure the lesion size after 12 days.

[0086] (3) Disease resistance phenotype determination: Figure 1 Figure A shows a comparison of leaves of wild-type Nipponbare and transgenic Nipponbare rice 12 days after inoculation with Xoo. Figure 1 It can be seen that 12 days after inoculation with Xoo pathogen, the length of the lesions on the knockout rice leaves (KO-LOC_Os02g41680) was significantly greater than that on the wild-type Nipponbare leaves, while the lesions on the overexpression rice leaves (OX-LOC_Os02g41680) were significantly smaller than those on the wild-type Nipponbare leaves.

[0087] In addition, rice lines overexpressing the LOC_Os02g41680 gene in agronomic traits ( Figure 2 C), taller plant height and fewer tillers than the wild type, with no significant differences in seed size, seed length, seed width, seed thickness, and thousand-grain weight ( Figure 3), therefore, the present invention can be used as rice germplasm resources and intermediate materials for disease-resistant rice breeding.

[0088] The above is only the optimal implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be understood and thought of by any person familiar with the technical field within the technical scope disclosed by the present invention should be included in the scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the claims.

[0089] Sequence Listing:

[0090]

[0091]

[0092] The protein sequence encoded by a rice defense gene LOC_Os02g41680 consists of 713 amino acids, and its amino acid sequence is as shown in SEQ ID NO.3 (MECENGRVSANGMSGLCVAAPRADPLNWGKATEEMTGSHLDEVKRMVAEYRQPLVKIEGASLR IAQVAAVAAAGEARVELDESARERVKASSDWVMNSMMNGTDSYGVTTGFGATSHRRTKEGGALQRELIRFLNAGAFGTGTDGHVLPAEATRAAMLVRINTLLQGYSGIRFEILEAIAKLLNANVTPCLPLRGTITASGDLVPLSYIAGLVTGRENAVAVAPDGSKVNAAEAFKIAGIQGGFFELQPKEGLAMVNGTAVGSGLASTVLFEANILAILAEVLSAVFCEVMNGKPEYTDHLTHKLKHHPGQIEAAAIMEHILEGSSYMKHAKKLGELDPLMKPKQDRYALRTSPQWLGPQIEVIRAATKSIEREINSVNDNPLIDVSRGKALHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLPSNLSGGRNPSLDYGFKGAEIAMASYCSELQFLGNPVTNHVQSAEQHNQDVNSLGLISSRKTDEAIDILKLMSSTFLIALCQAVDLRHIEENVKSAVKSCVMTVAKKTLSTNSTGDLHVARFCEKDLLKEIDREAVFAYADDPCSHNYPLMKKLRNVLVERALANGAAEFNADTSVFAKVAQFEEELRATLPGAIEAARAAVENGTAAIPSRITECRSYPLYRFVREELGTKYLTGEKTRSPGEELNKVLVAINEGKHIDPLLECLKEWNGEPLPIC).

Claims

1. Rice defense gene LOC_Os02g41680, whose nucleotide sequence is shown in SEQ ID NO.

1.

2. The rice gene LOC_Os02g41680 according to claim 1, characterized in that: The coding region sequence of the gene LOC_Os02g41680 is shown in SEQ ID NO.

2.

3. The rice gene LOC_Os02g41680 according to claim 1, characterized in that: The amino acid sequence of the protein encoded by the gene LOC_Os02g41680 is shown in SEQ ID NO.

3.

4. Use of the rice LOC_Os02g41680 gene according to any one of claims 1 to 3 in rice disease resistance.

5. The use according to claim 4, characterized in that: The application includes a recombinant expression vector, an expression box, a transgenic cell line or a genetically engineered bacterium of the rice LOC_Os02g41680 gene and a protein encoded by the gene.

6. The use according to claim 4 or 5, characterized in that: Including any one or more of the following applications in (A1)-(A5): (A1) Application in regulating rice resistance to bacterial blight; (A2) Use in the preparation of a product for regulating resistance of rice to bacterial blight; (A3) Application in breeding rice with high resistance to bacterial blight; (A4) Application in breeding products of rice with high resistance to bacterial blight; (A5) Application in breeding of rice with high resistance to bacterial blight; Wherein, the regulation is as follows (B1) or (B2): (B1) improving the resistance of rice to bacterial blight by up-regulating the expression level of the LOC_Os02g41680 gene, or up-regulating the content or activity of the protein encoded by the LOC_Os02g41680 gene; (B2) Reducing the resistance of rice to bacterial blight by inhibiting the expression level of the LOC_Os02g41680 gene, or inhibiting the content or activity of the protein encoded by the LOC_Os02g41680 gene.

7. The use according to claim 6, characterized in that: In the above (B1), the LOC_Os02g41680 gene in rice is overexpressed to improve the resistance of rice to bacterial blight; in the above (B2), the LOC_Os02g41680 gene in rice is knocked out to reduce the resistance of rice to bacterial blight.

8. A method for regulating rice resistance to bacterial blight, characterized in that: Knocking out the rice LOC_Os02g41680 gene reduces the resistance of rice to bacterial blight; overexpressing the rice LOC_Os02g41680 gene increases the resistance of rice to bacterial blight.

9. A method for constructing transgenic rice resistant to bacterial blight, characterized in that: The rice LOC_Os02g41680 gene is transferred into rice plants to obtain transgenic rice expressing the rice LOC_Os02g41680 gene at a high level.

10. The method for constructing a transgenic rice resistant to bacterial blight according to claim 9, characterized in that: The coding sequence of rice LOC_Os02g41680 gene was cloned into a plant expression vector, first transferred into Agrobacterium, and then transferred into rice cells through callus transformation to obtain transgenic rice that efficiently expressed rice LOC_Os02g41680 gene.

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