Application of OsHIR1 gene in improving resistance of rice to small brown planthopper

Abstract

The application discloses OsHIR1 Application of a gene in improving resistance of rice to a white-backed planthopper, and belongs to the technical field of agricultural biological gene engineering, OsHIR1 Application of a gene in improving resistance of rice to a white-backed planthopper, and the application OsHIR1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1. And a method for improving resistance of rice to a white-backed planthopper by using the gene comprises the following steps: OsHIR1 S1, cloning a rice OsHIR1 gene; S2, constructing OsHIR1 OsHIR1 an overexpression vector; S3, transforming the overexpression vector obtained in S2 into rice to obtain a transgenic plant with improved resistance of rice to a white-backed planthopper. The application provides a new selection for improving the resistance of rice to a white-backed planthopper, effectively improves the resistance of rice to a white-backed planthopper, and has great application value for breeding of rice resistant to a white-backed planthopper.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of agricultural bioengineering technology, and particularly relates to OsHIR1 Application of a gene in improving the resistance of rice to the white-backed planthopper. BACKGROUND

[0002] Rice is subjected to severe biological stress throughout its growth cycle and subsequent storage process, and among them, pest is one of the key factors restricting the yield and quality of rice.

[0003] In the plant stress resistance regulation network, plants can recognize pathogens through various mechanisms to trigger plant defense. One of the strongest responses of plant immunity is the hypersensitive response (HR), which can induce programmed cell death in plants. When certain parts of plants are infected by pathogens, the infected cells rapidly undergo programmed death to limit the spread of pathogens. Overexpression of the hypersensitive induced response (HIR) gene can also trigger HR in plants. HIR protein belongs to the proliferation, ion and death (PID) protein superfamily, and most members of the superfamily play an important role in cell proliferation, ion channel regulation and cell death. However, there is no report on HIR protein, especially OsHIR1 Application of a gene in the defense response of rice to the white-backed planthopper. SUMMARY

[0004] The application aims to provide OsHIR1 Application of a gene in improving the resistance of rice to the white-backed planthopper, which provides a new option for improving the resistance of rice to the white-backed planthopper. The application is to integrate the OsHIR1 gene into rice through an expression vector to effectively improve the resistance of rice to the white-backed planthopper, which has great application value for breeding rice resistant to the white-backed planthopper.

[0005] To solve the above technical problems, the technical scheme adopted by the application is as follows:

[0006] OsHIR1 Application of a gene in improving the resistance of rice to the white-backed planthopper, wherein OsHIR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0007] Preferably, the OsHIR1 gene encodes a protein with an amino acid sequence shown in SEQ ID NO. 2.

[0008] Preferably, the resistance of rice to the white-backed planthopper is regulated by adjusting the expression level of the OsHIR1 gene.

[0009] Preferably, a rice mutant sensitive to the white-backed planthopper is obtained by inhibiting the expression of the OsHIR1 gene in rice.

[0010] Preferably, the rice plant is improved in resistance to the white-backed planthopper by increasing the expression level of the gene. OsHIR1 Preferably, the rice plant is improved in resistance to the white-backed planthopper by increasing the expression level of the gene.

[0011] The application also provides a transgenic plant with improved resistance to the white-backed planthopper, wherein the transgenic plant comprises the overexpression vector. OsHIR1 The application also provides a transgenic plant with improved resistance to the white-backed planthopper, wherein the transgenic plant comprises the overexpression vector.

[0012] The application also provides a strain for improving the resistance of rice to the white-backed planthopper, wherein the strain comprises the overexpression vector.

[0013] The application also provides a method for improving the resistance of rice to the white-backed planthopper by using the gene, comprising the following steps: OsHIR1 The application also provides a method for improving the resistance of rice to the white-backed planthopper by using the gene, comprising the following steps:

[0014] S1, cloning the gene of rice; OsHIR1 S1, cloning the gene of rice;

[0015] S2, constructing an overexpression vector; OsHIR1 S2, constructing an overexpression vector;

[0016] S3, transforming the overexpression vector obtained in S2 into rice to obtain a transgenic plant with improved resistance to the white-backed planthopper.

[0017] Preferably, in S1, the cloning of the gene of rice is specifically as follows: OsHIR1 Preferably, in S1, the cloning of the gene of rice is specifically as follows:

[0018] extracting total RNA of rice and reverse transcribing the total RNA into cDNA as a template, and performing PCR amplification by using primers -F and -R to obtain the gene. OsHIR1 The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4. OsHIR1 The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4. OsHIR1 The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4. The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4.

[0019] The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4. OsHIR1 The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4. OsHIR1 The nucleotide sequence of the primer -F is shown in SEQ ID NO. 3, and the nucleotide sequence of the primer -R is shown in SEQ ID NO. 4.

[0020] Compared with the prior art, the application has the following advantages and technical effects:

[0021] The application discloses application of the gene in improving the resistance of rice to the white-backed planthopper. OsHIR1 The application discloses application of the gene in improving the resistance of rice to the white-backed planthopper. OsHIR1 The application discloses application of the gene in improving the resistance of rice to the white-backed planthopper. OsHIR1 The application discloses application of the gene in improving the resistance of rice to the white-backed planthopper. OsHIR1The homozygous plant overexpressing the gene and the mutant homozygous plant sensitive to the white-backed planthopper are obtained by the method. The method is suitable for monocotyledon plants, preferably Gramineae plants, more preferably Oryza plants, and most preferably rice plants. The method can improve the resistance of the plant to the white-backed planthopper and provide a new gene resource for insect-resistant breeding. In conclusion, the application is a potential biological engineering technology for improving the resistance of rice to the white-backed planthopper, which has great value for molecular breeding of rice resistant to the white-backed planthopper, helps to reduce the use of pesticides, improve the yield and quality of rice, and has important significance for sustainable development of agriculture.

[0022] The technical solutions of the application are further described in detail below with reference to the drawings and examples. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The wild type and OsHIR1 The overexpressed rice plant OsHIR1 The transcription level of the gene;

[0024] Figure 2 The wild type and OsHIR1 The target sequence edited by CRISPR / Cas9 in the mutant;

[0025] Figure 3 The overexpressed and mutant plants in Example 3 OsHIR1 The results of the resistance of the overexpressed and mutant plants to the white-backed planthopper, wherein Figure 3 A in the figure is the phenotype of the wild type plant (WT) and the OsHIR1 overexpressed plant OsHIR1-OX1 and OsHIR1- OX2 ) before and after treatment with the white-backed planthopper, OsHIR1 the mutant plant Oshir1-1 . Figure 3 B in the figure is the survival rate statistics of the wild type plant (WT) and the Os HIR1 overexpressed plant OsHIR1-OX1 and OsHIR1-OX2 ) and the mutant plant OsHIR1 after treatment with the white-backed planthopper. Oshir1-1 . DETAILED DESCRIPTION

[0026] The technical solutions of the application are further described in detail below with reference to the drawings and examples.

[0027] Unless otherwise defined, the technical terms or scientific terms used in the application shall have the usual meanings understood by those skilled in the art to which the application pertains.

[0028] Sources of test materials:

[0029] In the application, unless otherwise specified, other test materials and instruments and equipment are conventional test materials in the art and can be purchased through commercial channels.

[0030] Example 1

[0031] OsHIR1 The nucleotide sequence of the gene is shown in SEQ ID NO. 1.

[0032] SEQ ID NO. 1:

[0033] ATGGGTCAAGCACTCGGTTTGGTACAAGTAGATCAATCGACAGTAGCCATCAAGGAGTCCTTTGGGAAGTTTGATGAGGTTCTTGAGCCTGGATGCCACTTTTTACCCTGGTGCATAGGGAAGCAGATCGCTGGATATCTTTCCTTGCGTGTGCAGCAGCTGGATGTCCGTTGTGAAACAAAGACTAAGGACAATGTTTTTGTCAATGTTGTGGCATCTGTGCAATACCGTGCTCTTGCTGAGAAGGCATCAGACGCCTTTTACAGGCTTAGCAACACCAGGGAGCAAATCCAGTCGTATGTTTTTGATGTGATCAGGGCTAGTGTTCCAAAGATGAACTTGGATGATGCATTTGAGCAGAAGAATGAGATCGCAAAAGCTGTGGAAGATGAGCTTGAAAAGGCAATGTCAATGTATGGTTATGAGATTGTGCAAACTCTTATTGTTGATATTGAACCAGATGAGCATGTTAAGAGAGCAATGAATGAAATCAACGCAGCTGCTAGGCTGAGGGTGGCAGCCAATGAGAAAGCTGAAGCAGAGAAGATTCTTCAGATCAAGAGAGCTGAAGGAGATGCAGAATCCAAGTACTTGGCTGGTCTGGGTATCGCAAGGCAGCGTCAGGCTATAGTGGATGGGCTGAGAGACAGTGTTCTTGCCTTCTCCGAGAACGTGCCTGGGACCTCTGCCAAGGATGTCATGGATATGGTTCTGGTTACGCAATACTTCGACACCATGAAGGAGATAGGAGCCTCATCCAAGTCCTCTTCAGTGTTCATCCCTCACGGGCCAGGTGCCGTCAAAGATATCGCTGCGCAGATAAGAGATGGTCAGCTCCAGGCCAAATTGATCTAA.

[0034] OsHIR1 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2.

[0035] SEQ ID NO. 2:

[0036] MGQALGLVQVDQSTVAIKESFGKFDEVLEPGCHFLPWCIGKQIAGYLSLRVQQLDVRCETKTKDNVFVNVVASVQYRALAEKASDAFYRLSNTREQIQSYVFDVIRASVPKMNLDDAFEQKNEIAKAVEDELEKAMSMYGYEIVQTLIVDIEPDEHVKRAMNEINAAARLRVAANEKAEAEKILQIKRAEGDAESKYLAGLGIARQRQAIVDGLRDSVLAFSENVPGTSAKDVMDMVLVTQYFDTMKEIGASSKSSSVFIPHGPGAVKDIAAQIRDGQLQAKLI.

[0037] OsHIR1 Cloning of genes:

[0038] RNA extraction and cDNA synthesis:

[0039] (1) Grind the plant material in liquid nitrogen. Add 1 mL TRIZOL per 50 mg of material (the volume of material should be less than 10% of the volume of TRIZOL) and shake or pipette until there are no large particles.

[0040] (2) Incubate the suspension at 30°C for 5 minutes to completely dissociate the nucleic acid-protein complex. Add 0.2 mL chloroform / mL TRIZOL (initial) and shake vigorously by hand for 15 seconds, then incubate at 30°C for 2-3 minutes. Centrifuge at 12000 rpm, 2°C, for 15 minutes.

[0041] (3) Absorb the colorless supernatant (about 60% of the initial TRIZOL volume), add 0.5 mL isopropanol / mL TRIZOL (initial), and incubate at 30°C for 10 minutes. Centrifuge at 12000 rpm, 2°C, for 10 minutes.

[0042] (4) 75% ethanol wash (>1 mL ethanol / mL TRIZOL), 7500 rpm, 2°C, centrifuge for 5 minutes.

[0043] (5) Dry (briefly, complete drying will reduce the solubility of RNA), add DEPC-treated H2O, 60°C, 10 minutes, and obtain 100 μg total RNA per 100 mg of leaves.

[0044] cDNA synthesis:

[0045] (1) Take the total RNA of rice, pre-denature at 65°C for 5 minutes, and add the corresponding genomic enzyme reaction for 10 minutes to remove genomic DNA.

[0046] (2) The cDNA reaction system of rice was obtained by reverse transcription, as shown in Table 1.

[0047] Table 1 PCR reaction system

[0048]

[0049] Note: PCR reaction program: 37℃ pre-denaturation for 30 minutes.

[0050] OsHIR1 PCR amplification of genes:

[0051] Primer used OsHIR1 -F (SEQ ID NO. 3), OsHIR1 -R (SEQ ID NO. 4) to amplify OsHIR1 genes by PCR.

[0052] PCR amplification program: 94℃ pre-denaturation for 3 minutes; 98℃ denaturation for 15 seconds, 57℃ annealing for 10 seconds, 72℃ extension for 30 seconds (33 cycles); complete extension for 10 minutes; 16℃ preservation.

[0053] SEQ ID NO. 3:

[0054] cttctgcaggagctcggtaccATGGGTCAAGCACTCGGTTTG.

[0055] SEQ ID NO. 4:

[0056] tggagaagaaaccatggatccGATCAATTTGGCCTGGAGCTG.

[0057] OsHIR1 Construction of gene expression vector and transformation of Agrobacterium:

[0058] Construction of overexpression vector:

[0059] The OsHIR1 gene coding sequence DNA fragment was cloned into pUBI- OsHIR1 -GFP vector to obtain a recombinant plasmid, which was confirmed by sequencing.

[0060] Transformation of overexpression vector into Agrobacterium:

[0061] The recombinant vector was transformed into rice by Agrobacterium-mediated method to obtain OsHIR1 gene overexpression lines.

[0062] Screening of transgenic positive homozygous lines:

[0063] The seedlings of T0 generation plants were cultured to 2-leaf stage, and leaf RNA was extracted for detectionFigure 1 The expression levels of [the substance] were compared with those of wild-type (WT) plants, and the results were as follows: OsHIR1 As shown. The primer used is qp. OsHIR1 -F and qp OsHIR1-OX1 -R, select the two lines with the highest expression levels and name them OsHIR1-OX2 and OsHIR1 Among them, qp OsHIR1 The -F nucleotide sequence is shown in SEQ ID NO. 5, qp Figure 1 -R nucleotide sequences are shown in SEQ ID NO.6.

[0064] SEQ ID NO. 5: CCCTGGTGCATAGGGAAGCA.

[0065] SEQ ID NO. 6: CGTCTGATGCCTTCTCAGCAA.

[0066] Depend on OsHIR1 It can be seen that, OsHIR1-OX1 Overexpression plants ( OsHIR1-OX2 and OsHIR1 )middle OsHIR1 Gene expression levels were approximately six times higher than in wild-type plants, indicating that... OsHIR1-OX1 Overexpression plants ( OsHIR1-OX2 and OsHIR1 Successfully constructed.

[0067] T1 and T2 generation seeds were cultured on a medium containing herbicide. When the seedlings reached the 2-leaf stage, homozygous transgenic lines were screened by observing the survival rate after hygromycin treatment. Specifically, seeds were sown in a medium containing hygromycin and cultured until the 2-leaf stage. Seedlings whose growth was not inhibited by hygromycin were identified as transgenic positive plants.

[0068] Example 2

[0069] CRISPR / Cas9 OsHIR1 Vector construction and mutant identification:

[0070] Designed on the CRISPR-GE website (http: / / skl.scau.edu.cn / ). OsHIR1 After knocking out the target site and specific primers, a CRISPR / Cas9 vector named pYLCRISPR / Cas9Pubi was constructed, using primers A1-F and A1-R. Figure 2 CRISPR / Cas9 editing target sequences in mutants, such as Oshir1 As shown, the A1-F nucleotide sequence is shown in SEQ ID NO.7, and the A1-R nucleotide sequence is shown in SEQ ID NO.8.

[0071] Transform CRISPR / Cas9 vector into rice by Agrobacterium-mediated method to obtain mutant lines Oshir1 -1 and OsHIR1 -2, extract mutant plant DNA, perform PCR amplification and PAGE gel electrophoresis. Select samples with obvious differences from wild type bands and send for sequencing to confirm mutations.

[0072] Since the CRISPR / Cas9 technology obtains mutants, generally a few base deletions or insertions, it is difficult to screen using hygromycin and ordinary PCR. First, determine whether bases are inserted or deleted near the target point of the transgenic seedling, then determine the deleted or inserted bases by first-generation sequencing, and the detection method is as follows.

[0073] (1) PCR amplification of 50 bp upstream and downstream of the target point, so that the size of the amplification product is about 100 bp.

[0074] (2) Non-denaturing polyacrylamide gel (Table 2) electrophoresis of the amplification product, 4 hours of electrophoresis, constant voltage 180V.

[0075] (3) Peel the polyacrylamide gel from the glass plate (deionized water wash 2 times, about 5 seconds each time), and use 0.1% silver nitrate solution (209139-25G, Sigma) to stain the DNA with silver nitrate for 15 minutes. After staining, rinse the polyacrylamide gel with deionized water for 15 minutes and develop color.

[0076] (4) Soak the polyacrylamide gel in the developing solution (1% NaOH, 0.2% formaldehyde), and stop staining when there are obvious bands of the DNA band (in a fume hood).

[0077] (5) Compared with the wild type band, the different lines need to be selected, and the 1000 bp size DNA fragment (the target point is in the middle position of the fragment) is amplified by PCR again, and the amplification product is sent to the sequencing company for first-generation sequencing to determine whether the mutant is successfully constructed.

[0078] Table 2 12% non-denaturing polyacrylamide gel

[0079]

[0080] Note: 30% acrylamide: methylene bisacrylamide 19:1 (B546016, Sangon Biotech); N, N, N', N'-tetramethyl ethylenediamine (T9281-25ML, Sigma); ammonium persulfate (A3678, Sigma)

[0081] SEQ ID NO. 7: TCAATCGACAGTAGCCATCAAGG.​

[0082] SEQ ID NO. 8: TGCATAGGGAAGCAGATCGCTGG.

[0083] Example 3

[0084] Oshir1 Phenotypic analysis of rice planthopper resistance: wild-type NIP lines and mutant lines OsHIR1-OX1 -1 and overexpression homozygous lines OsHIR1-OX2 , Figure 3 Using rice seedlings as materials, the seedlings were cultured to the three-leaf stage. Different genotype rice seedlings were covered with planthopper treatment devices, with 120 planthoppers placed in each device. After 6-10 days of treatment, physiological indicators such as fresh weight, chlorophyll content, and plant survival rate were compared among the groups. The results are as follows: Figure 3 As shown.

[0085] Depend on OsHIR1 It can be seen that, OsHIR1 The overexpressing plants were more resistant to planthoppers than the wild type, and ​ The mutant is more susceptible to planthoppers.

[0086] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. OsHIR1 The use of genes in improving resistance of rice to the white-backed planthopper, characterized in that, The OsHIR1 The nucleotide sequence of the gene is shown as SEQ ID NO. 1; The OsHIR1 The amino acid sequence of the protein encoded by the gene is shown as SEQ ID NO. 2; By increasing OsHIR1 the expression level of the gene, the resistance of rice to the small brown planthopper is increased.

2. A method of increasing resistance of rice plant to Sogatella furcifera by using the recombinant DNA molecule of claim 1. OsHIR1 A method of increasing resistance of rice plant to Sogatella furcifera by using the recombinant DNA molecule of claim 1. comprising the following steps: S1, cloning of rice OsHIR1 genes; S2, construct OsHIR1 overexpression vector; S3, the overexpression vector obtained in S2 is transformed into rice to obtain transgenic plants with improved resistance to white-backed planthopper.

3. The method of claim 2, wherein, In S1, the cloned rice OsHIR1 The gene is specifically: Total RNA of rice was extracted and reversely transcribed into cDNA as template, which was amplified by PCR with primers OsHIR1 -F and OsHIR1 -R, and the products were cloned into pMD18-T vector. OsHIR1 Gene.

4. The method of claim 3, wherein, The nucleotide sequence of said primer OsHIR1 The nucleotide sequence of said primer -F is set forth in SEQ ID NO.

3. OsHIR1 The nucleotide sequence of said primer -R is set forth in SEQ ID NO. 4.

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