Application of OsNAC95 gene knockout in positive regulation of rice resistance to brown planthopper

Abstract

The present invention relates to the field of biological gene engineering technology, and in particular to the application of knocking out the OsNAC95 gene in positively regulating rice resistance to brown planthoppers. In the embodiment of the present invention, the OsNAC95 gene in the recipient rice is edited by CRISPR / Cas9 gene editing technology to obtain the transgenic plant OsNAC95-Cas edited by the OsNAC95 gene. The T2 generation homozygous transgenic plants were identified for brown planthopper resistance using the seedling group method. Compared with the transgenic recipient parent B1415 (control group, Bph14 / Bph15 polymerization system with Wushan Silk Seedling as the recurrent parent, denoted as B1415, the same below), OsNAC95-Cas is more insect-resistant, confirming that OsNAC95 is related to rice resistance to brown planthoppers and can be used to create new germplasm of rice resistance to brown planthoppers.

Description

Technical Field

[0001] The invention relates to the technical field of biological gene engineering, and in particular to application of knocking out OsNAC95 gene in positively regulating rice resistance to brown planthopper. Background Art

[0002] Brown planthopper (Nilaparvata lugens Brown planthoppers (BPH) are one of the main pests that affect rice production in my country. They are a pest that feeds exclusively on rice. They cause large areas of rice to fall over and die, resulting in reduced yields or even total crop failures through egg-laying, feeding, and the spread of rice diseases, which seriously threatens my country's food security. The use of pesticides to control them will increase production costs, pollute the environment and rice, and also make brown planthoppers resistant to pesticides.

[0003] Breeding and planting insect-resistant rice varieties containing brown planthopper resistance genes is an economical, effective, and environmentally friendly way to control brown planthoppers. Insect-resistant rice varieties bred by the combined use of brown planthopper resistance genes Bph14 and Bph15 can effectively improve rice's resistance to brown planthoppers. On this basis, further discovering and utilizing key resistance regulation genes and continuously improving rice varieties' insect resistance are necessary means to actively respond to the increasing harmfulness of brown planthoppers.

[0004] NAC( N AM, A TAF1 / 2, C The UC2) transcription factor family is one of the largest transcription factor families found in plant genomes. Its protein structure is characterized by a highly conserved NAC domain of about 150 amino acids at the N-terminus, which is involved in DNA binding, and a transcription activation domain at the C-terminus, which is involved in the transcriptional regulation of target genes. NAC transcription factors are involved in regulating a variety of biological processes in plants, including disease and insect resistance, growth and development, response to adverse stress, secondary growth, and hormone signal transduction. The rice OsNAC95 gene encodes a NAC transcription factor, which is significantly downregulated after being attacked by brown planthoppers. At present, there are no relevant research reports on the application of OsNAC95 in regulating rice resistance to brown planthoppers. Summary of the invention

[0005] In order to solve the above problems, the present invention provides an application of knocking out the OsNAC95 gene in positively regulating rice resistance to brown planthoppers. The resistance of rice plants to brown planthoppers is enhanced after OsNAC95 gene editing. It is a key gene for regulating rice resistance to brown planthoppers. The editing of this gene has important research value for breeding new rice varieties resistant to brown planthoppers.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides application of knocking out OsNAC95 gene in positively regulating rice resistance to brown planthopper.

[0008] Preferably, the CDS nucleotide sequence of the OsNAC95 gene is shown in SEQ ID No.1.

[0009] Preferably, the amino acid sequence of the protein encoded by the OsNAC95 gene is shown in SEQ ID No.2.

[0010] Preferably, the application comprises the following steps:

[0011] 1) Connect the pWMC025 vector and the target sequence to obtain a connection vector;

[0012] The target sequence includes target 1 and target 2, the nucleotide sequence of target 1 is shown in SEQ ID No.3, and the nucleotide sequence of target 2 is shown in SEQ ID No.4;

[0013] 2) transforming Escherichia coli with the ligated vector obtained in step 1) to extract the plasmid;

[0014] 3) transferring the plasmid obtained in step 2) into Agrobacterium to obtain transformed bacteria;

[0015] 4) Transforming rice with the transformed bacteria obtained in step 3) to obtain transgenic rice resistant to brown planthoppers edited with the OsNAC95 gene.

[0016] Preferably, the primer pair for amplifying the target sequence target in step 1) is a forward primer OsNAC95-F and a reverse primer OsNAC95-R; the nucleotide sequence of the forward primer OsNAC95-F is shown in SEQ ID No.5, and the nucleotide sequence of the reverse primer OsNAC95-R is shown in SEQ ID No.6.

[0017] Preferably, the system used for amplifying the target sequence is:

[0018] 1 μL of forward primer OsNAC95-F at a concentration of 10 μM, 1 μL of reverse primer OsNAC95-R at a concentration of 10 μM, 1 μL of pWMC025 vector at a concentration of 50 ng / μL, 5 μL of buffer, 4 μL of dNTP at a concentration of 2.5 mM, 1 μL of Pfu DNA polymerase, and 37 μL of ddH2O.

[0019] Preferably, the operating procedure for amplifying the target sequence is: 1) 95°C for 5 min; 2) 30 cycles: 95°C for 30 s, 58°C for 30 s, 72°C for 1 min; 3) 72°C for 5 min; 4) storage at 4°C.

[0020] Preferably, the pWMC025 vector and the target sequence are digested with BsaI enzyme and then ligated; T4 DNA ligase is used for ligation.

[0021] Preferably, the Escherichia coli is Escherichia coli DH5α; and the Agrobacterium is Agrobacterium EHA105.

[0022] The present invention also provides the use of the OsNAC95 gene described in the above technical solution in the breeding of rice resistant to brown planthoppers.

[0023] The beneficial effects of the present invention are:

[0024] In the embodiment of the present invention, the OsNAC95 gene in the recipient rice was edited by CRISPR / Cas9 gene editing technology to obtain the transgenic plant OsNAC95-Cas edited with the OsNAC95 gene. The T2 generation homozygous transgenic plants were identified for brown planthopper resistance using the seedling group method. Compared with the transgenic recipient parent B1415 (control group, Bph14 / Bph15 polymerized system with Wushan Silk Seedling as the recurrent parent, denoted as B1415, the same below), OsNAC95-Cas is more insect-resistant, confirming that OsNAC95 is related to rice resistance to brown planthoppers and can be used to create new rice germplasm resistant to brown planthoppers. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments are briefly introduced below.

[0026] Figure 1 Schematic diagram of CRISPR / Cas9 gene editing vector pWMC025;

[0027] Figure 2 Schematic diagram of the OsNAC95 gene structure; the blue box represents the 5'-UTR and 3'-UTR, the yellow box represents the exon region, the red line segment marks the gene editing target segment, and the gene length is shown in the scale bar, 20 bp;

[0028] Figure 3 The details of DNA sequence editing in the target region of the OsNAC95 gene-edited plant are shown; red letters indicate SG targeting sequences, bright blue letters indicate PAM sequences, and red lines indicate base deletions; compared with the transgenic receptor B1415, the OsNAC95-Cas-2 strain has 1 base and 1 base deletion in target regions 1 and 2, respectively, the OsNAC95-Cas-3 strain has 1 base and 2 base deletions in target regions 1 and 2, respectively, and the OsNAC95-Cas-9 strain has 53 bases and 1 base deletion in target regions 1 and 2, respectively;

[0029] Figure 4 This is the brown planthopper resistance phenotype of the T2 generation homozygous transgenic plants edited with the OsNAC95 gene;

[0030] Figure 5 Identification of the resistance level of T2 generation homozygous transgenic plants edited with OsNAC95 gene to brown planthoppers. DETAILED DESCRIPTION

[0031] The present invention provides an application of knocking out the OsNAC95 gene in positively regulating rice resistance to brown planthoppers. In the present invention, the CDS nucleotide sequence of the OsNAC95 gene is shown in SEQ ID No. 1, which is as follows:

[0032] ATGGAGCAGCAGCGGAGCAGGTCGACGGCGGCAGGAGGAGAGGTGGAG

[0033] GTGGAGCAGCTGCCGGGATTCAGGTTCCATCCGACGGAGGAGGAGCTGCT

[0034] GGAGTTCTACCTGAAGCAGGTGGTGCAGGGGAAGAAGCTCAAGTTCGACA

[0035] TCATCCCCACCGTCCACCTCTACCGCCACGACCCGCGCGAGCTCCCCGGA

[0036] CTCGCCCGCATCGGCGAGCGCGAGTGGTACTTCTTCGTCCCCCGCGACCGC

[0037] AAGCAGGCCACCGGCGGCGGCGGCGGC GGTAGGCCCAGCCGCACCA CGG

[0038] AGCGCGGCTTCTGGAAGGCCACCGGCTCCGACCGCGCCATCCGCTGCGCC

[0039] GCCGACCCCAAGCGCCTCATCGGCCTCAAGAAGACGCTCGTCTACTACGA

[0040] GGGCCGCGCCCCTCGCGGCACCAAGACCGACTGGGTCATGAACGAGTACC

[0041] GCCTCCCCGACGCCGCCGCCATTCCCGACACCATGCAATTGCAAATGCAAC

[0042] ACGACGACATGGTG CTCTGCAAGGTCTACCGGA AGGCGGTCTCCCTCAAG

[0043] GAGCTGGAGCAGAGGGTCGCCATGGAGGAGCTTGCACGCTCCACCACGTC

[0044] CTCCGGCACCCACAACACCGGCTCGCCCCTGCAGCAGGACTCCTCCTCCA

[0045] TCTCCATCTCCTCCTCCTCGGACGCCATGAAGAAAGAGGTGGTGGGGGTG

[0046] GATGAGGCGTCGGCGGCGGCGCATGAGCTGGTCCGGCCGGCGACGCTGA

[0047] GCCTGCCGCAGCTGGAGGTGGCGAGGCCGCAGAGCGGGCTGGAGTGGAT

[0048] GCAGGAGCCATTCCTGACGCAGCTGCGGAGCCCATGGATGGAGACCTGGTCGCCCTACTACGCCAGCGTCCTCAACTTCTGA, where the underlined sequences GGTAGGCCCAGCCGCACCA and CTCTGCAAGGTCTACCGGA are SG targeting sequences, and the bold sequences CGG and AGG are PAM sequences.

[0049] In the present invention, the amino acid sequence of the protein encoded by the OsNAC95 gene is shown in SEQ ID No. 2, which is as follows:

[0050] MEQQRSRSTAAGGEVEVEQLPGFRFHPTEEELLEFYLKQVVQGKKLKFDIIPTVHLYRHDPRELPGLARIGEREWYFFVPRDRKQATGGGGGGRPSRTTERGFWKATGSDRAIRCAADPKRLIGLKKTLVYYEGRAPRGTKTDWVM NEYRLPDAAAIPDTMQLQMQHDDMVLCKVYRKAVSLKELEQRVAMEELARSTTSSGTHNTGSPLQQDSSSISISSSSDAMKKEVVGVDEASAAAHELVRPATLSLPQLEVARPQSGLEWMQEPFLTQLRSPWMETWSPYYASVLNF.

[0051] In the present invention, the application preferably comprises the following steps:

[0052] 1) Connect the pWMC025 vector and the target sequence to obtain a connection vector;

[0053] The target sequence includes target 1 and target 2, the nucleotide sequence of target 1 is shown in SEQ ID No.3, and the nucleotide sequence of target 2 is shown in SEQ ID No.4;

[0054] 2) transforming Escherichia coli with the ligated vector obtained in step 1) to extract the plasmid;

[0055] 3) transferring the plasmid obtained in step 2) into Agrobacterium to obtain transformed bacteria;

[0056] 4) Transforming rice with the transformed bacteria obtained in step 3) to obtain transgenic rice resistant to brown planthoppers edited with the OsNAC95 gene.

[0057] The present invention connects the pWMC025 vector and the target sequence to obtain a connection vector; the target sequence includes target 1 and target 2, the nucleotide sequence of target 1 is shown in SEQ ID No.3, and the nucleotide sequence of target 2 is shown in SEQ ID No.4.

[0058] In the present invention, the pWMC025 vector is derived from Weimi Biotechnology Co., Ltd., and the relevant vector map is shown in Figure 1 In the present invention, the pWMC025 vector and the target sequence are preferably digested with BsaI enzyme before being connected. The present invention does not specifically limit the digestion method. A person skilled in the art can use conventional methods. The present invention preferably uses T4 DNA ligase for connection. The present invention does not specifically limit the connection method, and a conventional method can be used.

[0059] SEQ ID No.3: GGTAGGCCCAGCCGCACCA;

[0060] SEQ ID No. 4: CTCTGCAAGGTCTACCGGA.

[0061] In the present invention, the target 1 is contained in the forward primer OsNAC95-F, and the nucleotide sequence of the forward primer OsNAC95-F is shown in SEQ ID No. 5. In the present invention, the target 2 is contained in the reverse primer OsNAC95-R, and the nucleotide sequence of the reverse primer OsNAC95-R is shown in SEQ ID No. 6. In the present invention, the system used for the PCR amplification is: 1 μL of the forward primer OsNAC95-F with a concentration of 10 μM, 1 μL of the reverse primer OsNAC95-R with a concentration of 10 μM, 1 μL of the pWMC025 vector with a concentration of 50 ng / μL, 5 μL of buffer, 4 μL of dNTP with a concentration of 2.5 mM, 1 μL of Pfu DNA polymerase, and 37 μL of ddH2O. The PCR operation program is: 1) 95°C for 5min; 2) 30 cycles: 95°C for 30s, 58°C for 30s, 72°C for 1min; 3) 72°C for 5min; 4) stored at 4°C. The present invention preferably uses Weimi Biotechnology Co., Ltd. to perform target site sequence screening and primer design, and obtains the target site sequences GGTAGGCCCAGCCGCACCA and CTCTGCAAGGTCTACCGGA of the OsNAC95 gene through website screening, and then designs primers in combination with the restriction site.

[0062] SEQ ID No.5:

[0063] CCAATGGTCTCATGTGTG GGTAGGCCCAGCCGCACCA GTTTTAGAGC TAGAAATAG;

[0064] Primer description: The bold sequence is the BsaI restriction site, the sequence on the left of the BsaI restriction site is its protective base, the sequence on the right of the BsaI restriction site is the sticky linker connected to the pWMC025 vector, the underlined sequence is the target 1 sequence, and the right side of the target 1 sequence is the pWMC025 vector template sequence;

[0065] SEQ ID No.6:

[0066] ATTGGGGTCTCTAAAC TCCGGTAGACCTTGCAGAG CGCCACGGATCA TCTGCACA.

[0067] Primer description: The bold sequence is the BsaI restriction site, the sequence on the left of the BsaI restriction site is its protective base, the sequence on the right of the BsaI restriction site is the sticky linker connected to the pWMC025 vector, the underlined sequence is the target 2 sequence, and the right side of the target 2 sequence is the pWMC025 vector template sequence;

[0068] Amplification instructions: Use forward primer OsNAC95-F (SEQ ID No. 5) and reverse primer OsNAC95-R (SEQ ID No. 6), take pWMC025 vector as template, amplify the sequence from the first sgRNA to OsU3 promoter, cut through BsaI restriction site, and connect the whole into pWMC025 backbone vector to obtain the complete vector containing target 1 sequence and target 2 sequence.

[0069] The present invention transfers the obtained plasmid into Agrobacterium to obtain transformed bacteria. In the present invention, the Escherichia coli is preferably Escherichia coli DH5α. The present invention has no particular limitation on the method of transferring the plasmid into Agrobacterium, and conventional methods can be used.

[0070] The present invention transforms rice with the obtained transformant bacteria to obtain transgenic rice resistant to brown planthoppers edited with the OsNAC95 gene. In the present invention, the Agrobacterium is preferably Agrobacterium EHA105. The present invention has no particular limitation on the method of transforming rice with the transformant bacteria, and those skilled in the art can use conventional methods.

[0071] In order to further illustrate the present invention, the present invention is described in detail below in conjunction with embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0072] Example 1

[0073] Obtaining CRISPR / Cas9 gene-edited transgenic rice plant OsNAC95-Cas

[0074] (1) Obtaining transgenic rice

[0075] The target site sequence of OsNAC95 was designed through the website (http: / / crispr.hzau.edu.cn / CRISPR2 / ), and the forward primer OsNAC95-F (SEQ ID No. 5) containing target 1 was designed according to the vector restriction site: CCAATGGTCTCATGTGTGGGTAGGCCCAGCCGCACCAGTTTTAGAGCTAGA AATAG; the reverse primer OsNAC95-R (SEQID No. 6) containing target 2 was designed: ATTGGGGTCTCTAAACTCCGGTAGACCTTGCAGAGCGCCACGGATCATCTG CACA.

[0076] 1) Vector enzyme digestion: The enzyme digestion system is to use 1 μL BsaI, 5 μL Buffer, 5 μL of 200 ng / μL pWMC025 vector, 39 μL ddH2O, and incubate at 37°C for 1 hour.

[0077] 2) Vector purification and recovery: After the vector enzyme digestion and incubation are completed, the gel is cut by electrophoresis, and the large fragments are recovered using the DC301 DNA gel recovery kit (Nanjing Novozymes Biotech Co., Ltd.), and eluted with 40 μL of elution buffer to obtain the purified product after vector enzyme digestion.

[0078] 3) PCR amplification of target sequence: The system used for PCR amplification is 1 μL of forward primer OsNAC95-F at a concentration of 10 μM, 1 μL of reverse primer OsNAC95-R at a concentration of 10 μM, 1 μL of pWMC025 vector amplification template at a concentration of 50 ng / μL, 5 μL of buffer, 4 μL of dNTP at a concentration of 2.5 mM, 1 μL of Pfu DNA polymerase, and 37 μL of ddH2O. The PCR operation program is: 1) 95°C for 5 min; 2) 30 cycles: 95°C for 30 s, 58°C for 30 s, 72°C for 1 min; 3) 72°C for 5 min; 4) 4°C storage. 4) Purification and recovery of PCR products: After the PCR amplification reaction is completed, the gel is cut by electrophoresis, and the target fragment is recovered using the DC301 DNA gel recovery kit, and eluted with 40 μL of elution buffer to obtain the purified PCR product.

[0079] 5) Enzyme digestion of PCR products: The enzyme digestion system used was 1 μL BsaI, 5 μL Buffer, 40 μL of the purified PCR product obtained in step 4) above, 4 μL ddH2O, and incubated at 37°C for 1 h.

[0080] 6) Purification and recovery of PCR products after enzyme digestion: After the above-mentioned PCR product digestion and incubation is completed, gel electrophoresis is performed, and the target fragment is recovered using a DC301 DNA gel recovery kit and eluted with 40 μL of elution buffer to obtain a purified product after PCR product digestion.

[0081] 7) Ligation: Ligate the purified vector obtained in step 2) and step 6) with the PCR product. The ligation system is 2 μL of the purified product after vector digestion, 6 μL of the purified product after PCR digestion, 1 μL of T4 ligase, and 1 μL of buffer. Incubate at 4°C for 16 h.

[0082] 8) Transformation: The ligation product obtained in step 7) above was transformed into E. coli DH5α strain by heat shock method. Select clones for PCR amplification and sequencing verification. The sequencing forward primer QC1 (SEQ ID No. 7): CTGGCGAAAGGGGGATGTGCTGCAA and the reverse primer QC4 (SEQ ID No. 8): TTCAAACAAGTGTGACAAAAA Figure 1 As shown, the correctly aligned plasmid was transferred into Agrobacterium EHA105 strain, which was used to transform the Bph14 / Bph15 polymerization line with Wushansi Miao as the recurrent parent, and the T0 generation transgenic rice was obtained by hygromycin screening, which was the OsNAC95-edited transgenic strain, named OsNAC95-Cas.

[0083] (2) Identification of transgenic rice

[0084] The T2 transgenic plants were sequenced and analyzed: the forward primer OsNAC95-SF (SEQ ID No. 9): GGGGAAGAAGCTCAAGTTCGA and the reverse primer OsNAC95-SR (SEQ ID No. 10): TGGCTCCTGCATCCACTCC were designed on both sides of the target site, and the DNA of the transgenic plants was amplified and sequenced. The sequencing results are shown in Figure 3 As shown, nucleic acid sequence alignment showed that compared with the transgenic receptor B1415, the OsNAC95-Cas-2 strain had 1 base and 1 base deletion in target regions 1 and 2, respectively, the OsNAC95-Cas-3 strain had 1 base and 2 base deletions in target regions 1 and 2, respectively, and the OsNAC95-Cas-9 strain had 53 bases and 1 base deletion in target regions 1 and 2, respectively.

[0085] Example 2

[0086] The seedling group method was used to identify the brown planthopper resistance of OsNAC95-Cas transgenic rice and its transgenic receptor B1415. The identification work was assisted by Wuhan Hetaiqing Biotechnology Co., Ltd.

[0087] The brown planthopper sources used for identification were collected from rice fields under natural conditions and cultured on the brown planthopper-susceptible rice material "Taichung No. 1" from the Plant Genetics Center of Wuhan University.

[0088] The seeds of the test material were sown in a plastic cup with a diameter of 12 cm and a height of 15 cm. When the seedlings grew to the four-leaf stage, 10 brown planthoppers were inoculated into the second to third instar brown planthopper nymphs per seedling. The identification varieties were infested with brown planthoppers until the specific varieties died, and the damage degree of the identified individual plants was investigated, and the readings were graded. The individual plant grading evaluation criteria for rice seedling resistance identification are as follows:

[0089] Table 1 Evaluation criteria for individual plant grading in rice seedling resistance identification

[0090]

[0091]

[0092] The T2 generation transgenic homozygous strains edited by the OsNAC95 gene were identified for brown planthopper resistance. The results of the insect resistance identification showed that after the test material was inoculated with brown planthoppers, until the brown planthoppers fed on the transgenic recipient plant B1415 and caused most of the plants to die, the resistance score was 8.60. The three T2 generation transgenic strains edited by the OsNAC95 gene, OsNAC95-Cas-2, OsNAC95-Cas-3 and OsNAC95-Cas-9, only showed partial yellowing of leaves, and the resistance scores were 3.28, 3.34 and 3.53, respectively. The lower the resistance grade identification score, the stronger the plant resistance. The data are the means and standard deviations of three independent biological replicates. The data were analyzed using the t-test, and the P values ​​for the comparison between the experimental group and the control group are shown in the figure ( Figure 4 and 5 ).

[0093] The experimental results showed that gene editing of OsNAC95 can significantly enhance the resistance of B1415 plants to brown planthoppers, indicating that editing the OsNAC95 gene does regulate rice resistance to brown planthoppers.

[0094] Although the above embodiment describes the present invention in detail, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of knocking out rice OsNAC95 gene in positively regulating rice resistance to brown planthopper.

2. The use according to claim 1, characterized in that: The CDS nucleotide sequence of the rice OsNAC95 gene is shown in SEQ ID No.

1.

3. The use according to claim 1 or 2, characterized in that: The amino acid sequence of the protein encoded by the rice OsNAC95 gene is shown in SEQ ID No.

2.

4. Application of knocking out rice OsNAC95 gene in breeding rice resistant to brown planthopper.

Images