Application of uORF of OsTCP19 gene in improvement of rice stalk strength and lodging resistance

Abstract

The invention discloses an application of uORF of an OsTCP19 gene in improvement of rice stalk strength and lodging resistance. The invention provides an application of uORF for destroying the OsTCP19 gene or a substance for destroying the uORF in any one of the following aspects: A1) improving the lodging resistance of plants; a2) cultivating lodging-resistant plants; a3) preparing a product for improving the lodging resistance of the plants; the invention relates to a method for improving the strength of rice stalks, in particular to a method for improving the strength of rice stalks, which comprises the following steps: (1) preparing a product for improving the strength of rice stalks, (2) preparing a product for improving the strength of rice stalks, (4) improving the strength of plant stalks, (5) cultivating a plant with high strength of stalks, and (6) preparing a product for improving the strength of plant stalks, and the nucleotide sequence of uORF of the OsTCP19 gene is 5-AUGGCGUGA-3.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and relates to application of uORF of an OsTCP19 gene in improving stem strength and lodging resistance of rice. BACKGROUND

[0002] An upstream open reading frame (uORF) is a regulatory element widely existing in a 5'UTR region of a gene. The uORF contains a complete start codon (ATG) and a stop codon (TAA, TAG or TGA), and the start codon is generally located in the 5'UTR region. According to the length of the uORF sequence, the stop codon can be located in the 5'UTR region or the coding region of the gene. Generally, when mRNA of a gene is translated on a ribosome, the start codon (uATG) of the uORF sequence in the 5'UTR region is first recognized and translated by the ribosome, thereby hindering the combination of the start codon (mATG) of the downstream coding gene with the ribosome and correct and efficient translation. Therefore, the translation efficiency of a gene can be accurately regulated at the translation level by regulating the uORF through heredity.

[0003] High nitrogen fertilizer application is easy to cause lodging of crops. Rice is a major food crop, and dry matter accumulation in the late grain filling stage of rice contributes more than 60% to the grain yield. If lodging occurs at this time, the photosynthetic capacity and grain filling efficiency in this critical stage will be seriously affected, and the crop yield will be significantly reduced. At the same time, crop lodging is easy to cause sprouting of the panicle, reduce the mechanical harvesting efficiency, and further cause yield loss. In summary, improving the lodging resistance of crops is a key factor for improving yield under high nitrogen conditions, and is of great significance for ensuring food security. Stem mechanical strength is one of the most critical indicators for determining the lodging resistance of crops, which is mainly determined by the morphology of the secondary cell wall of the stem. SUMMARY

[0004] The technical problem solved by the present application is how to improve the stem strength and lodging resistance of rice.

[0005] To solve the above technical problem, the present application provides, in a first aspect, a substance for disrupting the uORF of an OsTCP19 gene or an application of the uORF disrupted as follows: A1) improving the lodging resistance of a plant; A2) breeding a lodging-resistant plant; A3) preparing a product for improving the lodging resistance of a plant; A4) improving the stem strength of a plant; A5) breeding a plant with high stem strength; A6) preparing a product for improving the stem strength of a plant; The OsTCP19 gene encodes a protein OsTCP19, which is any one of the following: B1) the protein comprises the amino acid residues shown in SEQ ID NO: 2; B2) the protein is a protein having more than 80% amino acid residue identity with the protein shown in B1) and having the same function; B3) the protein comprises a tag linked to the N-terminus or / and C-terminus of the protein shown in B1) or B2); The nucleotide sequence of the uORF of the OsTCP19 gene is 5'-AUGGCGUGA-3'. Wherein, A is adenine nucleotide, U is uracil nucleotide, G is guanine nucleotide, and C is cytosine nucleotide.

[0006] In the above, the OsTCP19 gene is derived from rice, and the nucleotide sequence thereof is any one of the following: C1) comprises the DNA molecule shown in SEQ ID NO: 1; C2) has more than 75% identity with the nucleotide sequence defined in C1) and encodes a protein having the same function; C3) hybridizes to the nucleotide sequence defined in C1) under stringent conditions and encodes a protein having the same function.

[0007] In the above, the nucleotide sequence of the DNA corresponding to the uORF of the OsTCP19 gene is 520-528 of SEQ ID NO: 1.

[0008] The above-mentioned disruption of the uORF of the OsTCP19 gene can be achieved by any means in the prior art, so that the start codon (ATG) of the uORF or the nucleotide residues containing the ATG are subjected to non-frameshift mutation, which can be deletion mutation (three or a multiple of three codons are deleted), base substitution mutation (one or more bases in ATG are replaced while maintaining non-frameshift), or complete deletion of the entire uORF sequence, thereby achieving non-translation of the uORF (520-528 of SEQ ID NO: 1) of the OsTCP19 gene. The non-frameshift mutation can be chemical mutagenesis, physical mutagenesis, RNAi, genome site-directed editing, or homologous recombination, etc.

[0009] In some embodiments, the base substitution mutation can be replacement of the three nucleotide residues (such as TAA) with non-ATG.

[0010] In some embodiments, the deletion mutation can be deletion of 24-bp from 504-527 of SEQ ID NO: 1.

[0011] In some embodiments, the deletion mutation can be deletion of 6-bp from 519-514 of SEQ ID NO: 1.

[0012] In the above-mentioned genome site-directed editing method, Zinc finger nuclease (ZFN) technology, Transcription activator-like effector nuclease (TALEN) technology or Clustered regularly interspaced short palindromic repeats / CRISPR associated (CRISPR / Cas9) system technology, and other technologies capable of achieving genome site-directed editing can be used. Regardless of which method is used, the uORF of the OsTCP19 gene described above is targeted, and the uORF of the OsTCP19 gene is disrupted.

[0013] In the above, the substance that disrupts the uORF of the OsTCP19 gene is a nucleic acid that targets the uORF, an expression cassette containing the nucleic acid, a recombinant vector containing the nucleic acid, a recombinant microorganism containing the nucleic acid, or a transgenic plant cell line, tissue or organ containing the nucleic acid.

[0014] In the above, the recombinant vector further contains a coding gene for Cas9.

[0015] In certain embodiments, the nucleic acid is an siRNA, shRNA, sgRNA, miRNA and / or antisense RNA.

[0016] In certain embodiments, the substance that disrupts the uORF of the OsTCP19 gene can be any one of the following: c1) an sgRNA that targets the uORF of the OsTCP19 gene; c2) an expression cassette containing the coding nucleic acid of the sgRNA of c1); c3) an expression cassette containing the coding nucleic acid of the sgRNA of c2); c4) a recombinant vector containing the coding nucleic acid of c2) or the expression cassette of c3); c5) a recombinant microorganism containing the coding nucleic acid of c2), the expression cassette of c3), or the recombinant vector of c4); c6) a transgenic plant cell line containing the coding nucleic acid of c2), the expression cassette of c3), or the recombinant vector of c4); c7) a transgenic plant tissue containing the coding nucleic acid of c2), or a transgenic plant tissue containing the expression cassette of c3), or a transgenic plant tissue containing the recombinant vector of c4); c8) a transgenic plant organ containing the coding nucleic acid of c2), or a transgenic plant organ containing the expression cassette of c3), or a transgenic plant organ containing the recombinant vector of c4).

[0017] In some embodiments, the target site of the sgRNA is the sequence from 521 to 540 of SEQ ID NO: 1. In some embodiments, the sgRNA is the RNA encoded by the sequence from 521 to 540 of SEQ ID NO: 1.

[0018] In some embodiments, the expression cassette of c3) is the expression cassette fragment prepared in the Examples containing the U3 promoter driving the uORF3-U3F / R dimer product.

[0019] In some embodiments, the recombinant vector of c4) is a uORF3-CRISPR knock-out vector.

[0020] In a second aspect, the present application provides the use of the uORF of the OsTCP19 gene as a regulatory element or as a target to regulate the expression of the OsTCP19 gene in at least one of the following: A1) improving the lodging resistance of a plant; A2) breeding a lodging-resistant plant; A3) preparing a product for improving the lodging resistance of a plant; A4) improving the stem strength of a plant; A5) breeding a plant with high stem strength; A6) preparing a product for improving the stem strength of a plant.

[0021] In some embodiments, the regulation of the expression of the OsTCP19 gene is the regulation of the translation of the OsTCP19 gene.

[0022] In some embodiments, the regulation of the translation of the OsTCP19 gene is the improvement of the translation efficiency of the downstream gene or the enhancement of the function of the protein encoded by the downstream gene after the uORF of the OsTCP19 gene is disrupted.

[0023] In a third aspect, the present application provides a method for improving the lodging resistance of a plant, comprising the step of disrupting the uORF of the OsTCP19 gene in a plant of interest to improve the lodging resistance of the plant of interest; the plant of interest contains the OsTCP19 gene and the uORF.

[0024] In a fourth aspect, the present application provides a method for improving stem strength of a plant, comprising the following steps: disrupting the uORF of the OsTCP19 gene in a plant of interest, so as to improve the stem strength of the plant of interest; wherein the plant of interest comprises the OsTCP19 gene and the uORF.

[0025] In a fifth aspect, the present application provides a method for breeding a plant with lodging resistance, comprising the following steps: disrupting the uORF of the OsTCP19 gene in a plant of interest, so as to obtain a plant with lodging resistance. The plant with lodging resistance has a higher lodging resistance than the plant of interest.

[0026] In a sixth aspect, the present application provides a method for breeding a plant with high stem strength, comprising the following steps: disrupting the uORF of the OsTCP19 gene in a plant of interest, so as to obtain a plant with high stem strength. The plant with high stem strength has a higher stem strength than the plant of interest.

[0027] In some embodiments, the disruption of the uORF of the OsTCP19 gene can be achieved by gene editing.

[0028] In some embodiments, the gene editing can be achieved by introducing a nucleic acid encoding an sgRNA targeting the uORF into a recipient plant via a recombinant vector.

[0029] In some embodiments, the recombinant vector is a uORF3-CRISPR knockout vector.

[0030] In the above, the lodging resistance is lodging resistance in a high-nitrogen planting environment.

[0031] In some embodiments, the lodging resistance is lodging resistance in a high-nitrogen planting environment. That is, the plant with lodging resistance has a higher lodging resistance than the plant of interest in a high-nitrogen planting environment.

[0032] In the above, the plant is any one of the following: D1) a plant of the family Poaceae; D2) a plant of the genus Oryza; D3) a rice plant.

[0033] In a seventh aspect, the present application provides a product, which is any one of the following: 1) the substance of the first aspect; 2) the uORF of the OsTCP19 gene of the first aspect.

[0034] uORF editing is an effective means of slightly or moderately increasing (up-regulating) gene expression. Three possible uORFs were found in the 5' UTR region of OsTCP19, as predicted on the website (http: / / www.rnairport.com:443 / ). Using a transcriptional activation reporter system, the transcriptional inhibition effect of the three uORFs on OsTCP19 was detected in rice protoplasts. Only the start codon (uATG) of the third uORF was mutated (to AAA by site-directed mutagenesis), which resulted in an increase of ~1.5-fold in the transcriptional activation activity. Therefore, the third uORF (uORF3) is a target for increasing the translation efficiency of downstream genes.

[0035] The present application uses the CRISPR-Cas9 system to perform site-directed knockout of the start codon of uORF3 in the 5' UTR region of OsTCP19 gene in rice plants to disrupt the translation of uORF3. Two independent whole-code mutated uORF3 editing materials (6 bp and 24 bp deletion, respectively) were successfully created, and the two mutations did not affect the translation termination sites of other uORFs. Phenotypic observation found that, compared with the wild type, the uORF3 edited lines showed more compact plant type, thicker secondary cell wall thickness, stronger stem strength and higher resistance to lodging. This result shows that uORF3 can improve the stem strength and lodging resistance of rice by gene editing. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 To detect the effect of uORF mutation using a luciferase reporter system.

[0037] Figure 2 For the mutation region sequencing results and phenotypes of T1 generation mutant transgenic homozygous rice lines.

[0038] Figure 3 For the cell wall detection results of T1 generation mutant transgenic homozygous rice lines.

[0039] Figure 4 For the lodging incidence detection results of T1 generation mutant transgenic homozygous rice lines. DETAILED DESCRIPTION

[0040] The present application will be further described in detail below in conjunction with the specific embodiments. The examples provided below are only to illustrate the present application, and are not intended to limit the scope of the present application. The examples provided below can serve as a guide for further improvement by those of ordinary skill in the art, and do not in any way constitute a limitation on the present application.

[0041] The experimental methods in the following examples are all routine methods, unless otherwise specified, which are carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained commercially.

[0042] The quantitative tests in the following examples, unless otherwise specified, are set up with three repeated experiments, and the results are averaged.

[0043] Example 1, Cloning of uORF in 5' UTR Region of Rice OsTCP19 Gene and Evaluation of Inhibition Effect on Downstream Genes 1. Cloning of OsTCP19 Gene 5' UTR Region Fragment and uORF The genomic nucleotide sequence of rice OsTCP19 gene is Sequence 1, wherein the 1-536th position of Sequence 1 is the DNA corresponding to the 5' UTR region (536-bp), the 537-1700th position is the CDS region of OsTCP19 gene (1164-bp), and the 1701-1950th position is the DNA corresponding to the 3' UTR region (250-bp).

[0044] The primer sequences for amplifying the full-length 5' UTR region of OsTCP19 gene are as follows: OsTCP19 uORF-F: 5'-CAGCCCGGG GGATCC AATCCCGACAAAGTACACACACC-3'; OsTCP19 uORF-R: 5'-ATGTTTTTGGCGTCTT CCATGG CTACTCTCACGCCATG-3'.

[0045] The 5' end of the above-mentioned OsTCP19 uORF-F primer is added with a BamHI restriction site, and the 5' end of the OsTCP19 uORF-R primer is added with a NcoI restriction site (indicated by underlining). The bolded part of the OsTCP19 uORF-F and OsTCP19 uORF-R primer sequences is the sequence on the vector, which is used for subsequent homologous recombination to construct the vector. The non-bolded part of the OsTCP19 uORF-F is the 1-23th nucleotide sequence of Sequence 1; and the non-bolded black part of the OsTCP19 uORF-R is the reverse complementary nucleotide sequence of the 519-534th position of Sequence 1.

[0046] The genomic DNA of Zhonghua 11 (ZH11) was extracted as a template, and PCR amplification was performed with the primer combination of OsTCP19 uORF-F / OsTCP19 uORF-R (the primer combination contains two enzyme cutting site adapters of BamHI and NcoI and vector sequences for subsequent homologous recombination) to obtain a 536-bp 5' UTR region of OsTCP19 gene (wild-type 5' UTR sequence without mutation).

[0047] After sequencing, the nucleotide sequence of the DNA corresponding to the 5' UTR region of the OsTCP19 gene is sequence 1, positions 1-536. The 5' UTR region of the rice OsTCP19 gene contains three uORFs, which are sequentially named uORF1, uORF2, and uORF3. The nucleotide sequence of the DNA corresponding to uORF1 is sequence 1, positions 46-174; the nucleotide sequence of the DNA corresponding to uORF2 is sequence 1, positions 91-174; and the nucleotide sequence of the DNA corresponding to uORF3 is sequence 1, positions 520-528.

[0048] The nucleotide sequence of uORF3 is 5´-AUGGCGUGA-3´, wherein A is an adenine nucleotide, U is a uracil nucleotide, G is a guanine nucleotide, and C is a cytosine nucleotide.

[0049] 2. Construction of a transcription activation expression vector of the wild-type sequence The 536-bp 5' UTR region of OsTCP19 gene obtained in Step 1 was replaced into the fragment between the BamHI and Ncol enzyme sites of the vector pGreenII 0800-LUC (the vector is described in the following article: Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005, 1: 13. (doi: 10.1186 / 1746-4811-1-13.) which is named pGreenII 0800-LUC in the article) to obtain the recombinant expression vector pGreenII 0800-uOsTCP19-LUC, in which the 5' UTR region of OsTCP19 gene is located upstream of the CDS region of the reporter gene LUC, and the 3 uORF regulatory elements of the 5' UTR region are located downstream of the LUC gene.

[0050] The vector construction was verified by sequencing.

[0051] The vector pGreenII 0800-uOsTCP19-LUC contains the wild-type 5' UTR sequence without mutation, and the vector is referred to as the WT vector.

[0052] 3. Construction of three uORF start codon mutation vectors Subsequently, the three uORF start codons (ATG) on the pGreenII 0800-uOsTCP19-LUC vector were mutated to AAA by using a site-directed mutation kit (NEB, E0554S).

[0053] Using the pGreenII 0800-uOsTCP19-LUC vector as a template, the following primers (used to mutate uORF1) were used to amplify the uORF1-mutated vector pGreenII 0800-uOsTCP19-1m-LUC (referred to as uORF1-m): uORF1m-F: 5'-CCTCCTGCCTCGAAACTAGCTCTCCCA-3'; uORF1m-R: 5'-GGGAGACGATGGTGTGTGTA-3'.

[0054] After sequencing, the mutant vector uORF1-m is the vector obtained by mutating the promoter ATG of uORF1 in the 5' UTR region of the OsTCP19 gene in the recombinant expression vector pGreenII 0800-uOsTCP19-LUC into AAA (the 46-48 of SEQ ID NO: 1), and keeping other nucleotides unchanged.

[0055] The following primers (for mutating uORF2) are used to amplify the uORF2 mutant vector pGreenII 0800-uOsTCP19-2m-LUC (abbreviated as uORF2-m) using the pGreenII 0800-uOsTCP19-LUC vector as a template: uORF2m-F: 5'-TCCTATCATAAACTCCTCCCTTCTCCACT-3'; uORF2m-R: 5'-GGGAGAGTGGAGAGATGGGA-3'.

[0056] After sequencing, the mutant vector uORF2-m is the vector obtained by mutating the promoter ATG of uORF2 in the 5' UTR region of the OsTCP19 gene in the recombinant expression vector pGreenII 0800-uOsTCP19-LUC into AAA (the 91-93 of SEQ ID NO: 1), and keeping other nucleotides unchanged.

[0057] The following primers (for mutating uORF3) are used to amplify the uORF3 mutant vector pGreenII 0800-uOsTCP19-3m-LUC (abbreviated as uORF3-m) using the pGreenII 0800-uOsTCP19-LUC vector as a template: uORF3m-F: 5'-ATATATATACCAAAGCGTGAGAGTAGCCATGGAT-3'; uORF3m-R: 5'-ACACACACACCCACACAACC-3'.

[0058] After sequencing, the mutant vector uORF3-m is the vector obtained by mutating the promoter ATG of uORF3 in the 5' UTR region of the OsTCP19 gene in the recombinant expression vector pGreenII 0800-uOsTCP19-LUC into AAA (the 520-522 of SEQ ID NO: 1), and keeping other nucleotides unchanged.

[0059] 4. Detection of the regulatory effect of three uORFs on downstream coding genes The transcriptional activation reporter vectors of WT, mutant uORF1-m, mutant uORF2-m and mutant uORF3-m obtained in the above 3 were introduced into the rice protoplast system, and the downstream coding gene activation effect of the wild type sequence vector WT and the three uORF start codon mutant vectors was detected. Each vector was counted for 5 independent biological repeats.

[0060] The specific process is as follows: 1) Preparation of rice protoplasts: The above-ground part of Zhonghua 11 rice seedlings after germination for about 10 days was cut off, and the stem base (2-4 cm) of the seedlings was cut into 0.5-1.0 mm thin sections with a sharp knife, and then quickly transferred to the prepared enzyme solution (100 plants / 50 mL enzyme solution). The main components of the enzyme solution include 0.6 M Mannitol, 10 mM MES (pH 5.7), 1.5% Cellulase (m / v), 0.75% Macerozyme (m / v), 0.12% BSA, 3.4 mM CaCl2, 5 mM β-mercaptoethanol. Avoid light treatment and place in a 28°C shaker at a speed of 60 rpm for 4 h to prepare rice protoplasts.

[0061] 2) Transformation of rice protoplasts: 10 μg of WT, mutant uORF1-m, mutant uORF2-m and mutant uORF3-m obtained in the above 3 were added to 100 μL of protoplasts, respectively. Then an equal volume of 40% PEG solution (40% PEG formula: 40% PEG 4000 (m / v), 0.2 M Mannitol, 100 mM CaCl2, 65°C water bath heating and dissolving) was added to the centrifuge tube, gently mixed, and then placed in room temperature for 15 min. The transformation reaction was terminated, and then 750 μL of W5 buffer (W5 buffer formula: 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MES, pH adjusted to 5.7) was used to resuspend the protoplasts, and the transformed vectors were expressed in the rice protoplasts after overnight culture at 28°C for about 12 h.

[0062] 3) Detection of uORF mutation effect by luciferase reporter system The overnight cultured protoplasts were subjected to 250g, 5 min low-speed centrifugation and the supernatant was removed. The protoplasts were resuspended with 100 μL 1x lysis solution in a dual luciferase assay kit (Promega, E1960). After vortexing to break the cells, 100 μL of firefly luciferase (LUC) substrate was added, and the enzyme activity LUC value was detected using a single-tube chemiluminescence detector (model: GLOMAX 20 / 20, Promega). Then 100 μL of Renilla reniformis (REN) substrate was added, and the enzyme activity REN value was detected as an internal reference. The ratio of LUC / REN represents the activation effect of the translation level of the downstream coding gene under different mutant vectors (denoted as transcriptional activation activity).

[0063] The results are shown in Table 1. Figure 1 As shown in Table 1, a is the position of the mutation, and b is the transcriptional activation activity. It can be seen that, compared with the wild-type 5' UTR, the start codon mutations of uORF1 and uORF2 have no activation effect on the downstream coding gene LUC, and only the start codon of uORF3 is mutated to AAA, which can cause a 1.5-fold increase in the transcriptional activation activity of the downstream gene (improve the translation efficiency or protein level of the downstream coding gene).

[0064] The above results show that uORF3 can inhibit the translation efficiency of the gene located downstream thereof, and gene editing (point mutation or base substitution) of the start codon of uORF3 in the 5' UTR region of the OsTCP19 gene can appropriately increase the expression of the OsTCP19 protein at the translation level.

[0065] Example 2, Obtaining and detecting of the uORF3 gene editing strain of OsTCP19 gene I. Obtaining of the gene editing strain 1. Design of sgRNA Using sequence 1 as a reference sequence, a pair of sgRNA primer sequences was designed near the start codon of uORF3 in the 5' UTR region of the OsTCP19 gene, named uORF3-U3F / uORF3-U3R.

[0066] The target point of the sgRNA is sequence 1, position 521-540.

[0067] The primer sequence of the sgRNA is as follows uORF3-U3F: 5'-ggcaCCATGGCTACTCTCACGCCA-3'; uORF3-U3R: 5'-aaacTGGCGTGAGAGTAGCCATGG-3'; For the convenience of subsequent vector connection, the ggca linker is added to the 5' end of the uORF3-U3F primer, and the aaac linker is added to the 5' end of the uORF3-U3R primer.

[0068] The uORF3-U3F is the reverse complement sequence of the nucleotide sequence at positions 521-540 in Sequence 1, and the uORF3-U3R is the nucleotide sequence at positions 521-540 in Sequence 1.

[0069] 2. Construction of knockout vector The primer pair uORF3-U3F / uORF3-U3R is denatured and annealed to obtain the dimeric product of uORF3-U3F / R.

[0070] The dimeric product of uORF3-U3F / R is then inserted into the BsaI enzyme cleavage site of the pYLgRNA-U3 vector to obtain an intermediate vector. The intermediate vector is used as a template to perform two rounds of nested PCR amplification to obtain an expression cassette fragment containing the U3 promoter driving the uORF3-U3F / R dimeric product. The primers used in the first round of PCR amplification are U-F: 5'-CTCCGTTTTACCTGTGGAATCG-3' and gRNA-R: 5'-CGGAGGAAAATTCCATCCAC-3', and the template is the intermediate vector. The primers used in the second round of PCR are B1': 5'-TTCAGAggtctcTctcgCACTGGAATCGGCAGCAAAGG-3' and BL: 5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3', and the template is the amplification product of the first round of PCR.

[0071] The expression cassette fragment is then connected into the BsaI enzyme cleavage site of the pYLCRISPR / Cas9-MH final vector using the method of cutting and connecting at the same time to obtain a single-target uORF3 CRISPR / Cas9 knockout vector, named uORF3-CRISPR.

[0072] The above pYLgRNA-U3 vector and pYLCRISPR / Cas9-MH vector are described in the following documents, the pYLgRNA-U3 is named pYLsgRNA-OsU3 in the document, the pYLCRISPR / Cas9-MH vector is named pYLCRISPR / Cas9Pubi-H in the document; the reference is Xie X, Ma X, Zhu Q, Zeng D, Li G, Liu Y.-G. 2017. CRISPR-GE: A Convenient Software Toolkit for CRISPR-Based Genome Editing. Mol. Plant (DOI: http: / / dx.doi.org / 10.1016 / j.molp.2017.06.004).

[0073] The uORF3-CRISPR knockout vector is a vector obtained by inserting a fragment containing an expression cassette fragment of uORF3-U3F / R dimer product driven by the U3 promoter into the fragment between the BsaI enzyme cutting sites of pYLCRISPR / Cas9-MH.

[0074] 3. Obtaining of the gene edited strain The knockout vector uORF3-CRISPR is transformed into Agrobacterium tumefaciens strain AGL1 by heat shock method, and a recombinant Agrobacterium strain containing the recombinant plasmid TRN1-CRISPR is screened. The recombinant Agrobacterium strain containing the recombinant plasmid uORF3-CRISPR is used to infect callus differentiated from seed embryo tissue culture of Zhonghua 11 (hereinafter referred to as wild type rice), and the specific transformation and screening method is described in the document "Yi Zili, Cao Shuyun, Wang Li, He Sujie, Chu Chengcai, Tang Zuoshun, Zhou Pohua, Tian Wenzhong. Study on improving the frequency of Agrobacterium-mediated transformation of rice. Acta Genetica Sinica, 2001, 28(4): 352-358". Finally, transgenic seedlings are obtained, and the transgenic rice into which the uORF3-CRISPR is inserted is T0 generation. The seed of the T0 generation is harvested to obtain T1 generation.

[0075] II. Detection of the gene edited strain T0 generation transgenic rice carrying uORF3-CRISPR from step 3 of the above-mentioned one was extracted for genomic DNA. PCR identification was performed with primers uORF3-cr F and uORF3-cr R against the OsTCP19 gene, and the PCR product was sequenced, resulting in two independent mutant transgenic rice lines with uORF3 start codon frame mutations (6-bp and 24-bp deletion, respectively) of uORF3, named uORF3-cr1 and uORF3-cr2. uORF3-cr F: 5'-TCTTTCTAGCTCTACCGGCG-3'; uORF3-cr R: 5'-GTGTGCCGGTCCTTCGTC-3'. wherein uORF3-cr F is the nucleotide sequence of 348-367 in SEQ ID NO: 1, and uORF3-cr R is the reverse complement of the nucleotide sequence of 710-727 in SEQ ID NO: 1.

[0076] T1 generation seeds of the T0 generation mutant transgenic rice lines uORF3-cr1 and uORF3-cr2 were harvested, and after sowing, T1 generation mutant transgenic homozygous rice lines uORF3-cr1 and uORF3-cr2 were obtained.

[0077] Genomic DNA of the T1 generation mutant transgenic homozygous rice lines uORF3-cr1 and uORF3-cr2 was extracted, and PCR identification was performed with uORF3-cr F and uORF3-cr R, respectively. Sequencing of the PCR amplification product showed that the mutant region of uORF3-cr1 was Figure 2 a), as follows: Sequencing of the PCR product of the T1 generation mutant transgenic homozygous rice line uORF3-cr1 showed that compared with uORF3 of the OsTCP19 gene in the Zhonghua 11 rice genome, uORF3 of the OsTCP19 gene on the homologous chromosome on the uORF3-cr1 genome was changed as follows: 24-bp was deleted from 504-527 of SEQ ID NO: 1, causing a frame mutation, resulting in deletion of the uORF3 start codon, uORF3 nucleic acid unable to be translated, and uORF3 translation being disrupted.

[0078] Sequencing of the PCR product of the T1 generation mutant transgenic homozygous rice line uORF3-cr2 showed that compared with uORF3 of the OsTCP19 gene in the Zhonghua 11 genome, uORF3 of the OsTCP19 gene on the homologous chromosome on the uORF3-cr2 genome was changed as follows: 6-bp was deleted from 519-514 of SEQ ID NO: 1, causing a frame mutation, resulting in deletion of the uORF3 start codon, uORF3 nucleic acid unable to be translated, and uORF3 translation being disrupted.

[0079] III. Phenotype identification of uORF3 gene edited lines 1. Phenotype observation The seeds of T1 generation mutant transgenic pure line uORF3-cr1 and uORF3-cr2 were sowed, and T1 generation mutant transgenic lines uORF3-cr1 and uORF3-cr2 were obtained after sowing. The wild type rice was used as a control.

[0080] The plant phenotypes were observed 130 days after sowing, and the results are shown in Figure 2 b. It can be seen that, compared with the wild type rice, the T1 generation mutant transgenic pure line uORF3-cr1 and uORF3-cr2 both showed a slightly compact phenotype.

[0081] 2. Stem strength detection of uORF3 gene edited lines The stem bending force of T1 generation mutant transgenic pure line uORF3-cr1 and uORF3-cr2, Zhonghua 11 control plant (WT) planted under high nitrogen condition (300 kg / ha) was counted, and 14 single plants were counted for each material.

[0082] The stem bending force determination method is as follows: to measure the maximum bending force (or breaking force), the second internode below the panicle of different genetic materials was collected 40 days after the rice heading, and a stem breaking force detector (model: INSTRON 5582) was used to detect the maximum bending force that different rice stems could withstand using a three-point bending test with an 8 cm support span.

[0083] The results are shown in Figure 2 c. Compared with the Zhonghua control plant, the maximum bending force of the uORF3-cr1 and uORF3-cr2 homozygous mutant plant stem was significantly increased, indicating that the destruction of the uORF3 start codon (destruction of uORF3 nucleic acid translation) led to the increase of the rice stem strength.

[0084] 3. Cell wall thickness detection of uORF3 gene edited lines The cross-section of the stem of T1 generation mutant transgenic pure line uORF3-cr1 and control plant Zhonghua 11 (WT) was observed by scanning electron microscope to detect whether there was a change in secondary cell wall thickness; 84 cells of the control plant Zhonghua 11 (WT) and 66 cells of the T1 generation mutant transgenic pure line uORF3-cr1 were counted.

[0085] For scanning electron microscopy analysis of cell wall thickness, the second internode below the panicle of WT and T1 generation mutant transgenic homozygous rice strain uORF3-cr1 was sampled, and fresh tissues were transversely cut into about 3 cm thick sections and fixed with 4% (w / v) paraformaldehyde (PFA). The samples were dehydrated by ethanol gradient, critical point dried, and then sprayed with gold particles for scanning electron microscopy observation.

[0086] The results are shown in Figure 3 , a is the scanning electron microscopy observation result, b is the statistical result of a, compared with the control plant Zhonghua 11 (WT), the cell wall thickness of T1 generation mutant transgenic homozygous rice strain uORF3-cr1 is significantly increased, indicating that the increase of cell wall thickness leads to the increase of stem strength and lodging resistance.

[0087] 4. Lodging rate detection of uORF3 gene edited strain To determine the field lodging resistance of uORF3 gene edited strain, low nitrogen (50 kg / ha) and high nitrogen (300 kg / ha) two nitrogen fertilizer gradients were set, and control plant Zhonghua 11 (WT) and T1 generation mutant transgenic homozygous rice strain uORF3-cr1 were planted in the field. The lodging rate of different materials was counted at the mature stage.

[0088] The rice lodging occurrence rate (referred to as lodging rate) is defined as the ratio of the number of lodging plants to the total number of plants per unit area at the mature stage, and the lodging plant refers to a plant single plant with a bending of more than 45°. At 150 days after rice sowing (or 60 days after heading), the lodging rate of different materials in the unit area plot was counted, and each material contained three independent biological repeats.

[0089] The results are shown in Figure 4 , it can be seen that the control plant Zhonghua 11 and the gene edited material uORF3-cr1 did not occur lodging under low nitrogen conditions; the lodging rate of the control plant Zhonghua 11 was as high as 75% under high nitrogen conditions, while the lodging rate of the gene edited strain uORF3-cr1 was only 5.56%. It is shown that the uORF3 gene edited strain (destroying uORF3 nucleic acid translation) significantly improves the lodging resistance of rice under high nitrogen conditions.

[0090] The above has been described in detail. For those skilled in the art, without departing from the purpose and scope of the present application, and without unnecessary experiments, the present application can be implemented in a wide range under the same parameters, concentrations and conditions. Although the present application gives a special example, it should be understood that further improvements can be made to the present application. In general, according to the principle of the present application, this application intends to include any change, use or improvement of the present application, including changes made by conventional techniques known in the art, which deviates from the range disclosed in the present application. Some basic features can be applied within the scope of the following attached claims.

Claims

1. A substance which destroys the uORF of the OsTCP19 gene or an application of the substance which destroys the uORF of the OsTCP19 gene in at least one of the following: A1) increasing the lodging resistance of a plant; A2) breeding a lodging resistant plant; A3) preparing a product for increasing the lodging resistance of a plant; A4) increasing the stem strength of a plant; A5) breeding a plant with high stem strength; A6) preparing a product for increasing the stem strength of a plant; the OsTCP19 gene encodes a protein OsTCP19, the protein OsTCP19 is any one of the following: B1) the protein comprises the amino acid residues shown in SEQ ID NO: 2; B2) the protein is a protein having more than 80% amino acid residue identity with the protein shown in B1) and having the same function; B3) the protein comprises a tag connected to the N-terminus or / and C-terminus of the protein shown in B1) or B2); the nucleotide sequence of the uORF of the OsTCP19 gene is 5'-AUGGCGUGA-3'.

2. Use according to claim 1, characterized in that: the substance which destroys the uORF of the OsTCP19 gene is a nucleic acid targeting the uORF, an expression cassette containing the nucleic acid, a recombinant vector containing the nucleic acid, a recombinant microorganism containing the nucleic acid, or a transgenic plant cell line, tissue or organ containing the nucleic acid.

3. Use according to claim 2, characterized in that: the nucleic acid is siRNA, shRNA, sgRNA, miRNA and / or antisense RNA.

4. An application of the uORF of the OsTCP19 gene as a regulatory element or as a target for regulating the expression of the OsTCP19 gene in at least one of the following: A1) increasing the lodging resistance of a plant; A2) breeding a lodging resistant plant; A3) preparing a product for increasing the lodging resistance of a plant; A4) increasing the stem strength of a plant; A5) breeding a plant with high stem strength; A6) preparing a product for increasing the stem strength of a plant.

5. A method for increasing the lodging resistance of a plant, comprising the step of destroying the uORF of the OsTCP19 gene in a plant of interest to increase the lodging resistance of the plant of interest; the plant of interest contains the OsTCP19 gene and the uORF.

6. A method for increasing the stem strength of a plant, comprising the step of destroying the uORF of the OsTCP19 gene in a plant of interest to increase the stem strength of the plant of interest; the plant of interest contains the OsTCP19 gene and the uORF.

7. A method for breeding a lodging resistant plant, comprising the step of destroying the uORF of the OsTCP19 gene in a plant of interest to obtain a lodging resistant plant; the lodging resistant plant has greater lodging resistance than the plant of interest.

8. A method for breeding a plant with high stem strength, comprising the step of destroying the uORF of the OsTCP19 gene in a plant of interest to obtain a plant with high stem strength; the plant with high stem strength has greater stem strength than the plant of interest.

9. Use according to any one of claims 1 to 4 or method according to any one of claims 5 to 8, characterized in that: the lodging resistance is lodging resistance in a high-nitrogen planting environment.

10. A product, being any one of: 1) the substance of any one of claims 1 to 4; 2) the uORF of the OsTCP19 gene as described in any one of claims 1 to 4.

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