Application of protein OsEAR9 in regulating rice plant height and yield

Abstract

This invention discloses the application of the protein OsEAR9 in regulating rice plant height and yield. The amino acid sequence of the protein OsEAR9 is shown in SEQ ID NO:1. Experiments have shown that overexpression of the OsEAR9 gene in rice yields transgenic rice; this transgenic rice exhibits significantly increased plant height, panicle length, number of primary branches, number of secondary branches, number of grains per panicle, and yield per plant. Inhibition of the OsEAR9 gene in rice yields OsEAR9 knockout rice, i.e., OsEAR9 homozygous mutants; the OsEAR9 homozygous mutants show significantly reduced plant height, panicle length, number of secondary branches, number of grains per panicle, and thousand-grain weight, but do not affect yield per plant. Therefore, the protein OsEAR9 can regulate rice plant height, yield, and panicle shape. This invention has significant application value.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically involving the application of protein OsEAR9 in regulating rice plant height and yield. Background Technology

[0002] In recent years, with researchers’ in-depth study of the molecular mechanisms of high yield in plants, the introduction of exogenous genes into plants using genetic engineering methods such as transgenics to increase yield has become one of the new ways to cultivate high-yielding plant varieties, which is of great significance to considerable and sustainable socio-economic and environmental development.

[0003] Rice is one of the world's most important food crops. With a continuously increasing population and a decreasing arable land area, increasing rice yield is a powerful measure to ensure global food security. However, rice production has stagnated for the past 20 years. Rice agronomic traits include plant height, tillering, grain size, and heading time. Generally speaking, shorter plant height increases the harvest index (seed-to-stalk ratio) and yield; the number of tillers directly affects the number of panicles, thus directly impacting yield; grain size, as a major factor determining seed weight, is also a crucial agronomic trait for rice yield; and heading time also determines whether rice can maximize yield. Furthermore, earlier heading time can help prevent the spread of plant diseases under certain conditions, avoiding yield losses. Therefore, improving rice agronomic traits has significant potential for improving rice quality and yield. Numerous genes related to rice agronomic traits have been reported, and these genes play an important role in improving rice quality. Summary of the Invention

[0004] The purpose of this invention is to increase rice yield.

[0005] This invention first protects the application of protein OsEAR9, which may be at least one of A1)-A6):

[0006] A1) Increase rice yield;

[0007] A2) Increase rice plant height;

[0008] A3) Change the panicle shape of rice;

[0009] A4) Developing genetically modified rice with increased yield;

[0010] A5) Developing transgenic rice with increased plant height;

[0011] A6) Develop transgenic rice with altered panicle shape.

[0012] The protein OsEAR9 described above can be b1), b2), b3), or b4):

[0013] b1) The amino acid sequence is that of the protein shown in SEQ ID NO:1;

[0014] b2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of b1);

[0015] b3) Proteins derived from rice and associated with rice yield, plant height and / or panicle shape, obtained by substituting and / or deleting and / or adding one or more amino acid residues as shown in b1) or b2).

[0016] b4) is a protein derived from rice and associated with rice yield, plant height and / or panicle shape, having 80% or more homology with the amino acid sequence defined by b1) or b2).

[0017] SEQ ID NO:1 consists of 105 amino acid residues.

[0018] To facilitate the purification of the protein in b1), a tag as shown in Table 1 can be attached to the amino or carboxyl terminus of the protein shown in SEQ ID NO:1.

[0019] Table 1. Sequence of Labels

[0020]

[0021]

[0022] The protein in b3) above, wherein the substitution and / or deletion and / or addition of one or more amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0023] The proteins mentioned in b3) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0024] The gene encoding the protein in b3) above can be obtained by deleting one or more amino acid residues from the codons in the DNA sequence shown in SEQ ID NO:3, and / or by performing a missense mutation on one or more base pairs, and / or by attaching the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0025] This invention also protects the application of nucleic acid molecules encoding any of the aforementioned proteins OsEAR9, which may be at least one of A1)-A6):

[0026] A1) Increase rice yield;

[0027] A2) Increase rice plant height;

[0028] A3) Change the panicle shape of rice;

[0029] A4) Developing genetically modified rice with increased yield;

[0030] A5) Developing transgenic rice with increased plant height;

[0031] A6) Develop transgenic rice with altered panicle shape.

[0032] In the above applications, the nucleic acid molecule may be a DNA molecule as shown in c1), c2), c3), c4), or c5):

[0033] c1) The coding region is the DNA molecule shown in SEQ ID NO:3;

[0034] c2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:3;

[0035] c3) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:2;

[0036] c4) has 75% or more homology with the nucleotide sequence defined by c1) or c2) or c3), and is derived from a rice DNA molecule that encodes any of the proteins described above, OsEAR9.

[0037] c5) hybridizes under strict conditions with a nucleotide sequence defined by c1) or c2) or c3) of a DNA molecule derived from rice that encodes any of the proteins described above, namely OsEAR9.

[0038] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0039] Of these, SEQ ID NO:2 consists of 452 nucleotides and SEQ ID NO:3 consists of 318 nucleotides. The nucleotides of SEQ ID NO:3 or SEQ ID NO:2 encode the amino acid sequence shown in SEQ ID NO:1.

[0040] Those skilled in the art can readily mutate the nucleotide sequence encoding the protein OsEAR9 of this invention using known methods, such as directed evolution and point mutation. Any artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the protein OsEAR9 of this invention, as long as they encode the protein OsEAR9, are derived from and equivalent to the nucleotide sequence of this invention.

[0041] As used herein, the term "identity" refers to sequence similarity to a natural nucleic acid sequence. "Identity" includes nucleotide sequences having 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein OsEAR9, which encodes the amino acid sequence shown in SEQ ID NO:1 of this invention. Identity can be evaluated visually or using computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0042] In any of the above applications, the yield may be the yield per plant.

[0043] In any of the above-described applications, the change in ear shape may manifest as an increase in ear length, number of primary branches, number of secondary branches, and / or number of grains in the main ear.

[0044] The present invention also protects a method for cultivating transgenic rice A, which may include the following steps: increasing the expression level and / or activity of any of the proteins OsEAR9 described above in recipient rice to obtain transgenic rice A; compared with recipient rice, transgenic rice A has increased yield, increased plant height and / or changed panicle shape.

[0045] In the above method, the yield can be the yield per plant.

[0046] In the above method, the change in ear shape can be manifested as an increase in ear length, number of primary branches, number of secondary branches, and / or number of grains in the main ear.

[0047] In the above method, the expression level and / or activity of any of the proteins OsEAR9 in the recipient rice can be increased by methods well known in the art, such as transgenic technology, multiple copies, alteration of promoters, and regulatory factors, to achieve the effect of increasing the expression level and / or activity of any of the proteins OsEAR9 in the recipient rice.

[0048] In the above method, increasing the expression level and / or activity of any of the proteins OsEAR9 in the recipient rice can be achieved by introducing a nucleic acid molecule encoding the protein OsEAR9 into the recipient rice.

[0049] In the above method, the nucleic acid molecule encoding the protein OsEAR9 (i.e., the gene encoding protein OsEAR9, the OsEAR9 gene) can be a DNA molecule as shown in c1), c2), c3), c4), or c5):

[0050] c1) The coding region is the DNA molecule shown in SEQ ID NO:3;

[0051] c2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:3;

[0052] c3) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:2;

[0053] c4) has 75% or more homology with the nucleotide sequence defined by c1) or c2) or c3), and is derived from a rice DNA molecule that encodes any of the proteins described above, OsEAR9.

[0054] c5) hybridizes under strict conditions with a nucleotide sequence defined by c1) or c2) or c3) of a DNA molecule derived from rice that encodes any of the proteins described above, namely OsEAR9.

[0055] The stringent conditions were: hybridization in a solution of 2×SSC and 0.1% SDS at 68°C, followed by two washes of 5 min each, and then hybridization in a solution of 0.5×SSC and 0.1% SDS at 68°C, followed by two washes of 15 min each.

[0056] The nucleic acid molecule can be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA or hnRNA.

[0057] The introduction of nucleic acid molecules encoding the protein OsEAR9 into the recipient rice can be achieved by introducing a recombinant vector containing any of the aforementioned nucleic acid molecules into the recipient rice.

[0058] The recombinant vector containing any of the above-mentioned nucleic acid molecules can be a recombinant plasmid obtained by inserting the OsEAR9 gene shown in SEQ ID NO:3 into the multiple cloning site of the expression vector.

[0059] The recombinant vector containing any of the above-mentioned nucleic acid molecules may specifically be a recombinant plasmid.

[0060] pCAMBIA1300-pUbi::EAR9. The recombinant plasmid pCAMBIA1300-pUbi::EAR9 can be used for...

[0061] The recognition site of the restriction endonuclease BamHI of the pCAMBIA1300 vector is inserted into the DNA molecule shown from position 1 to 315 from the 5' end of SEQ ID NO:3, and the small DNA fragment between the restriction endonucleases Pst I and Sal I is replaced with the DNA molecule shown in SEQ ID NO:4 to obtain the recombinant plasmid.

[0062] The present invention also protects a method for cultivating transgenic rice B, which may include the following steps: reducing the expression level and / or activity of any of the proteins OsEAR9 described above in recipient rice to obtain transgenic rice B; compared with recipient rice, transgenic rice B has reduced plant height, reduced thousand-grain weight and / or altered panicle shape.

[0063] In the above method, preferably, the change in ear shape can be manifested as a decrease in ear length, number of secondary branches and / or number of grains in the main ear.

[0064] In the above method, the reduction of the expression level and / or activity of any of the proteins OsEAR9 in the recipient rice can be achieved by mutating the encoding gene of the protein OsEAR9 in the recipient rice (i.e., the OsEAR9 gene).

[0065] In the above method, preferably, the gene encoding the protein OsEAR9 in the mutant recipient rice can be mutated by changing the OsEAR9 gene shown in SEQ ID No: 2 to OsEAR9 / -4bp / -3bp or OsEAR9 / -2bp / +1bp.

[0066] The OsEAR9 / -4bp / -3bp is a DNA molecule obtained by deleting positions 85 to 88 from the 5' end of SEQ ID No: 2 and positions 230 to 232 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

[0067] The OsEAR9 / -2bp / +1bp is a DNA molecule obtained by deleting positions 87 to 88 from the 5' end of SEQ ID No: 2 and inserting one nucleotide t between positions 230 and 231 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

[0068] In the above method, the encoding gene of the protein OsEAR9 in the mutant recipient rice is achieved by introducing a gene editing system into the recipient rice. The gene editing system may include a recombinant expression vector. The recombinant expression vector may contain a DNA molecule expressing gRNA targeting the encoding gene of the protein OsEAR9.

[0069] In the above method, preferably, the target sequence of the gRNA may be as shown in SEQ ID No: 2, positions 73-91 from the 5' end or SEQ ID No: 2, positions 227-245 from the 5' end.

[0070] In the above method, the recombinant expression vector may be the recombinant plasmid pYLCRISPR-EAR9 mentioned in the examples.

[0071] This invention also protects the use of material encoding the OsEAR9 protein (i.e., the OsEAR9 gene) in mutant recipient rice in the cultivation of transgenic rice B; compared with recipient rice, transgenic rice B has reduced plant height, reduced thousand-grain weight and / or altered panicle shape.

[0072] In the above applications, preferably, the change in ear shape can be manifested as a decrease in ear length, number of secondary branches, and / or number of grains in the main ear.

[0073] In the above applications, preferably, the mutation can be to mutate the OsEAR9 gene shown in SEQ ID No: 2 to OsEAR9 / -4bp / -3bp or OsEAR9 / -2bp / +1bp.

[0074] The OsEAR9 / -4bp / -3bp is a DNA molecule obtained by deleting positions 85 to 88 from the 5' end of SEQ ID No: 2 and positions 230 to 232 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

[0075] The OsEAR9 / -2bp / +1bp is a DNA molecule obtained by deleting positions 87 to 88 from the 5' end of SEQ ID No: 2 and inserting one nucleotide t between positions 230 and 231 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

[0076] In the above applications, preferably, the substance encoding the gene of any of the proteins OsEAR9 in the mutant receptor rice is as follows (B1) or (B2):

[0077] B1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the protein OsEAR9;

[0078] B2) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B1).

[0079] In the above applications, preferably, the nucleic acid molecule in B1) can be a DNA molecule expressing gRNA that targets the gene encoding the protein OsEAR9 or gRNA that targets the gene encoding the protein OsEAR9.

[0080] In the above applications, preferably, the target sequence of the gRNA may be as shown in SEQ ID No: 2, positions 73-91 from the 5' end or SEQ ID No: 2, positions 227-245 from the 5' end.

[0081] In the above applications, the recombinant vector mentioned in B2) can be the recombinant plasmid pYLCRISPR-EAR9 mentioned in the examples.

[0082] The recipient rice mentioned above can be the rice variety Kongyu 131 or the rice variety Nipponbare.

[0083] The genetically modified rice A mentioned above can be OE#1 or OE#2 mentioned in the examples.

[0084] The genetically modified rice B mentioned above may be cr-1 or cr-2 mentioned in the examples.

[0085] Experiments have shown that overexpression of the OsEAR9 gene in rice produces transgenic rice with significantly increased plant height, panicle length, number of primary branches, number of secondary branches, number of grains per panicle, and yield per plant. Suppressing the OsEAR9 gene in rice yields OsEAR9 knockout rice, i.e., OsEAR9 homozygous mutants; these mutants show significantly reduced plant height, panicle length, number of secondary branches, number of grains per panicle, and thousand-grain weight, but do not affect yield per plant. Therefore, the OsEAR9 protein can regulate rice plant height, yield, and panicle shape. This invention has significant application value. Attached Figure Description

[0086] Figure 1 To detect the relative expression level of the OsEAR9 gene in T2 generation homozygous transgenic OsEAR9 rice using real-time quantitative PCR.

[0087] Figure 2 This study aimed to identify agronomic traits in T2 generation homozygous transgenic OsEAR9 rice. Here, 'a' represents the mature plant phenotype and panicle phenotype, with a scale bar of 20 cm; 'bi' represents the statistical results of plant height, number of tillers per plant, panicle length, number of primary branches per panicle, number of secondary branches per panicle, number of grains per main panicle, 1000-grain weight, and yield per plant in T2 generation homozygous transgenic OsEAR9 rice. Values ​​are presented as mean ± standard deviation, with three technical replicates, each using 30 mature grains. A two-tailed t-test was used to analyze significant differences (* indicates a significant difference (P<0.05), ** indicates an extremely significant difference (P<0.01), and *** indicates a highly extremely significant difference (P<0.001)).

[0088] Figure 3 Partial sequencing results of the Cas9 target sequence for OsEAR9 homozygous mutants cr-1 and cr-2.

[0089] Figure 4This study aimed to identify the agronomic traits of homozygous OsEAR9 mutants. Here, 'a' represents the mature rice plant phenotype and panicle phenotype, with a scale bar of 20 cm; 'bi' represents the statistical results of plant height, number of tillers per plant, panicle length, number of primary branches per panicle, number of secondary branches per panicle, number of grains per main panicle, 1000-grain weight, and yield per plant for the homozygous OsEAR9 mutants. Values ​​are displayed as mean ± standard deviation, with three technical replicates, each using 30 mature grains. A two-tailed t-test was used to analyze significant differences (* indicates a significant difference (P<0.05), ** indicates an extremely significant difference (P<0.01), and *** indicates a highly extremely significant difference (P<0.001)). Detailed Implementation

[0090] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0091] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0092] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0093] The rice variety Nipponbare is described in the following literature: International Rice Genome Sequencing Project & Takuji Sasaki. (2005) The map-based sequence of the rice genome. Nature 436, 793-800. In the following examples, the rice variety Nipponbare is referred to as Nipponbare or NPB.

[0094] The rice variety Kongyu 131 is described in the following literature: Chang, Y., Nguyen, BH, Xie, YJ, Xiao, BZ, Tang, N., Zhu, WL, Mou, TM, Xiong, LZ (2017). Co-overexpression of the Constitutively Active Form of OsbZIP46 and ABA-Activated Protein KinaseSAPK6 Improves Drought and Temperature Stress Resistance in Rice. Front Plant Sci 8:1102. In the following examples, the rice variety Kongyu 131 is referred to as KY131.

[0095] The overexpression vector pCAMBIA1300 is described in the following literature: Roberts, C., Rajagopal, S., Smith, LM, Nguyen, TA, Yang, W., Nugrohu, S., Ravi, KS, Vijayachandra, K., Harcourt, RL, Dransfield, L., et al. (1997). A comprehensive set of modular vectors for advanced manipulations and efficient transformation of plants. pCAMBIA Vector Release Manual.

[0096] The knockout vector pYLCRISPR / Cas9-MH is described in the following literature: Ma X., Zhang Q., Zhu Q., Liu W., Chen Y., Qiu R., Wang B., Yang Z., Li H., Lin Y., Xie Y., Shen R., Chen S., Wang Z., Chen Y., Guo J., Chen L., Zhao X., Dong Z., and Liu Y.G. (2015). Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicotplants. Mol.Plant.doi:10.1016 / j.molp.2015.04.007.

[0097] Example 1: Discovery of the protein OsEAR9 and its encoding gene

[0098] The inventors of this application discovered the OsEAR9 gene in the genome of the rice variety Kongyu 131 through extensive experiments.

[0099] The nucleotide sequence of the OsEAR9 gene in the genomic DNA of rice variety Kongyu 131 is shown in SEQ ID NO:2.

[0100] The nucleotide sequence of the OsEAR9 gene in the cDNA of rice variety Kongyu 131 is shown in SEQ ID NO:3.

[0101] The OsEAR9 gene encodes the protein OsEAR9. The amino acid sequence of the protein OsEAR9 is shown in SEQ ID NO:1.

[0102] Example 2: Obtaining and identifying agronomic traits of T2 generation homozygous transgenic OsEAR9 rice.

[0103] 1. Construction of recombinant plasmid pCAMBIA1300-pUbi::EAR9

[0104] (1) Total RNA was extracted from rice variety Kongyu 131, followed by reverse transcription to obtain cDNA of rice variety Kongyu 131. Then, using the cDNA of rice variety Kongyu 131 as a template, pCAMBIA1300-EAR9-F:5'-GACTCTAGA was used for transcription. GGATCC ATGGCGGGAGGAGGGGTGTG-3' (underlined is the recognition site of the restriction endonuclease BamHI) and pCAMBIA1300-EAR9-R:

[0105] 5'-GCCGGACTT GGATCC PCR amplification was performed using a primer pair consisting of GGTGTGGAGGTCGACGACCT-3' (the underlined part is the recognition site of the restriction endonuclease BamHI), and approximately 350 bp of PCR amplification product 1 was recovered.

[0106] The PCR amplification product 1 contains the DNA molecule shown in SEQ ID NO:3 from position 1 to 315 starting from the 5' end.

[0107] (2) Genomic DNA was extracted from rice variety Kongyu 131 and used as a template. Primers pUbi F: 5'-CCAAGCTTGCATGC were used. CTGCAG TGCAGCGTGACCCGGTCGTG-3' (underlined is the recognition site of restriction endonuclease Pst I) and primer pUbi R:

[0108] 5'-ATGGATCCTCTAGA GTCGAC PCR amplification was performed using a primer pair consisting of AAGTAACACCAAACAACAGG-3' (the underlined part is the recognition site of restriction endonuclease Sal I), and approximately 2021 bp of PCR amplification product 2 was recovered.

[0109] PCR amplification product 2 contains the DNA molecule shown in SEQ ID NO:4, which is the Ubiquitin promoter.

[0110] (3) Homologous recombination was performed on the overexpression vector pCAMBIA1300, PCR amplification product 1 and PCR amplification product 2 to obtain the recombinant plasmid pCAMBIA1300-pUbi::EAR9.

[0111] The recombinant plasmid pCAMBIA1300-pUbi::EAR9 was sequenced. Sequencing results showed that the recombinant plasmid pCAMBIA1300-pUbi::EAR9 was obtained by inserting the DNA molecule shown in positions 1 to 315 from the 5' end of SEQ ID NO:3 into the recognition site of the restriction endonuclease BamHI in the pCAMBIA1300 vector, replacing the small DNA fragment between the restriction endonucleases Pst I and Sal I with the DNA molecule shown in SEQ ID NO:4.

[0112] The recombinant plasmid pCAMBIA1300-pUbi::EAR9 expresses the protein OsEAR9, whose amino acid sequence is shown in SEQ ID NO:1.

[0113] 2. Obtaining recombinant Agrobacterium

[0114] The recombinant plasmid pCAMBIA1300-pUbi::EAR9 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium, which was named EHA105 / pCAMBIA1300-pUbi::EAR9.

[0115] 3. Obtaining T2 generation homozygous transgenic OsEAR9 rice

[0116] EHA105 / pCAMBIA1300-pUbi::EAR9 was transformed into the rice variety Kongyu 131 using Agrobacterium infection. After screening, differentiation, and rooting, T0 generation OsEAR9 transgenic rice was obtained. The T0 generation OsEAR9 transgenic rice was self-pollinated and screened to obtain T1 generation OsEAR9 transgenic rice. The T1 generation OsEAR9 transgenic rice was self-pollinated and screened to obtain T2 generation homozygous OsEAR9 transgenic rice. The specific transformation and screening methods are referenced in the following literature: Yi Zili, Cao Shouyun, Wang Li, He Sijie, Chu Chengcai, Tang Zuoshun, Zhou Puhua, Tian Wenzhong. Study on increasing the frequency of Agrobacterium transformation in rice. Acta Genetica Sinica, 2001, 28(4): 352-358.

[0117] Two of the T2 generation homozygous transgenic OsEAR9 rice lines were named OE#1 and OE#2, respectively.

[0118] 4. Real-time quantitative PCR detection of the relative expression level of the OsEAR9 gene in T2 generation homozygous transgenic OsEAR9 rice.

[0119] The rice varieties to be tested were Kongyu 131, OE#1, or OE#2.

[0120] (1) Total RNA was extracted from the rice to be tested, and then reverse transcriptase was used to reverse transcribe the cDNA of the rice to be tested.

[0121] (2) Real-time quantitative PCR was used to detect the relative expression level of the OsEAR9 gene in the cDNA of the rice sample (with the OsActin1 gene as the internal reference gene).

[0122] The primers used to detect the OsActin1 gene are 5'-CAACACCCCGCTATGTACC-3' and 5'-CATCACCAGAGTCCAACACAA-3'.

[0123] Using the relative expression level of the OsEAR9 gene in rice variety Kongyu 131 as 1, the relative expression levels of the OsEAR9 gene in OE#1 and OE#2 were calculated respectively.

[0124] Test results are shown Figure 1 (KY131 is the rice variety Kongyu 131). The results showed that, compared with the rice variety Kongyu 131, the relative expression level of the OsEAR9 gene in OE#1 and OE#2 was significantly increased.

[0125] 5. Identification of agronomic traits in T2 generation homozygous transgenic OsEAR9 rice

[0126] Rice varieties (Kongyu 131, OE#1, or OE#2) were planted. The plant phenotype and panicle phenotype were observed at maturity. The plant height, number of tillers per plant, panicle length, number of primary branches per panicle, number of secondary branches per panicle, number of grains per main panicle, thousand-grain weight, and yield per plant were recorded at maturity. The sample size was 30 plants.

[0127] The experimental results are shown in Figure 2 (KY131 is the rice variety Kongyu 131). The results showed that, compared with the rice variety Kongyu 131, T2 generation homozygous transgenic rice varieties (such as OE#1 and OE#2) exhibited significantly increased plant height, panicle length, number of primary branches, number of secondary branches, number of grains per main panicle, and yield per plant. This indicates that increasing the expression level of the OsEAR9 protein in the rice variety Kongyu 131 can increase plant height, improve yield, and alter panicle shape; the changes in panicle shape were manifested in a significant increase in panicle length, number of primary branches, number of secondary branches, and / or number of grains per main panicle.

[0128] Example 3: Obtaining and identifying agronomic traits of the OsEAR9 homozygous mutant.

[0129] I. Obtaining the OsEAR9 homozygous mutant

[0130] Two target sites were selected for the experiment. The sequence of target site 1 is 5'-TTCAGGAACAACGGGGTGA-3' (i.e., SEQ ID No: 2, positions 73-91 from the 5' end), and the sequence of target site 2 is 5'-CCCTGCAGCTCCACAGGCG-3' (i.e., SEQ ID No: 2, positions 227-245 from the 5' end). The corresponding target gene for both is the OsEAR9 gene.

[0131] 1. Obtaining the recombinant plasmid pYLCRISPR-EAR9

[0132] (1) Primer EAR9-T1-F was designed and synthesized based on target 1:

[0133] 5'-GCCGTTCAGGAACAACGGGGTGA-3' and primer EAR9-T1-R: 5'-AAACTCACCCCGTTGTTCCTGAA-3' were used. Primers EAR9-T1-F and EAR9-T1-R were diluted to 100 μM with deionized water to obtain EAR9-T1-F and EAR9-T1-R dilutions, respectively. Annealing was then performed to form target fragment 1. Primer EAR9-T2-F was designed and synthesized based on target 2.

[0134] 5'-GTTGCGCCTGTGGAGCTGCAGGG-3' and primer EAR9-T2-R: 5'-AAACCCCTGCAGCTCCACAGGCG-3' were diluted to 100 μM with deionized water to obtain primer EAR9-T2-F dilution and primer EAR9-T2-R dilution, respectively; then annealing reaction was performed to form target fragment 2.

[0135] (2) Insert target fragment 1 into the recognition site of restriction endonuclease BsaI in plasmid pYLsgRNA-OsU6a to obtain intermediate vector 1. Insert target fragment 2 into the recognition site of restriction endonuclease BsaI in plasmid pYLsgRNA-OsU6b to obtain intermediate vector 2.

[0136] Plasmids pYLsgRNA-OsU6a and pYLsgRNA-OsU6b are described in the following literature: Ma X., Zhang Q., Zhu Q., Liu W., Chen Y., Qiu R., Wang B., Yang Z., Li H., Lin Y., Xie Y., Shen R., Chen S., Wang Z., Chen Y., Guo J., Chen L., Zhao X., Dong Z., and Liu YG (2015). Arobust CRISPR / Cas9 system for convenient, high-efficiency multiplex genomeediting in monocot and dicotplants. Mol.Plant.doi:10.1016 / j.molp.2015.04.007.

[0137] (3) Using intermediate vector 1 as a template, PCR amplification was performed using primer pair consisting of primer Uctcg-B1': 5'-TTCAGAGGTCTCTCTCGACTAGTGGAATCGGCAGCAAAGG-3' and primer gRctga-B2: 5'-AGCGTGGGTCTCGTCAGGGTCCATCCACTCCAAGCTC-3', and the PCR amplification product 1, approximately 629 bp, was recovered. Using intermediate vector 2 as a template, PCR amplification was performed using primer pair consisting of primer Uctga-B2': 5'-TTCAGAGGTCTCTCTGACACTGGAATCGGCAGCAAAGG-3' and primer gRcggt-BL: 5'-AGCGTGGGTCTCGACCGACGCGTCCATCCACTCCAAGCTC-3', and the PCR amplification product 2, approximately 515 bp, was recovered.

[0138] (4) Simultaneously insert PCR amplification product 1 and PCR amplification product 2 into the recognition site of the restriction endonuclease BsaI of the knockout vector pYLCRISPR / Cas9-MH to obtain the recombinant plasmid pYLCRISPR-EAR9.

[0139] 3. Obtaining the OsEAR9 homozygous mutant strain

[0140] Because rice is a diploid plant, when Cas9 begins to edit specific genes, both alleles on the two homologous chromosomes within the same cell can be edited, producing the same or different types of mutations. Therefore, two alleles in a plant are considered two gene editing events. A homozygous mutant is one where the OsEAR9 gene on both homologous chromosomes of the plant has the same mutation. A biallelic mutant is one where the OsEAR9 gene on both homologous chromosomes of the plant has been mutated, but in different forms. A heterozygous mutant is one where the OsEAR9 gene on one of the two homologous chromosomes of the plant has been mutated, while the OsEAR9 gene on the other homologous chromosome has not been mutated. A wild-type is one where the OsEAR9 gene on neither of the two homologous chromosomes of the plant has been mutated.

[0141] (1) The recombinant plasmid pYLCRISPR-EAR9 was introduced into Agrobacterium tumefaciens EHA105 to obtain recombinant Agrobacterium.

[0142] (2) Recombinant Agrobacterium was transformed into the rice variety Nipponbare using the Agrobacterium infection method. After screening, differentiation and rooting, T0 generation transgenic rice was obtained. The specific transformation and screening methods are referenced in the following literature: Yi Zili, Cao Shouyun, Wang Li, He Sijie, Chu Chengcai, Tang Zuoshun, Zhou Puhua, Tian Wenzhong. Study on increasing the frequency of Agrobacterium transformation in rice. Acta Genetica Sinica, 2001, 28(4): 352-358.

[0143] (3) Using genomic DNA from leaves of T0 generation transgenic rice as templates, the target region was amplified by PCR to obtain PCR amplification products. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with the Cas9 target sequence of the OsEAR9 gene (SEQ ID No: 2) to identify mutation types. Several heterozygous mutant strains were obtained.

[0144] (4) Self-pollinate the heterozygous mutant obtained in step (3) to obtain the seeds, which are the T1 generation seeds. The plants grown from the T1 generation seeds are the T1 generation plants. Self-pollinate the T1 generation plants to obtain the seeds, which are the T2 generation seeds. The plants grown from the T2 generation seeds are the T2 generation plants.

[0145] (5) Using genomic DNA from leaves of T2 generation plants as templates, the target region was amplified by PCR to obtain PCR amplification products. The PCR amplification products were then subjected to Sanger sequencing. The sequencing results were compared with the Cas9 target sequence of the OsEAR9 gene (SEQ ID No: 2) to identify mutation types.

[0146] Two homozygous OsEAR9 mutant strains were ultimately obtained, named cr-1 and cr-2, respectively. Partial sequencing results for cr-1 and cr-2 are shown below. Figure 3 (NPB stands for Nipponbare rice variety).

[0147] The OsEAR9 gene on both homologous chromosomes of cr-1 has the same mutation. Specifically, the OsEAR9 gene on both homologous chromosomes has a deletion of 4 nucleotides "gggg" (i.e., deletion at positions 85 to 88 from the 5' end in SEQ ID No: 2) and a deletion of 3 nucleotides "tgc" (i.e., deletion at positions 230 to 232 from the 5' end in SEQ ID No: 2), which causes a frameshift and results in the loss of function of the protein OsEAR9.

[0148] The OsEAR9 gene on both homologous chromosomes of cr-2 has the same mutation. Specifically, the OsEAR9 gene on both homologous chromosomes has a deletion of 2 nucleotides "gg" (i.e., deletion at positions 87 to 88 from the 5' end of SEQ ID No: 2) and an insertion of a single nucleotide "t" (i.e., insertion between positions 230 and 231 from the 5' end of SEQ ID No: 2), which causes a frameshift and results in the loss of function of the protein OsEAR9.

[0149] II. Identification of agronomic traits of OsEAR9 homozygous mutants

[0150] The rice varieties to be tested (Nipponbare, CR-1 or CR-2) were planted. The plant phenotype and panicle phenotype of the rice were observed at maturity. The plant height, number of tillers per plant, panicle length, number of primary branches per panicle, number of secondary branches per panicle, number of grains per main panicle, thousand-grain weight and yield per plant were recorded. The sample size was 30 plants.

[0151] The experimental results are shown in Figure 4 (NPB represents the rice variety Nipponbare). The results showed that, compared to the rice variety Nipponbare, homozygous OsEAR9 mutants (such as cr-1 and cr-2) exhibited significantly reduced plant height, panicle length, number of secondary branches per panicle, number of grains per main panicle, and thousand-grain weight, but did not affect yield per plant. Therefore, reducing the expression level of the protein OsEAR9 in the rice variety Nipponbare can reduce plant height, reduce thousand-grain weight, and alter panicle shape; the changes in panicle shape manifested as a significant reduction in panicle length, number of secondary branches per panicle, and / or number of grains per main panicle.

[0152] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of protein OsEAR9 is at least one of A1)-A6): A1) Increase rice yield; A2) Increase rice plant height; A3) Change the panicle shape of rice; A4) Developing genetically modified rice with increased yield; A5) Developing transgenic rice with increased plant height; A6) Developing transgenic rice with altered panicle shape; The protein OsEAR9 is as follows (b1), (b2), (b3), or (b4): b1) The amino acid sequence is that of the protein shown in SEQ ID NO:1; b2) The fusion protein obtained by attaching a tag to the N-terminus and / or C-terminus of b1); b3) Proteins derived from rice and associated with rice yield, plant height and / or panicle shape, obtained by substituting and / or deleting and / or adding one or more amino acid residues as shown in b1) or b2). b4) is a protein derived from rice and associated with rice yield, plant height and / or panicle shape, having 80% or more homology with the amino acid sequence defined by b1) or b2).

2. The application of a nucleic acid molecule encoding the protein OsEAR9 described in claim 1, comprising at least one of A1)-A6): A1) Increase rice yield; A2) Increase rice plant height; A3) Change the panicle shape of rice; A4) Developing genetically modified rice with increased yield; A5) Developing transgenic rice with increased plant height; A6) Develop transgenic rice with altered panicle shape.

3. The application according to claim 2, characterized in that: The nucleic acid molecule is a DNA molecule as shown in c1), c2), c3), c4), or c5): c1) The coding region is the DNA molecule shown in SEQ ID NO:3; c2) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:3; c3) The nucleotide sequence is the DNA molecule shown in SEQ ID NO:2; c4) has 75% or more homology with the nucleotide sequence defined by c1) or c2) or c3), and is derived from rice and is a DNA molecule encoding the protein OsEAR9 of claim 1. c5) hybridizes under stringent conditions with a nucleotide sequence defined by c1) or c2) or c3) of a DNA molecule derived from rice and encoding the protein OsEAR9 of claim 1.

4. The application according to any one of claims 1 to 3, characterized in that: The yield mentioned is the yield per plant; The changes in ear shape are manifested as an increase in ear length, number of primary branches, number of secondary branches, and / or number of grains in the main ear.

5. A method for cultivating transgenic rice A, comprising the following steps: increasing the expression level and / or activity of the protein OsEAR9 described in claim 1 in recipient rice to obtain transgenic rice A; compared with recipient rice, transgenic rice A has increased yield, increased plant height and / or changed panicle shape; Preferably, the yield is the yield per plant; Preferably, the change in ear shape is manifested as an increase in ear length, number of primary branches, number of secondary branches, and / or number of grains in the main ear.

6. The method according to claim 5, characterized in that: The improvement in the expression level and / or activity of the protein OsEAR9 of claim 1 in the recipient rice is achieved by introducing a nucleic acid molecule encoding the protein OsEAR9 into the recipient rice.

7. A method for cultivating transgenic rice B, comprising the following steps: reducing the expression level and / or activity of the protein OsEAR9 described in claim 1 in recipient rice to obtain transgenic rice B; compared with recipient rice, transgenic rice B has reduced plant height, reduced thousand-grain weight and / or altered panicle shape; Preferably, the change in ear shape is manifested as a decrease in ear length, number of secondary branches, and / or number of grains in the main ear.

8. The method according to claim 7, characterized in that: The reduction of the expression level and / or activity of the protein OsEAR9 in claim 1 in the recipient rice is achieved by mutating the gene encoding the protein OsEAR9 in the recipient rice. Preferably, the gene encoding the protein OsEAR9 in the mutant receptor rice is a mutation of the OsEAR9 gene shown in SEQ ID No: 2 to OsEAR9 / -4bp / -3bp or OsEAR9 / -2bp / +1bp; The OsEAR9 / -4bp / -3bp is a DNA molecule obtained by deleting positions 85 to 88 from the 5' end of SEQ ID No: 2 and deleting positions 230 to 232 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged; The OsEAR9 / -2bp / +1bp is a DNA molecule obtained by deleting positions 87 to 88 from the 5' end of SEQ ID No: 2 and inserting one nucleotide t between positions 230 and 231 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged.

9. The method according to claim 8, characterized in that: The gene encoding the protein OsEAR9 in the mutant recipient rice was obtained by introducing a gene editing system into the recipient rice; the gene editing system includes a recombinant expression vector; the recombinant expression vector contains a DNA molecule that expresses gRNA targeting the gene encoding the protein OsEAR9; Preferably, the target sequence of the gRNA is as shown in SEQ ID No: 2, positions 73-91 from the 5' end or SEQ ID No: 2, positions 227-245 from the 5' end.

10. The application of the material encoding the protein OsEAR9 described in claim 1 in mutant recipient rice in the cultivation of transgenic rice B; compared with recipient rice, transgenic rice B has reduced plant height, reduced thousand-grain weight and / or altered panicle shape; Preferably, the change in ear shape is manifested as a decrease in ear length, number of secondary branches and / or number of grains in the main ear; Preferably, the mutation is to mutate the OsEAR9 gene shown in SEQ ID No: 2 to OsEAR9 / -4bp / -3bp or OsEAR9 / -2bp / +1bp; The OsEAR9 / -4bp / -3bp is a DNA molecule obtained by deleting positions 85 to 88 from the 5' end of SEQ ID No: 2 and deleting positions 230 to 232 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged; The OsEAR9 / -2bp / +1bp is a DNA molecule obtained by deleting positions 87 to 88 from the 5' end of SEQ ID No: 2 and inserting one nucleotide t between positions 230 and 231 from the 5' end of SEQ ID No: 2, while keeping the other nucleotide sequences of SEQ ID No: 2 unchanged; Preferably, the substance encoding the protein OsEAR9 of claim 1 in the mutant receptor rice is as follows (B1) or (B2): B1) Nucleic acid molecules that inhibit or reduce the expression of the gene encoding the protein OsEAR9; B2) Expression cassettes, recombinant vectors, recombinant microorganisms, or transgenic plant cell lines containing the nucleic acid molecules described in B1); Preferably, B1) the nucleic acid molecule is a DNA molecule that expresses gRNA that targets the gene encoding the protein OsEAR9 or is gRNA that targets the gene encoding the protein OsEAR9. Preferably, the target sequence of the gRNA is as shown in SEQ ID No: 2, positions 73-91 from the 5' end or SEQ ID No: 2, positions 227-245 from the 5' end.

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