Protein for controlling grain number per ear of rice and coding gene thereof

Abstract

The invention discloses a protein for controlling the grain number per ear of rice and a coding gene thereof, and belongs to the technical field of plant genetic engineering. The method comprises the following steps: carrying out chemical mutagenesis on an indica rice restorer line 'Lehui 188' to obtain a mutant rub1, and carrying out high-throughput sequencing and MutMap analysis to obtain a candidate gene LOCOs04g41030 (OsBIR3). Researches show that the OsBIR3 gene significantly affects the number of branches on the upper portion of a rice panicle, and further affects the number of grains per panicle and the yield of rice. It is found for the first time that the OsBIR3 gene has the functions of positively regulating and controlling the number of branches on the upper portion of a rice spike, the number of grains per spike, the plant height, the maturing rate, the grain length and the thousand grain weight. The method lays a theoretical foundation for plant type improvement and genetic engineering breeding of rice, and has very important theoretical value and practical significance.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to an application of an OsBIR3 gene and / or a protein encoded by it in regulating the number of branches and stalks on the upper part of a rice panicle, the number of grains per panicle and the height of a plant. Background Art

[0002] Increasing rice yield per unit area is the primary measure to cope with the challenges of increasing world population, decreasing available arable land, and frequent extreme weather. The three factors of rice yield include the number of effective panicles, the number of grains per panicle, and grain weight, so the number of grains per panicle is one of the most important factors determining grain yield (Wang and Li, 2011).

[0003] Many rice panicle grain number genes have been cloned. These genes are involved in hormone synthesis and signaling pathways, transcriptional regulation or other metabolic pathways. For example, Gn1a encodes cytokinin oxidase / dehydrogenase (OsCKX2). Reduced expression of this gene leads to the accumulation of cytokinin in the inflorescence meristem, producing more primary branches, thereby increasing rice yield (Ashikari et al., 2005). GNP1 encodes rice gibberellin (GA) biosynthesis protein GA20 oxidase 1. Increased transcription level of GNP1 causes increased cytokinin content, leading to increased panicle grain number (Wu et al., 2016a). TSG1 is involved in the biosynthesis of auxin and also affects the signal transduction and transport of auxin during rice development. The auxin-deficient mutant tsg1 shows a reduction in panicle grain number (Guo et al., 2020). CPB1 is involved in the biosynthesis of brassinosteroids (BR), and the cpb1 mutant produces abnormal panicles with clustered primary branches in rice (Wu et al., 2016b). OsBAK1 is involved in BR signal transduction, regulating plant architecture, panicle size and number (Li et al., 2009; Shimin et al., 2014; Yuan et al., 2017).

[0004] BRs are a class of steroidal compounds that play an important role in regulating plant growth and development (Nolan et al., 2020; Xiong et al., 2022). In crops, BRs regulate many important agronomic traits, including plant height, tillering, leaf angle, grain size, flowering, senescence, and stress response (Tong and Chu, 2018). In rice, BR biosynthesis mutants such as brd1, d2, brd2, d11, cpb1, osdwarf4, oscpd1, and oscpd2 (Hong et al., 2002; Mori et al., 2002; Hong et al., 2003; Hong et al., 2005; Tanabe et al., 2005; Sakamoto et al., 2006; Zhan et al., 2022) and BR signaling mutants such as d61, tbp1 / sg2, dlt, dlt2smos1, and glw10 have been identified to date (Yamamuro et al., 2000; Zhao et al., 2013; Yuan et al., 2017; Lin et al., 2017; Tong et al., 2009; Zou et al., 2023; Qiao et al., 2024). 2017; Yuan et al., 2022). These BR-deficient and BR-insensitive rice mutants exhibit similar pleiotropic phenotypes, such as dwarfing, dark green leaves, curled leaves, reduced leaf angle, reduced fertility, and shortened grains (Tong and Chu, 2018). Nevertheless, among rice BR-related mutants, no mutants with significantly reduced number of branches on the panicle and thus significantly reduced number of grains per panicle have been reported.

[0005] The present invention discovered a gene OsBIR3 that regulates the number of rice grains per panicle and is related to the BR signaling pathway, cloned the gene, and studied its genetic and developmental mechanisms. After searching, the gene was not cloned in indica rice, and no related reports were found that OsBIR3 regulates the number of branches and stalks on the upper part of the rice panicle, thereby affecting the number of grains per panicle. The technical solution of the present invention lays a solid theoretical foundation for increasing rice yield, and has very important theoretical value and practical significance. Summary of the invention

[0006] In the present invention, the inventors conducted chemical mutagenesis on the indica rice restorer line "Lehui 188" (188R) and obtained a mutant rub1 (reduced upper branch 1) with a reduced number of upper branches on the panicle. The mutant showed a significant reduction in the number of upper branches on the panicle, resulting in a very significant reduction in the number of grains per panicle and a significant reduction in yield. Studies have found that the phenotype of the mutant is controlled by a pair of recessive nuclear genes. Gene cloning, transgenic complementation, gene knockout and other experiments have shown that the gene controlling the mutant trait is LOC_Os04g41030 (OsBIR3), which encodes an inactive receptor kinase protein (inactive receptor kinase precursor) without kinase activity and participates in the BR signaling pathway.

[0007] Based on this, the present invention provides the use of OsBIR3 gene and / or its encoded protein, expression vector containing OsBIR3 gene, and engineering bacteria containing the expression vector in regulating the number of branches and stalks on rice panicle, the number of grains per panicle and the plant height; the second object of the present invention is to provide a method for regulating the number of branches and stalks on rice panicle, the number of grains per panicle and the plant height; the third object of the present invention is to provide a method for increasing the number of branches and stalks on rice panicle, the number of grains per panicle and the plant height.

[0008] The present invention includes the following technical solutions:

[0009] In a first aspect, the present invention provides a method for detecting an OsBIR3 gene and / or its encoded protein for use in any of the following:

[0010] (a1) Application in prediction of rice traits or in the preparation of products for predicting rice traits;

[0011] (a2) Application in screening or assisting in screening individual rice plants, strains or varieties with a large number of grains per ear.

[0012] The DNA sequence of the OsBIR3 gene is selected from any one of the following nucleotide sequences:

[0013] (b1) the nucleotide sequence shown in SEQ ID NO: 1;

[0014] (b2) Adding and / or substituting and / or deleting one or several nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 to form a mutant gene or allele that can encode a protein with the same function.

[0015] The cDNA sequence of the OsBIR3 gene is shown in SEQ ID NO:2.

[0016] The amino acid sequence of the protein encoded by the OsBIR3 gene is selected from any one of the following amino acid sequences:

[0017] (c1) the amino acid sequence shown in SEQ ID NO: 3;

[0018] (c2) a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:3;

[0019] (c3) differs from the sequence shown in SEQ ID NO:3 by no more than 5, 4, 3, 2 or 1 amino acids;

[0020] (c4) A variant of SEQ ID NO: 3, wherein the variant differs from SEQ ID NO: 3 in that it has a substitution, a deletion and / or an insertion of one or more amino acid residues in the sequence or at least one N- / C-terminal extension.

[0021] The substance for detecting the OsBIR3 gene of the present invention is any one of the following:

[0022] (d1) a combination of PCR primers shown in SEQ ID NO: 5 and SEQ ID NO: 6;

[0023] (d2) a PCR reagent containing the PCR primer combination described in (d1);

[0024] (d3) A kit comprising the PCR primer composition described in (d1) and / or the PCR reagent described in (d2).

[0025] The substance for detecting the protein encoded by the OsBIR3 gene described in the present invention includes a reagent for detecting the protein encoded by the OsBIR3 gene by immunohistochemistry, or includes a reagent that can detect the expression amount of a nucleic acid related to the protein encoded by the OsBIR3 gene or the content of the protein encoded by the OsBIR3 gene, and the reagent includes but is not limited to an antibody, polypeptide, protein or nucleic acid molecule that binds to the protein encoded by the OsBIR3 gene.

[0026] In a specific embodiment of the present invention, the method for predicting rice traits comprises at least one of the following:

[0027] (e1) If a certain base deletion in the OsBIR3 gene leads to a frameshift mutation, the number of branches on the upper part of the rice panicle will decrease, the number of grains per panicle will decrease, and the plant height will decrease;

[0028] (e2) If the 104th base G of the OsBIR3 gene mutates to A, the number of branches on the upper part of the rice panicle decreases, the number of grains per panicle decreases, and the plant height decreases.

[0029] In a specific embodiment of the present invention, the method for screening or auxiliary screening of rice plants, strains or varieties with a large number of grains per panicle comprises: eliminating plants with a base deletion in the OsBIR3 gene resulting in a frameshift mutation or a mutation of the 104th base G to A, retaining plants with normal or high expression of the OsBIR3 gene, and selecting rice plants with a large number of branches on the upper part of the panicle, a large number of grains per panicle, and increased plant height.

[0030] In a second aspect, the present invention provides an expression vector containing an OsBIR3 gene for use in any of the following:

[0031] (f1) Application in regulating the number of branches on the upper part of rice panicle;

[0032] (f2) Application in regulating the number of grains per panicle in rice;

[0033] (f3) Application in regulating the growth of rice plants;

[0034] (f4) Application in screening or assisting in screening individual rice plants, strains or varieties with a large number of grains per ear;

[0035] (f5) Application in rice hybrid breeding;

[0036] (f6) Application in rice genetic engineering breeding.

[0037] In a third aspect, the present invention provides an application of an engineered bacterium in any of the following:

[0038] (g1) Application in regulating the number of branches on the upper part of rice panicle;

[0039] (g2) Application in regulating the number of grains per panicle in rice;

[0040] (g3) Application in regulating the growth of rice plants;

[0041] (g4) Application in screening or assisting in screening individual rice plants, strains or varieties with a large number of grains per ear;

[0042] (g5) Application in rice hybrid breeding;

[0043] (g6) Application in rice genetic engineering breeding.

[0044] The engineered bacteria comprises the OsBIR3 gene described in the first aspect of the present invention or the expression vector described in the second aspect of the present invention.

[0045] Specifically, the engineered bacteria are prepared by transforming the expression vector described in the second aspect of the present invention into a host bacteria, and the host bacteria include but are not limited to Escherichia coli or Agrobacterium.

[0046] The present invention constructs a functional complementary transgenic vector for the mutant rub1, namely an OsBIR3 overexpression vector, and transfects it into the mutant rub1, and the mutant rub1 has improved traits such as the number of branches on the panicle, the number of grains per panicle, and the plant height. Further, the present invention knocks out the OsBIR3 gene in rice based on the CRISPR / Cas9 gene editing system, and the number of branches on the panicle, the number of grains per panicle, the plant height, the fruit setting rate, the grain length, and the thousand-grain weight of the gene-knockout plants are significantly reduced, indicating that the OsBIR3 gene has the function of positively regulating the above traits of rice.

[0047] Based on this, in a fourth aspect of the present invention, the present invention provides a method for regulating the number of branches and stalks on the upper part of a rice panicle, the number of grains per panicle and the plant height, the method comprising transforming a rice cell using any of the following, and cultivating the transformed rice cell into a plant:

[0048] (h1) the OsBIR3 gene described in the first aspect of the present invention;

[0049] (h2) an expression vector containing the OsBIR3 gene described in (h1);

[0050] (h3) An engineered bacterium containing the expression vector described in (h2).

[0051] In a fifth aspect, the present invention provides a method for increasing the number of branches and stalks on the upper part of a rice panicle, the number of grains per panicle and the plant height, the method comprising the following steps:

[0052] (i1) Cultivation of rice embryonic callus;

[0053] (i2) The rice embryonic callus obtained in step (i1) was infected with an engineered bacterium containing an OsBIR3 gene overexpression vector by Agrobacterium transformation, and transgenic embryonic callus was obtained by resistance screening; transgenic embryonic callus was obtained by screening, differentiation, rooting and seedling hardening.

[0054] (i3) inducing the transgenic embryonic callus to differentiate, root, and harden the seedlings to obtain rice plants with increased number of branches on the panicle, number of grains per panicle, and plant height.

[0055] The technical solution provided by the present invention has the following technical advantages:

[0056] The inventors conducted chemical mutagenesis on the indica rice restorer line "Lehui 188" (188R) and obtained the mutant rub1, which showed a significant reduction in the number of branches on the panicle, resulting in a very significant reduction in the number of grains per panicle and a significant reduction in yield. Through high-throughput sequencing and MutMap analysis, the candidate gene LOC_Os04g41030 (OsBIR3) was obtained. According to research, the mutant phenotype is controlled by a pair of recessive nuclear genes. Cloning of the gene, construction of a transgenic complementation vector, and gene knockout experiments based on CRISPR / Cas9 showed that the OsBIR3 gene has the function of regulating the number of branches on the panicle of rice, thereby affecting the number of grains per panicle and yield of rice. In addition, the trait survey showed that the OsBIR3 gene also has an important effect on plant height, fruit setting rate, grain length and 1000-grain weight. The present invention first discovered that the OsBIR3 gene has the function of positively regulating the number of branches on the panicle of rice, the number of grains per panicle, plant height, fruit setting rate, grain length and 1000-grain weight. The research results of the present invention provide a theoretical basis and scientific basis for rice plant type improvement and genetic engineering breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 Phenotypic characteristics of rub1 mutants. (A, B) Plant and ear phenotypes of wild type (WT) 188R and mutant rub1, bar = 20 cm (A) or 5 cm (B). (C-I) Agronomic traits of wild type and mutants, including ear length, number of grains per ear, plant height, number of effective ears per plant, seed setting rate, 1000-grain weight, and grain length. (J) Morphology of young ears at different stages of wild type and mutants (ear lengths of ~1, ~2, ~3, ~7, ~10, ~16, and ~23 cm, respectively), bar = 5 cm. (K) Spike axis of wild type and mutants, red arrows indicate primary stalk nodes, bars = 5 cm. (L) Statistical analysis of the number of stalks per ear, the X-axis indicates the order of primary stalks from the bottom to the top of the ear, and the Y-axis indicates the number of secondary stalks on each primary stalk. (M, Q) Scanning electron micrographs of the apical meristem (SAM) of the wild type and mutants during the transition from vegetative growth to reproductive growth, Bars = 100 μm. (N, R) Scanning electron micrographs of the formation of primary branch primordium (PBP) of the wild type and mutants, Bars = 100 μm. (O, P, S, T) Scanning electron micrographs of the formation and development of primary branch primordium (PBP) of the wild type and mutants, Bars = 100 μm.

[0058] Figure 2MutMap analysis and molecular marker linkage mapping of the rub1 gene locus. (A) SNP index scatter plot showing that the rub1 mutation site is linked to the long arm of rice chromosome 4, and the red arrow indicates the mutation site. (B) 244 mutant plants were selected from the F2 population of rub1 and the japonica rice variety Nipponbare, and the rub1 locus was located in the region between the SSR marker RM303 and the InDel marker W1. (C) The rub1 locus was located in the region between the InDel markers W3 and W4. (D) The region between W3 and W4 is 280 kb long and contains the candidate gene LOC_Os04g41030. (E) The gene structure of LOC_Os04g41030, and the red arrow indicates that the mutation occurred at position 104 of LOC_Os04g41030, and the base G mutated to A, resulting in the mutation of the encoded amino acid cysteine ​​(Cys) to tyrosine (Tyr).

[0059] Figure 3 Verification of transgenic functional complementation. (A, B) Plant and ear phenotypes of wild type (WT), mutant rub1 and transgenic functional complementation (TP). (C) Sequencing detection of transgenic plants. (DI) Statistical analysis and comparison of the main agronomic traits (grains per ear, plant height, number of effective ears per plant, seed setting rate, 1000-grain weight, and grain length) of wild type (WT), mutant rub1 and transgenic functional complementation (TP) plants.

[0060] Figure 4 Gene knockout experiment. (A, B) Location and sequence of the knockout target in OsBIR3 (LOC_Os04g41030). (C) Sequencing alignment of the target sequence of japonica rice variety Zhonghua 11 (ZH11) and the knockout mutant (ko), showing that the knockout plant lacked 1 bp in the target sequence. (D-E) Plant and panicle phenotypes of ZH11 and ko. (FL) Statistical analysis and comparison of the main agronomic traits of ZH11 and ko plants (grains per panicle, plant height, number of effective panicles per plant, seed setting rate, grain length, grain width, and 1000-grain weight, respectively). DETAILED DESCRIPTION

[0061] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only some embodiments of the present invention, not all. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0062] Example 1 Gene Discovery

[0063] Phenotypic characteristics of mutant rub1

[0064] The present invention technicians conducted chemical mutagenesis on the indica rice restorer line "Lehui 188" (188R) and obtained a mutant rub1 (reduced upper branch 1) with a reduced number of upper branches on the panicle. The mutant showed a significant reduction in the number of upper branches on the panicle, resulting in a very significant reduction in the number of grains per panicle and a significant reduction in yield. In addition, the mutant also showed multiple phenotypes such as a significant reduction in plant height, fruit setting rate, grain length and 1,000-grain weight ( Figure 1 AI).

[0065] To understand the cause of abnormal ear development, we compared the morphology of the ears at different developmental stages (i.e., ear lengths of approximately ~1, ~2, ~3, ~7, ~10, ~16, and ~23 cm). Observations showed that at the ~1 to ~3 cm stage, the mutant ear tip was smaller; at the ~7 to ~10 cm stage, the branches on the upper part of the mutant ear were significantly sparse ( Figure 1 J). As the ear develops, the phenotypic differences of the branches on the ear become more and more obvious ( Figure 1 J). After heading, the internode length of adjacent primary branches on the rub1 spike axis was shorter than that of the wild type ( Figure 1 K). At the same time, no degeneration of the apical stalks was observed in rub1. Subsequently, we counted the number of secondary stalks on each primary stalk on the main spike axis. The results showed that there was no significant difference in the number of secondary stalks between the mutant and the wild type on the 1st to 11th primary stalks (from the bottom to the top of the spike), but the number of secondary branches on the 12th and 13th primary stalks decreased by about 2 to 3; on the 14th and above primary stalks, the rub1 mutant had no secondary stalks ( Figure 1 L).

[0066] In addition, the panicle formation process of wild-type and rub1 rice mutants was observed using scanning electron microscopy (SEM). The observations found that there was no significant difference in the morphology of the stem apical meristem (SAM) between the wild-type and rub1 mutants when the SAM transitioned from vegetative growth to reproductive growth ( Figure 1 M, Q), but the number of primary branch primordia produced by rub1 was 2-3 less than that of the wild type ( Figure 1 NP, RT).

[0067] Identification of rub1 candidate genes

[0068] Genetic analysis of the F1 and F2 generations of the hybrid of rub1 and 188R showed that the rub1 mutant phenotype was controlled by a pair of recessive nuclear genes. Twenty-five mutant plants with similar rub1 phenotypes were selected from the F2 population, and genomic DNA was extracted from equal amounts of leaves for high-throughput sequencing and MutMap analysis, resulting in the candidate gene LOC_Os04g41030 ( Figure 2A), which encodes an inactive receptor kinase precursor with no kinase activity. This protein is homologous to Arabidopsis BIR3, so the candidate gene is temporarily named OsBIR3. OsBIR3 is located on the long arm of chromosome 4, with a DNA sequence of 1869 bp and a cDNA of 1869 bp. It has no introns, encodes 622 amino acids, and has a molecular weight of about 66 kD. In the rub1 mutant, the 104th base of the OsBIR3 gene coding sequence mutated from G to A, resulting in the 35th amino acid sequence of the encoded protein mutated from cysteine ​​(Cys) to tyrosine (Tyr), and the Cys at this site is highly conserved in plants ( Figure 2 E). Subsequently, 244 mutant plants were selected from the F2 population of rub1 and the japonica rice variety Nipponbare. DNA was extracted from leaves of each plant and molecular genetic linkage analysis was performed using SSR and InDel molecular markers. The results showed that the rub1 locus was located in the 280kb interval between InDel markers W3 and W4 ( Figure 2 BD), further indicating that the candidate gene LOC_Os04g41030 is likely to be the target gene of rub1.

[0069] Example 2 Cloning of Rice OsBIR3 Gene

[0070] The following primer pairs were synthesized:

[0071]

[0072] DNA extracted from leaves of the wild-type parent 188R was used as a template, and primers P1 (underlined is the XbaⅠ restriction site) and P2 (underlined is the SalⅠ restriction site) were used for PCR amplification to obtain a fragment of about 1800 bp. The fragment was sequenced using primers P1-P4, and the correctly sequenced fragment was connected to the expression vector pMD-19T that was double-digested with XbaⅠ and SalⅠ, and the positive clones were screened and sequenced. The sequencing results showed that the OsBIR3 fragment obtained had the sequence shown in SEQ ID NO:1 in the sequence list, and its coding region was from position 15 to position 1883. The sequenced fragment was named OsBIR3.

[0073] Example 3 Obtaining the cDNA sequence of OsBIR1 gene

[0074] Subsequently, leaves of the wild-type parent 188R were taken at the tillering stage, and total RNA was extracted from the leaf samples using an RNA extraction kit, and then reverse transcription was performed using a reverse transcription kit to obtain cDNA.

[0075] Reverse transcription reaction system based on R223-01 kit:

[0076] ① Genomic DNA removal:

[0077]

[0078] After mixing by pipetting with a non-enzyme pipette, incubate at 42°C for 2 min in a PCR instrument, then immediately take out and place on ice;

[0079] ②Prepare the reverse transcription experimental system:

[0080]

[0081] After mixing by pipetting with an enzyme-free pipette, incubate at 50°C for 15 min and then at 85°C for 5 sec in a PCR instrument to obtain the first-strand cDNA.

[0082] The cDNA of the gene was amplified by RT-PCR using primers P1 and P2 to obtain a fragment of about 1800 bp, which was sequenced using primers P1-P4, and the correctly sequenced fragment was connected to the expression vector pC2300-35S-eGFP that had been double-digested with XbaⅠ and SalⅠ to construct the pC2300-35S-eGFP-OsBIR3 vector, and the positive clones were screened and sequenced. The sequencing results showed that the OsBIR3 fragment obtained had the sequence shown in SEQ ID NO:2 in the sequence table, and its coding region was from position 15 to position 1883, encoding 622 amino acids shown in SEQ ID NO:3 in the sequence table, and the molecular weight was about 66 kD.

[0083] Example 4 Construction of functional complementary transgenic vector

[0084] The following primer pairs were synthesized:

[0085]

[0086] The wild-type 188R DNA was used as a template and primers P5 and P6 (underlined are BamHI restriction sites) were used to amplify the gene DNA. The total length of the fragment was 6132 bp, and the specific sequence was shown in SEQ ID NO:4, including 3192 bp of OsBIR3 upstream sequence, 1869 bp of OsBIR3 gene sequence and 1071 bp of downstream sequence. The target fragment obtained by PCR was sequenced; then, the correctly sequenced target fragment was ligated to the expression vector pCAMBIA1300 digested with BamHI by homologous recombinase to construct a transgenic expression vector pCAMBIA1300-proOsBIR3-OsBIR3.

[0087] Example 5 Obtaining OsBIR3 transgenic functional complementary plants

[0088] The constructed transgenic expression vector pCAMBIA1300-proOsBIR3-OsBIR3 was used to infect the callus tissue of mutant rub1 seeds through Agrobacterium-mediated transformation. After screening, differentiation, rooting and seedling hardening, transgenic T0 seedlings were obtained. 19 positive transgenic plants were obtained, and the phenotypes of the positive transgenic plants in the offspring were restored to the level of wild type ( Figure 3 AC), such as the number of branches above the ear, the number of grains per ear, and the plant height, showed no significant differences from the wild type ( Figure 3 DI).

[0089] Example 6 Obtaining CRISPR / Cas9 knockout plants

[0090] Based on the nucleotide sequence of OsBIR3 gene, a knockout target site was designed at 182-201 from the gene start site ( Figure 4 AB). The sequence of OsBIR3 sgRNA is: GAGCCTGTGCCTGCTGTCG (SEQ ID NO: 11). The designed OsBIR3 sgRNA primers were annealed to form double strands and then connected to the CRISPR / Cas9 vector, and then OsBIR3 sgRNA::Cas9 was connected to the expression vector pCAMBIA1300. The constructed pCAMBIA1300:: OsBIR3 sgRNA::Cas9 expression vector was transferred into the japonica rice variety Zhonghua 11 (ZH11), and the OsBIR3 knockout transgenic lines obtained were sequenced and showed that these plants all lacked 1bp in the target sequence ( Figure 4 C). The knockout plants and wild-type ZH11 were similar to rub1 in terms of the number of branches above the panicle, the number of grains per panicle, and the plant height ( Figure 4 DE). Trait investigation showed that the number of grains per ear, plant height, fruit setting rate, grain length and 1000-grain weight of knockout transgenic plants were significantly reduced ( Figure 4 FL). This further proves the function of OsBIR3 gene, indicating that OsBIR3 regulates the number of branches on the upper part of rice panicle, the number of grains per panicle and other traits, thus significantly affecting rice yield.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the technical solutions described in the above embodiments may still be modified, or some or all of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a substance for detecting OsBIR3 gene and / or its encoded protein in any of the following: (a1) Application in prediction of rice traits or in the preparation of products for predicting rice traits; (a2) Application in screening or assisting in screening individual rice plants, strains or varieties with a large number of grains per ear.

2. The use according to claim 1, characterized in that: The DNA sequence of the OsBIR3 gene is selected from any one of the following nucleotide sequences: (b1) the nucleotide sequence shown in SEQ ID NO: 1; (b2) adding and / or substituting and / or deleting one or more nucleotides in the nucleotide sequence shown in SEQ ID NO: 1 to form a mutant gene or allele that can encode a protein with the same function; The amino acid sequence of the protein encoded by the OsBIR3 gene is selected from any one of the following amino acid sequences: (c1) the amino acid sequence shown in SEQ ID NO: 3; (c2) a sequence having a degree of identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:3; (c3) differs from the sequence shown in SEQ ID NO:3 by no more than 5, 4, 3, 2 or 1 amino acids; (c4) A variant of SEQ ID NO: 3, wherein the variant differs from SEQ ID NO: 3 in that it has a substitution, a deletion and / or an insertion of one or more amino acid residues in the sequence or at least one N- / C-terminal extension.

3. The use according to claim 1, characterized in that: The substance used to detect the OsBIR3 gene is any one of the following: (d1) a combination of PCR primers shown in SEQ ID NO: 5 and SEQ ID NO: 6; (d2) a PCR reagent containing the PCR primer combination described in (d1); (d3) a kit containing the PCR primer composition described in (d1) and / or the PCR reagent described in (d2); The substance used to detect the protein encoded by the OsBIR3 gene includes a reagent for detecting the protein encoded by the OsBIR3 gene by immunohistochemistry, or includes a reagent that can detect the expression amount of a nucleic acid related to the protein encoded by the OsBIR3 gene or the content of the protein encoded by the OsBIR3 gene, and the reagent includes an antibody, polypeptide, protein or nucleic acid molecule that binds to the protein encoded by the OsBIR3 gene.

4. The use according to claim 1, characterized in that: The method for predicting rice traits comprises at least one of the following: (e1) If a certain base deletion in the OsBIR3 gene leads to a frameshift mutation, the number of branches on the upper part of the rice panicle will decrease, the number of grains per panicle will decrease, and the plant height will decrease; (e2) If the 104th base G of the OsBIR3 gene mutates to A, the number of branches on the upper part of the rice panicle decreases, the number of grains per panicle decreases, and the plant height decreases.

5. The use according to claim 1, characterized in that: The method for screening or auxiliary screening of rice plants, strains or varieties with a large number of grains per panicle comprises: eliminating plants with a base deletion in the OsBIR3 gene leading to a frameshift mutation or a mutation of the 104th base G to A, retaining plants with normal or high expression of the OsBIR3 gene, and selecting plants with a large number of branches and stalks on the upper part of the rice panicle, a large number of grains per panicle, and increased plant height.

6. Use of an expression vector containing the OsBIR3 gene according to claim 1 in any of the following: (f1) Application in regulating the number of branches on the upper part of rice panicle; (f2) Application in regulating the number of grains per panicle in rice; (f3) Application in regulating the growth of rice plants; (f4) Application in screening or assisting in screening individual rice plants, strains or varieties with a large number of grains per ear; (f5) Application in rice hybrid breeding; (f6) Application in rice genetic engineering breeding.

7. Use of an engineered bacterium in any of the following: (g1) Application in regulating the number of branches on the upper part of rice panicle; (g2) Application in regulating the number of grains per panicle in rice; (g3) Application in regulating the growth of rice plants; (g4) Application in screening or assisting in screening individual rice plants, strains or varieties with a large number of grains per ear; (g5) Application in rice hybrid breeding; (g6) Application in rice genetic engineering breeding; The engineered bacteria comprises the OsBIR3 gene described in claim 1 or the expression vector described in claim 6.

8. The use according to claim 7, characterized in that: The engineered bacteria are prepared by transforming the expression vector described in claim 6 into a host bacterium, and the host bacterium includes Escherichia coli or Agrobacterium.

9. A method for regulating the number of branches and stalks on the upper part of a rice panicle, the number of grains per panicle and the plant height, the method comprising transforming a rice cell using any of the following, and cultivating the transformed rice cell into a plant: (h1) the OsBIR3 gene described in the first aspect of the present invention; (h2) an expression vector containing the OsBIR3 gene described in (h1); (h3) An engineered bacterium containing the expression vector described in (h2).

10. A method for increasing the number of branches and stalks on the upper part of a rice panicle, the number of grains per panicle and the plant height, the method comprising the following steps: (i1) Cultivating rice embryonic callus; (i2) The rice embryonic callus obtained in step (i1) was infected with an engineered bacterium containing an OsBIR3 gene overexpression vector by Agrobacterium transformation, and transgenic embryonic callus was obtained by resistance screening; transgenic embryonic callus was obtained by screening, differentiation, rooting and seedling hardening. (i3) inducing the transgenic embryonic callus to differentiate, root, and harden the seedlings to obtain rice plants with increased number of branches on the panicle, number of grains per panicle, and plant height.

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