Rice sbn1 protein, gene encoding same and use thereof

Abstract

The present application relates to rice SBN1 protein and its coding gene and application, and belongs to the technical field of genetic engineering. The present application provides application of overexpressed rice SBN1 gene in improving rice yield. The nucleotide sequence of the rice SBN1 gene is shown as SEQ ID NO. 3. The rice SBN1 gene is a key positive regulatory factor for controlling the development of primary branch, secondary branch and grain number per panicle. As an important gene resource, it not only helps to develop molecular markers to identify economically important alleles in rice germplasm in the practice of ideotype molecular breeding, but also provides a benchmark research example for improving rice grain yield.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to the rice SBN1 protein, its encoding gene, and its applications. Background Technology

[0002] Rice ( Rice As a major global food crop, rice provides staple food for more than half the population. Faced with the dual challenges of population growth and decreasing arable land, increasing rice yield per unit area is crucial for ensuring food security. During the development of the rice panicle, the inflorescence meristem forms multiple primary and secondary branches, and the number of branches directly affects the number of grains, ultimately determining rice yield. Therefore, increasing yield by enhancing the number of panicle branches has become an important research direction. Currently, [the text abruptly ends here, likely due to an incomplete sentence or missing information]. LAX1, LAX2, OSPL9 Single ear gene ( MOC1 ), Seed number gene ( RGN1 ), abnormal rachis tissue genes ( APO Ideal plant structure genes IPA1 ), Seed number 1a gene ( GN1a ), dense rachis genes ( DEP1 The study has identified several genes that regulate panicle branching, and has made breakthrough progress in areas such as screening of rice panicle mutants, gene cloning, and hormone regulation.

[0003] The rice growth cycle can be divided into a vegetative growth period and a reproductive growth period. During the vegetative growth period, leaf primordia continuously develop in the inner region of the shoot apical meristem (SAM) and differentiate into leaves. When photoperiod-induced conditions change, rice transitions from the vegetative growth period to the reproductive growth period. At this time, the spirally arranged shoot apical meristem guides the formation of the primary branch meristem (PBM), and then the secondary branch meristem (SBM) begins to differentiate in a double-row staggered pattern. Subsequently, the panicle meristem (SM) germinates on the primary and secondary branch primordia, including lateral panicle primordia and terminal panicle primordia. After developing to a certain extent, the inflorescence apical meristem stops growing and degenerates into a point (DP). Subsequently, secondary glumes and inner glumes differentiate sequentially within the panicle primordia.

[0004] Rice panicle structure exhibits diverse morphological characteristics in natural populations. Based on panicle weight, rice panicle structure can be classified into dense, short, erect, and sparse types. Based on the degree of neck curvature, it can be classified into curved panicles, semi-erect panicles, and erect panicles. With the development of genetics and molecular biology, specific genes or quantitative trait loci (QTLs) that regulate rice panicle structure have been discovered, and the genetic mechanisms of rice panicle development are gradually being elucidated.

[0005] The transition from meristem formation to spikelet meristem formation contributes to the shaping of the panicle rachis morphology. If this transition occurs prematurely, it leads to fewer branches, shorter branches, and a smaller panicle rachis; conversely, if the transition is delayed, it results in more branches, longer branches, and a larger panicle rachis. Once the panicle rachis-related meristems have differentiated, the elongation and development of branches then begin. Although the developmental mechanisms of branch meristems have been extensively studied, research on the elongation growth of rice branches remains very limited. Summary of the Invention

[0006] The purpose of this invention is to provide the rice SBN1 protein, its encoding gene, and its applications, in order to address the lack of rice-based technologies in existing fields. SBN1 The application of genes in improving rice yield and other aspects.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the rice SBN1 protein, its encoding gene, and its applications. SBN1 The nucleotide sequence of the gene is shown in SEQ ID NO.2; or Nucleotide sequences obtained by replacing, deleting, or adding one or more bases to the nucleotide sequence shown in SEQ ID NO.2; or A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO.2.

[0008] Preferably, increasing rice yield includes one or more of the following: increasing the number of primary branches, increasing the number of secondary branches, and increasing the number of grains per panicle.

[0009] The present invention also provides a recombinant plasmid, the recombinant plasmid comprising rice SBN1 Genes and empty vectors; The rice SBN1 The nucleotide sequence of the gene is shown in SEQ ID NO.2; or A nucleotide sequence obtained by replacing, deleting, or adding one or more bases to a nucleotide sequence as shown in SEQ ID NO.2; or A nucleotide sequence that is at least 90% identical to the nucleotide sequence shown in SEQ ID NO.2.

[0010] The present invention also provides a biomaterial comprising the recombinant plasmid described above.

[0011] Preferably, the biomaterial includes one or more of transgenic cell lines, engineered bacteria, host cells, and gene expression cassettes; The host cell can be a prokaryotic cell, yeast cell, insect cell, plant cell, or animal cell.

[0012] The present invention also provides the application of the aforementioned biomaterial in increasing rice yield, wherein increasing rice yield includes one or more of the following: increasing the number of primary branches, increasing the number of secondary branches, and increasing the number of grains per panicle of rice.

[0013] This invention also provides the application of the aforementioned biomaterials in rice marker-assisted breeding, cultivation and / or screening of high-yielding rice lines.

[0014] This invention also provides rice SBN1 Application of SNP sites in rice to improve rice yield, wherein the SNP sites are rice SBN1 The splicing site of exon 2, 8, or 12 of a gene; The second exon splicing site is located in rice. SBN1 The 26,398,319th position of the gene has an allele of C / T. The 8th exon splicing site is located in rice. SBN1 The 26,297,097th position of the gene has an allele of C / T. The splicing site of exon 12 is located in rice. SBN1 The 26401510th position of the gene has the C / T allele.

[0015] Preferably, increasing rice yield includes one or more of the following: increasing the number of primary branches, increasing the number of secondary branches, and increasing the number of grains per panicle.

[0016] This invention also provides rice SBN1 Application of SNP loci in marker-assisted breeding, cultivation, and / or screening of high-yielding rice lines in rice, wherein the SNP loci are rice SBN1 The splicing site of exon 2, 8, or 12 of a gene; The second exon splicing site is located in rice. SBN1 The 26,398,319th position of the gene has an allele of C / T. The 8th exon splicing site is located in rice. SBN1 The 26,297,097th position of the gene has an allele of C / T. The splicing site of exon 12 is located in rice. SBN1 The 26401510th position of the gene has the C / T allele.

[0017] The present invention has the following technical effects and advantages: This invention is the first to identify and confirm the rice SBN1Genes are key positive regulators controlling the development of primary and secondary branches and the number of grains per panicle in rice, and this has been fully verified from both positive and negative perspectives. SBN1 The function of genes is well-documented. Rice. SBN1 Spatial specificity of genes can promote branching, increase grain number, improve leaf morphology, increase stem size, and alter panicle type. This applies to rice. SBN1 After the gene was introduced into different rice varieties, the rice yield increased significantly, indicating that rice... SBN1 Genes can be used for the targeted design of rice panicle structure. As an important genetic resource, they not only help to develop molecular markers to identify economically important alleles in rice germplasm in ideal molecular breeding practices, but also provide benchmark research examples for improving rice grain yield. Attached Figure Description

[0018] Figure 1 It is a sparse spike mutant sbn1 The spike phenotype compared to wild-type SD16; Figure 2 For rice SBN1 A schematic diagram of gene location and structure; Figure 3 The vector is pCAMBIA1305.1- APFHN::SBN1 A schematic diagram of the structure; Figure 4 The vector is pCAMBIA1305.1- APFHN::SBN1 Transformation of sparse ear mutant sbn1 Allelic mutants SD33M808 Its phenotype can be restored; Figure 5 The vector is pCAMBIA1305.1- APFHN::SBN1 Transformation of sparse spike mutant sbn1 Allelic mutants SD33M1016 Its phenotype can be restored; Figure 6 The vector is pCAMBIA1305.1- APFHN::SBN1 Transformation of sparse ear mutant sbn1 Allelic mutants SD33M1060 Its phenotype can be restored; Figure 7 For the carrier CRSPR / Cas9:: SBN1 Structural diagram; Figure 8 For the carrier CRSPR / Cas9:: SBN1 Rice obtained by converting wild-type SD16 SBN1 Ear phenotypes of gene knockout lines B1015-1 and B1015-2; Figure 9 For rice SBN1Results of gene expression patterns in various tissues of rice. Detailed Implementation

[0019] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0020] The test material for this invention is the japonica rice variety SD16, which was obtained from the Institute of Crop Science, Chinese Academy of Agricultural Sciences. In the reagents of this invention, the qPCR high-script III cDNA synthesis kit was purchased from Thermo Fisher Scientific, Inc.

[0021] Example 1: Acquisition and Phenotypic Analysis of Mutants Three novel allelic sparse spikelet mutants were screened from the ethyl methanesulfonate (EMS) mutagenesis library of wild-type japonica rice SD16. sbn1 ( SD33M808、SD33M1016、SD33M1060 ),like Figure 1 As shown, wild-type SD16 and various sparse-ear mutants were recorded at the maturity stage. sbn1 The ear type traits, including ear length, number of primary branches, number of secondary branches, and number of grains per ear, are as follows: Figure 1 As shown.

[0022] The results showed that, compared with wild-type SD16, each sparse-ear mutant sbn1 The panicle exhibited the following significant characteristics: a significant reduction in the number of primary branches, secondary branches, and grains per panicle, resulting in a change from a dense panicle to a sparse panicle. This indicates that the three sparse-panicle mutants obtained through screening... sbn1 They all exhibit a similar phenotype with a reduced number of secondary branches.

[0023] Example 2: Obtaining candidate genes for rice Further, each sparse spike mutant sbn1 The wild-type SD16 was crossed with the wild-type SD16 to obtain F1 and F2 generations, and field surveys and sister line hybridization were conducted for verification. The wild-type SD16 was then crossed with various sparse-ear mutants. sbn1 Wild-type SD16 and various sparse-ear mutants were extracted after cultivation under natural field conditions. sbn1 The ear DNA was pooled and then subjected to batch separation and sequencing analysis (BSA sequencing analysis). The results are as follows: Figure 2 As shown.

[0024] Field surveys revealed that the secondary branching of the inflorescences in the F1 generation population developed normally, while the wild-type SD16 and various sparse-spike mutants in the F2 generation population showed different characteristics. sbn1 The segregation ratio is 3:1, indicating that the allelic mutants... SD33M808, SD33M1016, SD33M1060 The three corresponding recessive mutation sites regulate the number of secondary branches in rice; The results of sister line hybridization verification showed that each sparse-ear mutant sbn1 No new phenotypic differentiation appeared in the offspring, indicating that the above three recessive mutation sites are located at the same gene locus; BSA sequencing analysis results showed that each sparse spike mutant sbn1 Compared with wild-type SD16, it shows a high degree of differentiation on chromosome 2. SNP index, used to identify candidate genes LOC_Os02g43750 It is located on chromosome 2.

[0025] Example 3: Rice SBN1 Gene confirmation To verify LOC_Os02g43750 Whether gene mutations lead to changes in rice panicle morphology was investigated by constructing a complementary vector pCAMBIA1305.1 containing the promoter and full-length genomic DNA sequence within the pCAMBIA1305.1 vector. APFHN::SBN1 ,like Figure 3 As shown, it was transformed into the sparse-ear mutant using Agrobacterium-mediated transformation. sbn (1 SD33M808、SD33M1016、SD33M1060 Phenotypic complementary lines B1009(SBN1-1), B1010(SBN1-2), and B1011(SBN1-3) were obtained from the callus tissue of ), such as Figure 4~6 As shown.

[0026] LOC_Os02g43750 The full-length genomic DNA sequence of the gene is shown in SEQ ID NO.1.

[0027] SEQ ID NO.1: rice SBN1 The CDS sequence of the gene is shown in SEQ ID NO.2.

[0028] SEQ ID NO.2: rice SBN1 The amino acid sequence encoded by the gene is shown in SEQ ID NO.3.

[0029] SEQ ID NO.3: MSTQLTEHDHQPEQEPPHSENHLKQAEPNSFQFAEKETGYAGLQNFTGPKVDVGQTSGEQQHVKQMVGQQAPPGAQDARKRGYQPSIPFNMLIPILQAHLDRDKDMQLQTVWAKLRRNEVHKDDFLRVIRNIVGDQMLKQAAHKVFAQMQAQAQRSGQANANQANANQYSLQSQVSSSGSA QLHDQQVHVSTTPNQGQKNQALSSSQTFVQSGTQVQSSMTAHDNSIQRDAKGMHVTPNRPPVMNSAISAQTMNKQQQPTQVQQASQQIYGTTNRPDQPYTRPIGGSTPLSSLSSESEIRPSSHPAKMEILPSHPMTQQNAAAQQMQQNKDVKTNASNPRSNAKQDSGTGKGRAVGTGGSST KSQGKQGPPNFSTPPAAKSNKKTAGQKKSLETSGSTPPPPSKKQKTSGTFQEQSFDQLNDVTAVSGVNLREEEEQLLSAPKEESWASEEARKIAQEEDGKLFLQKGPLLKKLAAIVPKCNLKSIGGDVEHCLSMCVEERLRRFISTLIRVSKQRIDTEKSGHQLVITSDVGRQILRMNQKAK EEWDKKQAEETDKNKKQNEVDGGGTVELDKEKEETRSKNAKPNKEEDDKMRTTAANVAARQAVGGSDMLSKWQLMAEQARQKREGLDLAASSQRGTASRSHMAGKGPTDHHEASKRTHSAAFGTGGMNRQGRGPFAASHPKGPQRTISMKDVICVLEREPQMTKSRLIYRLYERLPGDSTRD The results showed that, compared with each sparse-ear mutant sbn1 In comparison, the number of primary branches, secondary branches, and grains per ear in each phenotypic complementary line were restored, reaching the level of wild-type SD16. (Note: This likely refers to a specific line or series of lines.) LOC_Os02g43750 Genes regulate various sparse spike mutants sbn1 The key gene for phenotype was named rice. SBN1 Gene; By comparing various sparse-ear mutants sbn1 By comparing the genome sequence with that of wild-type SD16, various sparse-ear mutants were discovered. sbn1 rice SBN1 The gene undergoes C→T base transitions at splice sites in exons 2, 8, and 12. These three SNP sites are located in rice. SBN1 The 26,398,319th, 26,297,097th, and 26,401,510th positions of the gene.

[0030] Example 4: Wild-type SD16 SBN1 gene knockout The sgRNA sequence was predicted using CRISPR-P software. The sequence was guide-9, with a prediction score of 99 and a maximum error of 0.3, located on the CDS negative strand at locus 26401683 on chromosome 2. The sgRNA expression cassette was obtained by ligating it into the intermediate vector pYLsgRNA-OsU3. Then, following the Golden Gate ligation method published in *Molecular Biology: A Laboratory Manual*, the sgRNA expression cassette was cloned into the plant binary vector pYLCRISPR / Cas9Pubi-H, resulting in the vector CRISPR / Cas9:: SBN1 ,like Figure 7 As shown; 12 rice variants were obtained by introducing Agrobacterium-mediated transformation into wild-type SD16. SBN1 Gene knockout lines B1015-1 to B1015-12, results are as follows Figure 8 As shown in Table 1, the primers for amplifying the sgRNA expression cassette used in the Golden Gate clone are also shown.

[0031] Table 1 Sequences of primers for sgRNA expression cassette amplification The results showed that, compared with wild-type SD16, rice SBN1 In the gene knockout lines, the number of primary branches, secondary branches, and grains per ear were significantly reduced in B1015-1 and B1015-2 lines, reaching the levels of the sparse-ear mutants. sbn1 The phenotypic level of rice proves SBN1 The key role of genes in the regulation of rice panicle type.

[0032] Example 5: Rice SBN1 Identification of gene expression patterns RNA libraries were constructed from vegetative tissues (roots, leaves, petioles, leaf sheaths, stems, tillers) and reproductive tissues (0.5cm, 1-3cm, and <5cm young panicles and spikelets) of wild-type SD16 at different developmental stages. cDNA was synthesized using the QuantScript III cDNA synthesis kit, and real-time quantitative PCR was used to detect the presence of cDNA in each tissue. SBN1 The transcriptional level of genes, the results are as follows Figure 9 As shown in Table 2. The primers for real-time quantitative PCR are listed below.

[0033] Table 2. Sequences of primers for real-time quantitative PCR The results showed that rice SBN1 The gene is expressed in all tissue-specific organs, but the expression levels differ between different tissues. In the later stages of rice development... SBN1 The transcriptional level of genes reaches its highest point in roots, leaf sheaths, young panicles, and mature seeds, indicating that rice... SBN1 Genes positively regulate the number of secondary branches in the spike through their specific activation in the secondary branch meristem.

[0034] As can be seen from the above embodiments, the present invention provides the rice SBN1 protein, its encoding gene, and its applications. The present invention confirms that rice... SBN1 Genes and their encoded proteins play a crucial role in regulating rice panicle structure. They are key positive regulators controlling the development of primary and secondary branches and the number of grains per panicle; loss of function leads to a significant reduction in the number of panicle branches and grains. As an important genetic resource, rice... SBN1 Genes not only help in developing relevant molecular markers for selecting superior alleles in molecular breeding, but also provide a theoretical basis and research paradigm for improving rice yield by regulating panicle type.

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Overexpression in rice SBN1 The application of genes in increasing rice yield is characterized by, The rice SBN1 The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The aforementioned method of increasing rice yield includes one or more of the following: increasing the number of primary branches, increasing the number of secondary branches, and increasing the number of grains per panicle.

3. The application of a biomaterial in increasing rice yield, characterized in that, The biomaterial includes recombinant plasmids, which include rice. SBN1 Genes and empty vectors; The rice SBN1 The nucleotide sequence of the gene is shown in SEQ ID NO.2; The aforementioned method of increasing rice yield includes one or more of the following: increasing the number of primary branches, increasing the number of secondary branches, and increasing the number of grains per panicle.

4. The application of a biomaterial in cultivating high-yield rice lines, characterized in that, The biomaterial includes recombinant plasmids, which include rice. SBN1 Genes and empty vectors; The rice SBN1 The nucleotide sequence of the gene is shown in SEQ ID NO.2.

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