GmSPO11-1 protein and its coding gene in apomixis breeding and sterile line creation

Abstract

This invention discloses the application of the GmSPO11-1 protein and its encoding gene in apomixis breeding and the creation of sterile lines. This invention utilizes CRISPR-Cas9 gene editing technology to knock out the GmSPO11-1 protein in soybeans. GmSPO11-1 Genes, Discovery GmSPO11-1 The mutant exhibits abnormal meiosis in its anthers or ovules, specifically due to... GmSPO11- 1 Gene knockout inhibits the formation of DSBs during meiosis and causes abnormal spindle assembly. The findings of this invention demonstrate that knocking out genes in soybeans… GmSPO11-1 GmSPO11-1 The gene can be used for apomixis breeding or to prepare sterile lines, and has significant application value for hybridization breeding and heterosis fixation of soybeans.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering technology, and in particular relates to the application of GmSPO11-1 protein and its encoding gene in apomixis breeding and the creation of sterile lines. Background Technology

[0002] In recent years, fully utilizing heterosis in the breeding of new soybean varieties has been crucial for further improving soybean quality and yield. Existing methods for utilizing soybean heterosis mainly fall into two categories: 1. The "three-line method" for soybean hybrid seed production and application: This method involves using a combination of sterile, restorer, and maintainer lines, aided by pollinating insects such as leafcutter bees, to produce hybrid seeds. 2. The "one-line method" for directly fixing soybean heterosis: One approach to fixing heterosis using the "one-line method" is synthetic apomixis—a special reproductive method that modifies the meiotic process to produce diploid gametophytes with undiminished chromosomes, allowing them to directly develop into embryos or seeds. Offspring produced using the "one-line method" have genotypes consistent with the maternal parent, ultimately achieving heterosis fixation. The production of diploid gametophytes requires alterations to three key aspects of the meiotic process: 1. Preventing homologous recombination between homologous chromosomes; 2. Premature separation of sister chromatids during the first meiotic division; 3. Skipping the second meiotic division. The changes in these three steps are crucial for the successful production of diploid gametes that are completely identical to the maternal parent's genetic material. Significant progress has been made in the research of the "three-line method" and the "one-line method" in rice, laying a solid foundation for the application of heterosis breeding in other crops.

[0003] The first step in diploid gamete induction is the suppression of homologous recombination, which is the molecular basis for ensuring the correct segregation of chromosomes and the generation of genetic diversity in eukaryotes. Homologous recombination begins with the formation of DNA double-strand breaks (DSBs) mediated by the topoisomerase SpO11. SpO11 belongs to the VIA family of archaea topoisomerase II. During meiosis, SpO11 forms a dimer with MTOPVIB from the same family to cut double-stranded DNA.

[0004] Soybeans are one of the world's most important dual-purpose crops (food and oil), and a vital source of plant protein and feed. As a strictly self-pollinating crop, soybean breeding has long relied on conventional hybridization. Research and utilization of the three-line and one-line methods for soybeans are crucial for accelerating the breeding of high-yield and high-quality new varieties. However, most existing germplasm resources cannot be directly used for the three-line system, particularly the limited availability of sterile lines, which severely restricts the research and utilization of the three-line method. Furthermore, genes related to the one-line method, especially those applicable to diploid gametes, are rarely reported, necessitating the discovery of relevant gene resources. Discovering more genes applicable to both the three-line and one-line methods will provide new gene resources for the seed production and application of three-line hybrid soybeans and for the fixation of heterosis in the one-line method. Summary of the Invention

[0005] This invention discovers GmSPO11-1 Mutations in this gene can prevent the formation of dimorphic spindle cells (DSBs) during soybean meiosis and lead to abnormal spindle assembly. It also causes the loss of homologous recombination and abnormal gamete division. Because this gene affects the initiation of homologous recombination and spindle assembly, cloning this gene can be linked to other genes in soybean. REC8 and TAM Gene combinations can be applied to the diploid gamete induction stage in synthetic apomixis or used alone to create sterile lines in the "three-line method" of hybrid seed production.

[0006] The first objective of this invention is to knock out GmSPO11-1 The application of genes in soybean apomixis breeding or the preparation of soybean sterile lines, the aforementioned GmSPO11-1 The gene encodes a protein with the amino acid sequence shown in SEQ ID NO.2.

[0007] Preferably, the aforementioned GmSPO11-1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Preferably, the application is knockout. GmSPO11-1 When applying genes in soybean apomixis breeding, knockout GmSPO11-1 Genes that inhibit DNA double-strand breaks during meiosis and reduce the number of homologous recombinations enable soybean apomixis breeding; The application described is the knockout of the GmSPO11-1 gene in the preparation of soybean male-sterile lines. GmSPO11-1 Genes cause abnormal spindle assembly during meiosis in soybeans, resulting in female sterility and / or male sterility.

[0009] Preferably, the knockout GmSPO11-1 The gene was knocked out using CRISPR-Cas9 gene editing technology. GmSPO11- 1 Gene.

[0010] Preferably, the knockout using CRISPR-Cas9 gene editing technology GmSPO11-1 The gene, whose sgRNA contains a guide sequence of nucleotides as shown in SEQ ID NO.3.

[0011] The beneficial effects of this invention are: This invention clones DSBs involved in the formation of soybean meiosis in soybean. GmSPO11-1 Gene. Knockout GmSPO11-1 Genes can suppress the formation of DSBs during plant meiosis. GmSPO11-1 Gene cloning provides new genetic resources for soybean apomixis breeding and has significant application value in this field. Specifically, knocking out or knocking down genes in soybeans... GmSPO11-1 Genes and soybeans REC8 and TAM The mutants are combined to induce diploid gametes that replace meiosis (MiMe) with mitosis, and hybrid vigor can be fixed by combining them with parthenogenesis or haploid induction.

[0012] Furthermore, this invention is the first to discover in soybeans... GmSPO11-1 Genes are involved in the spindle apparatus. This is achieved by reducing... GmSPO11-1 Gene expression levels can affect the normal spindle assembly process in plants, providing a new technical direction for the preparation of soybean male-sterile lines and having significant application value in three-line hybridization breeding. Attached Figure Description

[0013] Figure 1 To knock out Glyma.02G299600 ( GmSPO11-1 The effects of (a) on soybean development; where (a) is Glyma.02G299600 Gene structure, wild-type Huachun 6 (WT) and Gmspo11-1 (a) Genotype diagram of the mutant; (b) wild-type Huachun 6 and Gmspo11-1 Phenotypes of mutants during vegetative growth and development, scale bar 10 cm; (c) wild-type Huachun 6 and Gmspo11-1 The phenotype of the mutant during the reproductive development stage, specifically the phenotype of the pods, with a scale bar of 2 cm; (d)-(f) are wild-type Huachun 6, Gmspo11-1-1、Gmspo11-1-2 Alexandrine staining of anthers of the mutant, scale bar 100 µm; (g)-(i) are wild-type Huachun 6 and Gmspo11-1-1、Gmspo11-1-2 Toluidine blue staining of meiotic cells from mutant tetrads, scale bar 100 µm; (j) shows wild-type Huachun 6 and... Gmspo11-1-1、 Gmspo11-1-2 Statistics of different diatoms during the tetrad stage of the mutant; (k)-(m) represent wild-type Huachun 6 and... Gmspo11-1-1、Gmspo11-1-2 Image showing the results of transparent observation of the ovules of the mutant, with a scale bar of 25 µm.

[0014] Figure 2 For wild-type Huachun 6 and Gmspo11-1 Observation results of chromosome behavior of mutants; where (a)-(e) and (p)-(t) represent the chromosome behavior of wild-type Huachun 6, and (f)-(o), (u)-(z), and (aa)-(ad) represent the chromosome behavior of wild-type Huachun 6. Gmspo11-1 Mutant chromosome behavior; scale bar is 10 µm.

[0015] Figure 3 For wild-type Huachun 6 and Gmspo11-1 Immunofluorescence phenotypic analysis results of DSBs of mutants; where (a)-(f) show the autoantibody GmSPO11-1 in (a) early meristem stage of wild type, (b) late meristem stage of wild type, (c) zygote stage of wild type, (d) pachytene stage of wild type, and (e) other stages, respectively. Gmspo11-1-1 Thin period 、 (f) Gmspo11-1-2 Immunofluorescence during the leptomeningeal stage; (g)-(i) represent wild-type, Gmspo11-1-1 and Gmspo11-1-2 Immunofluorescence of the DSBs indicator marker protein γH2AX during the telogen phase; (j)-(l) represent wild-type, Gmspo11-1-1 and Gmspo11-1-2 Immunofluorescence observation results of GmDMC1 in the zygote phase, scale bar is 10 µm.

[0016] Figure 4 For wild-type Huachun 6 and Gmspo11-1 The results of spindle assembly observation in the mutant; where (a)-(c) are the results of spindle assembly observation in metaphase I, metaphase II, and telophase II of wild-type Huachun 6, respectively; (d)-(f) are the results of spindle assembly observation in the mutant, respectively. Gmspo11-1 Observation results of spindle assembly during metaphase I, metaphase II, and telophase II of the mutant meiosis. Detailed Implementation

[0017] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0018] The following embodiments are described GmSPO11-1 The gene number is Glyma.02G299600 The nucleotide sequence of its coding region is shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.2.

[0019] Example 1: GmSPO11-1 Acquisition of gene mutants Using the soybean variety Huachun 6 as an example, sgRNA containing a guide sequence such as SEQ ID NO.3 was designed using CRISPR-Cas9 gene editing technology for knockout. GmSPO11-1 Genes were used to obtain the corresponding mutants, and these mutants were named. Gmspo11-1 The following examples use two Gmspo11-1 Mutants, respectively Gmspo11-1-1 and Gmspo11-1-2, The specific mutation details of the obtained mutants are as follows: Figure 1 As shown in (a) above. During the vegetative growth and development stage, soybeans... Gmspo11-1 The vegetative growth of the mutant was not significantly different from that of the wild-type Huachun 6. Figure 1 (b)). During the reproductive development stage, the mutant is sterile, and the pods it produces are small fleshy pods ( Figure 1 (c)). Fertility analysis of the anthers using Alexandrine staining showed that: compared to wild-type Huachun 6 ( Figure 1 Compared to (d) in the middle, soybeans Gmspo11-1 mutant ( Figure 1 Pollen from (e) to (f) showed a complete sterility phenotype. Toluidine blue was used to investigate the sterility of wild-type Huachun 6 and soybean. Gmspo11-1 Fertility analysis of meiotic cells in the tetrad stage of the mutant showed that: soybean Gmspo11-1 The tetrad stage of the mutant exhibits a multidual morphology. Figure 1 (g)-(j)). Using ovule transparency observation technique to compare wild-type Huachun 6 and soybean. Gmspo11-1 The female fertility of the mutant was observed and analyzed, and the results showed that: soybean Gmspo11-1 The mutant's embryonic sac degenerates ( Figure 1 (k)-(m)). The above observations indicate that soybeans Gmspo11-1 The mutant exhibits abnormal development of male and female fertility, and the abnormality of the tetrad suggests that the mutant's meiotic process is abnormal.

[0020] Example 2: Gmspo11-1 Cellular phenotypic analysis of mutants To verify soybeans GmSPO11-1 Whether a gene participates in the meiotic process and thus affects the normal development of male and female gametophytes is investigated in this embodiment, focusing on wild-type Huachun 6 and... Gmspo11-1 The chromosome behavior of the mutant during meiosis was observed, and the procedure was as follows: 1. Wild-type Huachun 6 and 6, with lengths ranging from 1.3 to 1.8 mm and at different developmental stages, were selected respectively. Gmspo11-1The flower buds of the mutant were fixed in Carnoy's fixative at room temperature until completely decolorized.

[0021] 2. Wash the fixed flower buds three times with distilled water, place all the flower buds on lint-free paper to absorb the liquid, and then put them into PCR tubes containing enzymatic hydrolysate (3% pectinase (w / v), 3% cellulase (w / v), 3% cleavage enzyme (w / v), and the remainder is water). Incubate at 37°C for 90 min.

[0022] 3. Use a pipette to remove the enzymatic hydrolysate, wash the flower buds three times with distilled water, transfer them to a glass slide, and dissect the anthers under a microscope. Add an appropriate amount of distilled water to keep the anthers moist, and use tweezers to thoroughly crush the anthers to form a cell suspension.

[0023] 4. Place the slide on a 45°C heated stage, add 20 μL of 60% acetic acid aqueous solution, and gently stir with a pipette tip for 2 min. Add 2 drops of pre-chilled Carno fixative at -20°C to the center of the cell suspension (as high as possible) to allow it to diffuse. Once the slide is completely dry, mark the diffused cell area with an immunohistochemical pen (BC003, Biosharp) and store at -80°C. Alternatively, add 6-7 μL of DAPI (Vector Laboratories, H-1200-10), cover with a coverslip, and incubate in the dark for 5-10 min before microscopic examination and photography.

[0024] The results are as follows Figure 2 As shown, the prophase I of meiosis I, from the leptotene stage to the even-tene stage, Gmspo11-1 The mutant's chromosomal behavior is similar to that of the wild type. Figure 2 (a)-(b), (f)-(g), (k)-(l)). During pachytene, wild-type chromosomes complete synapsis. Figure 2 (c) in the middle, and Gmspo11-1 The mutant chromosomes failed to synapse. Figure 2 (h) and (m) in the diplotene stage). During the diplotene stage, the wild type exhibits distinct chamfered segment characteristics. Figure 2 (d) in the middle, and Gmspo11-1 The mutant showed that no crossing occurred among all homologous chromosomes. Figure 2 (i) and (n) in the text). During diakinesis, it can be observed that all homologous chromosomes in the wild type are connected by crossing over (i) Figure 2 (e) in Gmspo11-1 In the mutant, all homologous chromosomes are separated from each other. Figure 2 (j) and (o)). During metaphase I of meiosis, wild-type chromosomes are able to align orderly on the equatorial plate. Figure 2In (p), the mutant chromosomes cannot be arranged in an orderly manner on the equatorial plate, but instead exhibit a disordered or randomly dispersed distribution. Figure 2 In late I, wild-type sister chromatids correctly separate to the opposite poles of the spindle, ensuring accurate distribution of genetic material. Figure 2 (q) in, however, in Gmspo11-1 In the mutant, due to abnormal spindle assembly, sister chromatids cannot separate correctly. Figure 2 (v) and (aa)). During the second meiotic division, the wild-type sister chromatids separate to form a tetrad of four microspores. Figure 2 (r)-(t)), however, in Gmspo11-1 In the mutant, due to abnormal spindle assembly, multi-differences were formed ( Figure 2 (w)-(y), (ab)-(ad)).

[0025] Example 3: Gmspo11-1 Immunofluorescence phenotypic analysis of DSBs of mutants The slides prepared in the same steps 1-3 of Example 2 were dried on a 45°C hot plate. The cell diffusion areas were marked with an immunohistochemical pen and then placed in a wash tank containing Wash Buffer I (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, with the remainder being 1% Triton X-100 aqueous solution by volume) and soaked for 1 h.

[0026] Quickly add 100 μL of blocking buffer to the labeled area of ​​the slide and cover with a 24 mm × 32 mm coverslip. Incubate in a humidified chamber for 60 min. Dilute the corresponding primary antibodies (GmSPO11-1 antibody from rats, prepared in-house; γH2AX antibody (AF5836) from mice, purchased from Beyotime Biotechnology Co., Ltd.; and GmDMC1 antibody from rabbits, prepared in-house) with blocking buffer at a volume ratio of 1:400 to prepare antibody working solutions. Remove the coverslips and aspirate the buffer from the slides. Quickly add 70 μL of antibody working solution to the chromosome fragment labeling area of ​​each slide using the chromosomal fragmentation method. Cover with coverslips and incubate at 4°C for 48 h.

[0027] After incubation, remove the coverslips and wash them three times with Wash buffer II (137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.8 mM KH₂PO₄, with the remainder being 0.1% Tween 20 aqueous solution), 15 min each time. Then, add 100 μL of blocking buffer and block at 37°C for 1 h. Dilute the corresponding secondary antibodies 1:500 to obtain the working solutions. The rat (A48262), mouse (A48262), and rabbit (A11008) secondary antibodies were all purchased from Thermo Fisher Scientific. Add 70 μL of the working solution to each slide, cover with a coverslip, and incubate at 37°C for 1 h. Remove the coverslips and wash them three times with Wash buffer II, 15 min each time.

[0028] After slightly drying the slide in a dark room, add 9 μL of lipid-soluble DAPI, cover with a coverslip, and observe.

[0029] The results are as follows Figure 3 As shown, in wild-type Huachun 6, a large number of punctate signals of GmSPO11-1 autoantibodies can be clearly observed in the early stage of the thin line. Figure 3 (a) in the middle reaches its peak in the late stage of fine lines ( Figure 3 In (b) of the middle, the subsequent even-to-thickness and thoraco-to-thickness phases gradually decrease ( Figure 3 (c) and (d) in the text. And in... Gmspo11-1 and Gmspo11-2 No obvious GmSPO11-1 signal was observed during the thin-line period ( Figure 3 (e) and (f) in the text). Subsequently, the signal of the DSBs indicator protein γH2AX (when DNA double-strand breaks occur, histone H2AX is rapidly phosphorylated to γ-H2AX, forming signal spots around the break point) was observed. In wild-type Huachun 6, a large number of punctate signals of γH2AX antibody were clearly observed during the leptostectial stage. Figure 3 (g) in, while Gmspo11-1 and Gmspo11-2 There were no numerous dot-like signals during the thin-line phase. Figure 3 (h) and (i) in the text. Finally, we observed the signal of GmDMC1 (DMC1 (Disrupted Meiotic cDNA 1) is a recombinase belonging to the RecA family, responsible for catalyzing the invasion of single-stranded DNA into homologous double-stranded DNA to initiate homologous recombination). In wild-type Huachun 6, a large number of punctate signals of GmDMC1 antibody were clearly observed during the zygote stage. Figure 3 (j) in, while Gmspo11-1 and Gmspo11-2 No large number of point-like signals appeared during the even-term. Figure 3 (k) and (l) in the text. The immunofluorescence results of the autoantibodies GmSPO11-1, γ-H2AX, and GmDMC1 demonstrate that... Gmspo11-1 During meiosis in mutants, homologous stem cells (DSBs) cannot form. DSB formation is a prerequisite for homologous recombination, and the number of DSBs affects the quantity and distribution of homologous recombinations in the genome. For example, when the number of DSBs decreases, the number of recombinations also decreases accordingly, and their distribution changes, which can be used for apomixis (fixed heterosis) breeding in plants.

[0030] Example 4: Gmspo11-1 Spindle assembly phenotypic analysis of mutants Flower buds with a length of 1.5–2.0 mm were selected and fixed in a 2% formaldehyde solution for one hour, then washed three times with distilled water. The fixed buds were then placed in an enzymatic digestion solution (3% pectinase (w / v), 3% cellulase (w / v), 5% snailase (w / v), with the remainder being water) and incubated at 37°C for 2 hours, followed by washing three times with distilled water. Meiotic cells were dissected and extracted from the buds on a glass slide, and then crushed with forceps to prepare a cell suspension. The cell suspension was evenly spread on a glass slide using a syringe needle and air-dried at room temperature for later use.

[0031] First, immerse the slides in Wash buffer I (same as in Example 3) for 30 min. Then, add 100 μL of blocking buffer and block at 37°C for 1 h. Dilute β-Tubulin antibody (for labeling spindle fibers, AF2835, purchased from Beyotime Biotechnology Co., Ltd.) at a ratio of 1:2000, add 70 μL of the diluted β-Tubulin antibody to each slide, cover with a coverslip, and incubate at 4°C for 16 h.

[0032] Remove the coverslips and wash them three times with Wash Buffer II (same as in Example 3), 15 min each time. Then add 100 μL of blocking buffer and block at 37°C for 1 h. Dilute the mouse secondary antibody (same as in Example 3) at a ratio of 1:500, add 70 μL of the diluted secondary antibody to each slide, cover with coverslips, and incubate at 37°C for 1 h. Remove the coverslips and wash them three times with Wash Buffer II, 15 min each time.

[0033] After slightly drying the slide in a dark room, add 9 μL of lipid-soluble DAPI, cover with a coverslip, and observe.

[0034] The results are as follows Figure 4As shown, in wild-type Huachun 6, during metaphase I of meiosis in wild-type cells, chromosomes are neatly arranged on the equatorial plate and exhibit a clear bipolar spindle morphology. Figure 4 (a) in the middle; during metaphase II of meiosis, two bipolar spindles with normal orientation are visible ( Figure 4 (b)); In late stage II, the four separated nuclei assemble into a radial spindle structure to promote the formation of a normal tetrad. Figure 4 In contrast, in (c)). Gmspo11-1-2 In the mutant, the metaphase I spindle retains its bipolar structure, but its morphology changes from the normal spindle shape to an elliptical shape. Figure 4 (d)); Entering the mid-stage II, three spindle-shaped structures of different sizes and with disordered orientations appear ( Figure 4 (e)); By late stage II, abnormally oriented radial spindle structures form between the cell nuclei ( Figure 4 (f) in the middle. This proves that GmSPO11-1 Knockout of this substance can lead to abnormal spindle assembly during meiosis, and therefore can be applied to the creation of soybean male-sterile lines.

[0035] The above embodiments were created with Huachun-6 as the background. GmSPO11-1 The loss-of-function material of the gene demonstrates that this gene is involved in the formation of DSBs during meiosis, providing a basis for utilizing soybean... GmSPO11-1 This gene-based method provides a novel approach to creating diploid soybean gametes and offers new genetic resources for immobilizing heterosis in soybeans through apomixis, holding significant application value in soybean variety breeding. Furthermore, the above embodiments also demonstrate… GmSPO11-1 It plays an important role in the assembly of the spindle apparatus and affects the normal development of the gametophyte; therefore, soybeans are used in this process. GmSPO11-1 Genetically created soybean male-sterile lines have certain application potential and provide new genetic resources for three-line hybridization of soybeans, which has important application value in the breeding of new soybean varieties.

Claims

1. Knockout GmSPO11-1 The application of genes in soybean apomixis breeding or the preparation of soybean sterile lines is characterized by, The aforementioned GmSPO11-1 The gene encodes a protein with the amino acid sequence shown in SEQ ID NO.

2.

2. The application according to claim 1, characterized in that, The aforementioned GmSPO11-1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. The application according to claim 1, characterized in that, The application described is knockout. GmSPO11-1 When applying genes in soybean apomixis breeding, knockout GmSPO11-1 Genes that inhibit DNA double-strand breaks during meiosis and reduce the number of homologous recombinations enable soybean apomixis breeding.

4. The application according to claim 1, characterized in that, The application described is knockout. GmSPO11-1 When applying the gene to the preparation of soybean male-sterile lines, knockout GmSPO11-1 Genes cause abnormal spindle assembly during meiosis in soybeans, resulting in female sterility and / or male sterility.

5. The application according to claim 1, characterized in that, The aforementioned knockout GmSPO11-1 The gene was knocked out using CRISPR-Cas9 gene editing technology. GmSPO11-1 Gene.

6. The application according to claim 5, characterized in that, The aforementioned knockout using CRISPR-Cas9 gene editing technology GmSPO11-1 The gene, whose sgRNA contains a guide sequence of nucleotides as shown in SEQ ID NO.3.

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