Method for regulating meiotic crossover recombination based on HEI10 phosphorylation

Abstract

This invention discloses a method for regulating meiotic crossing recombination based on HEI10 phosphorylation. The invention replaces S221, T224, S250, and S287 in the disordered region of HEI10 with alanine to construct a HEI10 phosphorylation-deficient mutant. It was found that HEI10 phosphorylation-deficient mutants inhibit phase separation and aggregation dynamics on chromosomes, significantly increasing recombination quantity, promoting the redistribution of recombination to centromere neighborhoods, and eliminating recombination interference effects. This invention is the first to reveal that a single phosphorylation regulation process can simultaneously control the quantity, distribution, and interference of recombination. The method provided by this invention does not rely on disrupting the synaptic complex structure and is applicable to Arabidopsis thaliana and various crops (including rice, maize, soybean, and wheat), possessing significant application value in improving genetic recombination efficiency, breaking linkage arrest, and accelerating breeding processes.

Description

Technical Field

[0001] This invention belongs to the fields of molecular genetics, meiosis regulation mechanisms and crop genetics and breeding technology, specifically involving a method for regulating meiotic crossing recombination based on HEI10 phosphorylation. Background Technology

[0002] Chromosomal crossing over and recombination during meiosis are crucial for the proper segregation of homologous chromosomes and the generation of genetic diversity. Although numerous DNA double-strand breaks (DSBs) occur during meiosis, only a small fraction are repaired as recombination events, and this process is tightly regulated.

[0003] Existing research indicates that meiotic recombination has the following typical characteristics: First, the number of recombinations is strictly limited, with each pair of homologous chromosomes generally having 1-3 recombination events; second, the distribution of recombinations on chromosomes is significantly non-random, usually concentrated in the distal regions of chromosomes, while being significantly suppressed in the centromere neighborhood; in addition, there is a "recombination interference" phenomenon, that is, the formation of a recombination event will inhibit the re-occurrence of recombination in its neighboring regions, thus resulting in a spatially spaced distribution of recombinations.

[0004] HEI10, a core member of the ZMM protein family, plays a crucial role in the formation of Class I recombinations (COs), and its expression level is positively correlated with the number of recombinations. In Arabidopsis thaliana, under wild-type conditions, female gametes produce an average of approximately 2.8 recombinations, while males produce approximately 5.2. Increasing HEI10 expression levels can boost the number of recombinations to approximately 6.2 in females and 9.9 in males, but this strategy only achieves a limited increase and still retains a significant recombination interference effect.

[0005] Furthermore, the number of recombinations can also be increased by regulating other recombination-related factors. For example, the synaptonemal complex (SC) components... ZYP1 In the mutants, females and males had approximately 7.1 and 7.0 recombinations, respectively, and recombination interference disappeared; anti-recombination factor RECQ4 mutants ( recq4ab In this study, females had approximately 17.5 recombinants, and males approximately 14 recombinants; the highest reported level of recombinants to date originated from... zyp1 recq4ab The mutants showed recombination counts of 34.7 in females and 23.5 in males. However, the above strategies generally share a common problem: recombination is mainly concentrated in the distal regions of chromosomes and is difficult to effectively extend to low-recombination regions such as near the centromere.

[0006] Therefore, simply by increasing HEI10 While increasing the expression levels of recombination inhibitors and synaptic complex factors (such as RECQ4 and ZYP1) can improve recombination rates, the following problems still exist: (1) Genetic manipulation is complex: it usually requires the superposition of multiple genes or multiple pathway regulation; (2) There is an upper limit to the increase in the number of recombinations: even in the context of multiple mutants, it is still difficult to make further breakthroughs; (3) Recombination distribution is limited: Crossover is still mainly limited to the distal region of chromosomes and is difficult to enter the near centromere region; (4) Reorganization intervention is difficult to control precisely: some strategies only weaken rather than completely eliminate the intervention; (5) Dependence on structural disruption: Some methods achieve regulation by disrupting the synaptic complex, which may affect chromosome structural stability and the meiotic process.

[0007] On the other hand, recent studies have shown that post-translational modifications of proteins (such as phosphorylation) and phase separation of biomolecules may play important roles in the regulation of meiosis. However, a unified molecular regulatory mechanism that can simultaneously explain and regulate the amount and distribution of recombination and the mechanism of interference formation is still lacking.

[0008] Therefore, there is an urgent need to develop a new technological approach to achieve efficient regulation of the quantity, distribution, and interference of meiotic recombination without damaging chromosome structure, and to overcome the limitations of existing technologies. Summary of the Invention

[0009] The purpose of this invention is to provide a method for synergistically regulating the amount of recombination (CO), reshaping the recombination distribution, and eliminating recombination interference by adjusting the phosphorylation state of HEI10 protein and changing its phase separation behavior, thereby overcoming the limitations of existing technologies such as limited recombination quantity, constrained distribution, and difficulty in eliminating interference.

[0010] Therefore, the first object of the present invention is to provide the application of reducing the phosphorylation level of HEI10 protein in promoting crossover recombination of chromosomes during meiosis in plants, the application of which does not depend on the formation of DNA double-strand breaks or the assembly of synaptic complexes.

[0011] Preferably, the application is the use of reducing HEI10 protein phosphorylation level to increase the number of whole chromosome crossover recombinations during plant meiosis, promote the extension of crossover recombination from the distal centromere region to the procentric region of chromosomes, and / or reduce recombination interference effects.

[0012] Preferably, the plant is Arabidopsis thaliana, rice, corn, wheat, or soybean.

[0013] A method for promoting meiotic chromosome crossing over recombination in Arabidopsis thaliana includes the step of reducing the phosphorylation level of HEI10 protein, wherein the HEI10 protein is a protein with the amino acid sequence shown in SEQ ID NO.5.

[0014] Preferably, the HEI10 protein is a protein encoded by a gene with a nucleotide sequence as shown in SEQ ID NO.6.

[0015] Preferably, the method for reducing the phosphorylation level of HEI10 protein is at least one of the following: (1) Mutate one or more phosphorylation sites in the disordered region of the HEI10 protein at a specific site, wherein the phosphorylation sites are serine at position 221, threonine at position 224, serine at position 250, and serine at position 287 in the amino acid sequence shown in SEQ ID NO.5, thereby inhibiting the phosphorylation of HEI10. (2) Inhibit the formation of CDKA;1-SDS complex between CDKA;1 protein and SDS protein, wherein the CDKA;1 protein is a protein with an amino acid sequence as shown in SEQ ID NO.1, and the SDS protein is a protein with an amino acid sequence as shown in SEQ ID NO.3.

[0016] Preferably, the site-directed mutation of one or more phosphorylation sites in the disordered region of the HEI10 protein involves replacing serine or threonine at the phosphorylation site with alanine.

[0017] Preferably, the inhibition of CDKA;1 protein and SDS protein forming the CDKA;1-SDS complex is achieved by knocking out... CDKA; 1 Gene or knockout SDS Gene-based; as described CDKA;1 The nucleotide sequence of the gene is shown in SEQ ID NO.2, which encodes the CDKA;1 protein; SDS The nucleotide sequence of the gene is shown in SEQ ID NO.4, which encodes the SDS protein.

[0018] Preferred, knockout SDS Gene knockout SDS The N-terminus of the gene, as described SDS The N-terminal nucleotide sequence of the gene is 1-1080 bp as shown in SEQ ID NO.4. The beneficial effects of this invention are: 1. Provide a unified and controllable restructuring regulation mechanism. This invention achieves synergistic regulation of the number, spatial distribution, and interference effects of meiotic crossing recombination by modulating the phosphorylation state of the HEI10 protein. Unlike existing technologies that employ multiple intervention pathways such as regulating protein expression levels, disrupting the synaptic complex, or inhibiting anti-recombination factors, this invention achieves multi-dimensional regulation based on a single molecule modification, establishing a unified and predictable regulatory mechanism.

[0019] 2. Significantly exceeds the upper limit of recombination quantity (superior to all existing single-gene or combination strategies) Compared with the prior art, the present invention does not increase HEI10 At the expression level, a significant increase in the number of cross-recombinations can be achieved simply by reducing its phosphorylation level. Specifically: (1) Compared with the wild type (approximately 2.8 recombinants in females and approximately 5.24 recombinants in males), the present invention can increase the occurrence of recombination by 7-8 times; (2) Significantly higher than HEI10 Overexpression (approximately 6.2 in females and 9.9 in males); (3) Surpass recq4ab Mutants (approximately 17.5 females and 14 males) and zyp1 recq4ab The level of improvement achieved by the mutants (approximately 34.7 females and 23.5 males).

[0020] These results demonstrate that the present invention breaks through the recombination limit achievable through traditional expression regulation or combinatorial mutation, and has significant technical advantages.

[0021] 3. Achieve reshaping of the reorganized distribution This invention not only increases the number of recombinations, but also significantly alters their distribution pattern on chromosomes: (1) Promotes recombination from the distal region of the chromosome to the region near the centromere; (2) Significantly increases the frequency of recombination events in traditional recombination inhibition regions (such as centromere neighborhoods).

[0022] Compared with existing technologies (including) HEI10 Overexpression, recq4ab and zyp1 recq4ab While mutants are still mainly confined to the distal regions of chromosomes, this invention achieves the reshaping of recombination distribution, thus providing a key technical means to break genetic linkage.

[0023] 4. Eliminating recombination interference without disrupting the synaptic complex. In existing technologies, eliminating recombination interference typically relies on disrupting the synaptic complex (e.g., zyp1 (Mutation), but this strategy affects chromosome structural stability. This invention achieves this by regulating the phosphorylation state of HEI10, while maintaining the structural integrity of the synaptic complex: (1) Complete elimination or significant reduction of recombination interference; (2) Removal of spatial constraints between recombination sites.

[0024] This feature has not been reported in existing technologies and has significant innovation and practical value.

[0025] 5. Reveal and utilize novel molecular regulatory mechanisms (phase separation-driven recombination control) This invention is the first to be proposed and verified: (1) The phosphorylation state of HEI10 protein regulates its phase separation behavior (condensation and coarsening kinetics). (2) This affects the competitive relationship and selection process among recombination sites; (3) The final decision is made on the number, distribution and interference patterns of cross-recombinations.

[0026] This mechanism breaks through the traditional regulatory framework centered on gene expression or structural proteins, and establishes a new regulatory paradigm of "post-translational modification-phase separation-recombination mode".

[0027] 6. The technical approach is simplified and easy to implement. Compared to existing methods that require multiple gene stacking (such as...) recq4ab , zyp1 Compared with complex genetic operations (such as those involving genetic manipulation), this invention has the following advantages: (1) Significant effects can be achieved with modification of only a single gene locus; (2) It can be achieved through gene editing (such as CRISPR / Cas), protein engineering or chemical regulation; (3) It is easy to operate, efficient and highly repeatable.

[0028] 7. Widely applicable to a variety of crops, with significant breeding application value. Because HEI10 protein is highly conserved in plants, the technology of this invention can be widely applied to major crops such as rice, corn, wheat, and soybeans, as well as other plants with HEI10 homologous proteins.

[0029] By increasing the recombination frequency and breaking the chain reaction barrier, this invention can: (1) Accelerate the recombination and aggregation of desirable traits; (2) Improve breeding efficiency; (3) Shorten the breeding cycle; (4) Improve crop yield and adaptability. Attached Figure Description

[0030] Figure 1The diagrams show the results of the interaction between CDKA;1, SDS, and HEI10. AC represents the results of the fibrillation luciferase complementation experiment in tobacco leaves, detecting the interaction between CDKA;1 and SDS-N (A) and HEI10 and SDS-N (B). D represents the results of the yeast two-hybrid experiment, detecting the interaction between HEI10 and SDS-N. EF represents the results of the affinity purification pull-down experiment, verifying the relationship between His-HEI10 and MBP-SDS-N and GST-CDKA;1. G is the structural modeling diagram of the interaction between CDKA;1, SDS, and HEI10.

[0031] Figure 2 This is a diagram demonstrating that HEI10 promotes the formation of co-localized aggregates of the CDKA;1-SDS complex in tobacco cells. A shows the results of predicting the disordered regions of CDKA;1 and SDS using PrDOS; B shows the immunofluorescence staining of CDKA;1 and HEI10 in tobacco cells with and without MMS treatment, with a scale bar of 5 μm; C shows the experimental results verifying that HEI10 can form aggregates with CDKA;1 and SDS in vitro, with scale bars of 20 μm and 10 μm respectively.

[0032] Figure 3 This demonstrates that the CDKA;1-SDS complex promotes HEI10 phosphorylation; where A is the detection... ProAct7:: HEI10-Flag / Col-0 and ProAct7::HEI10-Flag / sds The results of protein phosphorylation levels are shown in Figure B; B is the result of protein phosphorylation levels. ProAct7::HEI10-Flag / Col-0 and ProAct7::HEI10-Flag / sds Statistical analysis of protein phosphorylation levels, *** indicates that the two-tailed Student's t test showed a large P < 0.001; C is: in vitro phosphorylation results of MBP-SDS, GST-CDKA;1 and His-HEI10, with GST-CDKF;1 used to activate CDKA;1 kinase activity; D is the results of the in vitro phosphorylation experiments of MBP-SDS, GST-CDKA;1 and His-HEI10. ProAct7::Flag / Col-0、 ProCDKA;1::CDKA;1-Flag / cdka;1 and ProSDS::SDS-Flag / sds Immunoprecipitation of Flag, CDKA;1-Flag, and SDS-Flag proteins was performed, followed by a semi-in vivo phosphorylation experiment with co-incubation with His-HEI10; E is a schematic diagram of the HEI10 phosphorylation site; F is from... ProAct7::HEI10 S70F / 4A / 4D -Flag / hei10 Immunoprecipitates HEI10 and HEI10 in transgenic Arabidopsis plants S70F HEI10 4A and HEI10 4DThe results of in vivo phosphorylation detection of proteins; the numbers represent the intensity of the migration bands where phosphorylation has occurred.

[0033] Figure 4 This is a graph showing the results of verifying the effect of HEI10 dephosphorylation mutation on meiotic crossing recombination; where A is in ProHEI10::HEI10-geno / hei10 A) Graph showing the results of HEI10 protein level detection in the central inflorescence of transgenic plants, using anti-HEI10 antibody detection, with Tubulin as an internal control; B) Graph showing HEI10 immunofluorescence staining results in Twt (WT-150) and T4A (4A-7), scale bar 5 μm; C) Statistical analysis of the number of HEI10 focal points on chromosomes during diakinesis of Col-0 and HEI10 transgenic lines (Twt: WT-150, WT-163; T4A: 4A-7, 4A-15), **** indicates P<0.0001; D) Chromosomal behavior in metaphase I of Col-0, Twt (WT-150), and T4A (4A-7) observed using centromere FISH probes, scale bar 5 μm.

[0034] Figure 5 This study verifies that the HEI10 dephosphorylation mutation significantly increases the number of crossover recombinations in male and female gametes. A shows the experimental design; B shows the CO count for each gamete, with each point representing a BC1 individual (corresponding to one gamete); C shows a schematic diagram of the representative chromatid structure, with green and purple representing Col and L, respectively. er The color transition points indicate the CO position, and white dots represent centromeres; D represents the average CO number of pseudo F2 individuals (the sum of the mean CO values ​​of males and females); E represents the correlation analysis between the number of COs on a single chromatid and chromosome length (Mb), and the error bars represent the 90% confidence interval.

[0035] Figure 6 This is a recombination distribution and interference analysis; where AC represents the distribution of genome-wide CO in females, males, and pseudo F2 (sliding window: 1 Mb, step size 50 kb); D represents the CO frequency distribution on the normalized chromosome axis from telomere (TEL) to centromere (CEN); E represents CoC curve analysis: the chromosome is divided into 13 intervals, and the average overlap coefficient of each pair of intervals is calculated.

[0036] Figure 7 It is a meiotic crossover and recombination model based on phase separation; where A represents mRFP-HEI10 and mRFP-HEI10 4A Images of in vitro phase separation experiments taken after 15 min and 30 min of incubation, scale bar 50 μm; B shows the statistical results of droplet number and average size (n=5, mean ± SD); C shows the FRAP analysis of HEI10 and HEI10. 4AThe results of droplet dynamics are shown in the figure with a scale bar of 1 μm; D is the quantitative analysis of fluorescence recovery in the FRAP experiment (n=10); E is the result of the yeast double-hybrid experiment to verify the self-interaction of HEI10.

[0037] Figure 8 This is a schematic diagram of a model revealing the involvement of HEI10 phosphorylation in meiotic crossing over and phase separation; where A is a schematic diagram of the involvement of HEI10 in meiotic crossing over and phase separation in the normal phosphorylation state (wild type), and B is a schematic diagram of the involvement of HEI10 in meiotic crossing over and phase separation in the unphosphorylated state. Detailed Implementation

[0038] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0039] The following examples all use the HEI10 protein from Arabidopsis thaliana as the subject of experiments. All Arabidopsis thaliana species used in the examples (…) A. thaliana Both the mutants and transgenic lines are based on the Columbia (Col-0) ecotype, unless otherwise specified. Information on the mutants used is as follows: hei10 For SALK_014624, sds For SAIL_129_F01, cdka;1 For SALK_106809, spo11 The tobacco material used is SALK_146172. Nicotiana benthamiana .

[0040] The following are examples CDKA;1 The gene, whose nucleotide sequence is shown in SEQ ID NO.2, encodes a protein whose amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the self-promoter used to construct the vector is shown in SEQ ID NO.7; SDS The gene, whose nucleotide sequence is shown in SEQ ID NO.4, encodes a protein with the amino acid sequence shown in SEQ ID NO.3, and the nucleotide sequence of the self-promoter used to construct the vector is shown in SEQ ID NO.8; HEI10 The gene, whose nucleotide sequence is shown in SEQ ID NO.6, encodes a protein with the amino acid sequence shown in SEQ ID NO.5, and the nucleotide sequence of the self-promoter used to construct the vector is shown in SEQ ID NO.9.

[0041] Example 1: Validation of the interaction between HEI10 and the CDKA;1-SDS kinase complex In the initial phase, IP-MS was used to identify potential interacting proteins of HEI10. CDKA;1 (Cyclin-dependent kinase A;1) was identified as a candidate interacting protein. This protein is a cyclin-dependent kinase essential for meiotic crossing (CO) formation. Normally, the kinase activity of CDKs is regulated by forming active complexes with cyclins. Furthermore, previous studies have shown that CDKA;1 can interact with the meiosis-specific cyclin SDS (Solo Dancers). Therefore, it is speculated that the CDKA;1-SDS kinase complex is likely an upstream regulator of HEI10 phosphorylation.

[0042] To verify whether HEI10 is regulated by the CDKA;1-SDS complex, the split luciferase complementation (SLC), yeast two-hybrid (Y2H), and in vitro pull-down assays were performed.

[0043] 1. SLC Experiment Will CDKA; 1、HEI10 The N-terminus of SDS (as shown in nucleotide sequence 1-1080 bp in SEQ ID NO.3) was constructed into NLuc and Cluc vectors, respectively, and transiently expressed in tobacco leaves using Agrobacterium GV3101. Luciferase signal was detected 36 hours after treatment. The results showed that significant fluorescence signals were produced when the N-terminal region of SDS was co-expressed with either CDKA;1 or HEI10. Figure 1 (A and B in the text), while CDKA;1 and HEI10 did not produce a significant fluorescence signal ( Figure 1 (C in the text). This indicates that SDS acts as a bridging protein, mediating the formation of a complex between HEI10 and CDKA;1 via its N-terminus.

[0044] 2. Y2H Experiment Will CDKA;1 , HEI10 The pGADT7 trap vector (AD) was constructed, transformed into the Y187 yeast strain, and cultured on SD / -Leu medium for 3 days; the N-terminus and C-terminus of SDS (as shown in nucleotide sequence 1081-1737bp in SEQ ID NO.3) were constructed into the pGBKT7 bait vector (BD), transformed into the Y2H gold yeast strain, and cultured on SD / -Trp medium for 3 days.

[0045] AD and BD bacteria were crossbred on YPDA plates for 24 hours and then amplified in liquid SD / -Trp-Leu (DDO) medium for 36 hours. The yeast culture was diluted and spotted onto SD / -Trp-Leu or SD / -His-Ade-Trp-Leu (QDO) solid medium and cultured for 3-5 days. Protein interactions were assessed with or without abamin A (AbA, to enhance selection strength) and X-α-Gal (for color development; blue colonies indicate a positive interaction). Results showed that HEI10 interacted stably with the N-terminus of SDS (…). Figure 1 (D in the middle).

[0046] 3. Pull-down experiment HEI10 was fused with a His tag and cloned into the pET28a vector; SDS was fused with an MBP tag at the N-terminus and cloned into the pMAL-c5X vector; and CDKA;1 was fused with a GST tag and cloned into the pGEX-4T-1 vector. GST-CDKA;1, MBP-SDS-N, His-HEI10, and the corresponding control proteins were expressed and purified in *E. coli* BL21(DE3), and binding assays were performed after incubation at 4°C for 2 hours. The results further showed that HEI10 could directly bind to SDS-N, but not directly to CDKA;1. Figure 1 (EF in the text).

[0047] Furthermore, structural modeling using AlphaFold 3 (https: / / alphafoldserver.com / ) further supports this interaction and identifies a cluster of interface residues primarily located in the N-terminal region of the SDS. Figure 1 (G in the middle).

[0048] The above results indicate that SDS, as a bridging protein, mediates the formation of a complex between HEI10 and CDKA;1 through its N-terminus, providing a molecular basis for subsequent phosphorylation regulation.

[0049] Example 2: HEI10 promotes phase separation of CDKA;1-SDS complex Example 9 has demonstrated that HEI10 can undergo liquid-liquid phase separation (LLPS). To determine whether the components of the CDKA;1-SDS complex have similar properties, we analyzed the sequence characteristics of CDKA;1 and SDS using the Protein Disorder Prediction System (PrDOS). The results showed that CDKA;1 did not contain obvious intrinsically disordered regions (IDRs), suggesting that it is unlikely to undergo phase separation independently. Figure 2 (A) Therefore, we tested whether HEI10 could recruit CDKA;1 into the condensate. To analyze the role of HEI10 in protein aggregation, experiments were conducted using a transient tobacco expression system and an in vitro recombinant protein system.

[0050] In in vivo experiments, HEI10-Flag and CDKA;1-GFP expression vectors were constructed under the 35S promoter and transformed into Agrobacterium GV3101 competent cells. The bacterial culture was then cultured to OD100. 600 The concentration was 0.8-1.0. After centrifugation, the samples were collected and resuspended in a suspension solution (10 mM MME (pH 5.7), 10 mM MgCl2, 200 μM acetylsyl syringone). The bacterial suspension was injected into tobacco leaves for 36 hours. Leaf samples were then harvested, cut into small pieces, and incubated in 100 mg / mL methyl methanesulfonate (MMS) solution in the dark for 1.5-2 hours to induce DNA damage. Immunostaining was then performed after the incubation period.

[0051] Immunofluorescence assays and microscopic observation revealed that under MMS treatment, HEI10 and CDKA;1 formed distinct co-localized aggregates in the cell nucleus, while without MMS treatment, HEI10 and CDKA;1 showed diffuse distribution; and CDKA;1, when expressed alone, still showed diffuse distribution under MMS treatment. Figure 2 (B in the middle).

[0052] In in vitro experiments, C-terminal fusion of GFP, mRFP, HEI10-IDR sequence (i.e., nucleotide sequence 310-912 bp as shown in SEQ ID NO. 6) with a GFP tag (HEI10-IDR-GFP), C-terminal fusion of SDS-N sequence with GFP (SDS-N-GFP), and C-terminal fusion of CDKA;1 sequence with mRFP (CDKA;1-mRFP) were constructed in the pET28a vector. N-terminal fusion of HEI10 sequence with mRFP (mRFP-HEI10) was constructed in the pet50b-NusA vector. Prokaryotic protein expression and purification were performed to obtain GFP, mRFP, HEI10-IDR-GFP, mRFP-HEI10, SDS-N-GFP, and CDKA;1-mRFP proteins. Purified HEI10-IDR-GFP protein was mixed with CDKA;1-mRFP to a final concentration of 10 μM for both, and incubated in a buffer solution (50 mM Tris-HCl pH 7.5, 150 mM NaCl, and 10% PEG8000, with the remainder being water). The results showed that HEI10 could drive the formation of CDKA;1 droplet-like aggregates; furthermore, when SDS-N-GFP protein was added to mRFP-HEI10 protein under the same experimental conditions, SDS-N-GFP was also recruited into the HEI10 aggregates. Figure 2 (C in the middle).

[0053] The above results indicate that HEI10, as the core component for phase separation, drives the formation of CDKA;1-SDS condensates, providing a physical basis for its functional regulation.

[0054] Example 3: HEI10 as a direct phosphorylation substrate for the CDKA;1-SDS complex In the in vivo phosphorylation detection experiment, construct ProAct7::HEI10-Flag The recombinant vectors were transferred into wild-type Arabidopsis thaliana (Col-0) or... sds- / + Heterozygous mutant ( sds Mutant homozygous sterility, transgenic to sds In heterozygous mutants, genotypes are identified, and samples are collected from contemporary isolates. sds In experiments conducted on Arabidopsis thaliana under a homozygous mutant background, proteins were extracted from the central inflorescences (inflorescences from stages 1 to 9, including all meiotic stages), and HEI10-Flag protein was immunoprecipitated. Phos-tag and SDS-PAGE were used to separate the proteins, and HEI10 protein was detected using an anti-Flag antibody. Phosphorylation levels were detected using an anti-pS / pT antibody. Results showed that HEI10 exhibited a distinct phosphorylation migration band in the wild-type, while... sds The signal is significantly weakened in the background. Figure 3 (A and B in the text).

[0055] In the in vitro phosphorylation detection experiment, CDKA;1 and CDKF;1 The coding sequence of (Cyclin-Dependent Kinase F;1, a CDK-activated kinase for activating CDKA;1) was fused with a GST tag and cloned into the pGEX-4T-1 vector. The coding sequence of SDS was fused with an MBP tag and cloned into the pMAL-c5X vector. These were then expressed and purified in *E. coli* BL21(DE3). 1 μg of purified GST-CDKA;1, MBP-SDS, His-HEI10, and GST-CDKF;1 proteins were added to phosphorylation buffer (50 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 mM ATP, balance water) and incubated at 30°C for 30-60 minutes before phosphorylation detection. The results showed that in the presence of CDKF;1, the CDKA;1-SDS complex, as a complete complex, strongly phosphorylated HEI10, while no phosphorylation was detected in the absence of CDKF;1. Figure 3 (C in the middle).

[0056] In the in vivo phosphorylation detection experiment, a system was constructed. ProAct7::Flag / Col-0 , ProCDKA;1::CDKA;1- Flag / cdka;1 and ProSDS::SDS-Flag / sds -Arabidopsis complementary lines were used, and central inflorescences (inflorescences from stage 1 to 9) were collected and proteins were extracted. Flag, CDKA;1-Flag, and SDS-Flag proteins were obtained by immunoprecipitation. Phosphorylation buffer containing 1 μg of His-HEI10 recombinant protein was added to each protein, and after incubation at 30°C for 1 hour, the phosphorylation level of HEI10 was detected using an anti-pS / pT antibody. Results are as follows: Figure 3 As shown in D, the CDKA;1-Flag and SDS-Flag complex obtained by immunoprecipitation can effectively phosphorylate recombinant HEI10, while the control immunoprecipitate cannot effectively phosphorylate recombinant HEI10.

[0057] The results of this embodiment indicate that HEI10 is a direct phosphorylation substrate of the CDKA;1-SDS complex.

[0058] Example 4: Identification and Functional Analysis of HEI10 Phosphorylation Sites Group-based Prediction System (GPS) 5.0 was used to predict potential CDK-like kinase phosphorylation sites in Arabidopsis HEI10. Five candidate sites with high scores were identified: S70, S221, T224, S250, and S287. S221, T224, S250, and S287 are all located within the disorder region (IDR) of HEI10, while S70 is located outside this region. Figure 3 (E in the text). Therefore, by replacing Ser(S) at the S70 site with Phe(F), the vector was constructed. ProAct7::HEI10 S70F -Flag The remaining four phosphorylation sites were then subjected to site-directed mutagenesis, replacing Ser (S, serine) / Thr (T, threonine) with Ala (A, alanine) or Asp (D, aspartic acid) to construct the vector. ProAct7::HEI10 4A -Flag , ProAct7::HEI10 4D -Flag The constructed vectors were introduced into Arabidopsis thaliana. hei10 The mutant was analyzed by extracting proteins from the central inflorescences (inflorescences 1 to 9), and the phosphorylation level was determined by SDS-PAGE analysis with 100 μM Phos-tag. Results ( Figure 3 The F in the text displays HEI10-Flag and HEI10. S70F There was no significant difference in phosphorylation levels among the -Flags, indicating that S70 had no significant effect on HEI10 phosphorylation. The substitution of the four phosphorylation sites in the IDR region with Ala-based HEI10... 4A This leads to a significant decrease in phosphorylation levels, HEI10 4DThe mutations in almost all proteins showed upward band migration compared to the HEI10 base protein, indicating that these four sites jointly bear most of the phosphorylation of HEI10.

[0059] Furthermore, we found that this phosphorylation process occurs in spo11 (Mostly mediates the formation of DSB during meiosis) mutants and zyp1 The fact that it still occurs in mutants indicates that it does not depend on DSB formation or synaptic complex assembly.

[0060] The above results indicate that phosphorylation of the IDR region is a key molecular basis for HEI10 regulation.

[0061] Example 5: HEI10 dephosphorylation significantly increased cross-recombination sites To determine how HEI10 phosphorylation affects meiotic recombination, a study driven by its own promoter will be conducted. HEI10 Gene or HEI10 4A The expression carriers are imported respectively hei10 Arabidopsis mutants were used to obtain HEI10 expression restorer lines. ProHEI10:: HEI10-geno / hei10 - Twt) and HEI10 phosphorylation deletion mutant ( ProHEI10::HEI10 4A -geno / hei10 - T4A). Two independent transgenic lines, WT-150 and WT-163, with HEI10 protein expression levels close to endogenous levels were selected from the Twt material. Similarly, two independent transgenic lines, 4A-7 and 4A-15, with comparable HEI10 protein levels were selected from the T4A material. Figure 4 In the A section, statistical analysis was performed on the number of HEI10 focal points on diplotene chromosomes.

[0062] The HEI10 focal count results showed that during meiotic diakinesis, Twt recovered to approximately 13 HEI10 focals / cell, while T4A significantly increased to approximately 30 focals / cell. Figure 4 (C in the text). Further analysis showed that multiple HEI10 foci in T4A could be concentrated on the same chromosome, and the interfoci spacing was significantly shortened, showing a random distribution trend, indicating that the normal spatial spacing constraint was disrupted. Figure 4 In cytological analysis during metaphase I of meiosis, elongated bivalents and increased cross-like structures were also observed in TA4 (B); Figure 4 The D in the figure is consistent with the increase in the number of crossovers, indicating that the T4A mutant has more CO, and the loss of phosphorylation of HEI10 protein promotes CO formation.

[0063] In summary, these results indicate that HEI10 protein dephosphorylation significantly promotes cross-recombination and breaks spatial constraints.

[0064] Example 6: HEI10 dephosphorylation achieves unprecedented recombination enhancement To quantitatively assess the effect of HEI10 phosphorylation on the number and distribution of cross-links (CO), Arabidopsis L-type cross-links were first obtained using CRISPR Cas9. er Ecotype Knockout HEI10 The CRISPR mutant ( hei10 crispr ), and then with L er Ecological Arabidopsis thaliana was backcrossed and Cas9 background was removed, resulting in... hei10 crispr Heterozygous mutant Arabidopsis thaliana plants ( hei10 crispr- / + Meanwhile, we backcrossed HEI10 transgenic Arabidopsis with Col-0, obtaining... hei10 Arabidopsis thaliana heterozygous for T-DNA insertion mutation and transgene heterozygous ( ProHEI10::HEI10 WT / 4A -geno / hei10 - / + Then use hei10 crispr- / + and ProHEI10::HEI10 WT / 4A -geno / hei10 - / + These two strains were hybridized, and the offspring were segregated by genotyping to obtain strains carrying the desired genetic information. HEI10 Genetically modified heterozygotes hei10 T-DNA mutation heterozygote, hei10 crispr Mutant heterozygote, possessing both Col and L er Background strains ( ProHEI10::HEI10 WT / 4A -geno / hei10 - / + hei10 crispr- / + As the experimental group, and as the group with no genetic modification or genetically modified organisms (GMOs) at all. hei10 Mutant, possessing both Col and L er The background plants served as a control group. These two strains were then crossed reciprocally with Col-0, and the resulting pseudo F2 population was used as the final sequencing population. Figure 5 (A in the middle).

[0065] For the pseudo F2 group and zyp1 recq4Genome recombination was performed on the mutants. The pseudo F2 population included 215 females and 142 males in the WT group; 215 females and 144 males in the Twt group; and 215 females and 144 males in the T4A group. zyp1 recq4 The mutants consisted of 186 male and 186 female individuals. Figure 5 (B in the sequence). The sequencing depth was approximately 8×. Recombination sites were identified using a sliding window algorithm and inGAP-family.

[0066] Table 1. Recombination of male and female gametes from different materials Sequencing results (Table 1 and Figure 5 B) shows that in WT, an average of 3.68 COs per female gamete and 5.2 COs per male gamete were detected, consistent with previously reported heterologous exchange differences. As expected, Twt showed increased recombination, with an average of 6.88 COs per female and 10.15 COs per male, while still maintaining heterologous exchange differences, consistent with previously reported CO increase levels in HEI10 overexpressing plants, indicating a dose-response effect of HEI10. Recombination in T4A was significantly increased across the entire genome, with COs increasing dramatically to an average of 27.46 per female gamete and 43.24 per male gamete, approximately 7.5 times and 8.3 times that of the wild type, respectively. Despite this significant increase, heterologous exchange differences remained. The number of recombinations in T4A exceeded that of the strongest single-gene mutant previously reported. recq4ab The rate is 1.6 times higher in females and 3.1 times higher in males. In extreme cases, a single gamete can carry up to 56 (female) and 62 (male) COs, distributed across five chromosomes. Figure 5 The highest CO values ​​observed in WT were 8 and 10, respectively. Overall, the CO level in T4A was unprecedented, reaching approximately 70.7 CO / simulated F2 (estimated as the sum of female and male averages), exceeding all previously reported mutants, including... zyp1 recq4 (58.2 / simulated F2) Figure 5 (D in the text). In summary, these results indicate that the loss of HEI10 phosphorylation releases unprecedented levels of meiotic recombination, revealing phosphorylation as a key factor limiting the number of crossovers across the entire genome.

[0067] Example 7: Reorganization of distribution towards centromere region Furthermore, the distribution of CO on chromosomes was analyzed. From Figure 5 E and Figure 6As can be seen from the AD data, CO in female and male meiosis of WT and Twt is still preferentially enriched in the distal region, mainly concentrated in the distal region of the chromosome arm (i.e., far from the centromere); while CO is significantly increased throughout the entire chromosome in T4A, especially in the region near the centromere. This occurrence of CO is more obvious in male meiosis.

[0068] The pericentromere region is defined as the chromosomal segment surrounding the centromere, which is typically rich in DNA methylation and usually strongly suppresses CO formation, even in a high recombination background (e.g., zyp1 recq4 The same applies to T4A, where the CO frequency in the near-centromere region is significantly increased, indicating that the deletion of the phosphorylated portion of HEI10 overcomes the recombination inhibition in the near-centromere region, revealing that phosphorylation is an important factor limiting cross-distribution across the entire genome.

[0069] Example 8: HEI10 dephosphorylation eliminates recombination interference To determine how HEI10 phosphorylation affects CO interference, a coefficient of coincidence (CoC) analysis was performed. This index is defined as the ratio of the actual frequency of dual CO formation to the expected frequency based on independent CO formation in two intervals. A CoC value of 1 indicates no interference, while a value less than 1 indicates interference. Results ( Figure 6 E) shows that: in WT, the CoC curve of meiosis is below 1 (CoC<1) at short intervals, indicating strong cross-interference. As the interval distance increases, the CoC value gradually approaches 1, reaching the baseline level at about 13 Mb in females and about 10 Mb in males, confirming that the interference in female meiosis is stronger. In Twt, CO interference still exists, but its range of action is shortened in both females and males, indicating that the interference intensity is weaker than in WT. In T4A, the CoC curve is close to 1 and flat at all interval distances, indicating that CO interference is completely lost in male and female meiosis.

[0070] To further quantify the interference, we calculated the interference length (L_int), which reflects the spatial extent of the crossing pattern. Consistent with the CoC analysis, the L_int of females in the WT (0.475) was higher than that of males (0.318), confirming stronger interference in females. In the Twt, L_int decreased in both females (0.197) and males (0.166), indicating weakened interference. In the T4A, however, L_int was close to 0 (0.009 for females and 0.008 for males), consistent with the absence of detectable CO interference, suggesting that the inhibitory effect of a single exchange on a second exchange in a neighboring region is almost nonexistent.

[0071] The above results indicate that HEI10 phosphorylation is a key factor in the formation of recombination interference.

[0072] Example 9: Phase Separation and Kinetic Properties Regulated by HEI10 Phosphorylation Four predicted phosphorylation sites in HEI10 are located in its IDR region, suggesting that these four predicted phosphorylation sites may be involved in regulating phase separation. To verify this, we expressed and purified the HEI10 protein and the phosphorylation deletion mutant HEI10. 4A Both proteins are fused with mRFP tags. Droplet formation was detected by adding PEG8000 to a final concentration of 10% at a protein concentration of 5 μM. Both proteins formed aggregates under these conditions; however, HEI10 showed a different effect compared to HEI10. 4A Exhibiting faster and more extensive droplet formation ( Figure 7 (A in the text). Quantitative image analysis showed that at 15 minutes and 30 minutes, HEI10 formed significantly larger and more numerous droplets. Figure 7 In section B), at 30 minutes, the average droplet size of HEI10 was 40.85 ± 2.48 pixels, while HEI10... 4A The droplet size is 31.93 ± 1.51 pixels; at the same time, the number of droplets is significantly higher, with HEI10 having 718.0 ± 33.02 droplets. 4A The number of droplets was 414.8 ± 52.75.

[0073] To assess the material properties of these condensates, fluorescence photobleaching recovery (FRAP) experiments were performed. HEI10 droplets exhibited faster and more complete fluorescence recovery over a period of 240 seconds. Figure 7 The CD in the figure indicates that it has higher internal fluidity and fluidity, compared to HEI10. 4A The aggregates recovered slowly, exhibiting a more static and less kinetic state. The results indicate that phosphorylation enhances the aggregation ability of HEI10 and promotes the formation of dynamic, liquid-like structures.

[0074] To determine whether phosphorylation affects the self-interactions of HEI10, a yeast two-hybrid assay was performed using mutants with dephosphorylation sites. Wild-type HEI10 exhibited strong self-interactions. The S221A, T224A, and S250A mutations had relatively small effects on these interactions; in contrast, HEI10... 4A and HEI10 S287A It exhibited significantly reduced self-interactions and impaired binding ability with wild-type HEI10. Figure 7 The E in the figure indicates that dephosphorylation inhibits the self-interaction of HEI10, weakening its ability to form aggregates.

[0075] Conclusion: Phosphorylation enhances phase separation and molecular exchange in HEI10, thereby regulating recombination patterns.

[0076] Based on the results of all the above embodiments, a system as follows was constructed. Figure 8 The model diagram shown in Figure E illustrates that during meiosis, numerous small HEI10 foci are loaded onto the chromosome and undergo liquid-liquid phase separation (LLPS), subsequently coarsening to form larger aggregates that mark crossing over recombination sites. In the wild type, HEI10 diffuses along the chromosome, with some foci undergoing phosphorylation, enhancing their phase separation ability, promoting aggregation and molecular recruitment, ultimately forming a few spaced-apart CO sites exhibiting strong interference. (The last sentence appears to be incomplete and possibly refers to HEI10.) 4A In the dephosphorylation mutant, the lack of phosphorylation reduces the mobility and phase separation ability of HEI10, hindering the coarsening process and resulting in the continued presence of a large number of medium-sized foci, thereby increasing the number of CO sites and eliminating interference effects.

[0077] Because the HEI10 protein is highly conserved in plants, reducing HEI10 phosphorylation to promote meiotic crossing recombination can be widely applied in major crops such as rice, corn, wheat, and soybean, as well as other plants with HEI10 homologs. Reducing HEI10 phosphorylation can increase recombination frequency and break linkage blockade, thus showing profound application prospects in accelerating the recombination and aggregation of desirable traits, improving breeding efficiency, and shortening the breeding cycle.

Claims

1. Application of reducing HEI10 protein phosphorylation level in promoting chromosome crossing over and recombination during plant meiosis.

2. Use according to claim 1, characterized in that, To investigate the application of reducing HEI10 protein phosphorylation levels in increasing the number of whole chromosome crossing recombinations during plant meiosis, promoting the extension of crossing recombinations from the distal centromere region to the procentric region of chromosomes, and / or reducing recombination interference effects.

3. Use according to claim 1, characterized in that, The plants mentioned are Arabidopsis thaliana, rice, corn, wheat, or soybean.

4. A method of promoting meiotic chiasma recombination in Arabidopsis thaliana, characterized by, The method includes a step of reducing the phosphorylation level of the HEI10 protein, which is a protein with the amino acid sequence shown in SEQ ID NO.

5.

5. The method of claim 4, wherein, The HEI10 protein is a protein encoded by a gene whose nucleotide sequence is shown in SEQ ID NO.

6.

6. The method of claim 4, wherein, The method for reducing the phosphorylation level of HEI10 protein is at least one of the following: (1) Mutate one or more phosphorylation sites in the disordered region of the HEI10 protein at a specific site, wherein the phosphorylation sites are serine at position 221, threonine at position 224, serine at position 250, and serine at position 287 in the amino acid sequence shown in SEQ ID NO.5, thereby inhibiting the phosphorylation of HEI10. (2) Inhibit the formation of CDKA;1-SDS complex between CDKA;1 protein and SDS protein, wherein the CDKA;1 protein is a protein with an amino acid sequence as shown in SEQ ID NO.1, and the SDS protein is a protein with an amino acid sequence as shown in SEQ ID NO.

3.

7. The method of claim 6, wherein, The site-directed mutation of one or more phosphorylation sites in the disordered region of the HEI10 protein involves replacing serine or threonine at the phosphorylation site with alanine.

8. The method of claim 6, wherein, The inhibition of the CDKA;1 protein and the SDS protein from forming the CDKA;1-SDS complex is achieved by knocking out CDKA;1 the gene or knocking out SDS the gene; the CDKA;1 nucleotide sequence of the gene, as shown in SEQ ID NO. 2, encodes the CDKA;1 protein; the SDS nucleotide sequence of the gene, as shown in SEQ ID NO. 4, encodes the SDS protein.

9. The method of claim 8, wherein, knockout SDS gene is knocked out SDS N-terminal of the gene, the SDS The nucleotide sequence of the N-terminal of the gene is 1-1080 bp as shown in SEQ ID NO. 4.

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