Method for preparing angiosperm haploid based on egg cell elimination and application thereof

Abstract

The application discloses a method for preparing angiosperm haploid based on egg cell elimination and application, and belongs to the technical field of biological breeding. The application discloses a method for preparing angiosperm haploid based on egg cell elimination, which comprises the following steps: driving a lethal gene or a toxic gene to express by using an egg cell specific promoter, obtaining a transgenic angiosperm, namely a female haploid induction line; performing self-crossing on the female haploid induction line or offspring thereof or performing crossbreeding with other plant materials as a female parent, obtaining self-crossing offspring or crossbreeding offspring, namely the plant haploid. The angiosperm haploid preparation method provided by the application can completely retain the genetic background of a female parent, accelerate the breeding process, and completely avoid the trait separation problem caused by gene recombination in a traditional crossbreeding induction method, so that the homozygous period of excellent traits of crops can be greatly shortened, and the breeding process can be significantly accelerated.

Description

Technical Field

[0001] This invention belongs to the field of biological breeding technology, specifically relating to a method and application for preparing angiosperm haploids based on oocyte elimination. Background Technology

[0002] Pureline creation is a core component of modern biological breeding systems and a key rate-limiting step that has long constrained breeding efficiency. In traditional conventional breeding models, obtaining highly homozygous inbred lines typically requires more than eight generations of self-pollination and trait selection, resulting in a long cycle and low efficiency. In contrast, haploid breeding technology, by doubling the chromosomes of haploid plants induced by haploidization, can obtain trait-stable purelines in just two generations, significantly shortening the breeding cycle and improving selection efficiency. It has become an important technical means to accelerate the breeding of superior crop varieties.

[0003] In existing technologies, haploid induction mainly relies on in vitro regeneration pathways or in vivo induction strategies. In vitro pathways often involve the in vitro culture of explants such as pistils, ovules, anthers, or free immature pollen to induce haploid gametophyte cells into haploid plants. In vivo pathways include distant hybridization, pollination with haploid induction lines, and pollination after treating pollen with physical (radiation, high temperature, etc.) or chemical treatments (phosphatidylcholine, methimazole, etc.). In recent years, with the maturation of gene editing technology, the preparation of haploids through targeted editing of specific genes has become a research hotspot. Most current haploid-inducing genes are paternally specific genes, such as AtDMP8 / 9 in Arabidopsis thaliana and ZmPLD3 and ZmPLA1 in maize. Some patents also disclose that the cell-specific gene DMP can induce haploid production in Arabidopsis thaliana and tomato (CN111996209A), and that PLA mutations can produce haploids. Furthermore, haploids can also be produced by gene editing of the egg cell-specific gene ECS1 (CN111073875A).

[0004] However, the aforementioned haploid induction technologies based on gene editing still have significant limitations in practical applications: most known haploid induction genes are expressed paternally specifically, and their loss of function or mutation is often accompanied by abnormal pollen development or decreased plant fertility, leading to reduced seed setting rate and limiting the efficiency of haploid induction. Although male sterility can be used for rapid haploid screening, male sterility induced by paternal genes significantly increases the difficulty of haploid screening. In addition, the effects of existing induction genes are mostly species-specific, making them difficult to apply across multiple crop species and lacking broad applicability.

[0005] Therefore, developing a maternal haploid induction pathway that does not rely on haploid induction genes has become an urgent need to be addressed in this field. Summary of the Invention

[0006] The purpose of this invention is to overcome the shortcomings of haploid induction technology in gene editing in practical applications, and to provide a method and application for preparing angiosperm haploids based on oocyte elimination.

[0007] In a first aspect, the present invention provides a method for preparing angiosperm haploid inducible lines based on oocyte elimination, comprising the following steps: using an oocyte-specific promoter to drive lethal gene expression to ablate the oocyte, thereby obtaining a transgenic angiosperm, which is the maternal haploid inducible line.

[0008] Preferably, the oocyte-specific promoter includes at least one of EC1.1, EC1.2, EC1.3, EC1.4, and EC1.5, and the nucleotide sequences of EC1.1 to EC1.5 are shown in SEQ ID NO:1 to 5.

[0009] Preferably, the lethal gene includes at least one of the following: a gene that can cause cell death, a gene whose expression product can cause cell death, and a modified gene that can cause cell death.

[0010] Preferably, the angiosperm is Arabidopsis thaliana, and the lethal gene includes at least one of the Barnase gene, the DTA gene, and the C-terminal truncated NTL11 gene.

[0011] The NTL11 gene is a membrane-bound NAC transcription factor that is normally anchored to the cell membrane and has no lethal activity. When the C-terminus of the NTL11 gene is truncated, the membrane localization domain is lost, and the gene abnormally enters the cell nucleus, activating downstream programmed cell death (PCD)-related signaling pathways and directly inducing programmed apoptosis. The nucleotide sequence of the NTL11 gene is shown in SEQ ID NO:6, and the nucleotide sequence of the C-terminated NTL11 gene is shown in SEQ ID NO:7. The Barnase gene encodes a nonspecific ribonuclease (RNase) that, in the absence of an intracellular inhibitor (Barstar), continuously degrades mRNA, rRNA, and tRNA, completely blocking protein synthesis and disrupting cellular function, ultimately leading to cell death. The nucleotide sequence of the Barnase gene is shown in SEQ ID NO:8.

[0012] The DTA (diphtheria toxin A subunit) is a potent ADP-ribosyltransferase that catalyzes irreversible ADP-ribosylation of EF-2 (elongation factor 2) intracellularly, completely inactivating EF-2 and halting translation. Even trace amounts can lead to cell death. The nucleotide sequence of the DTA gene is shown in SEQ ID NO:9.

[0013] Secondly, the present invention provides a method for preparing angiosperm haploids based on oocyte elimination, wherein the maternal haploid induction line or its offspring prepared by any of the above methods is self-pollinated or used as a maternal parent to hybridize with other plant materials to obtain self-pollinated offspring or hybrid offspring, which are the plant haploids.

[0014] Preferably, the method further includes the following steps: performing haploid trait identification and / or fluorescent labeling identification and / or leaf ploidy identification and / or chromosome staining identification on the self-pollinated offspring or the hybrid offspring, and selecting at least one of the above methods to identify the offspring as plant haploids.

[0015] Thirdly, the present invention provides a nucleic acid molecule encoding an oocyte-specific promoter and a lethal gene as described in any of the above claims, wherein the nucleic acid molecule includes a nucleic acid molecule encoding the oocyte-specific promoter, a nucleic acid molecule encoding the lethal gene, and a nucleic acid molecule encoding a terminator.

[0016] Fourthly, the present invention provides an expression vector comprising the above-mentioned nucleic acid molecule.

[0017] Fifthly, the present invention provides a host cell containing the above-described nucleic acid molecules or the above-described expression vectors.

[0018] In a sixth aspect, the present invention provides the application of using an egg cell-specific promoter to drive the expression of lethal genes in the preparation of plant maternal haploid induction lines or plant haploids.

[0019] Compared with the prior art, the present invention has the following advantages: The method for preparing angiosperm haploids provided by this invention can completely preserve the maternal genetic background, accelerate the breeding process, and completely avoid the problem of trait segregation caused by gene recombination in traditional hybridization induction methods. It can significantly shorten the homozygous cycle of excellent crop traits and significantly accelerate the breeding process.

[0020] The method for preparing angiosperm haploids provided by this invention has strong technical compatibility, does not affect the normal development and germination of pollen, and can be used in conjunction with existing mature paternal haploid induction systems to further improve haploid induction efficiency. It provides important theoretical support and practical guidance for optimizing crop haploid breeding technology and improving breeding efficiency.

[0021] The method for preparing angiosperm haploids provided by this invention has wide applicability and can be extended to various crops such as grains and vegetables across species, and has great versatility and promotional value.

[0022] The method for preparing haploids in angiosperms provided by this invention reveals a novel reproductive regulatory mechanism. The elimination of oocytes can induce the production of haploids, opening up a new research field for in-depth analysis of the interaction between oocytes and synergists and elucidating the molecular regulatory network of plant embryo sac development and fertilization. Attached Figure Description

[0023] Figure 1 This is a GFP fluorescence image of the oocytes in Example 1; Figure 2 The phenotypic diagram of the haploid in Example 1; Figure 3 This is a diagram showing the ploidy detection of the blades in Example 1; Figure 4 This is a chromosome staining detection diagram from Example 1. Detailed Implementation

[0024] To better understand the present invention, the following embodiments are further illustrations of the invention, but the scope of the invention is not limited to these embodiments. Unless otherwise specified, the experimental methods used in the embodiments of the present invention are conventional methods. The reagents and materials used in the embodiments are all commercially available, and the quantitative experiments involved in the embodiments are all performed in at least three replicates.

[0025] The present invention adopts the following technical solution: Expression vectors containing oocyte-specific promoters (including EC1.1, EC1.2, EC1.3, EC1.4, EC1.5, etc.) and lethal or toxic genes (including NTL11, DTA, Barnase, etc.) with pollen fluorescence were constructed, and stable transgenic plants were obtained through transgenic processes. These transgenic plants achieve specific elimination of oocytes in vivo because the expression of lethal or toxic genes is driven by the oocyte-specific promoters.

[0026] Transgenic T1 generation positive plants were screened based on the brightness of pollen fluorescence from positive plants. After emasculating the positive plants, RNA was extracted from the emasculated ovules 24 hours later, and the expression level of lethal genes was identified by qRT-PCR. Plants with higher expression levels were selected to determine the oocyte elimination line.

[0027] Seeds of oocyte-eliminating strains are cultured and obtained. Depending on the cultivation method of different plants, offspring seeds are obtained through artificial or natural pollination during the flowering period. Haploid phenotype identification, leaf ploidy identification, and chromosome staining identification are used to screen and identify plant haploids in individual offspring plants.

[0028] The effects of the present invention will be described in detail below with reference to the embodiments.

[0029] Example 1: Preparation of Arabidopsis haploids through oocyte elimination of plant haploid inbred lines Specifically, Arabidopsis haploids were prepared by using the oocyte-specific promoter EC1.2 to drive the C-terminally truncated NTL11 gene.

[0030] In this embodiment, all wild-type Arabidopsis thaliana seeds were obtained from the Arabidopsis Biological Resource Center (ABRC). Unless otherwise specified, Arabidopsis seeds were cultured on 1 / 2 MS medium, vernalized at 4°C for 3 days, and then transferred to the culture room. Seedlings were transplanted 7 days later. After transplanting, the seedlings were cultured in a 1:1 mixture of substrate and vermiculite with high-temperature insecticide treatment. After harvesting, the seeds were dried at 28°C for 2 days before storage or further germination on a substrate. The culture room was maintained at 22°C with a 16-hour light-8-hour dark environment.

[0031] 1. Construction of expression carriers 1.1 Amplification of the target sequence Using the Arabidopsis thaliana genome as a template, PCR amplification was performed using primer pairs EC1.2-S / EC1.2-A and TER-S / TER-A (Novozymes 2×Phanta Ultra Master Mix, catalog number P518 / P528-01) to obtain the sequences of the oocyte-specific promoter EC1.2 and terminator. cDNA was extracted from Arabidopsis leaves and used as a template for PCR amplification using primer pair 11ΔC-S / 11ΔC-A to obtain the C-terminal truncated sequence of the NTL11 gene. The amplified products were subjected to agarose gel electrophoresis. Gels containing the target band of the expected size were excised and purified using a gel extraction kit (Sangon Biotech, catalog number SK8131) to obtain the target band.

[0032] The sequences of primer EC1.2-S are shown in SEQ ID NO:11, primer EC1.2-A are shown in SEQ ID NO:12, primer TER-S are shown in SEQ ID NO:13, primer TER-A are shown in SEQ ID NO:14, primer 11ΔC-S are shown in SEQ ID NO:15, and primer 11ΔC-A are shown in SEQ ID NO:16. 1.2 Enzyme digestion and ligation reaction The purified terminator fragment and the P092 expression vector carrying LAT52pro::GFP were double-digested with AvrII and EcoRI restriction endonucleases (NEB, catalog numbers R0174V and R0101V), respectively, and incubated at 37°C for 150–180 min. The digestion products were then subjected to agarose gel electrophoresis, followed by gel extraction to purify the linearized vector and the target fragment using a gel extraction kit.

[0033] The reaction was prepared according to the T4 DNA ligase system (Thermo, catalog number EL0011), thoroughly mixed, and incubated overnight at 16°C to achieve ligation of the target fragment and the vector, thereby obtaining the recombinant ligation product.

[0034] 1.3 Construction of recombinant plasmids Take 2.5 μL of the recombinant ligation product and add it to DH5α E. coli competent cells (Sangon Biotech, catalog number B528413). Gently mix and place on ice for 30 min. Place the EP tube on a float plate and heat shock it in a 42°C water bath for 90 s, then quickly transfer it to ice for 2 min to cool. In a clean bench, add 800 μL of antibiotic-free LB liquid medium to the heat-shocked competent cells and incubate at 37°C and 180 r / min with shaking for 45 min. Spread the cultured bacterial solution onto the surface of 50 μg / mL spectinomycin-resistant LB solid medium plates, invert the plates, and incubate overnight at 37°C.

[0035] Remove the overnight LB agar plates and pick single colonies of suitable morphology and size, labeling them. Use a sterile toothpick to pick the labeled single colonies and perform PCR amplification using the primer pair TER-S / TER-A (Novozymes, catalog number P101-03). Detect the PCR products by agarose gel electrophoresis, selecting positive colonies with expected band size and bright bands. Use a sterile toothpick to pick the corresponding positive colonies and inoculate them into test tubes containing 5 mL of LB liquid medium. Incubate overnight at 37°C and 220 rpm with shaking.

[0036] Plasmids were extracted from *E. coli* using the Tiangen Plasmid Mini-Prep Kit (B0917A). 5 μL of plasmid was digested with restriction endonucleases (AvrII and EcoRI) for verification. Two specific bands observed during digestion indicated a positive result. Positive plasmid samples were sent for sequencing. Recombinant plasmids that aligned correctly with the sequencing data were identified as vectors containing terminators and stored at -20°C for later use.

[0037] The subsequent steps are the same as steps 1.2 and 1.3 above. The truncated C-terminal sequence of the NTL11 gene (double digested with XhoI and AvrII) and the EC1.2 promoter sequence (double digested with KpnI and XhoI) are sequentially ligated into the vector containing the terminator to complete the construction of the final expression vector and obtain the recombinant plasmid.

[0038] 2. Transgenic and screening processes in Arabidopsis thaliana 2.1 Agrobacterium-mediated transformation Agrobacterium GV3101 was picked and cultured in LB liquid medium at 28°C with shaking until the logarithmic growth phase (OD200). 600=0.5–0.6), centrifuge at 4°C to collect the bacterial pellet and discard the supernatant. Wash the bacterial cells 2–3 times with pre-chilled sterile 10% glycerol to remove salts, and finally resuspend the bacterial cells in a small amount of 10% glycerol to prepare electroporation competent Agrobacterium. Add the recombinant plasmid obtained in step 1 to the competent Agrobacterium and mix gently. Transfer to a pre-chilled electroporation cuvette for electroporation transformation. After electroporation, immediately place on ice for 3 min, and immediately add antibiotic-free LB liquid medium. Incubate at 28°C for 1 h. Spread the bacterial culture on LB agar plates containing 50 μg / mL spectinomycin and incubate at 28°C for 48 h to obtain positively transformed Agrobacterium.

[0039] 2.2 Transgenic method using flower immersion Wild-type Arabidopsis thaliana plants that have bolted and flowered were selected, and the initial main inflorescence (1-5 cm) was pruned. The plants were cultured for another 3 days, and thoroughly watered one day before transformation. The successfully transformed Agrobacterium tumefaciens obtained in step 2.1 was added to LB liquid medium and cultured with shaking at 28°C for 24 h. After centrifugation, the precipitate was collected, the supernatant was removed, and the bacteria were resuspended in inoculum solution (5% sucrose + 300 μL / L Silwet-77) to achieve an OD value of 0.8.

[0040] Remove the pods and pollinated flowers from the Arabidopsis thaliana plants, immerse the entire inflorescence in the bacterial solution for 1 minute, and gently shake. Cover the infected plants with plastic wrap to maintain humidity, incubate in the dark for 1 day, and then place them under normal culture conditions until the plants mature.

[0041] 2.3 Screening of oocyte ablation lines After the seeds of the aforementioned transgenic Arabidopsis thaliana matured, T0 generation seeds were harvested. The T0 generation seeds were disinfected with 2% sodium hypochlorite solution for 7 minutes and then rinsed three times with sterile water. Subsequently, the T0 generation seeds were evenly spread on 1 / 2 MS medium containing 50 μg / mL kanamycin and cultured in a 22℃ light-controlled medium for 7-12 days (16 hours light / 8 hours dark). The addition of kanamycin to the medium resulted in positive plants having green leaves and growing normally, while negative plants had yellow leaves. Positive plants were selected and transferred to soil, then cultured in a 22℃ artificial greenhouse (16 hours light / 8 hours dark) to obtain T1 generation transgenic plants.

[0042] After the T1 generation transgenic plants flowered, positive plants were screened using a fluorescence microscope. Plants with green fluorescent pollen were identified as positive. Flowers of positive plants that were showing white but not yet open were selected, and the petals and sepals were gently separated with tweezers. The anthers were removed for emasculation. The pistils were collected 24 hours after emasculation, and total RNA was extracted using the TaKaRa MiniBEST Plant RNA Extraction Kit (TaKaRa Bio).

[0043] Using total RNA as a template, real-time quantitative reverse transcription PCR (M-MLV reverse transcriptase, Invitrogen) was performed using primer pairs 11ΔC-qPCR-S / 11ΔC-qPCR-A, and quantitative detection and analysis were performed using the CFX Connect real-time quantitative PCR system (Bio-Rad). The sequence of primer 11ΔC-qPCR-S is shown in SEQ ID NO:17, and the sequence of primer 11ΔC-qPCR-A is shown in SEQ ID NO:18.

[0044] The strain with the highest expression level of the NTL11 C-terminated sequence was selected in real-time quantitative reverse transcription PCR. The selected strains were emasculated for 24 hours, and the elimination of oocytes was observed using GFP fluorescence.

[0045] Test results as follows Figure 1 As shown, the left side represents wild-type ovules, and the right side represents transgenic ovules. EC represents oocytes, CEC represents collapsed oocytes, CC represents central cells, and SC represents synergists. The scale bar is 20 μm. It can be seen that normal oocyte fluorescence can be observed in wild-type ovules, but in transgenic ovules, only the fluorescence remaining after oocyte rupture can be seen, indicating an oocyte ablation line.

[0046] 3. Preparation of haploids After self-pollination of the above-mentioned oocyte-ablation lines, seeds were harvested and cultured to obtain haploid plants.

[0047] Observe the fertility of offspring plants and the size of flowers, such as Figure 2 As shown, wild-type diploid plants are tall with large leaves, normal-sized flowers, and normal fertility; haploid plants are short with narrower leaves, cannot produce fruit normally, and have flowers that are significantly smaller than those of wild-type diploid plants.

[0048] 4. Flow cytometry for leaf ploidy detection Take two fresh young leaves from the haploid plant in step 3 and place them in a culture dish. Add 100 μL of nuclear extraction solution (CyStain UV P Kit, Sysmex, catalog number 05-5002) and thoroughly chop the leaves with a blade. Then add another 400 μL of nuclear extraction solution and incubate at room temperature for 5 min. Next, add 1 mL of staining solution (CyStain UV P Kit), mix well, and incubate for 1 min. Filter the mixture through a 40 μm nylon filter into a flow cytometer (FALCON, 352235) and perform haploid ploidy detection using a flow cytometer.

[0049] Test results as follows Figure 3As shown, the signal peak value of the wild-type diploid was set to 50 as a reference, and the corresponding peak value of the haploid plant was about 25, meaning that the genetic material content was about half that of the wild-type diploid.

[0050] 5. Chromosome staining identification The haploid plant inflorescences from step 3 were fixed under vacuum in Carnoy's fixative (ethanol:acetic acid = 3:1) for 1 hour, followed by rinsing twice with 10 mM citrate buffer (pH 4.5). Then, the plants were enzymatically digested for 1 hour with a solution containing 0.3% cellulase and 0.3% pectinase. After digestion, the plants were rinsed twice again with citrate buffer and stained with 10 μg / mL DAPI solution in the dark. After staining, slides were prepared, and the microsporocytes were gently pressed to release them. The chromosome number during the late meiotic phase I of the microsporocytes was observed using a laser confocal microscope under a 63× oil immersion microscope.

[0051] Test results as follows Figure 4 As shown, the wild-type diploid has 10 chromosomes at this stage, while the haploid plant microspore mother cells have 5 chromosomes.

[0052] Nucleotide sequence of promoter EC1.1 of SEQ ID NO:1 ATGACGGAATTAGCATATCTCATGCACGTTAAGACCTTGTCTAAAACTTCTCTTCTTCTCTTATATATTTATGTGTTATGAAACGCCTATCATGAATTAGCTCTACTAAATCTAGCAACCTTTCAAATTTGCAGTATTGCAGGTGTCTCTGTGTCTTTAAAATAGTTGCCTTATGATTTCTTCGGTTTCAAGATGATCAAATAGTTATAGATTTCATGCTCACACATGCTCATTAGATGTGTACATACTTTACTTACCCAAATCTATTTTCTCGCAAAGATTTTGATGGTAAAGCTGATTTGGTTCTATTGAACTAAATCAAACGAGTTTCAGACTGAGTGATTCTAATCCGGCCCATTAGCCCCTAAACAGACCCACTAATTACGCAGCTTTTAATAGAGTAATTACACCTAGTTTACCCACTAAACCACTAAGCACTAATTATCTCACAATCTAATGAGCTTCCCTCGTAATTACTTGGGCTTTCACTCTACCATTTATTTGTAACAGTCAAGTCTCTACTGTCTCTATATAAACTCTCTAAAGTTAACACACAATTCTCATCACAAACAAATCAACCAAAGCAACTTCTACTCTTTCTTCTTTCGACCTTATCAATCTGTTGAGAA Nucleotide sequence of promoter EC1.2, SEQ ID NO:2 AAGCATTTGCGTTTGGTTTATCATTGCGTTTATACAAGGACAGAGATCCACTGAGCTGGAATAGCTTAAAACCATTATCAGAACAAAATAAACCATTTTTTGTTAAGAATCAGAGCATAGTAAACAACAGAAACAACCTAAGAGAGGTAACTTGTCCAAGAAGATAGCTAATTATATCTATTTTATAAAAGTTATCATAGTTTGTAAGTCACAAAAGATGCAAATAACAGAGAAACTAGGAGACTTGAGAATATACATTCTTGTATATTTGTATTCGAGATTGTGAAAATTTGACCATAAGTTTAAATTCTTAAAAAGATATATCTGATCTAGATGATGGTTATAGACTGTAATTTTACCACATGTTTAATGATGGATAGTGACACACATGACACATCGACAACACTATAGCATCTTATTTAGATTACAACATGAAATTTTTCTGTAATACATGTCTTTGTACATAATTTAAAAGTAATTCCTAAGAAATATATTTATACAAGGAGTTTAAAGAAAACATAGCATAAAGTTCAATGAGTAGTAAAAACCATATACAGTATATAGCATAAAGTTCAATGAGTTTATTACAAAAGCATTGGTTCACTTTCTGTAACACGACGTTAAACCTTCGTCTCCAATAGGAGCGCTACTGATTCAACATGCCAATATATACTAAATACGTTTCTACAGTCAAATGCTTTAACGTTTCATGATTAAGTGACTATTTACCGTCAATCCTTTCCCATTCCTCCCACTAATCCAACTTTTTAATTACTCTTAAATCACCACTAAGCTTCGAATCCATCCAAAACCACAATATAAAAACAGAACTCTCGTAACTCAATCATCGCAAAACAAAACAAAACAAAACAAAAACCCCAAAAAGAAAGAATAATGGCTTCTAACACAAGT Nucleotide sequence of promoter EC1.3 of SEQ ID NO:3 Nucleotide sequence of SEQ ID NO:4 promoter EC1.4 Nucleotide sequence of promoter EC1.5 of SEQ ID NO:5 CACATTCATCATATCAGCCTCCACCACAGAAAACTGCAATTAGCTTGACATTAATGTAACACAAACTCCTAAAATTTGAAATCTCGTGTCTAATTCGATCGTTTATAGCTGCAAGCAAGCAACAAAAATCATTGAAACAACAAAAAGCTCGGGAATGTGAGATTCGTTCTCTTATGTACTCAATTTAAGCTGAAAGCAACAACAATCAACACACAGAACCAGAATCCAATAAATTTGGACTATCTAAACTTAAACTTAAACTGCAGAAATTGAATCAAAATCACGAATAGTTCAAAAGAAAAAACATCAGATGAGAAAGAGCTGATTTCATACGAAGTAACATGAACGTCGCGGACTTCGCGGCCATGAGATTTGTAGGTCTTGATTAAGATTCCACGGTGCGGGTTCCAGAGGCGGATAGTTCGATCCTTGCCACAGGTGAGAGCGTAGTTTCCGTCACCGTTAAACCTCGCCGCTAAAACCGCCCCCTCGTGGCCTTTAAGTATGTGTGCCTCCTTCGTCGGCAGCTCCGTCGCGCTCATCTCTCTCGCCTGAAACTTACAATCACGTTTCCATAGAACGGGCTTATACTAAACTAAACATAATAATTTATTTATTTTAATGGGTTTCCATAAAGCCCAATTTAGTTGGCCCAATAGCTTGCAAAACTGGGCGTAACGGATAATTTAATAAAGACACGGTAAATGCTTAGATAGAGGATTAGAGGGTAATTAAATTAACCACGATCACTGTGATAATTACTACAACATTAAACGACAAAAAAACTTTTCGTCTCCCTCATAATCTTCTACTATATATTCGTCACATCACACTCATAATCTCTTACAAAAAATCCATAACACAAAAAGAAGCA Nucleotide sequence of NTL11 gene of SEQ ID NO:6 SEQ ID NO:7 Nucleotide sequence of the C-terminus truncated NTL11 gene Nucleotide sequence of Barnase gene, SEQ ID NO:8 ATGGCACAGGTTATCAACACGTTTGACGGGGTTGCGGATTATCTTCAGACATATCATAAGCTACCTGATAATTACATTACAAAATCAGAAGCACAAGCCCTCGGCTGGGTGGCATCAAAAGGGAACCTTGCAGACGTCGCTCCGGGGAAAAGCATCGGCGGAGACATCTGTATGGATCTTTACTTGATGTTTATCATCTGCTCCTGTGTTCAAAGATAGAAACTATTGATTTCTCTCATGTAGTCTCAAACAGGGAAGGCAAACTCCCGGGCAAAAGCGGACGAACATGGCGTGAAGCGGATATTAACTATACATCAGGCTTCAGAAATTCAGACCGGATTCTTTACTCAAGCGACTGGCTGATTTACAAAACAACGGACCATTATCAGACCTTTACAAAAATCAGATAA Nucleotide sequence of DTA gene, SEQ ID NO:9 ATGGGCGCTGATGATGTTGTTGATTCTTCTAAATCTTTTGTGATGGAAAACTTTTCTTCGTACCACGGGACTAAACCTGGTTATGTAGATTCCATTCAAAAAGGTATACAAAAGCCAAAATCTGGTACACAAGGAAATTATGACGATGATTGGAAAGGGTTTTATAGTACCGACAATAAATACGACGCTGCGGGATACTCTGTAGATAATGAAAACCCGCTCTCTGGAAAAGCTGGAGGCGTGGTCAAAGTGACGTATCCAGGACTGACGAAGGTTCTCGCACTAAAAGTGGATAATGCCGAAACTATTAAGAAAGAGTTAGGTTTAAGTCTCACTGAACCGTTGATGGAGCAAGTCGGAACGGAAGAGTTTATCAAAAGGTTCGATGATGGTGCTTCGCGTGTAGTGCTCAGCCTTCCCTTCGCTGAGGGGAGTTCTAGCGTTGAATATATTAATAACTGGGAACAGGCGAAAGCGTTAAGCGTAGAACTTGAGATTAATTTTGAAACCCGTGGAAAACGTGGCCAAGATGCGATGTATGAGTATATGGCTCAAGCCTGTGCAGGAAATCGTGTCAGGCGATGA Nucleotide sequence of the terminator of SEQ ID NO:10 TTTATTTTCACTTTTCTGTTTTTTGGTTTGTTTTAGTATCTATTTCGTATTTTCCTTGTAATTTAGATTATCTGATTTAAACTCCAATGTTCTACTATTTTTATCTATTGTATTGTTAAATAAAATGTTGTGTTGATTCTATCTAGACTTGTTTGATAGGAGGAGTACATTATGACGTTAGTAAAAGCTTCCTCAATGCAGTACTTTTTTTGTTAAGATCTCAAACTATGCAATTAATTACCAATTGAGTCCGTGATGTAGTGGAGGCCAAAAAAAAAAAAAAGAGTCCGCGTGGGCTTTGTCCTTTTTGTTGTGTAGGCATGTCATGTTGGCCCAAAGTCTTAATCTCCACGTTGTGTTTTGTATCTATTTTATTTAGATTAGATTATGAGTTCTTTAGA SEQ ID NO:11 Primer EC1.2-S CAACGCGTTGGGAGCTCGGTACCAAGCATTTGCGTTTGGTTTATC SEQ ID NO:12 Primer EC1.2-A TTTTGTTCACCGTTCATCTCGAGACTTGTGTTAGAAGCCATTATTC SEQ ID NO:13 Primer TER-S NNNNCCTAGGTTTATTTTCACTTTTCTGTTTTTTG SEQ ID NO:14 Primer TER-A NNNNGAATTCTCTAAAGAACTCATAATCTAATC SEQ ID NO:15 Primer 11ΔC-S NNNNCTCGAGATGGGTCGTGGCTCAGTGACG SEQ ID NO:16 Primer 11ΔC-A NNNNCCTAGGCTATTGTTTTGAAGAAGATCCTTCGCC SEQ ID NO:17 Primer 11ΔC-qPCR-S ATCTCCGAGGAAGACTTGAACG SEQ ID NO:18 Primer 11ΔC-qPCR-A CGGTGACGGAAATAGCATCG The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A method for preparing angiosperm haploid inducible lines based on oocyte elimination, characterized in that, Includes the following steps: By using an egg cell-specific promoter to drive the expression of lethal genes and ablate the egg cells, transgenic angiosperms are obtained, which are the maternal haploid induction lines.

2. The method for preparing angiosperm haploid inducible lines based on oocyte elimination according to claim 1, characterized in that, The oocyte-specific promoter includes at least one of EC1.1, EC1.2, EC1.3, EC1.4, and EC1.

5.

3. The method for preparing angiosperm haploid inducible lines based on oocyte elimination according to claim 1, characterized in that, The lethal gene includes at least one of the following: a gene that can cause cell death, a gene whose expression product can cause cell death, and a gene that can cause cell death after modification.

4. The method for preparing angiosperm haploid inducible lines based on oocyte elimination according to claim 1, characterized in that, The angiosperm is Arabidopsis thaliana, and the lethal gene includes at least one of the Barnase gene, the DTA gene, and the C-terminal truncated NTL11 gene.

5. A method for preparing angiosperm haploids based on oocyte elimination, characterized in that, The haploid inducing line of the maternal parent prepared by any one of the methods of claims 1-4 or its offspring are self-pollinated or used as the maternal parent to hybridize with other plant materials to obtain self-pollinated offspring or hybrid offspring, which are the plant haploids.

6. The method for preparing angiosperm haploids based on oocyte elimination according to claim 5, characterized in that, The method further includes the following steps: performing plant haploid identification on individual plants of the self-pollinated offspring or the hybrid offspring, using at least one of the following identification methods: haploid trait identification, fluorescent labeling identification, leaf ploidy identification, and chromosome staining identification.

7. A nucleic acid molecule, characterized in that, The nucleic acid molecules include nucleic acid molecules encoding an oocyte-specific promoter as described in any one of claims 1-4, nucleic acid molecules encoding a lethal gene as described in any one of claims 1-4, and nucleic acid molecules encoding a terminator.

8. An expression carrier, characterized in that, Includes the nucleic acid molecule as described in claim 7.

9. A host cell, characterized in that, The host cell contains either the nucleic acid molecule of claim 7 or the expression vector of claim 8.

10. Application of using egg cell-specific promoters to drive lethal gene expression in the preparation of plant maternal haploid induction lines or plant haploids.

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