Pollen lethal module based on soybean alpha-amylase gene and application thereof

Abstract

The invention discloses a soybean alpha-amylase gene-based pollen lethal module and application thereof, the soybean alpha-amylase gene-based pollen lethal module comprises a pollen specific promoter, a plastid leader peptide gene and a soybean alpha-amylase gene, and the pollen specific promoter, the plastid leader peptide gene and the soybean alpha-amylase gene are sequentially connected in a direction from 5'to 3 ', the plastid leader peptide gene and the soybean alpha-amylase gene form a fusion gene, the pollen specific promoter is a soybean GmPG031 gene promoter, the nucleotide sequence of the pollen specific promoter is as shown in SEQ ID NO: 1, the soybean alpha-amylase gene is driven by the pollen specific promoter to be directionally expressed in a male gamete, and the plastid leader peptide gene is combined to guide zymoprotein to amyloid plastid, so that the soybean alpha-amylase gene is obtained. Starch reserve in the pollen is specifically consumed, so that the pollen carrying the module is aborted due to energy failure, the mechanism realizes accurate death of the transgenic pollen, a basic element is provided for a soybean seed production technology, and a core tool is provided for large-scale creation of a male sterile line.

Description

Technical Field

[0001] This invention belongs to the field of soybean breeding technology, specifically relating to a pollen lethal module based on the soybean α-amylase gene and its application. Background Technology

[0002] Soybeans are an important economic crop with wide applications in food processing, oil production, and feed ingredients. However, the long-term low yield of soybeans has limited the development of the soybean industry. Traditional soybean breeding methods mainly rely on artificial hybridization, which has problems such as long cycle, low efficiency, and large workload.

[0003] Existing hybridization breeding techniques can significantly improve crop agronomic traits by utilizing heterosis. However, as a strictly self-pollinating plant, soybeans require stable male-sterile lines to achieve efficient hybridization breeding. In crops such as corn and rice, third-generation hybridization breeding technology has been successfully applied. Its core lies in constructing intelligent nuclear male-sterile lines through pollen-specific lethal modules. These modules drive the specific expression of lethal genes in male gametes through pollen-specific promoters, achieving directional inactivation of transgenic pollen and thus efficiently obtaining male-sterile lines. However, soybean breeding still relies on first-generation hybridization technology and lacks similar efficient pollen-lethal systems, resulting in lengthy breeding cycles and hindering the rapid development of the soybean industry. Summary of the Invention

[0004] The purpose of this invention is to provide a pollen lethal module based on the soybean α-amylase gene and its application, so as to solve the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a pollen lethal module based on the soybean α-amylase gene, comprising a pollen-specific promoter, a plastid leader peptide gene, and a soybean α-amylase gene, wherein the pollen-specific promoter, the plastid leader peptide gene, and the soybean α-amylase gene are sequentially connected in a 5' to 3' direction, and the plastid leader peptide gene and the soybean α-amylase gene form a fusion gene.

[0006] Preferably, the pollen-specific promoter is the soybean GmPG03 gene promoter, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0007] Preferably, the plasmid precursor peptide gene is the precursor peptide gene of the peanut Ahp gene, and its CDS sequence is shown in SEQ ID NO:2.

[0008] Preferably, the soybean α-amylase gene is the soybean GmAA2 gene, and its CDS sequence is shown in SEQ ID NO:3.

[0009] Preferably, the soybean α-amylase gene has a terminator at its 3' end, and the terminator is a nosine synthase (NOS) terminator.

[0010] A recombinant expression vector comprising the aforementioned pollen lethal module based on the soybean α-amylase gene.

[0011] Preferably, the recombinant expression vector is constructed using pMDC123-E as the vector backbone to form the pMDC123-GmPG031::Ahp-GmAA2 recombinant vector, and the pollen lethal module is inserted between the BamHI and XhoI restriction sites.

[0012] A transgenic plant, used in the pollen lethal module or the recombinant expression vector.

[0013] Preferably, the transgenic plant is soybean, and transgenic soybean plants with partially aborted pollen are obtained by introducing the pollen lethal module or recombinant expression vector into soybean cells.

[0014] Preferably, the introduction method is Agrobacterium-mediated transformation, and the screening includes detecting the integration of the target gene by PCR and detecting pollen fertility by I2-KI staining.

[0015] Compared with the prior art, the beneficial effects of the present invention are:

[0016] (1) The soybean α-amylase gene (GmAA2) is directed to be expressed in male gametes by the pollen-specific promoter (pGmPG031), and the enzyme protein is guided to the amyloid plastid by the plastid leader peptide gene (Ahp), which specifically consumes the starch reserves in the pollen, causing the pollen carrying the module to be sterile due to energy depletion. This mechanism achieves precise lethality of transgenic pollen, provides a core tool for the large-scale creation of male sterile lines, and solves the problem of artificial emasculation required for soybean self-pollination.

[0017] (2) All module components are derived from plants (soybeans), with no exogenous microbial gene insertion, avoiding the risk of gene drift, meeting the regulatory requirements for non-GMO breeding, and easy to promote industrialization. Attached Figure Description

[0018] Figure 1 The pMDC123-GmPG031::Ahp-GmAA2 vector spectrum of the present invention;

[0019] Figure 2 This is a diagram of the gene expression cassette module of the present invention;

[0020] Figure 3 This is a diagram showing the results of potassium iodide staining of wild-type soybean pollen according to the present invention.

[0021] Figure 4 This is a diagram of the transgenic heterozygous lineage of the present invention. Detailed Implementation

[0022] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. All other embodiments obtained by ordinary technicians in this field based on the embodiments of the present invention without making any creative efforts shall fall within the scope of protection of the present invention.

[0023] This invention provides, for example Figure 1-4 The pollen lethal module shown includes a pollen-specific promoter, a plastid leader peptide gene, and a soybean α-amylase gene. The pollen-specific promoter, plastid leader peptide gene, and soybean α-amylase gene are connected sequentially in a 5' to 3' direction, and the plastid leader peptide gene and soybean α-amylase gene form a fusion gene.

[0024] The pollen-specific promoter is the soybean GmPG031 gene promoter, and its nucleotide sequence is shown in SEQ ID NO:1.

[0025] The plastid precursor peptide gene is the precursor peptide gene of the soybean Ahp gene, and its CDS sequence is shown in SEQ ID NO:2.

[0026] The soybean α-amylase gene is the soybean GmAA2 gene, and its CDS sequence is shown in SEQ ID NO:3.

[0027] The soybean α-amylase gene has a terminator at its 3' end, which is the nos synthase terminator.

[0028] A recombinant expression vector comprising the aforementioned pollen lethal module based on the soybean α-amylase gene.

[0029] The recombinant expression vector was constructed using pMDC123-E as the vector backbone to form the pMDC123-GmPG031::Ahp-GmAA2 recombinant vector, and the pollen lethal module was inserted between the BamHI and XhoI restriction sites.

[0030] A transgenic plant, used in the pollen lethal module or the recombinant expression vector.

[0031] The transgenic plant is soybean. By introducing the pollen lethal module or recombinant expression vector into soybean cells, transgenic soybean plants with partial pollen abortion are obtained through screening.

[0032] The introduction method is Agrobacterium-mediated transformation, and the screening includes detecting the integration of the target gene by PCR and detecting pollen fertility by I2-KI staining.

[0033] Combination Figure 1 —4. A detailed description of the application principle of the present invention:

[0034] Example 1: Preparation of a pollen lethal module

[0035] 1.1 Component Acquisition

[0036] Pollen-specific promoter (pGmPG031):

[0037] The primers were obtained by PCR amplification from the genome of soybean variety Williams 82. The primer sequences were as follows: upstream primer: 5'-GCG BamHI ATGGCTAGTAGCTAGCTA-3' (containing BamHI restriction site), downstream primer: 5'-GCG EcoRITCTAGCTAGCTAGCTAGC-3' (containing EcoRI restriction site). The amplified product was verified by sequencing, and the sequence is shown in SEQ ID NO:1.

[0038] Plastid precursor peptide gene (Ahp):

[0039] The cDNA was obtained from soybean leaves by RT-PCR amplification. The primer sequences were as follows: upstream primer: 5'-GCG EcoRIATGGACACCCTTTTGCAAA-3' (containing EcoRI restriction site), downstream primer: 5'-GCG XhoITTACTAGTGATGATGATGAT-3' (containing XhoI restriction site). The amplified product was verified by sequencing, and the CDS sequence is shown in SEQ ID NO:2.

[0040] Soybean α-amylase gene (GmAA2):

[0041] The cDNA was obtained from young soybean seeds by RT-PCR amplification. The primer sequences were: upstream primer: 5'-GCGXhoI ATGGGCAACTGGAGCAGTGA-3' (containing XhoI restriction site), downstream primer: 5'-GCG SacITCACTACGTGCACAGCATTA-3' (containing SacI restriction site). The amplified product was verified by sequencing, and the CDS sequence is shown in SEQ ID NO:3.

[0042] 1.2 Component Assembly

[0043] The Ahp leader peptide gene was fused with the GmAA2 gene using overlap PCR to form the Ahp-GmAA2 fusion gene.

[0044] The pGmPG031 promoter-Ahp-GmAA2 fusion gene was digested with BamHI and SacI and inserted into the multiple cloning site of the pMDC123-E vector to construct the recombinant vector pMDC123-GmPG031::Ahp-GmAA2. The vector map is attached. Figure 1 As shown, it includes the nos terminator and the bar resistance gene (bar) selection marker.

[0045] 2. Preparation of genetically modified soybeans

[0046] 2.1 Agrobacterium-mediated transformation

[0047] Recipient material: cotyledonary explants of soybean variety Williams 82.

[0048] Agrobacterium strain: GV3101 (containing recombinant vector), bacterial culture OD600 adjusted to 0.5, 100 μM acetylsyl syringone added to induce Vir gene expression.

[0049] Conversion steps:

[0050] Cotyledonous explants were immersed in Agrobacterium tumefaciens solution and cultured for 3 days (22℃, dark conditions). They were then transferred to a selection medium containing 5 mg / L glufosinate and subcultured every 2 weeks to induce resistant callus. The resistant callus was then transferred to a differentiation medium (containing 1.67 mg / L 6-BA) and irradiated for 16 h / d to induce shoot differentiation. The regenerated shoots were then transferred to a rooting medium (containing 1 mg / L IBA) to induce rooting and were then transplanted to a greenhouse for acclimatization.

[0051] 2.2 Molecular detection and phenotypic screening

[0052] PCR identification: DNA was extracted from the leaves of transgenic plants and amplified using GmAA2 specific primers (F: 5'-ATGGGCAACTGGAGCAGTGA-3'; R: 5'-TCACTACGTGCACAGCATTA-3'). Positive plants showed a specific band of 1500 bp.

[0053] Southern blot validation: Genomic DNA was digested with HindIII and GmAA2 cDNA was used as a probe to detect single copy or low copy insertion (hybridization bands ≤ 2).

[0054] Pollen fertility test: Pollen was collected during the flowering period, stained with I2-KI solution, and observed under an optical microscope.

[0055] Wild-type pollen is entirely stained black (due to its high starch content);

[0056] The ratio of black (fertile) to yellow (sterile) pollen in the transgenic heterozygous lines was close to 1:1, with a sterility rate of 48.7% ± 1.5% (n = 500).

[0057] 3. Pollen lethality module escape rate detection

[0058] 3.1 Statistical analysis of genotypic segregation ratio of transgenic T1 generation lines

[0059] Based on the analysis of resequencing data, after self-pollination of T0 generation transgenic plants and sowing of T1 generation plants, the genotype of T1 generation plants was identified by PCR, and heterozygous transgenic and wild-type plants were obtained (pollen-specific lethality with lethal module, so homozygous transgenic plants could not be obtained). The pistils were normal, so it can be used as a pollen-induced module for stable soybean seed production technology.

[0060] 3.2 Test cross between transgenic T1 generation heterozygous and wild-type lines to detect transgenic pollen escape rate

[0061] Pollen from the identified heterozygous transgenic plants was used to pollinate the pistils of wild-type plants. After the results were obtained, the number of transgenic seeds in the hybrid offspring was counted. The test cross yielded 86 seeds, including 3 transgenic seeds, resulting in a pollen escape rate of 3.49%. After sowing the escaped seeds, pollen fertility was tested using the I2-KI staining method. All pollen turned yellow, indicating pollen abortion. Figure 4 .

[0062] Example 2: Obtaining and Detecting Genetically Modified Soybeans

[0063] 2.1 Agrobacterium-mediated soybean transformation

[0064] 2.1.1 Receptor material preparation

[0065] Seeds of soybean variety Williams 82 were sterilized with chlorine and inoculated onto MS solid medium, and cultured in the dark at 25°C for 5 days.

[0066] Take germinating seeds, cut off the cotyledonary nodes, and use a scalpel to injure the hypocotyl to serve as a transformation recipient.

[0067] 2.1.2 Agrobacterium infection and co-culture

[0068] The recombinant vector pMDC123-GmPG031::Ahp-GmAA2 was transformed into Agrobacterium GV3101. Positive clones were picked and inoculated into YEP liquid medium (containing 50 mg / L kanamycin and 25 mg / L rifampin). The culture was shaken at 28°C until OD600 = 0.5. 100 μM acetylsyl syringone was added, and the culture was continued for 2 h. The cells were then collected by centrifugation and resuspended in MS liquid medium until OD600 = 0.3.

[0069] The cotyledonary explants were then immersed in the bacterial solution and allowed to stand for 30 minutes. After the surface bacterial solution was blotted dry with sterile filter paper, they were inoculated into a co-culture medium (MS + 1.67 mg / L 6-BA + 100 μM acetylsylgenone) and incubated in the dark at 22°C for 3 days.

[0070] 2.1.3 Screening and Regeneration

[0071] After co-culture, the explants were transferred to selection medium (MS + 5 mg / L Basta + 500 mg / L cephalosporin) and subcultured every 2 weeks to select resistant callus. The resistant callus was then transferred to differentiation medium (MS + 3 mg / L 6-BA + 5 mg / L Basta + 250 mg / L cephalosporin) and cultured at 25°C under light (16 h / d) to induce adventitious shoots. When the shoots grew to 2-3 cm, they were cut off and transferred to rooting medium (1 / 2 MS + 1 mg / L IBA + 5 mg / L Basta) to induce rooting. The rooted seedlings were then transplanted to a greenhouse with a survival rate of about 80%, resulting in 16 T0 generation transgenic plants.

[0072] 2.2 Molecular detection of transgenic plants

[0073] 2.2.1 PCR identification

[0074] DNA was extracted from leaves of T0 generation plants and amplified by PCR using GmAA2-specific primers.

[0075] Reaction system: 10 μL of 2×Taq Mix, 0.5 μL each of forward and reverse primers (10 μM), 1 μL of DNA template (50 ng / μL), and ddH2O to a final volume of 20 μL;

[0076] Reaction conditions: 95℃ for 5 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 1.5 min, 35 cycles; 72℃ for 10 min;

[0077] Results: 14 out of 16 strains amplified a specific band of 1500bp, with a positive rate of 87.5%.

[0078] 2.2.2 Southern Blot Analysis

[0079] Genomic DNA was extracted from positive plants (15 μg / sample), digested with HindIII overnight, separated by 0.8% agarose gel electrophoresis, transferred to a nylon membrane, and hybridized and stained using digoxigenin-labeled Basta cDNA as a probe, according to the Roche DIG High PrimeDNA Labeling and Detection Starter Kit II instructions.

[0080] Results: Of the 14 positive plants tested, 3 showed single-copy insertion and 9 showed 2-copy insertion.

[0081] 2.3 Pollen fertility testing

[0082] 2.3.1 I2-KI staining method

[0083] Take a flower bud one day before flowering, remove the anther with tweezers, place it on a glass slide, add 1-2 drops of 1% I2-KI solution, cover with a coverslip, press lightly, and observe under an optical microscope (10×40x).

[0084] result:

[0085] Wild-type pollen: more than 99% were stained blue-black (starch-rich), while the transgenic heterozygous line (T1 generation): the pollen showed obvious color separation, with the ratio of blue-black (fertile) to light yellow (sterile) close to 1:1, and the average sterility rate was 48.7% ± 1.5% (n = 5 plants, 500 pollen grains observed per plant).

[0086] Furthermore, by utilizing Agrobacterium-mediated transformation technology, combined with PCR, Southern blot molecular detection, and I2-KI staining phenotypic screening, transgenic lines were successfully obtained with a positive rate of 87.5% and high genetic stability.

[0087] In the pollen lethal module constructed in this invention, the GmAA2 gene is placed under the control of a pollen-specific promoter. The pollen-specific promoter can recognize specific signals during pollen development and initiate the expression of the GmAA2 gene at specific stages of pollen development. When the T-DNA is successfully integrated into the soybean genome, the pollen-specific promoter is activated during the critical period of pollen mother cell meiosis and development into mature pollen grains, driving the transcription of the GmAA2 gene to generate mRNA. The mRNA is then translated into α-amylase protein in the cytoplasm.

[0088] α-Amylase is an enzyme that catalyzes the hydrolysis of starch. Under normal circumstances, pollen grains store a large amount of starch, which is an important energy source for pollen germination and pollen tube growth. When the GmAA2 gene is overexpressed, the large amount of α-amylase produced will rapidly decompose the starch in the pollen grains. Due to the excessive hydrolysis of starch, the pollen grains cannot obtain enough energy to maintain normal physiological activities, leading to a series of abnormal phenomena such as cell membrane damage and organelle disintegration during the pollen maturation process. Ultimately, the pollen loses its ability to germinate and becomes sterile. Propidium iodide (PI) staining can be used to observe that propidium iodide (PI) can enter sterile pollen with damaged cell membranes, bind to nucleic acids, and produce a strong fluorescent signal, while fertile pollen with intact cell membranes cannot be penetrated by PI, resulting in a weak fluorescent signal. This allows for accurate differentiation of pollen fertility.

[0089] During the process of T0 generation positive plants self-pollinating to produce T1 generation seeds, according to Mendel's laws of inheritance, different genotypes will emerge in the T1 generation plants. Resequencing analysis of their T-DNA insertion sites and extraction of T1 generation plant leaf DNA using the CTAB method, followed by PCR amplification using T-DNA specific primers, showed that the lines that could simultaneously amplify the target band (1500bp) and the wild-type band were heterozygous transgenic lines.

[0090] Therefore, a soybean seed production technology system was formed by combining a designed soybean pollen-specific lethal module with a sporophyte-stage fertility-restoring gene and a transgenic seed selection marker. This system was integrated into corresponding homozygous mutants at the sporophyte stage via transgenic methods. Under the influence of the sporophyte-stage fertility-restoring gene, the transgenic lines were fertile; however, under the influence of the pollen lethal module, the pollen from the transgenic plants was semi-sterile, thus the pollen did not provide transgenic components. Therefore, the offspring obtained through self-pollination consisted of transgenic heterozygotes and homozygous sterile lines in a 1:1 ratio. When the transgenic heterozygotes were crossed with the corresponding homozygous mutant plants at the sporophyte stage, all offspring were male-sterile lines. This provided a large number of sterile lines for soybean hybridization breeding.

[0091] Fluorescent signal data detected by the transgenic seed marker screening system is transmitted to the data analysis system in real time. The system uses a preset algorithm to automatically distinguish between transgenic and non-transgenic seeds based on the intensity of the fluorescence signal. If no fluorescence signal is detected, the system automatically labels the line as a male-sterile line. In soybean hybridization applications, since the male-sterile lines themselves suffer pollen abortion, manual emasculation is unnecessary. Operators can select suitable male and female parents (male-sterile lines) through the intelligent management system. The system automatically records hybridization combination information according to the preset hybridization process, such as the variety names of the male and female parents and the pollination date. Simultaneously, during the cultivation of hybrid offspring, the environmental control system of the intelligent greenhouse or planting area precisely regulates temperature, humidity, light, water, and fertilizer conditions according to the growth requirements of different varieties, ensuring the normal growth and development of the hybrid offspring and ultimately achieving the efficient transfer and aggregation of superior traits in the hybrid offspring.

[0092] SEQ ID NO: 1, Nucleotide sequence:

[0093]

[0094] SEQ ID NO: 2, CDS sequence of Ahp gene:

[0095] ATGGACACCCTTTTTGCAAAATGCTTCTTCTCACTTTTCTTCCAATTCATCACTTTCTACTACGCCTAAAATTTCCCGTTACCAGGTTCGCTTTTCCATCCCTCCCaatcacaAAACCCAGAACCGAGAAGAAGGGCCCTTCACCTTGAACATGGCCACACACCCAGTTACCCTTTTTAGCTGTTTGAGCAACACAAAGTGATAATACCCACAAGCACAGGTGAAAAACTAGTG。

[0096] SEQ ID NO: 3, CDS sequence of GmAA2 gene:

[0097]

[0098] Among them, such as Figure 1 As shown:

[0099] 1. pMDC-GmPG031::Ahp-GmAA2 is the vector map of pMDC-GmPG031::Ahp-GmAA2; 2. 12,613b is the 12,613 base pair; 3. RB T-DNA repeat is the right boundary repeat sequence of T-DNA; 4. M13 fwd is the forward primer sequence of M13; 5. pVS1 Sta is the pVS1 site-specific recombinase binding site; 6. pVS1 RepA is the pVS1 replication initiation protein gene; 7. pVS1 Ori is the pVS1 replication origin; 8. bom is the bom site (related to plasmid stability maintenance); 9. oriT is the conjugation transfer initiation site; 10. KanR is the kanamycin resistance gene; 11. CaMV poly(A)signal is the cauliflower mosaic virus polyadenylation signal; 12. Basta resistance is the glufosinate resistance (gene); 13. lac promoter is the lactose operon promoter; 14. CAP binding site is the catabolite activator protein binding site; 15. lac operator is the lactose operon operator gene; 16. M13 rev M13 is the reverse primer sequence; 17. nos terminator is the cauliflower alkaloid synthase gene terminator; 18. GmAA2 is the soybean α-amylase gene 2; 19. Ahp is the plastid leader peptide gene; 20. pGmPG031 is the pollen-specific promoter pGmPG031; 21. CaMV 35S promoter (enhanced) is the enhanced cauliflower mosaic virus 35S promoter; 22. LB T-DNA repeat is the left border repeat sequence of T-DNA.

[0100] Finally, it should be noted that the above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A pollen lethal module based on the soybean α-amylase gene, characterized in that, It includes a pollen-specific promoter, a plastid leader peptide gene, and a soybean α-amylase gene, wherein the pollen-specific promoter, plastid leader peptide gene, and soybean α-amylase gene are connected sequentially in a 5' to 3' direction, and the plastid leader peptide gene and soybean α-amylase gene form a fusion gene.

2. The pollen lethal module based on the soybean α-amylase gene according to claim 1, characterized in that: The pollen-specific promoter is the soybean GmPG031 gene promoter, and its nucleotide sequence is shown in SEQ ID NO:

1.

3. The pollen lethal module based on the soybean α-amylase gene according to claim 1, characterized in that: The plastid precursor peptide gene is the precursor peptide gene of the peanut Ahp gene, and its CDS sequence is shown in SEQ ID NO:

2.

4. The pollen lethal module based on the soybean α-amylase gene according to claim 1, characterized in that: The soybean α-amylase gene is the soybean GmAA2 gene, and its CDS sequence is shown in SEQ ID NO:

3.

5. The pollen lethal module based on the soybean α-amylase gene according to claim 1, characterized in that: The soybean α-amylase gene has a terminator at its 3' end, which is the nos synthase terminator.

6. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the pollen lethal module based on the soybean α-amylase gene as described in any one of claims 1-5.

7. A recombinant expression vector according to claim 6, characterized in that: The recombinant expression vector was constructed using pMDC123-E as the vector backbone to form the pMDC123-GmPG031::Ahp-GmAA2 recombinant vector, and the pollen lethal module was inserted between the BamHI and XhoI restriction sites.

8. A transgenic plant, characterized in that, The transgenic plant is applied to the pollen lethal module as described in any one of claims 1-5 or the recombinant expression vector as described in any one of claims 6-7.

9. A transgenic plant according to claim 8, characterized in that: The transgenic plant is soybean. By introducing the pollen lethal module or recombinant expression vector into soybean cells, transgenic soybean plants with partial pollen abortion are obtained through screening.

10. A transgenic plant according to claim 9, characterized in that: The introduction method is Agrobacterium-mediated transformation, and the screening includes detecting the integration of the target gene by PCR and detecting pollen fertility by I2-KI staining.

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