A method for targeted expression of the AtTPS1 gene to reduce plant fertility

Abstract

This invention provides a directional expression AtTPS1 A method to reduce plant fertility using genes. This method will... AtTPS1 Genes can be operatively linked to pOLE1 Promoter, construct recombinant expression vector, and transform into plant cells to enable... pOLE1 Promoters drive in plant reproductive organs AtTPS1 Gene expression. Experiments showed that the pod abortion rate of transgenic plants was significantly higher than that of wild-type plants, and the chlorophyll fluorescence parameter Fv / Fm was significantly reduced during the reproductive growth stage. AtTPS1 The expression level of the gene in the pods was significantly increased. This invention provides an efficient and stable technical means for the cultivation of artificial sterile lines, the reduction of hybrid seed production costs, and the expansion of hybrid breeding resources.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and specifically relates to a directed expression method. AtTPS1 Methods to reduce plant fertility through gene therapy. Background Technology

[0002] Hybrid breeding is a core approach in modern agriculture to improve crop production efficiency and quality. Compared to conventional varieties, hybrids exhibit significant advantages in yield increase, enhanced stress resistance, and expanded adaptability. In the selection and large-scale production of hybrids, the cultivation and utilization of plant sterile lines play a crucial role. By utilizing sterile lines, breeders can precisely control the hybridization process, ensuring the purity and consistency of hybrid seeds, thereby shortening the breeding cycle of new varieties. However, current plant hybrid breeding methods still face many technical bottlenecks: ① Insufficient stability of sterile lines: Some existing sterile lines are susceptible to environmental factors (such as temperature and light), leading to fertility recovery and severely affecting the purity of hybrid seeds; ② Limited genetic resources: Naturally existing sterile mutant resources are limited, making it difficult to cover all superior varieties and restricting the selection range of hybrid combinations; ③ Complex and costly seed production technology: Traditional sterile line maintenance and seed production procedures are complex, requiring significant investment of manpower and resources, resulting in high seed production costs. AtTPS1 (trehalose-6-phosphate synthase 1) is a key enzyme involved in the regulation of plant sugar metabolism. Although its important role in plant development is known, how to use this gene to achieve precise regulation of plant fertility and apply it to overcome the aforementioned seed production challenges remains an urgent problem to be solved in the field of breeding technology.

[0003] The information disclosed in this background section is intended only to enhance the understanding of the overall background of the invention and should not be construed as an admission or in any way implying that the information constitutes prior art known to those skilled in the art. Summary of the Invention

[0004] To address the shortcomings of the prior art, this invention provides a method utilizing a specific promoter ( pOLE1 ) Targeted expression AtTPS1 Genetic approach. This method significantly reduces plant fertility (increases pod abortion rate) by interfering with the normal physiological balance of plants in specific tissues and stages, thus providing an efficient and stable technical means for cultivating new artificial sterile lines, reducing seed production costs, and expanding hybridization breeding resources.

[0005] The first aspect of the present invention provides a directional expression AtTPS1 Methods that reduce plant fertility through gene manipulation have the following characteristics: AtTPS1 Genes can be operatively linked to pOLE1Promoter, construct recombinant expression vector; transform recombinant expression vector into plant cells, and enable... pOLE1 The promoter drives the plant reproductive organs. AtTPS1 Gene expression, thereby reducing plant fertility.

[0006] Preferably, the reduction in plant fertility is manifested in an increased rate of pod abortion.

[0007] Preferably, the plant is Arabidopsis thaliana.

[0008] Preferably, the recombinant expression vector comprises an operably linked pOLE1 promoter, AtTPS1 Genes and transcription terminators.

[0009] Preferably, pOLE1 The promoter is Arabidopsis thaliana olein 1 promoter, and its nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing.

[0010] Preferably, AtTPS1 The gene is the Arabidopsis thaliana trehalose-6-phosphate synthase 1 gene, and its coding sequence is shown in SEQ ID NO: 2 in the sequence listing.

[0011] Preferably, the recombinant expression vector comprises an operably linked pOLE1 promoter, AtTPS1 Genes and transcription terminators.

[0012] Preferably, the transcription terminator is the Nos terminator.

[0013] Preferably, the recombinant expression vector further comprises a selection marker gene.

[0014] Preferably, the screening marker gene is a kanamycin resistance gene.

[0015] Preferably, the transformation is performed using an Agrobacterium-mediated flower soaking method.

[0016] A second aspect of the present invention provides a recombinant expression vector for reducing plant fertility, the recombinant expression vector comprising... pOLE1 promoters and AtTPS1 Gene, pOLE1 promoter and AtTPS1 Genes can be linked maneuverably.

[0017] Preferably, pOLE1 The nucleotide sequence of the promoter is shown in SEQ ID NO: 1 in the sequence listing. AtTPS1 The coding sequence of the gene is shown in SEQ ID NO: 2 in the sequence listing.

[0018] The third aspect of the present invention provides the method of the first aspect of the present invention, and the application of the recombinant expression vector of the second aspect in plant hybridization breeding, which has the feature of reducing plant fertility.

[0019] The fourth aspect of the present invention provides amplification for constructing vectors. pOLE1 The promoter primer pair, pOLE1 The promoter is as described in the second aspect of the present invention. pOLE1 The promoter has the following characteristics, and the sequences of the primer pairs are shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing.

[0020] The fifth aspect of the present invention provides amplification for constructing vectors. AtTPS1 The primer pair of the gene, AtTPS1 The gene is as described in the second aspect of this invention. AtTPS1 The gene has such characteristics that the primer pair sequences are shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing.

[0021] The role and effect of invention Using strong promoter expression AtTPS1 While it can increase plant resistance, it can also cause side effects such as premature aging of plants. This invention uses " pOLE1 promoter+ AtTPS1 The transgenic plant (pOLE1::AtTPS1) showed no significant differences from the wild type in vegetative growth indicators such as the number, diameter, and number of branches, and its effects were limited to the seed and embryonic development stages. Plants with a high sterility rate were artificially created without altering other phenotypes. This invention is the first to clearly demonstrate the ability to directionally enhance [the genetic makeup of] Arabidopsis seeds. AtTPS1 The expression, without any change in other phenotypes, significantly increased the pod abortion rate by artificially creating plants with a high abortion rate.

[0022] Compared with the prior art, the present invention has at least the following beneficial effects: 1. At the end of the experiment, on day 184, the statistical results showed that the transgenic type ( pOLE1::AtTPS1 There were no significant differences between the wild type (Col-0) and the wild type in terms of the number of rosette leaves, the diameter of the rosette leaves, and the number of branches; 2. At the end of the experiment, on day 184, the statistical results showed that the transgenic type ( pOLE1::AtTPS1 The pod abortion rate of the 0 type was significantly higher than that of the wild type (Col-0), which was statistically highly significant (P<0.05). 3. After day 107 of the experiment, the transgenic type ( pOLE1::AtTPS1The Fv / Fm (chlorophyll fluorescence parameter) value of transgenic plants began to be lower than that of wild-type plants (Col-0), and this indicator can be used as a physiological marker of reduced fertility in transgenic plants. 4. Semi-quantitative analysis of RNA extracted from mature pods confirmed that the transgenic... AtTPS1 The gene expression level was significantly higher than that of the wild type, which proves that the abortion phenotype is indeed caused by... AtTPS1 The effect is caused by targeted overexpression, rather than random insertion, which ensures the reproducibility of the technique. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the carrier construction in an embodiment of the present invention; Figure 2 This is a schematic diagram of the carrier structure in an embodiment of the present invention; Figure 3 These are the experimental results of chlorophyll fluorescence parameters in the embodiments of the present invention; Figure 4 These are the experimental results of the number of rosette leaves in the embodiments of the present invention; Figure 5 These are experimental results regarding the diameter of the rosette leaves in embodiments of the present invention; Figure 6 These are the experimental results of the number of branches in the embodiments of the present invention; Figure 7 These are the experimental results of the sterility rate in the embodiments of the present invention; Figure 8 This is an image showing the agarose gel electrophoresis results in an embodiment of the present invention.

[0024] in, Figure 1 In Chinese, the alphabetical order is as follows: 1. CaMV 35S promoter (Cauliflower Mosaic Virus 35S promoter) indicates the 35S promoter of cauliflower mosaic virus; 2. EGFP (Enhanced Green Fluorescent Protein) indicates enhanced green fluorescent protein; 3. EcoRI (Escherichia coli Restriction enzyme RI) represents a type II restriction endonuclease in Escherichia coli; 4. KpnI (Klebsiella pneumoniae restriction endonuclease I) indicates Klebsiella pneumoniae restriction endonuclease I; 5. Nos (Nopaline Synthase terminator) indicates the terminator of nopaline synthase; 6. OLE1 promoter (Oleosin 1 promoter) indicates the oleosin 1 promoter; 7. pCambia1300-35S-EGFP indicates a vector with pCambia1300 as the backbone and the CaMV 35S promoter driving the EGFP reporter gene. 8. pCambia1300-OLE1-EGFP indicates that the 35S promoter of the pCambia1300-35S-EGFP vector has been replaced with the OLE1 promoter; 9. pCambia1300-OLE1-TPS-EGFP indicates the final constructed expression vector, which is driven by the OLE1 promoter to express the TPS1 gene and carries an EGFP tag; 10. SpeI (Sphaerotilus natans restriction endonuclease I) indicates Sphaerotilus natans restriction endonuclease I; 11. TPS1 (Trehalose-6-Phosphate Synthase 1) represents trehalose-6-phosphate synthase 1.

[0025] Figure 2 In Chinese, the alphabetical order is as follows: 1. R-Border (Right Border) represents the right boundary sequence; 2. EcoRI (Escherichia coli Restriction enzyme RI) represents a type II restriction endonuclease in Escherichia coli; 3. pOLE1 (Oleosin 1 promoter) indicates the oleosin 1 promoter; 4. KpnI (Klebsiella pneumoniae restriction endonuclease I) indicates Klebsiella pneumoniae restriction endonuclease I; 5. AtTPS1 (Arabidopsis thaliana Trehalose-6-Phosphate Synthase 1) represents the Arabidopsis thaliana trehalose-6-phosphate synthase 1 gene; 6. SpeI (Sphaerotilus natans restriction endonuclease I) indicates Sphaerotilus natans restriction endonuclease I; 7. EGFP (Enhanced Green Fluorescent Protein) indicates enhanced green fluorescent protein; 8.tNos (Nopaline Synthase terminator) represents the terminator of nopaline synthase; 9. L-Border (Left Border), left border sequence.

[0026] Figures 3-7 In Chinese, the alphabetical order is as follows: 1. Col-0 (Columbia-0) represents the Columbia-0 ecotype; 2. pOLE1::AtTPS1 This indicates that an Arabidopsis thaliana line with the "pOLE1 promoter driving the TPS1 gene" was introduced via transgenic technology under the Col-0 background; 3. Fv / Fm (Variable Fluorescence / Maximum Fluorescence) represents the variable fluorescence / maximum fluorescence ratio.

[0027] Figure 8 In Chinese, the alphabetical order is as follows: 1. AtActin2 (Arabidopsis thaliana Actin 2) represents the Arabidopsis thaliana actin 2 gene; 2. AtTPS1 (Arabidopsis thaliana Trehalose-6-Phosphate Synthase 1) represents the Arabidopsis thaliana Trehalose-6-phosphate synthase 1 gene; 3. cDNA (complementary DNA) represents complementary DNA; 4. Col-0 (Columbia-0) indicates the Columbia-0 ecotype; 5. Marker (DNA Marker / Molecular Weight Marker) represents a DNA molecular weight marker; 6. pOLE1::AtTPS1 This indicates that "" was introduced through transgenic technology under the Col-0 background. pOLE1 Startup driver TPS1 Arabidopsis strains with "genes". Detailed Implementation

[0028] To make the technical means, creative features, objectives, and effects of this invention easier to understand, the following description, in conjunction with embodiments and accompanying drawings, provides a directional representation of this invention. AtTPS1 The methods for reducing plant fertility through gene therapy are explained in detail.

[0029] Unless otherwise specified, all reagents and raw materials used in this invention are commercially available products.

[0030] The following are some of the raw materials and their sources: Table 1. Pharmaceutical Reagents and Manufacturers

[0031] Table 2 Equipment and Manufacturers

[0032] <Example> 1. Preparation of Escherichia coli DH5α competent cells 1.1 DH5α competent cells were re-stripeped from the frozen glycerol stock solution onto LB solid medium and incubated at 37°C for 12 h; 1.2 Pick a single colony and inoculate it into 10 ml of LB liquid medium. Incubate the LB liquid medium on a shaker at 37°C for 12 h to obtain the culture. 1.3 Add 1 ml of culture to 100 ml of LB liquid medium; 1.4 The LB liquid medium obtained in 1.3 was incubated on a shaker at 37°C for 2.5 h; 1.5 Incubate the LB liquid medium obtained in 1.4 on ice for about 15 minutes; 1.6 Centrifuge the LB liquid culture medium obtained in 1.5 at 4500 rpm for 5 min and transfer the cells to 4 pre-cooled sterile test tubes; 1.7 Then pour out the LB liquid culture medium from the sterile test tubes and add 10 ml of 50 mM CaCl2 pre-cooled at 4°C to each tube to resuspend the cells; 1.8 Place the cells on ice for a 30-minute ice bath; 1.9 To obtain a sufficient number of cells, repeat steps 1.6–1.8 once; 1.10 Combine all the cells obtained above into one tube (approximately 8 ml in total) and add 3.5 ml of sterile 50% glycerol; 1.11 Dispense the cells into sterile EP tubes (400 μl per tube) and store at -80°C.

[0033] 2. Plant DNA Extraction 2.1 When the Arabidopsis seedlings have grown to 4 to 6 leaves, select the tenderest leaves, collect them in EP tubes and immediately pour in liquid nitrogen; 2.2 The Arabidopsis thaliana leaf material was ground into a fine powder with no obvious leaf tissue particles, no fibrous lumps, and a dry, uniform powder using a small pestle; 2.3 Vortex the fine powdered Arabidopsis leaf material obtained in 2.2 with 400 μl of DNA extraction buffer (200 mM Tris-HCl, 250 mM NaCl, 25 mM EDTA and 0.5% SDS) until homogeneous, and incubate the EP tube at room temperature for 1 h. 2.4 Place the EP tube into a centrifuge and centrifuge at full speed for 1 minute to precipitate the Arabidopsis leaf material; 2.5 Transfer 300 μl of supernatant to a new EP tube; 2.6 Add 300 μl of isopropanol to the collected supernatant and mix. Incubate the mixture at room temperature for 2 min. 2.7 Centrifuge the EP tube at full speed for 5 minutes in a centrifuge; 2.8 Discard the supernatant and allow the precipitate to dry at room temperature; 2.9 Add 100 μl of ultrapure water to the precipitate that has been dried at room temperature, and gently shake to mix. 2.10 Agarose gel electrophoresis was performed using an electrophoresis tank and a three-constant multi-purpose electrophoresis system (Beijing Liuyi Instrument Factory) to check the quality and quantity of DNA. The extracted Arabidopsis genomic DNA was detected by agarose gel electrophoresis; the bands were clear, regular, and uniform without diffusion or tailing; there were no bright spots of impurities remaining in the sample wells, and no protein or polysaccharide residues; there were no obvious diffuse RNA bands at the bottom of the gel. This indicates that the extracted DNA was of good integrity, high purity, free from degradation contamination, and of suitable concentration, fully meeting the requirements for subsequent experiments.

[0034] 3. Plant RNA extraction 3.1 RNA was extracted from Arabidopsis leaves using the same method as in 2.1 using the Plant RNA Extraction Kit (Plant RNA Kit R6827) (OMEGA, USA); 3.2 Agarose gel electrophoresis was performed using an electrophoresis tank and a three-constant multi-purpose electrophoresis apparatus (Beijing Liuyi Instrument Factory) to check the quality and quantity of RNA. Electrophoresis can observe characteristic RNA bands, the presence of degradation tails, and contamination by genomic DNA and proteins. Clear and sharp bands, a balanced ratio of 28S to 18S band brightness, no diffuse tails, and no residual bands in the sample wells were considered acceptable standards. This was used to determine whether the RNA integrity, purity, and content met the requirements for subsequent experiments.

[0035] 4. Reverse transcription 4.1 Using a reverse transcription kit (PrimeScript) TMThe RT Reagent Kit with gDNA Eraser (Takara, Japan) reverse transcribed the plant RNA extracted from 3 into cDNA, and the Arabidopsis cDNA product was aliquoted and stored at -20℃.

[0036] 5. AtTPS1 Gene, pOLE1 Promoter amplification 5.1 Using primers TPS1-F and TPS1-R, a sequence of approximately 2.826 kb in length was... AtTPS1 The nucleic acid sequence was amplified from Arabidopsis cDNA. AtTPS1 The nucleic acid sequence was transformed into the insertion vector (PMD18-T vector) according to the method specified in TaKaRa Code: D101A. 5.2 Using primers KpnⅠ-TPS1-F and SpeⅠ-TPS1-R to insert the vector (PMD18-T vector) AtTPS1 Nucleic acid sequences are amplified; 5.3 The amplification processes described in 5.1 and 5.2 above were performed using an Eppendorf polymerase chain reaction (PCR) system from Eppendorf China Ltd. The first PCR amplification system and amplification program are shown in Table 3 below: Table 3. First PCR amplification system

[0037] The first PCR amplification procedure is as follows: 94℃ for 4 minutes 94℃ for 30 seconds 54℃ for 30 seconds, 35 cycles in total 72℃ 120s 72℃ for 10 minutes 5.4 Using primers pOLE1-F and pOLE1-R, the pOLE1 promoter nucleic acid sequence with a length of approximately 0.926 kb was amplified from Arabidopsis DNA and transformed into the insertion vector (PMD18-Tvector) according to the method specified in TaKaRa Code: D101A. 5.5 The pOLE1 promoter nucleic acid sequence of the insert vector (PMD18-T vector) was amplified using primers EcoRⅠ-pOLE1-F and KpnⅠ-pOLE1-R; 5.6 The amplification processes described in 5.4 and 5.5 above were performed using an Eppendorf China Polymerase Chain Reaction System. The second PCR amplification system and amplification program are shown in Table 4 below.

[0038] Table 4. Second PCR amplification system

[0039] The pOLE1 promoter PCR amplification program is as follows: 94℃ for 4 minutes 94℃ for 30 seconds 58℃ for 30 seconds, 35 cycles in total 72℃ 120s 72℃ for 10 minutes The primer sequences mentioned above are shown in Table 5.

[0040] Table 5. PCR primer sequences

[0041] 6. Gene transformation 6.1 Take 1.11 of competent E. coli cells from the refrigerator and thaw them on ice. Under aseptic conditions, take 5 μL of the insert vector obtained in 5.1 or 5.4 and mix it with the competent cells by pipetting and aspiration. Incubate on ice for 30 min. Quickly transfer the EP tube to a 42°C water bath and heat shock for 90 s. Incubate on ice for 2 min. Add 150 μL of LB liquid medium, mix well, and incubate at 37°C and 180 rpm for 1 h to revive the cells. Spread the cells evenly on LB solid medium containing 100 μg / mL ampicillin (CAS: 7177-48-2) and incubate at 37°C with the plates inverted for 12 h. Pick a single colony with a sterilized toothpick and inoculate it into LB liquid medium containing 100 μg / mL ampicillin. Incubate at 37°C and 180 rpm for 12 h with shaking in a constant temperature shaking incubator.

[0042] 7. Construction of pOLE1::AtTPS1 vector 7.1 The BioVector plasmid vector for bacterial strain cell protein antibody gene preservation center was used with the pCAMBIA2300-eGFP vector; 7.2 pCAMBIA2300-eGFP and EcoRI-pOLE1-KpnI were double-digested with EcoRI and KpnI respectively in a 20 μL reaction system at 37℃ for 3 h. After the reaction, the enzyme digestion was checked by agarose gel electrophoresis. The conditions for the first double digestion reaction are shown in Table 6.

[0043] Table 6. First double enzyme digestion system

[0044] 7.2 The pCAMBIA2300-eGFP fragment and pOLE1 promoter fragment from the agarose gel electrophoresis in 7.2 were recovered using the MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0 (Takara Bio Engineering (Dalian) Co., Ltd.); 7.3 The recovered promoter fragment was ligated to the pCAMBIA2300-eGFP fragment in a 10 μL volume and ligated at 4 °C for 12 h to obtain the ligation product; the ligation reaction conditions are shown in Table 7.

[0045] Table 7. Connection System

[0046] 7.4 Take 2 μL of the ligation product and add it to 20 μL of EDH5α competent cells obtained in 1.11. Incubate on ice for 30 min, then incubate in a 42°C water bath for 45 seconds. Immediately return the cells to ice and incubate for 2 min. 7.5 The pOLE1-eGFP vector and KpnⅠ-AtTPS1-SpelⅠ were constructed by double digestion with KpnI and SpelI respectively. The reaction system was 20 μL, and the digestion was carried out at 37℃ for 3 h. After the reaction was completed, the results were checked by agarose gel electrophoresis. The conditions for the second double digestion reaction are shown in Table 8.

[0047] Table 8. Second double enzyme digestion system

[0048] 7.6 The pOLE1-eGFP fragment from 7.5 agarose gel electrophoresis was recovered using a gel extraction kit (MiniBEST Agarose Gel DNA Extraction Kit Ver. 3.0) (Takara Bio Engineering (Dalian) Co., Ltd.). AtTPS1 Gene fragments; 7.7 will be recycled AtTPS1 The gene fragment was ligated to the pOLE1-eGFP fragment in a 10 μL volume at 4°C for 12 h. 7.8 Identification was performed using double digestion with EcoRI and KpnI, and KpnI and SpelI. 7.7 The ligation vector was obtained to construct a positive clone of the pOLE1::AtTPS1 vector.

[0049] A schematic diagram of the pOLE1::AtTPS1 vector construction method is shown below. Figure 1 As shown, the structure is as follows Figure 2 As shown. The EcoRⅠ-pOLE1::AtTPS1-SpeⅠ sequence is as follows: 8. Preparation of Agrobacterium competent cells 8.1 First, the resistance of Agrobacterium strain GV3101 to 50 μg / ml gentamicin (CAS: 1403-66-3) and 10 μg / ml rifampicin (CAS: 13292-46-1) was tested; 8.2 Single colonies of Agrobacterium strain GV3101 were cultured in LB liquid medium containing 30 μg / ml gentamicin and 10 μg / ml rifampicin at 28°C with shaking for 12 h to obtain the culture; 8.3 Add 4 ml of culture to a sterile 500 ml flask containing 100 ml of LB medium, and shake well at 250 rpm at 28°C until the culture reaches an OD600 of approximately 0.6 to 0.8; 8.4 Cool the culture medium from 8.3 on ice and centrifuge it at 3000g at 4℃ for 5 min; 8.5 Discard the supernatant and resuspend the cells in 1 ml of pre-cooled 20 mM CaCl2 solution; 8.6 Aliquot the cells into pre-chilled 1.5ml EP tubes in 0.1ml portions.

[0050] 8.7 The cells were frozen in liquid nitrogen and stored at -80°C. The cells obtained were named Agrobacterium competent cells.

[0051] 9. AtTPS1 Plasmid transformation of Agrobacterium competent cells 9.1 The frozen Agrobacterium competent cells obtained in 8.7 were transferred to ice for thawing; 9.2 Add 1 μg of the pOLE1::AtTPS1 vector obtained in 7.8 to 100 μl of Agrobacterium competent cells, gently pipette 4 to 5 times to mix it evenly, and then put it back on ice; 9.3 Incubate the above mixture in a water bath at 37°C for 5 minutes; 9.4 Place the mixture back on ice for 30 minutes; 9.5 Add the above mixture to 1 ml of sterile LB liquid medium and shake well at 28°C (150 rpm) for 4 h; 9.6 Spread the bacterial culture evenly on LB solid medium containing rifampicin (10 μg / mL), gentamicin (30 μg / mL) and kanamycin (CAS: 8063-07-8) (50 μg / mL); 9.7 After incubation at 28℃ for 48 hours, the transformed colonies appeared on the plate, yielding Agrobacterium-positive single colonies containing the 7.8 vector.

[0052] 10. Agrobacterium-mediated flower soaking transformation of Arabidopsis thaliana 10.1 Wild-type Arabidopsis thaliana (Col-0) seeds purchased from the Arabidopsis Information Resource were sterilized and then sown in a solution containing 0.3% agarose, incubated at 4°C in the dark for 4 days. The seeds were then pipetted into prepared flowerpots. All flowerpots were incubated at 20°C with a 12-hour light-dark cycle and a light intensity of 130 μmol·m⁻¹. -2 ·s -1 Only five seedlings are kept in each pot; the rest are discarded. Water regularly to keep the compost moist. To ensure uniform inflorescence growth, primary inflorescences are manually removed. After two months of growth, when almost all Arabidopsis plants have uniform inflorescence development, the flower soaking method is used to transform the Arabidopsis. The specific steps are as follows: 10.2 Inoculate the Agrobacterium positive single colony obtained in 9.7 into 5 ml of LB liquid medium containing rifampicin and kanamycin, shake well at 28°C and 200 rpm for 48 h to obtain Agrobacterium culture solution; 10.3 Then, 2 ml of Agrobacterium culture was inoculated into 200 ml of LB liquid medium containing rifampicin and kanamycin antibiotics, and the medium was shaken at 28°C and 250 rpm for 24 h. 10.4 Centrifuge the Agrobacterium culture medium from 10.3 at 3500g for 10 min at room temperature and discard the supernatant; 10.5 Using a pipette, gently resuspend the precipitated cells in 5% (w / v) sucrose to achieve an OD600 of 0.6-0.8. Add 0.04% of the organosilicone surfactant (Silwet L-77) (Beijing Solarbio Science & Technology Co., Ltd.) and mix gently. 10.6 Transform the healthy Arabidopsis thaliana inflorescences from 10.1 by immersing them in the resuspended cells from 10.5 for 30–60 seconds; After transformation, Arabidopsis thaliana was managed normally and harvested after the seeds matured.

[0053] 11. AtTPS1 Screening of genetically modified plants 11.1 After maturity, the seeds were harvested and T0 generation seeds were sown on MS solid medium containing 50 μg / mL kanamycin. Transgenic positive Arabidopsis thaliana could germinate and grow normally on MS solid medium containing kanamycin; non-transgenic seeds turned yellow, wilted and could not root and grow true leaves after germination. T1 generation transgenic positive Arabidopsis thaliana was obtained by screening and then transferred to flower pots. The above steps were repeated until homozygous transgenic plants were screened.

[0054] 12. PCR method for verifying transgenic plants 12.1 The selected transgenic plants were subjected to PCR to verify their transgenicity. The primers are shown in Table 9 below: Table 9. Primer sequences for PCR verification of transgenic plants

[0055] 13. Determination of chlorophyll fluorescence parameters in plants 13.1 On days 37, 44, 51, 58, 65, 72, 79, 86, 93, 100, 107, 114, 121, 128, 135, 142, 149, 156, 163, 170, 177, and 184 after transplanting Arabidopsis thaliana into pots, chlorophyll fluorescence parameters were measured using a PAM-2500 pulse-modulated fluorometer (PAM 2500, Walz Inc., Effeltrich, Germany). The procedure is as follows: First, Arabidopsis thaliana leaves were allowed to undergo dark adaptation for 30 minutes. Mature, healthy, functional leaves were selected as the measurement subjects, and measurements were taken at approximately the same location on each leaf. A diode with a wavelength of 650 nm was chosen as the light source. The chlorophyll fluorescence parameter was measured as Fv / Fm. The Fv / Fm value was calculated using the formula Fv / Fm=(Fm-Fo) / Fm.

[0056] 14. Statistical analysis of plant morphological traits 14.1 On day 184, morphological characteristics of cultured Arabidopsis thaliana were statistically analyzed, including the number of rosette leaves, rosette leaf diameter, number of branches, and pod abortion rate.

[0057] 15. Experimental Data Analysis 15.1 Experimental data were entered using Excel 2023 (Microsoft, USA). SPSS version 21.0 (IBM, USA) was used to calculate the mean and standard deviation of each indicator. One-way ANOVA was performed using the least significant difference (LSD) method. Different letters indicate significant differences, with P < 0.05. Graphs were plotted using SigmaPlot 12.0 (Systat, USA). Experimental results are as follows: Figures 3-7 As shown.

[0058] like Figure 3 As shown, on day 37 of the experiment, the wild-type (Col-0) and transgenic (Col-0) pOLE1::AtTPS1 The Fv / Fm ratio was lower in the wild type than in the transgenic type as the experiment progressed. From day 93 to day 107, the wild type and the transgenic type were roughly equal. After day 107, the wild type was higher than the transgenic type.

[0059] like Figure 4As shown, at the end of day 184 of the experiment, there was no significant difference in the number of rosette leaves between the wild type and the transgenic type.

[0060] like Figure 5 As shown, at the end of day 184 of the experiment, there was no significant difference in the diameter of rosette leaves between the wild type and the transgenic type.

[0061] like Figure 6 As shown, at the end of day 184 of the experiment, there was no significant difference between wild-type and transgenic branches.

[0062] like Figure 7 As shown, at the end of day 184 of the experiment, the abortion rate of the wild type was significantly lower than that of the transgenic type.

[0063] 16. AtTPS1 Semi-quantitative gene expression analysis experiment At the end of day 184 after transplanting Arabidopsis thaliana 13.1 into flowerpots, mature and healthy pods were selected, and RNA was extracted according to method 3. The RNA was then used to obtain corresponding Arabidopsis thaliana cDNA according to method 4. The cDNA was then used in method 5, employing TPS1-F, TPS1-R, AtACTIN2-F (5′-GGTAACATTGTGCTCAGTGGTGG-3′), and AtACTIN2-R (5′-AACGACCTTAATCTTCATGCTGC-3′) from method 12, for PCR amplification according to method 5. The PCR products were then subjected to agarose gel electrophoresis, and the band brightness was observed in the electrophoresis image. The experimental results showed that in the pod cDNA, the AtTPS1 band of the transgenic pOLE1::AtTPS1 was significantly brighter than that of the wild type, indicating a significantly increased expression level. (See attached image). Figure 8 .

[0064] The above embodiments are preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention.

Claims

1. A type of directed expression AtTPS1 The method for reducing plant fertility through gene therapy is characterized by, include: Will AtTPS1 Genes can be operatively linked to pOLE1 Promoter, construct recombinant expression vector; transform the recombinant expression vector into plant cells, and enable the... pOLE1 The promoter drives the plant reproductive organs. AtTPS1 Gene expression, thereby reducing plant fertility.

2. The method according to claim 1, characterized in that, The decrease in plant fertility is reflected in the increased rate of pod abortion.

3. The method according to claim 1, characterized in that, The recombinant expression vector includes operably linked pOLE1 promoter, AtTPS1 Genes and transcription terminators.

4. A recombinant expression vector that reduces plant fertility, characterized in that, The recombinant expression vector contains pOLE1 promoters and AtTPS1 Genes, the ones mentioned pOLE1 promoter and the AtTPS1 Genes can be linked maneuverably.

5. The recombinant expression vector according to claim 4, characterized in that, The pOLE1 The nucleotide sequence of the promoter is shown in SEQ ID NO: 1 in the sequence listing, and the coding sequence of the AtTPS1 gene is shown in SEQ ID NO: 2 in the sequence listing.

6. The method as described in any one of claims 1-3, and the application of the recombinant expression vector as described in claim 4 or 5 in plant hybridization breeding, characterized in that, Used to reduce plant fertility.

7. Used for amplification pOLE1 The promoter primer pair, the pOLE1 The promoter is as described in claim 4 pOLE1 The promoter is characterized in that, The sequences of the primer pairs are shown in SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence listing.

8. Used for amplification AtTPS1 The primer pairs for the gene, as described AtTPS1 The gene is as described in claim 4 AtTPS1 Genes, characterized by, The sequences of the primer pairs are shown in SEQ ID NO: 5 and SEQ ID NO: 6 in the sequence listing.

Images