Pollen specific promoter proGmMS1 and application thereof

Abstract

The invention discloses a pollen specific promoter proGmMS1 and application thereof, the pollen specific promoter proGmMS1 is cloned from a soybean gene GmMS1, and has a nucleotide sequence as shown in SEQ ID NO.1; or has a nucleotide sequence complementary to the nucleotide sequence shown as SEQ ID NO.1. The invention also discloses a preparation method of the gene. The pollen specific promoter proGmMS1 can accurately regulate and control expression of a target gene in a pollen development stage, creates a new soybean male sterile line, provides an efficient tool for soybean cross breeding, reduces ecological risks of transgenic plants, and has important theoretical and practical values.

Description

Technical Field

[0001] The present application belongs to the field of plant genetic engineering technology, and specifically relates to a pollen-specific promoter proGmMS1 and its application. The pollen-specific promoter is cloned from the soybean (Glycine max) genome, can drive the specific expression of the target gene in pollen, and is suitable for the creation of plant male sterile lines, the construction of hybrid breeding systems and the safety control of transgenic organisms. Background Art

[0002] Transgenic technology is an important tool for studying plant gene function. Promoter is an important element for regulating gene expression. The choice of promoter type determines the time and location of gene expression. Therefore, in plant genetic engineering, choosing a suitable promoter is crucial to achieving specific expression of exogenous genes.

[0003] According to the degree of regulation of the promoter on the transcription level, it can be divided into weak promoters and strong promoters. In reference to the transcription pattern, promoters can be divided into: constitutive promoter, tissue specific promoter and inducible promoter. Among them, tissue specific promoter is a promoter that can drive gene expression in specific organs or tissues. Its regulatory effect makes the gene often only expressed in certain specific organs or tissues, and shows the characteristics of developmental regulation. This makes them have important applications in genetic engineering, metabolic engineering and biotechnology, and can achieve precise regulation of gene expression, with the advantages of high ecological safety and low metabolic burden.

[0004] Among tissue-specific promoters, pollen-specific promoters are an important type, which can drive genes to be specifically expressed in plant pollen, and thus can be widely used in crop genetic improvement and male sterile line breeding. At present, relatively few pollen-specific promoters derived from soybean genes have been reported. For example, a soybean pollen-specific promoter GmLat52P is disclosed in a Chinese patent application with publication number CN117025661A, which can drive the specific expression of the pollen-lethal gene GmAMY1 in soybean flowers, and can destroy the vigor of transgenic plant pollen, so that it can be determined that the pollen-lethal gene expression cassette can be applied to the creation and cultivation of soybean intelligent nuclear sterile lines. However, the ratio of fertile pollen grains to aborted pollen grains in heterozygous transgenic plants driven by the promoter GmLat52P to GmAMY1 expression is close to 1:1. In other words, although the expression of GmAMY1 driven by the pollen-specific promoter GmLAT52P can inactivate transgenic soybean pollen, the effect is not significant, which shows that the driving force of the promoter GmLat52P on the target gene is very limited. In addition, pollen-specific promoters of other species (such as maize Zm13, etc.) are highly applicable in monocotyledons due to species compatibility limitations, but have insufficient activity or poor specificity in dicotyledons. Summary of the invention

[0005] In view of this, the primary purpose of the present application is to provide a pollen-specific promoter proGmMS1, which can accurately regulate the expression of the target gene during the pollen development stage, and has a strong driving force on the target gene and significant effect. It has important theoretical and practical value for creating new soybean male sterile lines, providing an efficient tool for soybean hybrid breeding, and reducing the ecological risks of transgenic plants.

[0006] In order to achieve the above objectives, this application adopts the following technical solutions: One aspect of the present application provides a pollen-specific promoter proGmMS1, which has a nucleotide sequence as shown in SEQ ID NO.1; or, has a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.1.

[0007] Another aspect of the present application provides an expression cassette comprising the pollen-specific promoter proGmMS1 described above.

[0008] Another aspect of the present application provides a recombinant expression vector containing the pollen-specific promoter proGmMS1 described above.

[0009] Another aspect of the present application provides a host cell comprising the pollen-specific promoter proGmMS1 described above.

[0010] Another aspect of the present application provides the use of the pollen-specific promoter proGmMS1 as described above, or the expression cassette as described above, or the recombinant expression vector as described above, or the host cell as described above in at least one of the following (1)-(3): (1) Cultivation of male-sterile plant varieties or lines; (2) Cultivating plant varieties or strains with enhanced pollen activity; (3) Cultivate plant varieties or strains with reduced pollen activity.

[0011] Beneficial effects of this application: The pollen-specific promoter proGmMS1 provided in the present application can drive the specific expression of exogenous target genes in plant pollen, and has a strong driving force on the exogenous target genes and a significant effect, which is of great significance for the creation of plant male sterile lines, the construction of hybrid breeding systems and the safety control of transgenic organisms. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The expression pattern analysis of GmMS1 in different soybean tissues in Example 1, wherein: Figure 1 a in the figure is the real-time fluorescence quantitative qRT-PCR result of GmMS1 in different soybean tissues and flower stages; Figure 1 (b) shows the semi-quantitative RT-PCR results of GmMS1 in different soybean tissues and at different flower stages.

[0013] Figure 2 This is the pHZM33::GUS vector map in Example 3.

[0014] Figure 3 This is the proGmMS1::GUS vector map in Example 3.

[0015] Figure 4 This is the GUS staining image of the T1 generation proGmMS1::GUS transgenic Arabidopsis flower tissue in Example 3, wherein: Figure 4 a in the figure shows GUS staining of the whole flower of two transgenic Arabidopsis lines: wild type WT and proGmMS1::GUS, and the scale bar is 1 mm; Figure 4 b shows GUS staining of anthers, anther walls and pollen of wild-type WT and proGmMS1::GUS transgenic Arabidopsis, with scale bars of 75 μm, 45 μm and 40 μm, respectively.

[0016] Figure 5 This is the vector map of pCAMBIA1300-AMY1 in Example 4.

[0017] Figure 6This is the vector map of pCAMBIA1300-proGmMS1::GmAMY1 in Example 4.

[0018] Figure 7 This is the vector map of pCAMBIA1300-proGmLAT52::GmAMY1 in Example 4.

[0019] Figure 8 The result of proGmMS1 in Example 4 initiating heterologous expression of the pollen lethal gene GmAMY1, wherein: Figure 8 a in the figure shows the Alexander staining results of anthers of wild-type Arabidopsis, proGmLat52::GmAMY1 and proGmMS1::GmAMY1, and the scale bar is 75 μm; Figure 8 b shows the DAPI staining results of wild-type Arabidopsis, proGmLat52::GmAMY1 and proGmMS1::GmAMY1 pollen, the scale bar is 10 μm; Figure 8 c in the figure shows the SEM results of pollen of wild-type Arabidopsis, proGmLat52::GmAMY1 and proGmMS1::GmAMY1, and the scale bar is 25 μm; Figure 8 d in the figure are the statistical results of pollen viability determination of wild-type Arabidopsis, proGmLAT52::GmAMY1 and proGmMS1::GmAMY1. DETAILED DESCRIPTION

[0020] The following will clearly and completely describe the implementation methods of the present application. The technical solutions in the implementation methods described below are exemplary and are only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can fully combine the implementation methods of the present application to obtain other implementation methods without creative work, and these implementation methods are also within the scope of protection of the present application.

[0021] The first aspect of the present application discloses a pollen-specific promoter proGmMS1, which is cloned from GmMS1 and can drive the specific expression of a target gene in pollen.

[0022] GmMS1 is a soybean nuclear male sterility gene, and male sterility is mostly caused by normal pistil development, but abnormal stamen development or pollen abortion. The applicant's previous studies have shown that mutations in the GmMS1 gene can lead to soybean infertility, abnormal pollen development, extrusion and excessive vacuolation of the tapetum cells, resulting in failure of microspore release and callus degradation (GmMs1 encodes a kinesin-like protein essential for male fertility in soybean (Glycine maxL.) [J]. Journal of Integrative Plant Biology, 2021. DOI: 10.1111 / jipb.13110.). Therefore, it is reasonable to suspect and speculate that the promoter of the GmMS1 gene plays a key role in the pollen development process. In this application, by analyzing the spatiotemporal expression pattern of GmMS1, it was found that the GmMS1 gene was highly expressed in flowers and gradually increased as the pollen matured. Based on this, this application is proposed.

[0023] In the present application, the pollen-specific promoter proGmMS1 has the nucleotide sequence shown in SEQ ID NO.1, or has a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.1.

[0024] Through software analysis, the pollen-specific promoter proGmMS1 contains cis-acting elements GTGA-box, AGAAA-box and TCATTT-box related to pollen-specific expression, and also contains TGTGG-box that enhances pollen-specific expression. The pollen-specific promoter can drive the specific expression of the target gene in pollen with strong driving force and significant effect, thus providing a new tool for soybean genetic engineering research and application.

[0025] The second aspect of the present application discloses an expression cassette, which contains the pollen-specific promoter proGmMS1 described in the first aspect of the present application.

[0026] In the present application, the expression cassette refers to the minimum nucleotide sequence framework required for gene expression, which includes at least three key elements, namely a promoter, a target gene and a terminator.

[0027] In some examples, the target gene is a reporter gene. It is well known to those skilled in the art that the expression pattern and intensity of the promoter are generally detected by a reporter gene. In the present application, there are no special requirements for the reporter gene. The reporter gene commonly used in plant promoter research known in the art can be used. Specific examples include β-glucuronidase gene (GUS), green fluorescent protein gene (GFP), etc., and other fluorescent proteins derived from the above two fluorescent proteins; in addition, it can also be a luciferase gene (LUC), etc., but it is not limited thereto. In some preferred examples, the reporter gene is GUS.

[0028] In some examples, a reporter gene is constructed according to the "promoter-reporter gene-terminator" vector construction method to obtain an expression cassette, the promoter and reporter gene are operably connected and transformed, and the spatiotemporal expression and expression intensity of the promoter can be determined based on the results of plant tissue staining.

[0029] In the present application, the transformation system may adopt the protoplast transformation system, gene gun bombardment or Agrobacterium-mediated transient expression system, and transgenic plant stable expression system well known in the art, without particular limitation.

[0030] It is understandable that the construction of the specific expression cassette and the transformation method are all carried out using methods well known to those skilled in the art, and those skilled in the art have such capabilities.

[0031] The third aspect of the present application discloses a recombinant expression vector comprising the pollen-specific promoter proGmMS1 described in the first aspect of the present application.

[0032] In the present application, the recombinant expression vector refers to a DNA molecule that has been transformed and recombined by means of genetic engineering, etc., which can introduce foreign genes into host cells and achieve their efficient expression. For recombinant expression vectors, their core functions rely on the synergistic effect of multiple functional elements, specifically, including promoters, terminators, and regulatory sequences that are operably connected to the upstream and downstream of the promoter and terminator or between the two. These regulatory sequences can be enhancers, transcription termination information, replication origin (Ori), polyadenylation sequences, multiple cloning sites (MCS), resistance markers, etc. In the present application, the promoter is the pollen-specific promoter proGmMS1 in the present application, and other elements can be designed accordingly as needed and operably connected to form a recombinant vector without special restrictions.

[0033] It is understood that in the present application, the term "operably connected (connected)" or "operably connected (connected)" refers to the functional spatial arrangement of two or more nucleic acid regions or nucleic acid sequences. For example, a promoter region is placed at a specific position relative to the target gene nucleic acid sequence, so that the transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operably connected" to the nucleic acid sequence.

[0034] In the present application, the target gene is a gene related to pollen, for example, it can be a pollen lethal gene, a pollen trait improving gene or a pollen trait suppressing gene.

[0035] In some examples, the target gene is a pollen lethal gene, such as GmAMY1, but is not limited thereto.

[0036] In other examples, the target gene is a pollen trait improvement gene, that is, any gene that has the ability to improve the pollen traits of plants to increase pollen activity. Specific examples include MAD transcription factors for the development of stamens and flower organs, flowering regulatory genes FT class, etc., but are not limited thereto.

[0037] In other examples, the target gene is a pollen trait suppressor gene, that is, any gene that has a suppressive function on the pollen trait of a plant to reduce pollen activity and reduce or avoid gene drift. Specific examples include barnase, DAM, etc., but are not limited thereto.

[0038] In the present application, the recombinant vector is a recombinant vector that uses genetic engineering to insert and recombinant pollen-specific promoter proGmMS1 and target gene into an expression vector. The expression vector forms here include but are not limited to plasmids, bacteriophages, viral vectors, etc. In some preferred examples, the expression vector used is a plant expression vector, such as pCAMBIA1300, etc., but is not limited thereto.

[0039] The fourth aspect of the present application discloses a host cell comprising the pollen-specific promoter proGmMS1 described in the first aspect of the present application.

[0040] In the present application, the host cell refers to a cell that can be transfected or transformed with the recombinant expression vector described in the present application, and enables the recombinant expression vector to achieve stable expression. It is understood that the source of the host cell includes plants, animals, bacteria, fungi, phages or viruses, etc., which can be selected as needed. The transformation method can be used according to the selection of the host cell, which is well known to those skilled in the art.

[0041] In some examples, the host cell is Agrobacterium. Specifically, the recombinant expression vector is transformed into Agrobacterium, and then the plant is transfected through Agrobacterium-mediated transfection.

[0042] The fifth aspect of the present application discloses the use of the pollen-specific promoter proGmMS1 as described in the first aspect of the present application, or the expression cassette as described in the second aspect of the present application, or the recombinant expression vector as described in the third aspect of the present application, or the host cell as described in the fourth aspect of the present application in at least one of the following (1)-(3): (1) Cultivation of male-sterile plant varieties or lines; (2) Cultivating plant varieties or strains with enhanced pollen activity; (3) Cultivate plant varieties or strains with reduced pollen activity.

[0043] In the present application, the above-mentioned pollen-specific promoter proGmMS1, expression cassette, recombinant expression vector, host cell, etc. are introduced into the starting plant through transgenic engineering technology, thereby obtaining the corresponding target plant, which is of great significance in the cultivation of male sterile plant varieties or lines, plant varieties or lines with enhanced pollen activity, and plant varieties or lines with reduced pollen activity.

[0044] In the present application, the plant is a dicotyledonous crop, which is mainly characterized by having two cotyledons (embryos) in the seed, and specific examples include at least one of soybean, Arabidopsis, tobacco, tomato, peanut, cotton, rapeseed, etc., but are not limited thereto. In some examples, the plant is one of soybean and Arabidopsis.

[0045] The following are specific embodiments of the present application. It should be noted that the following specific embodiments are only for illustrative purposes and do not limit the scope of the present application in any way.

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0047] In addition, unless otherwise specified, methods without specific conditions or steps are conventional methods, for example, referring to the conditions described in "Molecular Cloning Laboratory Manual" by Sambrook et al., or the conditions recommended by the manufacturer. The reagents and materials used can be obtained from commercial sources.

[0048] Example 1 Analysis of the expression pattern of GmMS1 in different soybean tissues The expression of genes is different at different stages of individual development and in different tissues and cell types of individuals, that is, gene expression is spatiotemporal, and the spatiotemporal characteristics of gene expression provide important information for the study of gene function. Therefore, in this example, by analyzing the spatiotemporal expression pattern of GmMS1 (Glyma.13G114200, Glycine max Wm82.a4.v1), it was determined that the expression of the GmMS1 gene in flowers gradually increases as pollen matures.

[0049] Specifically, in this example, the temporal and spatial expression characteristics of the GmMS1 gene were analyzed by semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR).

[0050] The steps are as follows: 1. Roots, stems, leaves, flowers at different opening stages and seeds of soybean were selected, and total RNA was isolated from soybean tissues using TaKaRa MiniBEST PlantRNA Extraction Kit-9769 (TaKaRa, Dalian).

[0051] (1) Quickly transfer the fresh plant tissue sample to a mortar pre-cooled with liquid nitrogen, and grind the tissue with a pestle until it is ground into a powder with no obvious visible particles. Place the ground sample (50-100 mg) in a 1.5 mL sterile tube and add 450 μL of 50× DTT Solution Buffer RL. Use a pipette to repeatedly pipette until there is no obvious precipitation in the lysate.

[0052] (2) Centrifuge the sample at 12,000 r / min and 4°C for 5 min and transfer the supernatant into a new 1.5 mL sterile tube.

[0053] (3) Add 1 / 2 volume of anhydrous ethanol to the supernatant (precipitate may appear at this time), and use a pipette to mix the solution evenly. Transfer the entire mixture (including the precipitate) to the RNA Spin Column (containing a 2mL Collection Tube). (If the volume of the mixture is greater than 600μL, add it in batches, and the volume added each time should not exceed 600μL.) (4) Centrifuge at 12000 r / min for 1 min and discard the filtrate. Place the RNA Spin Column back into the 2 mL Collection Tube.

[0054] (5) Add 500 μL of Buffer RWA to the RNA Spin Column, centrifuge at 12,000 rpm for 30 seconds, and discard the filtrate. (6) First, confirm that the specified volume of anhydrous ethanol has been added to Buffer RWB. Add 600 μL of Buffer RWB around the wall of the RNA Spin Column tube to help completely wash away the salt adhering to the tube wall. Centrifuge at 12,000 rpm for 30 seconds and discard the filtrate. (7) Repeat step (6). (8) Place the RNA Spin Column back on the 2 mL Collection Tube and centrifuge at 12,000 rpm for 2 min. (9) Place the RNA Spin Column on a 1.5 mL RNase Free Collection Tube, add 50-200 μL of Rase Free ddH2O or 0.1% DEPC-treated water to the center of the RNA Spin Column membrane, and let stand at room temperature for 5 min. (10) Centrifuge at 12000 rpm for 2 min to elute RNA. 2. After the integrity is detected by electrophoresis and 28S and 18S are clearly visible, the concentration is measured by a micro-spectrophotometer and stored in a -80°C refrigerator; the first-strand cDNA is synthesized using the PrimeScript™ RT reagent Kit with gDNA Eraser (PerfectReal Time) reverse transcription kit-RR047A (TAKARA, Dalian): (1) Remove residual genomic DNA from the extracted RNA: Add the following components to a 200 μL sterile RNase-free PCR tube: 5× gDNA Eraser Buffer (2 μL), gDNA Eraser (1 μL), Total RNA plus RNase Free ddH2O to make up the volume to 7 μL; reaction conditions: 42°C, 2 min; After obtaining soybean RNA, use Thermo Nanodrop to detect the RNA concentration, normalize the concentrations of all extracted soybean RNAs from different tissues, and then perform RNA reverse transcription. (2) For reverse transcription of RNA, add the following components into a 200 μL centrifuge tube: PrimeScript RT Enzyme MixI (1 μL), 5× PrimeScript Buffer 2 (for Real Time) (4 μL), RT Primer Mix*4 (1 μL), RNaseFree dH2O (4 μL); reaction conditions: 37°C (15 min), 85°C (5 s); the obtained cDNA is stored at -80°C for subsequent detection of target gene expression.

[0055] 3. Based on the reference sequence of the GmMS1 gene compared from the soybean reference genome database (https: / / Phytozome-next.jgi.doe.gov / ), primers were designed using Primer Premier 5. The specific primer information is as follows:

[0056] GmACTIN11 was used as the quantitative internal reference gene for qRT-PCR, and the key marker genes in soybean flowering development were used as references. AceQ Universal SYBR gPcR Master Mix-Q511-02 (Vazyme, Nanjing) was used for real-time fluorescence quantitative PCR: the following components (20 μL) were added to a 200 μL RNase-free PCR tube: ChamQ UniversalSYBR qPCR Master Mix (10 μL), upstream primer (0.4 μL), downstream primer (0.4 μL), cDNA (100 ng), and RNaseFree H2O was added to the volume to 20 μL. The real-time PCR reaction used a two-step method (2-3 steps set 35 cycles) with the following program: 95°C (20 s), 93°C (10 s), 65°C (20 s).

[0057] RT-PCR reaction system (25 μL): 2×Rapid Taq Master Mi-P222 enzyme (Vazyme, Nanjing) 12.5μL, H2O 9.5μL, upstream and downstream primers 1μL each, cDNA template 1μL.

[0058] PCR program: pre-denaturation at 95°C (5 min); denaturation at 95°C (30 sec), annealing at 60°C (30 sec), extension at 72°C (30 sec), 28 cycles; extension at 72°C (5 min); storage at 16°C.

[0059] Quantitative results using 2 -△△Ct The algorithm performs relative quantitative analysis on the expression of detected genes to determine the tissue-specific expression of target genes.

[0060] Figure 1The results of semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR) are shown in the figure. It can be seen that the expression level of GmMS1 in flowers gradually increases with the development and maturity of pollen. This indicates that the expression of GmMS1 is specifically regulated by the pollen development stage and may play an important role in the pollen development process. It may be involved in the formation of pollen wall, the development and improvement of various organelles in pollen cells, and the synthesis of pollen tube germination-related substances, so as to ensure that pollen has normal function and vitality.

[0061] Example 2 Cloning of GmMS1 promoter and analysis of cis-acting elements In this example, the CTAB method was first used to extract soybean Williams 82 leaf genomic DNA, and then the soybean Williams 82 genomic DNA was used as a template to clone the pollen-specific promoter sequence proGmMS1.

[0062] 1. Obtaining soybean Williams 82 genomic DNA Genomic DNA was extracted from fresh leaves of plants using the cetyltrimethylammonium bromide (CTAB) method. The specific method is as follows: (1) Take a 1 cm long leaf and place it in a 2 mL centrifuge tube. Add a small steel ball to the tube and freeze it in liquid nitrogen. Oscillate at 45 Hz for 60 seconds until it becomes powder. Then add 800 μL of CTAB extract. Mix by hand and place in a 65 °C water bath for 10 minutes. The liquid in the tube should be green in color. Invert and shake every 3 minutes.

[0063] (2) Add 800 μL of chloroform:isoamyl alcohol (volume ratio 24:1) in a fume hood and shake up and down for 1 min to fully extract.

[0064] (3) Centrifuge at 12000 rpm for 5 min and transfer approximately 600 μL of the supernatant into a new 2 mL centrifuge tube.

[0065] (4) Add double volume of anhydrous ethanol to precipitate the DNA, invert 8 to 10 times, and place in a -20°C refrigerator for 30 min.

[0066] (5) Centrifuge at 12000 rpm for 2 min, discard the supernatant, and the white flocs at the bottom of the tube are DNA. Air-dry in a fume hood for 2 h.

[0067] (6) Add 100 μL of ddH2O and store at -20°C for long term storage.

[0068] 2. Cloning of the pollen-specific promoter sequence proGmMS1 Using soybean Williams 82 genomic DNA as template, primers were designed and the pollen-specific promoter proGmMS1 was cloned from soybean Williams 82 genomic DNA.

[0069] The primer pairs used for amplification are as follows:

[0070] PCR reaction system (25 μL): 2× Rapid Taq Master Mi-P222 enzyme (Vazyme, Nanjing, China) 12.5 μL, H O 9.5 μL, 1 μL each of upstream and downstream primers, and 1 μL of DNA template.

[0071] PCR program: pre-denaturation at 95°C (5 min); denaturation at 95°C (30 sec), annealing at 60°C (30 sec), extension at 72°C (30 sec), 36 cycles; extension at 72°C (5 min); storage at 16°C.

[0072] The PCR product was recovered and sequenced, and the sequencing results showed that the nucleotide sequence of the pollen-specific promoter proGmMS1 was as shown in SEQ ID NO.1, and the length was 1622 bp.

[0073] 3. Analysis of cis-acting elements of pollen-specific promoter proGmMS1 The pollen-specific promoter proGmMS1 was uploaded to the software PlantCARE for analysis. The results showed that the pollen-specific promoter proGmMS1 contained the cis-acting elements GTGA-box, AGAAA-box and TCATTT-box related to pollen-specific expression. In addition, it also contained TGTGG-box which has the function of enhancing pollen-specific expression.

[0074] Example 3 Construction of proGmMS1::GUS expression cassette and genetic transformation of Arabidopsis 1. Construction of proGmMS1::GUS expression cassette The empty plasmid pHZM33::GUS involved in this example was prepared and stored by our laboratory. For details, please refer to pHZM33 disclosed in the Chinese patent application with publication number CN118028355A. The difference in this example is that GFP is replaced by GUS. The map of the empty plasmid pHZM33::GUS in this example can be found in Figure 2 .

[0075] The specific steps for constructing the proGmMS1::GUS expression cassette are as follows: (1) Select StuI and AvrII restriction endonucleases (TaKaRa) to double-digest the empty plasmid pHZM33::GUS.

[0076] Among them, the enzyme digestion system is 25μL: 1 μL each of restriction endonucleases, 2.5 μL of Cutsmart, 5.5 μL of H2O, and 15 μL of target vector.

[0077] Enzyme digestion program: 37°C, 3h; 80°C, 20min; storage at 16°C.

[0078] (2) The gel-recovered product of the proGmMS1 promoter sequence was connected to the vector after restriction digestion by homologous recombination method.

[0079] Among them, the connection system is 10μL: 2 μL of the digested vector, 3 μL of the gel-recovered product, and 5 μL of homologous recombination enzyme (ClonExpress MultiS One Step Cloning Kit) (Vazyme, Nanjing).

[0080] Connection procedure: 50℃, 50min; storage at 16℃.

[0081] (3) Transformation of Mach1-T1-DL1015M (Weidi Biotechnology, Shanghai) E. coli single clone selection transformation process is as follows: (a) Take out the competent March T1 E. coli from the -80℃ freezer and thaw it on ice. Gently tap it to accelerate the dissolution.

[0082] (b) Prepare a clean 1.5 mL centrifuge tube, add all the ligation products and 50 μL of dissolved competent E. coli (try to add to the bottom of the centrifuge tube), flick to mix, and place on ice for 30 min.

[0083] (c) The mixed sample was heat-shocked in a metal bath at 42 °C for 60 s and then placed on ice for 2 min.

[0084] (d) Open the centrifuge tube in the clean bench, add 500 μL of sterilized LB liquid culture medium (without antibiotics), place the centrifuge tube in a constant temperature shaker at 37°C, 220 rpm, and culture for 1 h.

[0085] (e) Prepare LB solid culture medium (containing 50 μg / L kanamycin) in advance in an ultra-clean bench, burn the coating stick with an alcohol lamp, place the centrifuge tube in the shaker in a centrifuge at 6000 rpm for 1 min, take out the tube and use a sterilized pipette in the ultra-clean bench to remove part of the supernatant, leaving 100 μL of the resuspended precipitate, and mix by pipetting.

[0086] (f) Spread the plate, mark the dish, and invert it in a 37°C incubator for overnight culture (12-16 h).

[0087] After successful sequencing and identification, the proGmMS1::GUS expression cassette was obtained, and its map can be found in Figure 3 .

[0088] 2. Genetic transformation of Arabidopsis thaliana Arabidopsis genetic transformation was performed using the floral dipping method, and the specific steps are as follows: (1) Take 1 μL proGmMS1::GUS expression cassette plasmid and add 50 μL GV3101 Agrobacterium AC1001 (Weidi Biotechnology, Shanghai). After fully mixing, place on ice for 5 minutes, in liquid nitrogen for 5 minutes, in a 37°C water bath for 5 minutes, and in an ice bath for 5 minutes. Add 500 μL of antibiotic-free LB liquid medium and shake in a shaker at 28°C and 220 rpm for 2-3 hours. Prepare LB solid medium (containing 50 μg / L rifampicin and 50 μg / L kanamycin) in advance in a clean bench. Burn the coating stick with an alcohol lamp and place the bacterial liquid in a centrifuge at 6000 rpm for 1 minute. After taking it out, use a sterilized pipette in a clean bench to remove part of the supernatant, leaving 100 μl of the resuspended precipitate, pipette and mix well, inoculate it on the LB solid medium, and culture it in the dark at 28°C for 48 hours. After a single colony grows, perform colony PCR screening, add 600 μL LB (containing 50 μg / L rifampicin and 50 μg / L kanamycin) liquid culture medium to the identified positive clone, and culture overnight at 28°C in a shaker at 220 rpm. The next day, preserve the bacteria with 50% glycerol and store at -80°C.

[0089] (2) Transfer 1 mL of bacterial solution into 10 mL of LB liquid medium (containing 50 μg / L rifampicin and 50 μg / L kanamycin) and culture at 28°C at 220 rpm until the bacterial solution concentration reaches OD 600 The value was 1.2-1.6, 5000r / min, centrifuged for 15min, discarded the supernatant, and resuspended the cells in 5% sucrose solution until OD 600 The value is about 1.3; before infection, add Silwet L-77 surfactant at a volume ratio of 0.05%, take Arabidopsis thaliana with good growth conditions and suitable period, remove the young pods and opened flowers on the inflorescence, and soak the inflorescence in the prepared bacterial solution for 1 minute; protect the infected plants from light, culture in the dark for 24 hours, take them out, and grow and culture them normally.

[0090] To improve the transformation efficiency, the new inflorescences were infected again every other week, for a total of three times; after harvesting the T0 generation seeds, positive plants were selected using 1 / 2MS solid medium containing hygromycin (50 μg / mL) resistance, and Arabidopsis seedlings were sprayed with Basta (1 / 2000) resistance (different vectors have different resistance) to select homozygous lines for experiments.

[0091] In this embodiment, LB medium is shown in the following table:

[0092] Note: Place in the autoclave at 121°C and sterilize at high temperature and high pressure for 20 min. Wait until it cools to the appropriate temperature, add the corresponding antibiotics, and pour the culture medium into a 9cm×9cm round plastic culture dish, with about 20mL of culture medium in each culture dish.

[0093] The configuration of 1 / 2MS culture medium is shown in the table below:

[0094] Note: Place in a sterilizer at 121°C and sterilize under high temperature and high pressure for 20 minutes. When cooled to the appropriate temperature, pour the culture medium into a 13cm×13cm square plastic culture dish, with about 40mL of culture medium poured into each culture dish.

[0095] 3. GUS detection of transgenic Arabidopsis Different tissues of transgenic Arabidopsis seedlings were stained with X-gluc (5-bromo-4-chloro-3-indolyl β- d -glucuronide), GUS staining buffer [containing 1 mM 5-bromo-4-chloro-3-indolyl β- d -glucuronidase (Gold BioTechnology, St. Louis, Missouri, USA), 100 mM sodium phosphate (pH 7.5), 0.5 mM potassium ferrocyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, and 0.1% (v / v) Triton X-100]. All treatments were incubated at 37°C for 4 h and destained with 70% (v / v) ethanol, and images were taken using a stereo microscope.

[0096] The results are as follows Figure 4 As shown in , proGmMS1::GUS was stained blue in transgenic Arabidopsis pollen, but not in wild-type pollen, indicating that proGmMS1 is a pollen-specific promoter.

[0097] Example 4 Construction and phenotypic identification of the recombinant expression vector pCAMBIA1300-proGmMS1::GmAMY1 1. Construction of recombinant expression vector pCAMBIA1300-proGmMS1::GmAMY1 (1) Construction of pCAMBIA1300-AMY1 vector The pCAMBIA1300 vector (Miaoling plasmid platform, P21036) was linearized by NcoI and XbaI restriction endonucleases (NEB), and the vector was prepared and stored in the laboratory. Primers were designed to clone the pollen lethal gene GmAMY1, and the pollen lethal gene GmAMY1 was connected to construct the recombinant expression vector pCAMBIA1300-AMY1. The specific operation steps were carried out with reference to the steps of enzyme digestion and homologous recombination in Example 3, wherein the primers involved are shown in the following table:

[0098] The constructed pCAMBIA1300-AMY1 vector map is shown in Figure 5 .

[0099] (2) Construction of the recombinant expression vector pCAMBIA1300-proGmMS1::GmAMY1 The pCAMBIA1300-AMY1 vector was linearized by BamHI digestion and ligated to the promoter proGmMS1 to construct the recombinant expression vector pCAMBIA1300-proGmMS1::GmAMY1 (see vector map for details). Figure 6 ); The specific operation steps are carried out with reference to the steps of enzyme digestion and homologous recombination in Example 3.

[0100] At the same time, the same steps and methods were used to connect the promoter GmLAT52 (sequence see CN117025661A) to the pCAMBIA1300-AMY1 vector to construct the recombinant expression vector pCAMBIA1300-proGmLAT52::GmAMY1 (vector map see Figure 7 The primers involved are shown in the following table:

[0101] (3) Transform Arabidopsis thaliana according to the steps in Example 3 and perform phenotypic identification.

[0102] Alexander staining: Take the white Arabidopsis flowers, use tweezers to peel off the calyx and petals, pick out the anthers and place them on a glass slide, drop 20-30 μL of Alexander stain (G3050, Beijing Solebow Technology Co., Ltd.) on the anthers, cover with a coverslip and stain for 10-20 minutes, and examine under a microscope.

[0103] DAPI staining: at 10 am, take the fully opened flowers of the day, use tweezers to peel off the sepals and petals, shake the pollen onto the slide, drop 10μL DAPI staining solution (SL7100, Beijing Coolbo Technology Co., Ltd.), cover with a coverslip and stain at room temperature for 3-5 minutes, and observe the staining results under a fluorescence microscope.

[0104] Scanning electron microscopy analysis: Pollen was fixed in 2.5% glutaraldehyde at 4 °C overnight, treated with 30%, 50%, 70%, 90%, and 100% ethanol for 10 min, transferred to isoamyl acetate for 20 min, critical point dried, gold coated for 30 s, and photographed under a scanning electron microscope (JSM-6390LV).

[0105] The results are as follows Figure 8 As shown in .

[0106] Alexander staining results showed that wild-type mature pollen grains were stained purple-red, while some aborted pollen grains in the overexpression were not stained ( Figure 8 a) in the figure.

[0107] The DAPI staining results showed that the mature pollen of Arabidopsis wild type could develop normally to the trinuclear stage, while no clear nuclear morphology could be observed inside some pollen grains of proGmMS1::GmAMY1 ( Figure 8 b) in the above.

[0108] Scanning electron microscopy analysis of pollen morphology showed that the outer walls of some proGmMS1::GmAMY1 pollen grains were wrinkled or collapsed, indicating that these pollen grains could not develop normally ( Figure 8 c) in.

[0109] The results of pollen viability test showed that the use of GmMS1 promoter to drive the expression of pollen lethal gene GmAMY1 significantly increased the pollen lethality rate (pollen viability decreased from 42.04±7.73% driven by the reported pollen-specific promoter LAT52 promoter to 8.72±5.76% driven by proGmMS1 promoter). The results showed that proGmMS1 promoter can drive the specific expression of target gene in pollen, and the driving force is strong and the effect is significant. This is of great significance for improving plant strains or varieties through genetic engineering ( Figure 8 d) in.

[0110] It should be noted that the present application is not limited to the above-mentioned embodiments. The above-mentioned embodiments are only examples, and the embodiments having the same structure as the technical idea and the same effect as the technical solution of the present application are all included in the technical scope of the present application. In addition, without departing from the scope of the main purpose of the present application, various modifications that can be thought of by those skilled in the art to the embodiments and other methods of combining some of the constituent elements in the embodiments are also included in the scope of the present application.

Claims

1. A pollen-specific promoter proGmMS1, characterized in that The nucleotide sequence thereof is the nucleotide sequence shown in SEQ ID NO.1; or a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.

1.

2. An expression cassette, characterized in that Contains the pollen-specific promoter proGmMS1 according to claim 1.

3. The expression cassette according to claim 2, characterized in that The expression cassette also contains a reporter gene.

4. The expression cassette according to claim 3, characterized in that The reporter gene is GUS, GFP or LUC.

5. A recombinant expression vector, characterized in that: Contains the pollen-specific promoter proGmMS1 according to claim 1.

6. The recombinant expression vector according to claim 5, characterized in that It also contains a target gene, which is a pollen lethal gene, and the pollen lethal gene is GmAMY1.

7. A host cell, characterized in that Contains the pollen-specific promoter proGmMS1 according to claim 1.

8. Use of the pollen-specific promoter proGmMS1 according to claim 1, or the expression cassette according to any one of claims 2 to 4, or the recombinant expression vector according to any one of claims 5 to 6, or the host cell according to claim 7 in breeding male sterile plant varieties.

9. The use according to claim 8, characterized in that The plant is a dicotyledonous crop; The dicotyledonous crop is at least one of soybean and Arabidopsis thaliana.

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