A pollen-specific promoter proGmMS1 and its application

Abstract

The present application discloses a pollen-specific promoter proGmMS1 and its application. This pollen-specific promoter proGmMS1 is cloned from the soybean gene GmMS1 and has a nucleotide sequence as shown in SEQ ID NO.1; alternatively, it has a nucleotide sequence complementary to the nucleotide sequence shown in SEQ ID NO.1. This pollen-specific promoter proGmMS1 can precisely regulate the expression of a target gene during the pollen development stage, create new soybean male sterile lines, provide an efficient tool for soybean hybrid breeding, and at the same time reduce the ecological risk of transgenic plants, having important theoretical and practical values.

Description

Technical Field

[0001] This application belongs to the technical field of plant genetic engineering, and specifically relates to a pollen-specific promoter proGmMS1 and its application. This pollen-specific promoter is cloned from the soybean (Glycine max) genome and can drive the specific expression of a target gene in pollen, and is applicable to the creation of plant male sterile lines, the construction of a hybrid breeding system, and the control of transgenic biosafety. Background Art

[0002] The transgenic technology is an important tool for plant gene function research. The promoter is an important element for gene expression regulation. The selection of the promoter type determines the expression time and location of the gene. Therefore, in plant genetic engineering, the selection of a suitable promoter is crucial for achieving the specific expression of foreign genes.

[0003] According to the degree of regulation of the promoter on the transcription level, it can be divided into a weak promoter and a strong promoter. With reference to the transcription pattern, the promoter can also be divided into: constitutive promoter, tissue-specific promoter, and inducible promoter. Among them, the 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 the fields of genetic engineering, metabolic engineering, and biotechnology, can achieve the precise regulated expression of genes, and has the advantages of high ecological safety and small metabolic burden.

[0004] Among tissue-specific promoters, pollen-specific promoters are an important type, which can drive genes to specifically express in plant pollen, and thus can be widely applied to crop genetic improvement and the cultivation of male sterile lines. Currently, relatively few pollen-specific promoters derived from soybean genes have been reported. For example, a Chinese patent application with the publication number CN117025661A discloses a soybean pollen-specific promoter GmLat52P, which can drive the specific expression of the pollen lethal gene GmAMY1 in soybean flowers and can disrupt the viability of transgenic plant pollen. Therefore, it can be determined that the pollen lethal gene expression cassette can be applied to the creation and cultivation of soybean intelligent nuclear male sterile lines. However, the ratio of fertile pollen grains to aborted pollen grains in heterozygous transgenic plants in which the promoter GmLat52P drives the expression of GmAMY1 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, indicating 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.) have limitations in species compatibility. They are highly applicable in monocotyledonous plants but have insufficient activity or poor specificity in dicotyledonous plants. Summary of the Invention

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

[0006] To achieve the above object, the present application adopts the following technical solutions:

[0007] One aspect of the present application provides a pollen-specific promoter proGmMS1, which 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.

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

[0009] Another aspect of the present application provides a recombinant expression vector containing the pollen-specific promoter proGmMS1 described above.

[0010] Another aspect of the present application provides a host cell containing the pollen-specific promoter proGmMS1 described above.

[0011] 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 any at least one of the following (1)-(3):

[0012] (1) Cultivating male sterile plant varieties or lines;

[0013] (2) Cultivating plant varieties or lines with enhanced pollen activity;

[0014] (3) Cultivating plant varieties or lines with reduced pollen activity.

[0015] Advantages of the present application:

[0016] The pollen-specific promoter proGmMS1 provided by the present application can drive the specific expression of exogenous target genes in plant pollen, and has a strong driving force for exogenous target genes, with significant effects. It is of great significance for the creation of plant male sterile lines, the construction of hybrid breeding systems, and the control of transgenic biosafety. Description of the drawings

[0017] Figure 1 For the analysis of the expression pattern of GmMS1 in different tissues of soybean in Example 1, where Figure 1 a in is the real-time fluorescence quantitative qRT-PCR result of GmMS1 in different tissues of soybean and different stages of flowers; Figure 1 b in is the semi-quantitative RT-PCR result of GmMS1 in different tissues of soybean and different stages of flowers.

[0018] Figure 2 For the vector map of pHZM33::GUS in Example 3.

[0019] Figure 3 For the vector map of proGmMS1::GUS in Example 3.

[0020] Figure 4 For the GUS staining map of the flower tissue of T1 generation proGmMS1::GUS transgenic Arabidopsis thaliana in Example 4, where Figure 4 a in is the GUS staining of the whole flower of two transgenic Arabidopsis thaliana lines of wild type WT and proGmMS1::GUS, and the scale bar is 1 mm; Figure 4 b in is the GUS staining of the anthers, anther walls and pollen of wild type WT and proGmMS1::GUS transgenic Arabidopsis thaliana, and the scale bars are 75 μm, 45 μm, 40 μm respectively.

[0021] Figure 5 For the vector map of pCAMBIA1300-AMY1 in Example 4.

[0022] Figure 6 It is the vector map of pCAMBIA1300-proGmMS1::GmAMY1 in Example 4.

[0023] Figure 7 It is the vector map of pCAMBIA1300-proGmLAT52::GmAMY1 in Example 4.

[0024] Figure 8 It is the result of heterologous expression of the pollen lethal gene GmAMY1 driven by proGmMS1 in Example 4. Among them, Figure 8 a in is the Alexander staining result of anthers of wild-type Arabidopsis, proGmLat52::GmAMY1 and proGmMS1::GmAMY1. The scale bar is 75 μm; Figure 8 b in is the DAPI staining result of pollen of wild-type Arabidopsis, proGmLat52::GmAMY1 and proGmMS1::GmAMY1. The scale bar is 10 μm; Figure 8 c in is the scanning electron microscopy result of pollen of wild-type Arabidopsis, proGmLat52::GmAMY1 and proGmMS1::GmAMY1. The scale bar is 25 μm; Figure 8 d in is the statistical result of pollen viability determination of wild-type Arabidopsis, proGmLAT52::GmAMY1 and proGmMS1::GmAMY1. Detailed implementation manners

[0025] The implementation manners of the present application will be clearly and completely described below. The technical solutions in the described implementation manners are exemplary and only possible technical implementations of the present application, not all possible implementations. Those skilled in the art can completely combine the implementation manners of the present application and obtain other implementation manners without creative labor, and these implementation manners are also within the protection scope of the present application.

[0026] 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.

[0027] GmMS1 is a soybean nuclear male sterility gene. Male sterility mostly means that the pistil develops normally, but the stamens develop abnormally or pollen abortion occurs. Previous studies by the applicant have shown that mutations in the GmMS1 gene can cause sterility in soybeans, abnormal pollen development, and tapetal cells being squeezed and overly vacuolated, resulting in failure of microspore release and callose degradation (GmMs1 encodes a kinesin‐like protein essential for male fertility in soybean (Glycine max L.)[J]. Journal of Integrative Plant Biology, 2021. DOI: 10.1111 / jipb.13110.). Therefore, it is reasonably suspected and speculated that the promoter of the GmMS1 gene plays a key role in pollen development. In this application, by analyzing the spatio-temporal expression pattern of GmMS1, it was found that the GmMS1 gene is highly expressed in flowers and gradually increases as pollen develops and matures. Based on this, this application was proposed.

[0028] In this 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.

[0029] Through software analysis, this pollen-specific promoter proGmMS1 contains cis-acting elements GTGA-box, AGAAA-box, and TCATTT-box related to pollen-specific expression. In addition, it also contains a TGTGG-box that has the effect of enhancing pollen-specific expression. This pollen-specific promoter can drive the specific expression of the target gene in pollen, and the driving force is strong and the effect is remarkable, thus providing a new tool for soybean genetic engineering research and application.

[0030] The second aspect of this application discloses an expression cassette, and the expression cassette contains the pollen-specific promoter proGmMS1 described in the first aspect of this application.

[0031] In this application, an expression cassette refers to the smallest nucleotide sequence framework required for gene expression, which at least includes three key elements, namely a promoter, a target gene, and a terminator.

[0032] In some examples, the target gene is a reporter gene. As is well known to those skilled in the art, the expression pattern and intensity of a promoter are generally detected through a reporter gene. In this application, there is no particular requirement for the reporter gene, and reporter genes commonly used in plant promoter research well known in the art can be adopted. Specific examples include β-glucuronidase gene (GUS), green fluorescent protein gene (GFP), etc., and can also be other fluorescent proteins derived from the above two fluorescent proteins; in addition, it can also be luciferase gene (LUC), etc., but is not limited thereto. In some preferred examples, the reporter gene is GUS.

[0033] In some examples, an expression cassette is constructed by building the reporter gene in the vector construction mode of "promoter - reporter gene - terminator", the promoter and the reporter gene are operably linked and transformed, and the spatiotemporality and expression intensity of the promoter expression can be judged according to the results of plant tissue staining.

[0034] In this application, the transformation system can adopt the protoplast transformation system, gene gun bombardment, Agrobacterium-mediated transient expression system, stable expression system of transgenic plants, etc. well known in the art, without particular limitation.

[0035] It can be understood that the construction of the specific expression cassette and the transformation method are both carried out by methods well known to those skilled in the art, and those skilled in the art have such capabilities.

[0036] The third aspect of this application discloses a recombinant expression vector containing the pollen-specific promoter proGmMS1 described in the first aspect of this application.

[0037] In this application, the recombinant expression vector refers to a DNA molecule modified and recombined by means of genetic engineering, etc., which can introduce an exogenous gene into a host cell and achieve its high-efficiency expression. For a recombinant expression vector, its core function depends on the synergistic action of multiple functional elements. Specifically, it includes a promoter, a terminator, and regulatory sequences operably linked upstream and downstream or between the promoter and the terminator. These regulatory sequences can be enhancers, transcription termination information, replication origin (Ori), polyadenylation sequence, multiple cloning site (MCS), resistance marker, etc. In this application, the promoter is the pollen-specific promoter proGmMS1 in this application. After other elements are designed accordingly as needed, they can be operably linked to form a recombinant vector, without particular limitation.

[0038] It is understood that in the present application, the term "operatively connected (linked)" or "operational connection (linkage)" 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 nucleic acid sequence of a target gene, such that the transcription of the nucleic acid sequence is guided by the promoter region, and thus the promoter region is "operatively connected" to the nucleic acid sequence.

[0039] 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 improvement gene, or a pollen trait suppression gene.

[0040] In some examples, the target gene is a pollen lethal gene, such as GmAMY1, but is not limited thereto.

[0041] In other examples, the target gene is a pollen trait improvement gene, that is, any gene capable of improving the pollen traits of plants to increase pollen activity. Specific examples include MAD transcription factors for stamen and floral organ development, flowering regulation genes such as FT-like, etc., but are not limited thereto.

[0042] In other examples, the target gene is a pollen trait suppression gene, that is, any gene with an inhibitory function on the pollen traits of plants to reduce pollen activity, reduce or avoid gene drift. Specific examples include barnase, DAM, etc., but are not limited thereto.

[0043] In the present application, the recombinant vector is constructed by inserting and recombining a pollen-specific promoter proGmMS1 and a target gene into an expression vector by means of genetic engineering. The forms of the expression vector here include but are not limited to plasmids, phages, 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.

[0044] The fourth aspect of the present application discloses a host cell containing the pollen-specific promoter proGmMS1 described in the first aspect of the present application.

[0045] 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 sources of the host cells include plants, animals, bacteria, fungi, phages, or viruses, etc., and can be specifically selected according to needs. The transformation method can be those well-known to those skilled in the art according to the selection of the host cell.

[0046] 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 transformation.

[0047] 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 any at least one of the following (1)-(3):

[0048] (1) Cultivating male sterile plant varieties or lines;

[0049] (2) Cultivating plant varieties or lines with enhanced pollen activity;

[0050] (3) Cultivating plant varieties or lines with reduced pollen activity.

[0051] In the present application, through genetic engineering techniques, the above-mentioned pollen-specific promoter proGmMS1, expression cassette, recombinant expression vector, host cell, etc. are introduced into the starting plants, so as to obtain the corresponding target plants, which is of great significance in cultivating male sterile plant varieties or lines, plant varieties or lines with enhanced pollen activity, and plant varieties or lines with reduced pollen activity.

[0052] In the present application, the plant is a dicotyledonous crop, and its main characteristic is that there are two cotyledons (embryonic leaves) in the seed. Specific examples that can be mentioned include at least one of soybean, Arabidopsis thaliana, tobacco, tomato, peanut, cotton, rapeseed, etc., but are not limited thereto. In some examples, the plant is one of soybean and Arabidopsis thaliana.

[0053] The following are specific examples of the present application. It should be noted that the following specific examples are only for illustrative purposes and do not limit the scope of the present application in any way.

[0054] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present application belongs. The terms used in the specification of the present application herein are only for the purpose of describing specific embodiments and are not intended to limit the present application.

[0055] In addition, unless otherwise specified, the methods without specific conditions or steps recorded are all conventional methods. For example, they refer to the conditions described in "Molecular Cloning: A Laboratory Manual" written by Sambrook et al., or the conditions recommended by the manufacturer. The reagents and materials used can be obtained from commercial sources.

[0056] Example 1 Analysis of the expression pattern of GmMS1 in different tissues of soybean

[0057] Gene expression varies at different stages of individual development, as well as in different tissues and cell types of an individual. That is, gene expression has spatio-temporal characteristics, and these characteristics provide important information for the study of gene function. Therefore, in this example, by analyzing the spatio-temporal expression pattern of GmMS1 (Glyma.13G114200, Glycine max Wm82.a4.v1), it was determined that the expression level of the GmMS1 gene gradually increases as pollen develops and matures in flowers.

[0058] Specifically, in this example, semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR) were used to analyze the spatio-temporal expression characteristics of the GmMS1 gene.

[0059] The operation steps are as follows:

[0060] 1. Select the roots, stems, leaves, flowers at different opening stages, and seeds of soybeans, and use the TaKaRa MiniBEST Plant RNA Extraction Kit - 9769 (TaKaRa, Dalian) to isolate total RNA from soybean tissues.

[0061] (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 without obvious visible particles. Place the powdered sample (50 - 100 mg) in a 1.5 mL sterilized tube and add 450 μL of Solution Buffer RL containing 50×DTT, and pipette repeatedly until there is no obvious precipitate in the lysate.

[0062] (2) Centrifuge the sample at 12000 r / min at 4°C for 5 min, and pipette the supernatant into a new 1.5 mL sterilized tube.

[0063] (3) Add an equal volume of absolute ethanol to the supernatant (precipitation may occur at this time), and use a pipette to mix the solution evenly. Transfer all of the mixed solution (including the precipitate) into an RNA Spin Column (containing a 2 mL Collection Tube). (If the volume of the mixed solution is greater than 600 μL, add it in batches, and the volume added each time should not be greater than 600 μL.)

[0064] (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.

[0065] (5) Add 500 μL of Buffer RWA to the RNA Spin Column, centrifuge at 12000 r / min for 30 s, and discard the filtrate.

[0066] (6) First, confirm that the specified volume of absolute ethanol has been added to Buffer RWB. Add 600 μL of Buffer RWB along the inner wall of the RNA Spin Column to help thoroughly rinse the salts adhering to the wall. Centrifuge at 12,000 r / min for 30 s and discard the filtrate.

[0067] (7) Repeat operation step (6).

[0068] (8) Place the RNA Spin Column back onto the 2 mL Collection Tube and centrifuge at 12,000 r / min for 2 min.

[0069] (9) Place the RNA Spin Column onto a 1.5 mL RnaseFree 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 it stand at room temperature for 5 min.

[0070] (10) Centrifuge at 12,000 r / min for 2 min to elute the RNA.

[0071] 2. After detecting the integrity by electrophoresis and clearly visualizing 28S and 18S, measure the concentration with a micro-spectrophotometer and then store it in a -80°C refrigerator. Use the PrimeScript™ RT reagent Kit with gDNA Eraser (PerfectReal Time) reverse transcription kit - RR047A (TAKARA, Dalian) to synthesize the first-strand cDNA:

[0072] (1) Remove the residual genomic DNA in the extracted RNA: Add the following components to a 200 μL sterile and RNase-free PCR tube: 5×gDNA Eraser Buffer (2 μL), gDNA Eraser (1 μL), and add RNase Free ddH2O to the Total RNA to make the volume up to 7 μL. Reaction conditions: 42°C, 2 min. After obtaining the soybean RNA, use a Thermo Nanodrop to detect the concentration of the RNA. After normalizing the concentrations of all the extracted soybean RNA from different tissues, perform reverse transcription of the RNA.

[0073] (2)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 was stored at -80 °C for later use in the detection of the expression level of the target gene.

[0074] 3. According to the reference sequence of the GmMS1 gene aligned 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 in the table:

[0075]

[0076] Using GmACTIN11 as the quantitative internal reference gene for qRT-PCR, taking the key marker genes during the soybean flowering and development process as references, AceQ Universal SYBR gPcR Master Mix-Q511-02 (Vazyme, Nanjing) was used for real-time fluorescence quantitative PCR: Add the following components (20 μL) into 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 used to make up the volume to 20 μL. The Real-time PCR reaction adopted a two-step method (35 cycles were set for steps 2 - 3) with the following procedure: 95 °C (20 s), 93 °C (10 s), 65 °C (20 s).

[0077] RT-PCR reaction system (25 μL):

[0078] 2×Rapid Taq Master Mi-P222 enzyme (Vazyme, Nanjing) 12.5 μL, H2O 9.5 μL, 1 μL each of the upstream and downstream primers, 1 μL of cDNA template.

[0079] PCR procedure: 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); Store at 16 °C.

[0080] The quantitative results were used with 2-△△Ct The algorithm detects the expression level of genes for relative quantitative analysis and determines the tissue-specific expression of target genes.

[0081] Figure 1 The results of semi-quantitative RT-PCR and quantitative real-time PCR (qRT-PCR) are shown. It can be seen that the expression level of GmMS1 in flowers gradually increases as pollen develops and matures. This indicates that the expression of GmMS1 is specifically regulated by the pollen development stage and may play an important role in the process of pollen development. It may be involved in the formation of the pollen wall, the development and improvement of various organelles in pollen cells, the synthesis of substances related to pollen tube germination, etc., to ensure that pollen has normal functions and vitality.

[0082] Example 2 Cloning and cis-acting element analysis of the GmMS1 promoter

[0083] In this example, genomic DNA of soybean Williams 82 leaves was first extracted using the CTAB method. Then, using the genomic DNA of soybean Williams 82 as a template, the pollen-specific promoter sequence proGmMS1 was cloned.

[0084] 1. Obtain genomic DNA of soybean Williams 82

[0085] Genomic DNA was extracted from fresh leaves of plants using the cetyltrimethylammonium bromide (CTAB) method. The specific method is as follows:

[0086] (1) Take a 1-cm-long leaf and place it in a 2-mL centrifuge tube. Add small steel beads to the tube, freeze it thoroughly in liquid nitrogen, shake it at 45 Hz for 60 s until it becomes powdery, then add 800 μL of CTAB extraction buffer, mix it well by hand, and place it in a 65°C water bath for 10 min. The liquid in the tube is best when it is turquoise green. In the middle, it needs to be inverted and shaken well every 3 min.

[0087] (2) Add 800 μL of chloroform:isoamyl alcohol (volume ratio 24:1) in the fume hood, shake it up and down for 1 min to fully extract.

[0088] (3) Centrifuge at 12000 rpm for 5 min, and pipette about 600 μL of the supernatant into a new 2-mL centrifuge tube.

[0089] (4) Add twice the volume of absolute ethanol to precipitate DNA, invert it 8 - 10 times, and place it in a -20°C refrigerator for 30 min.

[0090] (5) Centrifuge at 12000 rpm for 2 min, discard the supernatant, and the white flocculent substance at the bottom of the tube is DNA. Air-dry it in the fume hood for 2 h.

[0091] (6) Add 100 μL of ddH2O and store it at -20 °C for long-term preservation.

[0092] 2. Cloning of the pollen-specific promoter sequence proGmMS1

[0093] Using the genomic DNA of soybean Williams 82 as a template, primers were designed to clone the pollen-specific promoter proGmMS1 from the genomic DNA of soybean Williams 82.

[0094] The primer pairs used for amplification are as follows:

[0095]

[0096] PCR reaction system (25 μL):

[0097] 2×Rapid Taq Master Mi-P222 enzyme (Vazyme, Nanjing, China) 12.5 μL, H2O 9.5 μL, 1 μL of each upstream and downstream primer, and 1 μL of DNA template.

[0098] 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); store at 16 °C.

[0099] The PCR product was recovered and sequenced. The sequencing result showed that the nucleotide sequence of the pollen-specific promoter proGmMS1 is as shown in SEQ ID NO.1, with a length of 1622 bp.

[0100] 3. Cis-acting element analysis of the pollen-specific promoter proGmMS1

[0101] The pollen-specific promoter proGmMS1 was uploaded to the software PlantCARE for analysis. The results showed that the pollen-specific promoter proGmMS1 contains cis-acting elements GTGA-box, AGAAA-box, and TCATTT-box related to pollen-specific expression. In addition, it also contains the TGTGG-box that has the effect of enhancing pollen-specific expression.

[0102] Example 3 Construction of the proGmMS1::GUS expression cassette and genetic transformation of Arabidopsis thaliana

[0103] 1. Construction of the proGmMS1::GUS expression cassette

[0104] The empty plasmid pHZM33::GUS involved in this example was prepared and stored in our laboratory. Specifically, pHZM33 disclosed in the Chinese patent application with the publication number CN118028355A can be referred to. The difference in this example is that GFP in it is replaced by GUS. For the map of the empty plasmid pHZM33::GUS in this example, please refer to Figure 2 。

[0105] The specific steps for constructing the proGmMS1::GUS expression cassette are as follows:

[0106] (1)Select the restriction endonucleases StuI and AvrII (TaKaRa) to perform double digestion on the empty plasmid pHZM33::GUS.

[0107] Among them, the digestion system is 25 μL:

[0108] 1 μL of each restriction endonuclease, 2.5 μL of Cutsmart, 5.5 μL of H2O, and 15 μL of the target vector.

[0109] Digestion program: 37 °C, 3 h; 80 °C, 20 min; store at 16 °C.

[0110] (2)Ligate the gel recovery product of the proGmMS1 promoter sequence with the digested vector by homologous recombination method.

[0111] Among them, the ligation system is 10 μL:

[0112] 2 μL of the digested vector, 3 μL of the gel recovery product, and 5 μL of homologous recombination enzyme (ClonExpress MultiS One Step Cloning Kit) (Vazyme, Nanjing).

[0113] Ligation program: 50 °C, 50 min; store at 16 °C.

[0114] (3)The transformation process of picking monoclonal colonies from Mach1-T1-DL1015M (Vidi Biotechnology, Shanghai) Escherichia coli is as follows:

[0115] (a)Take out the Mach T1 Escherichia coli competent cells from the -80 °C refrigerator, place them on ice to melt, and gently flick them to accelerate dissolution.

[0116] (b)Prepare a clean 1.5 mL centrifuge tube, add all the ligation products and 50 μL of the dissolved Escherichia coli competent cells (try to add them to the bottom of the centrifuge tube), gently flick and mix, and place them on ice for 30 min.

[0117] (c)Place the mixed sample in a metal bath at 42 °C for heat shock for 60 s, and then place it on ice for 2 min.

[0118] (d) Open the centrifuge tube in a laminar flow hood, add 500 μL of sterilized LB liquid medium (without antibiotics), place the centrifuge tube in a constant temperature shaker, and incubate at 37 °C and 220 rpm for 1 h.

[0119] (e) Prepare LB solid medium (containing 50 μg / L kanamycin) in advance in the laminar flow hood, heat the spreader with an alcohol lamp, place the centrifuge tube in the shaker in a centrifuge at 6000 rpm for 1 min, take it out, and use a sterilized pipette in the laminar flow hood to aspirate part of the supernatant, leaving 100 μL to resuspend the precipitate, and pipette to mix evenly.

[0120] (f) Plate the cells, mark the Petri dish, and incubate it upside down in a 37 °C incubator overnight (12 - 16 h).

[0121] After successful sequencing identification, the proGmMS1::GUS expression cassette was obtained, and its map is shown in Figure 3 .

[0122] 2. Genetic transformation of Arabidopsis thaliana

[0123] The floral dip method was used for the genetic transformation of Arabidopsis thaliana, and the specific operations are as follows:

[0124] (1) Add 1 μL of the proGmMS1::GUS expression cassette plasmid to 50 μL of Agrobacterium tumefaciens GV3101 AC1001 (Vidi Biotechnology, Shanghai), mix well, and then incubate 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 successively. Add 500 μL of antibiotic-free LB liquid medium, and incubate with shaking at 28 °C and 220 rpm for 2 - 3 h. Prepare LB solid medium (containing 50 μg / L rifampicin and 50 μg / L kanamycin) in advance in the laminar flow hood, heat the spreader with an alcohol lamp, place the bacterial solution in a centrifuge at 6000 rpm for 1 min, take it out, and use a sterilized pipette in the laminar flow hood to aspirate part of the supernatant, leaving 100 μl to resuspend the precipitate, and pipette to mix evenly and inoculate on the LB solid medium, and incubate in the dark at 28 °C for 48 h. When single colonies grow out, perform colony PCR screening. The identified positive clones are added to 600 μL of LB (containing 50 μg / L rifampicin and 50 μg / L kanamycin) liquid medium, and incubated with shaking at 28 °C and 220 rpm overnight. The next day, preserve the bacteria with 50% glycerol and store at -80 °C.

[0125] (2) Transfer 1 mL of the bacterial solution into 10 mL of LB liquid medium (containing 50 μg / L rifampicin and 50 μg / L kanamycin), and incubate with shaking at 220 r / min at 28 °C until the bacterial solution concentration reaches an OD 600 value of 1.2 - 1.6, centrifuge at 5000 r / min for 15 min, discard the supernatant, add 5% sucrose solution to resuspend the bacterial cells until the OD600 The value is about 1.3; before infiltration, add Silwet L-77 surfactant at a volume ratio of 0.05%. Select Arabidopsis thaliana with good growth status and appropriate growth stage, remove the young pods and open flowers on the inflorescence, and soak the inflorescence in the prepared bacterial solution for 1 minute; keep the infiltrated plants in the dark for 24 hours, and then take them out and grow them normally.

[0126] To improve the transformation efficiency, infiltrate the new inflorescences again every other week for a total of three times; after harvesting the T0 generation seeds, use a 1 / 2MS solid medium containing hygromycin (50 μg / mL) resistance to screen for positive plants, and spray Arabidopsis thaliana seedlings with Basta (1 / 2000) resistance (the resistance of different vectors is different), and screen for homozygous lines for experiments.

[0127] In this example, the LB medium is shown in the following table:

[0128]

[0129] Note: Place it in an autoclave at 121°C and sterilize it at high temperature and high pressure for 20 minutes. Wait for it to cool to an appropriate temperature, add the corresponding antibiotics, and pour the medium into a 9 cm × 9 cm round plastic petri dish. Pour about 20 mL of medium into each petri dish.

[0130] The preparation of the 1 / 2MS medium is shown in the following table:

[0131]

[0132] Note: Place it in an autoclave at 121°C and sterilize it at high temperature and high pressure for 20 minutes. When it cools to an appropriate temperature, pour the medium into a 13 cm × 13 cm square plastic petri dish. Pour about 40 mL of medium into each petri dish.

[0133] 3. GUS detection of transgenic Arabidopsis thaliana

[0134] Stain different tissues of transgenic Arabidopsis thaliana seedlings with X-gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide) and 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 ferricyanide, 0.5 mM potassium ferrocyanide, 10 mM EDTA, and 0.1% (v / v) Triton X-100]. Incubate all treatments at 37°C for 4 hours, decolorize with 70% (v / v) ethanol, and take pictures with a stereomicroscope.

[0135] The results are as Figure 4As shown in [Figure 0], the transgenic Arabidopsis pollen of proGmMS1::GUS was stained blue, but there was no color in the wild-type pollen, indicating that proGmMS1 is a pollen-specific promoter.

[0136] Example 4 Construction and Phenotypic Identification of Recombinant Expression Vector pCAMBIA1300-proGmMS1::GmAMY1

[0137] 1. Construction of Recombinant Expression Vector pCAMBIA1300-proGmMS1::GmAMY1

[0138] (1) Construction of pCAMBIA1300-AMY1 Vector

[0139] The pCAMBIA1300 vector (Miaoling plasmid platform, P21036) was linearized by NcoI and XbaI restriction endonucleases (NEB). 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 ligated to construct the recombinant expression vector pCAMBIA1300-AMY1. The specific operation steps refer to the steps of restriction digestion and homologous recombination in Example 3. The primers involved are shown in the following table:

[0140]

[0141] The map of the constructed pCAMBIA1300-AMY1 vector is shown in Figure 5 .

[0142] (2) Construction of Recombinant Expression Vector pCAMBIA1300-proGmMS1::GmAMY1

[0143] The pCAMBIA1300-AMY1 vector was linearized by BamHI digestion and ligated with the promoter proGmMS1 to construct the recombinant expression vector pCAMBIA1300-proGmMS1::GmAMY1 (the vector map is shown in Figure 6 ); the specific operation steps refer to the steps of restriction digestion and homologous recombination in Example 3.

[0144] Meanwhile, using the same steps and methods, the promoter GmLAT52 (the sequence is shown in CN117025661A) was ligated into the pCAMBIA1300-AMY1 vector to construct the recombinant expression vector pCAMBIA1300-proGmLAT52::GmAMY1 (the vector map is shown in Figure 7 ). The primers involved are shown in the following table:

[0145]

[0146] (3)After transforming Arabidopsis thaliana according to the steps of Example 3, phenotypic identification was carried out.

[0147] Alexander staining: Take the Arabidopsis thaliana flowers showing white tips, use forceps to peel off the sepals and petals, pick the anthers and place them on a glass slide. Drop 20 - 30 μL of Alexander staining solution (G3050, Solarbio Science & Technology Co., Ltd., Beijing) on the anthers, cover with a cover slip and stain for 10 - 20 min, then examine under a microscope.

[0148] DAPI staining: At 10 am, take the fully opened flowers of the day, use forceps to peel off the sepals and petals, shake the pollen onto a glass slide, drop 10 μL of DAPI staining solution (SL7100, Coolaber Technology Co., Ltd., Beijing), cover with a cover slip and stain at room temperature for 3 - 5 min, then observe the staining result under a fluorescence microscope.

[0149] Scanning electron microscopy analysis: Fix the pollen in 2.5% glutaraldehyde at 4°C overnight, treat it successively with 30%, 50%, 70%, 90% and 100% ethanol for 10 minutes, transfer it to isoamyl acetate for 20 minutes, perform critical point drying, coat with gold for 30 seconds, and take pictures under a scanning electron microscope (JSM - 6390LV).

[0150] The results are as Figure 8 shown in

[0151] The results of Alexander staining showed that mature pollen grains of the wild type would be stained purple - red, while some aborted pollen grains in the over - expression group were not stained ( Figure 8 a in

[0152] The results of DAPI staining showed that mature pollen of wild - type Arabidopsis thaliana could normally develop to the trinuclear stage, while clear nuclear morphology could not be observed inside some pollen grains in proGmMS1::GmAMY1 ( Figure 8 b in

[0153] Analysis of pollen morphology by scanning electron microscopy showed that the outer walls of some pollen grains in proGmMS1::GmAMY1 had folds or collapses, indicating that these pollen grains could not develop normally ( Figure 8 c in

[0154] Results of pollen viability detection: Using the GmMS1 promoter to drive the expression of the 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 the proGmMS1 promoter). The results showed that the proGmMS1 promoter could drive the specific expression of the target gene in pollen, with strong driving force and significant effect. This is of great significance for improving plant strains or varieties through genetic engineering ( Figure 8 d in

[0155] It should be noted that this application is not limited to the above embodiments. The above embodiments are only examples, and embodiments having the same composition and the same function and effect as the technical idea within the scope of the technical solution of this application are all included in the technical scope of this application. In addition, within the scope of not departing from the gist of this application, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways constructed by combining some of the constituent elements in the embodiments are also included in the scope of this application.

Claims

1. A pollen-specific promoter proGmMS1, characterized in that, Its nucleotide sequence is the nucleotide sequence shown in SEQ ID NO.

1.

2. An expression cassette, characterized in that, It contains the pollen-specific promoter proGmMS1 described in claim 1.

3. The expression cassette according to claim 2, wherein, The expression cassette further 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, It contains the pollen-specific promoter proGmMS1 described in claim 1.

6. The recombinant expression vector according to claim 5, wherein It further contains a target gene, the target gene is a pollen lethal gene, and the pollen lethal gene is GmAMY1.

7. A host cell, characterized in that, It contains the pollen-specific promoter proGmMS1 described in claim 1.

8. Use of the pollen-specific promoter proGmMS1 according to claim 1, or the expression cassette according to any one of claims 2-4, or the recombinant expression vector according to any one of claims 5-6, or the host cell according to claim 7 in cultivating a male sterile plant variety, the plant being a dicotyledonous plant, the dicotyledonous plant being at least one of soybean and Arabidopsis thaliana.

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