A pollen-specific expression promoter and its application

Abstract

The present invention discloses a pollen-specific expression promoter p1755. The nucleotide sequence of the promoter p1755 is shown in Sequence Listing SEQ ID NO: 1. The present invention proves through RT-qPCR that the 1755 gene is a gene with high anther-specific expression, clones its promoter, and connects it to the 5' end of GUS to construct a vector. Through stable transformation, transgenic maize lines are obtained, and the GUS staining experiment is used to prove that the p1755 promoter is a gene with high pollen-specific expression, providing a molecular tool of a pollen-specific promoter for maize biological breeding, and also having great application potential in the subsequent creation of male sterile lines.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a pollen-specific expression promoter and its application. Background Art

[0002] Flower organs are crucial for plant reproduction and survival. Flower organs not only attract pollinators but also serve as the sites for plant sexual reproduction. Through flower organs, plants can complete the fertilization process, thus multiplying offspring and ensuring the continuation of the species. Flower-specific promoters can perform genetic operations on flower organs, thereby improving crop yield and quality (Li et al. 2019), and this process will not affect vegetative growth. In flowering plants, the normal development of pollen is crucial for ensuring the success of sexual reproduction and maintaining species continuity.

[0003] For crops, well-developed pollen is the key to ensuring high yields, exerting heterosis, and breeding new varieties. Therefore, researchers have been committed to finding tools that can drive the specific expression of target genes in pollen. They found that the OsSUT3 promoter still maintains strong and specific expression in pollen after entering a heterologous system (Li et al. 2020). This provides an important tool for subsequent driving the specific expression of target genes in pollen and has potential agricultural application value. Pollen-specific promoters, as key tools for molecular genetics research on pollen development, contribute to promoting the molecular breeding process of male sterile lines. Taking the male sterile AB line of Chinese cabbage as an example, researchers verified the anther specificity of its promoter through RNA-seq identification and RT-qPCR detection (Zhao et al. 2022). When this promoter drives the high-efficiency expression of GUS in the anthers of transgenic seedlings, it further proves its potential in practical applications.

[0004] In addition, researchers have also successfully used the tobacco anther-specific promoter TA29 to drive the expression of the ricin toxin gene (RTA) in tobacco (Lv et al. 2020). Compared with the control group plants, the anthers of transgenic tobacco not only showed a significant reduction in pollen release but also a substantial decline in pollen viability. Although these transgenic tobacco plants can flower normally, they cannot produce seeds, thus showing the characteristics of male sterility. This research result provides new ideas and methods for using anther-specific promoters to produce male sterile plants.

[0005] In the development process of transgenic crops, promoters play an important role. In the field of traditional genetic engineering, constitutive promoters have been favored for their stable ability to express foreign genes. However, with the continuous innovation of technology and the in-depth research, tissue-specific promoters have gradually attracted attention due to their unique application potential. Tissue-specific promoters have a high degree of spatio-temporal specificity and can achieve precise control of foreign genes within a specific time and space range. Compared with constitutive promoters, tissue-specific promoters not only avoid the problems of plant nutrient waste and unnecessary gene expression but also show unique advantages in practical applications. Therefore, exploring tissue-specific promoters and developing efficient, precise, and non-interfering multi-gene expression strategies are of great significance in applications such as biological breeding and rationally designing recombinant cell factories. Summary of the Invention

[0006] The object of the present invention is to provide a pollen-specific expression promoter and its application.

[0007] A pollen-specific expression promoter p1755, the nucleotide sequence of the promoter p1755 is shown in SEQ ID NO: 1 of the sequence listing.

[0008] A recombinant vector, expression cassette or recombinant bacterium containing the pollen-specific expression promoter p1755.

[0009] A method for creating transgenic plants, transferring the recombinant vector, expression cassette or recombinant bacterium into plants.

[0010] Preferably, the plant is maize.

[0011] An application of a recombinant vector, expression cassette or recombinant bacterium such as the pollen-specific expression promoter p1755 in initiating the expression of a target gene in plants.

[0012] Preferably, the target gene is specifically expressed in plant pollen.

[0013] Preferably, the plant is maize.

[0014] An application of a recombinant vector, expression cassette or recombinant bacterium such as the pollen-specific expression promoter p1755 in plant genetic improvement.

[0015] Preferably, the plant is maize.

[0016] Advantages of the present invention: The present invention proves through RT-qPCR that the 1755 gene is an anther-specific highly expressed gene, clones its promoter, connects it to the 5' end of GUS to construct a vector. Through stable transformation, transgenic maize lines are obtained, and the GUS staining experiment is used to prove that the p1755 promoter is a pollen-specific highly expressed gene, providing a molecular tool of pollen-specific promoter for maize biological breeding, and also having great application potential in the subsequent creation of male sterile lines. Brief Description of the Drawings

[0017] Figure 1 is the RT-qPCR result of the 1755 gene.

[0018] Figure 2 is the construction of the stable transformation vector;

[0019] In the figure, a: Map of the pBDRS-SP vector; b: Schematic diagram of the expression vector, "35S" is the cauliflower mosaic virus CaMV35S; "Bar" is the glufosinate screening marker gene; "GUS" is the β-glucuronidase gene.

[0020] Figure 3 is the detection of the GUS gene in transgenic plants.

[0021] Figure 4 is the detection of the Bar gene in transgenic plants.

[0022] Figure 5 is the GUS staining result of transgenic maize plants;

[0023] In the figure: Bar = 1 mm, and the blue color is the color marker of GUS expression.

[0024] Figure 6 is the GUS staining of maize anthers at different magnifications. Detailed Embodiments

[0025] To facilitate the understanding of the present invention, the present invention will be described more comprehensively below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. Embodiment

[0026] I. Experimental Methods

[0027] Extraction of Maize Tissue RNA:

[0028] (1) Take out the mortar sterilized by high temperature and high pressure, wash it with liquid nitrogen, take 80 mg of tissues such as the roots, stems and leaves of B73 maize, freeze them quickly with liquid nitrogen, and place them in the mortar for grinding.

[0029] (2) Transfer the above powder sample into a 1.5 mL EP tube, add 600 μL of Trizol, and let it stand at room temperature for 5 min to thoroughly separate the nucleoprotein complex.

[0030] (3) Add 600 μL of chloroform, vortex for 15 sec after covering the cap, and let it stand at room temperature for 5 min. Centrifuge at 4°C and 12,000 r / min for 15 min. After centrifugation, the liquid in the tube is divided into three layers. The lower red tissue sediment is dissolved in the phenol-chloroform phase, the middle layer is protein and DNA, and the upper layer is the colorless aqueous phase. RNA only exists in the aqueous phase, and the aqueous phase accounts for 50% of the total Trizol.

[0031] (4) Transfer the upper aqueous phase to another clean EP tube, add 0.5 mL of isopropanol, let it stand at room temperature for 10 min, and centrifuge at 4°C and 12,000 r / min for 10 min. After centrifugation, a white RNA precipitate can be seen on the side wall and bottom of the tube.

[0032] (5) Discard the supernatant, add 1.0 mL of 75% ethanol to wash the RNA precipitate, mix well with an oscillator, centrifuge at 4°C with a rotation speed of 12,000 r / min for 5 min to thoroughly wash away the organic solvent.

[0033] (6) Discard the supernatant, place it in the air for 8 min to dry the RNA precipitate. Do not completely dry the RNA precipitate, as this will greatly reduce its solubility.

[0034] (7) Resuspend the RNA precipitate with 50 μL of DEPC water, pipette it several times with a pipette tip, place it on ice, take 2 μL of RNA, prepare a 2% agarose gel with TBE (1×) solution, and electrophorese at 180 V for 10 min to check the quality of RNA extraction. Reverse transcribe RNA using the cDNA synthesis SuperMix kit, and perform PCR amplification with the Actin200 primer to check the quality of cDNA.

[0035] Reverse transcription of RNA into cDNA:

[0036] After the RNA extracted in the above experiment is qualified by electrophoresis detection, use the TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix kit from TransGen Biotech to reverse transcribe it into cDNA. The reverse transcription system used in this article is all 20 μL, and the reverse transcription program is carried out using a PCR instrument. The reverse transcription system is configured as follows (Table 1).

[0037] Table 1 Reverse transcription system

[0038]

[0039] Reverse transcription procedure:

[0040] Step 1: Incubate at 42 °C for 30 min;

[0041] Step 2: Heat at 85 °C for 5 sec to inactivate TransScript® RT / RI and gDNA Remover. After reverse transcription, check its quality by PCR amplification.

[0042] RT-qPCR:

[0043] Qualified cDNA was used to detect the expression sites and levels of candidate genes using an RT-qPCR kit. Gene sequence information was found using the MaizeGDB (https: / / www.MaizeGDB.org / ) and Gramene (https: / / www.gramene.org) websites respectively, and primer design was performed using the software primer 5. In this experiment, the maize Actin gene was used as an internal reference gene, and an ABI 7500 Real-Time PCR instrument was used. The system configuration is shown in Table 2.

[0044] Table 2 RT-qPCR system

[0045]

[0046] RT-qPCR procedure:

[0047] Step 1: Pre-denature at 94 °C for 30 sec;

[0048] Step 2: Denature at 94 °C for 5 sec;

[0049] Step 3: Anneal at 60 °C for 15 sec;

[0050] Repeat steps 1 - 3 for 40 cycles.

[0051] Promoter cloning: The candidate gene sequence was found through the MaizeGDB (https: / / www.MaizeGDB.org / ) website, and primer design was performed using the primer 5 software. In this study, a 50 µL system was used for promoter cloning. Using the B73 maize genome as a template, the primers listed in Table 3 were used to configure the system, which was placed in a PCR instrument and amplified using the high-fidelity enzyme KOD three-step method. The target fragment was recovered by gel electrophoresis. The system configuration is as follows:

[0052] Table 3 Promoter cloning system

[0053]

[0054] Linearized vector:

[0055] (1) Number the clean, dry, and sterilized 200 μL EP tubes. Using a micropipette, add 1 μg of DNA and 2 μL of the corresponding restriction endonuclease reaction 10× buffer to each tube. Then add ddH2O to make the total volume 19 μL. After mixing the solution in the tube, add 1 μL of the enzyme solution, gently flick the tube wall to mix, and then use a microcentrifuge to centrifuge so that the solution concentrates at the bottom of the tube. When using restriction endonucleases, try to minimize the time they are out of the refrigerator to avoid loss of activity.

[0056] (2) After mixing the reaction system, place the EP tube on a metal bath and incubate in a 37 °C water bath for 30 min to complete the digestion reaction.

[0057] (3) Incubate on ice to stop the reaction, and perform agarose gel electrophoresis at a voltage of 120 V and a concentration of 1.2%.

[0058] Homologous recombination: Perform gel electrophoresis on the linearized vector and PCR product, recover and purify them, and use the TransGen Biotech Basic Seamless Cloning and Assembly Kit for homologous recombination ligation. The homologous recombination primers in the experiment are shown in Table 4. The specific reaction system is as follows:

[0059] Table 4 Configuration of the homologous recombination system

[0060]

[0061] In a 10 μL reaction system, the amount of vector and each insert fragment added is 0.05 pms, and the molar ratio of vector to each insert fragment is 1:2. pmols = mass ng / (fragment length bp × 0.65 kDa)

[0062] Gently mix and centrifuge briefly. React at 50 °C for 20 min; after the reaction, place the centrifuge tube on ice to cool for a few seconds. The recombinant product can be stored at -20 °C or directly used for transformation.

[0063] Transformation of Escherichia coli:

[0064] In this experiment, Trelief® 5α competent cells from Tsingke Biotech Co., Ltd. were used. The specific operation is as follows:

[0065] (1) Take 100 μL of competent cells and place them in an ice bath to thaw.

[0066] (2) After the competent cells have thawed, add 1 ng of the recombinant to the competent cell suspension, gently flick to mix, and let it stand in an ice bath for 30 min.

[0067] (3) Heat shock at 42 °C for 2 min, then quickly transfer the centrifuge tube to an ice bath and let it stand for 4 min without shaking the centrifuge tube.

[0068] (4) Add 450 µL of sterile LB medium (without antibiotics) to each centrifuge tube, mix well, and place it on a shaker at 37 °C, shake culture at 200 r / min for 45 min to resuscitate the bacteria.

[0069] (5) Take 200 µL of the transformed competent cells and add them to the LB solid agar medium containing the corresponding antibiotics. Use a sterile spreader to evenly spread the cells. Place the plate at 37 °C until the liquid is absorbed, then incubate it upside down at 37 °C for 14 h.

[0070] Small-scale plasmid DNA extraction:

[0071] In this experiment, the TransGen Biotech EasyPure® Plasmid MiniPrep Kit was used. The specific experimental steps are as follows:

[0072] (1) Pipette 2 mL from the overnight culture into a centrifuge tube, centrifuge at 10,000 r / min for 1 min, discard the supernatant, invert the tube onto filter paper to remove the remaining liquid. If the amount of the bacterial solution is too large, it can be collected by centrifugation in multiple times.

[0073] (2) Add 250 µL of colorless RB solution (containing RNase A) to the centrifuge tube, shake to suspend the bacterial pellet. There should be no small bacterial clumps left, otherwise it will affect the subsequent lysis effect.

[0074] (3) Add 250 µL of blue LB solution to the centrifuge tube, gently invert the tube up and down 5 times to fully lyse the bacteria, forming a blue transparent solution. The color changes from semi-transparent to transparent blue, indicating complete lysis.

[0075] (4) Continue to add 350 µL of yellow NB solution to the centrifuge tube, gently mix 5 times (the color changes from blue to completely yellow, indicating uniform mixing and complete neutralization), until a compact yellow flocculent substance is formed, and let it stand at room temperature for 2 min.

[0076] (5) Centrifuge at 12,000 r / min for 5 min, carefully pipette the supernatant into the centrifuge column using a pipette gun. Centrifuge at 12,000 r / min for 1 min, discard the waste liquid in the collection tube. If the volume of the supernatant is greater than 800 µL, it can be added to the column in multiple times and centrifuged under the above conditions, and the waste liquid is discarded.

[0077] Add 650 µL of solution WB, centrifuge at 12,000 r / min for 1 min, discard the waste liquid in the collection tube; repeat the operation twice.

[0078] (7) Centrifuge at 12,000 r / min for 2 min to completely remove residual WB.

[0079] (8) Place the centrifuge column in a clean centrifuge tube, add 40 µL of Elution Buffer or ddH2O (pH>7.0) to the center of the column, and let it stand at room temperature for 1 min.

[0080] (9) Centrifuge at 10,000 r / min for 1 min to elute the DNA. The eluted DNA product was stored at -20°C.

[0081] Agrobacterium transformation:

[0082] This experiment uses the EHA105 competent cell from Qingke Biotechnology Co., Ltd. The specific steps are as follows:

[0083] (1) Place the competent Agrobacterium stored at -80°C at room temperature or pinch it with your fingertips for a while, wait for it to partially melt, and then place it on ice.

[0084] (2) Add 1 μg of target plasmid, mix gently, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0085] (3) Add 700 μL of sterile culture medium (YEB or LB) without antibiotics to the centrifuge tube, mix well, and culture at 28°C and 200 rpm for 2 h to allow the bacteria to recover.

[0086] (4) Pipette 400 μL of the resuscitation solution and evenly spread it on a YEB plate containing the corresponding antibiotics. Place the plate upside down in a 28°C incubator and culture for 2 days.

[0087] Agrobacterium-mediated stable transformation of maize:

[0088] (1) Spread the revived Agrobacterium evenly on solid YEB medium containing Kan and Rif dual resistance and culture at 28°C until corn is stably transformed.

[0089] (2) Soak 11 DAP corn ears in 5% NaClO + 0.02% Tween 20 for 30 min to disinfect them, and then rinse them three times with sterile water to remove the mixture on the surface of the ears.

[0090] (3) Remove the corn embryos completely and place them into 2 mL EP tubes (containing liquid infection medium), with 90 embryos placed in each tube.

[0091] (4) Agrobacterium infection and co-culture: Pick up Agrobacterium from the solid medium with an inoculation loop, put it into a 5 mL EP tube (containing liquid infection medium), place it in a shaker, incubate at 180 r / min for 3 h until the OD600 reaches 0.55.

[0092] (5) Wash the embryos in the 2.0 mL EP tube twice with the liquid infection medium, then suck dry the liquid with a pipette, add 1.8 mL of the Agrobacterium infection solution, and gently mix the liquid 20 times, and incubate in the dark at room temperature for 5 min.

[0093] (6) Then pour it on the sterilized filter paper to suck dry the excess infection solution, place the embryos with the scutellum facing up in the co-culture medium, and culture in the dark at 22 °C for 4 days;

[0094] Recovery culture: The recovery medium is cultured in the dark in an incubator at 28 °C for 7 days;

[0095] Screening for transformation events: Culture in the screening medium in the dark at 28 °C, once every 2 weeks, for 4 times;

[0096] Light culture of seedlings: Screen with the differentiation medium for 2 weeks;

[0097] Transfer the seedlings to the rooting medium. After normal and strong roots grow, transfer them to pots.

[0098] Select the seedlings with better development and culture them in the greenhouse until they grow and develop normally.

[0099] PCR identification of transgenic maize materials:

[0100] For the transgenic maize materials obtained by the above experimental method, it is necessary to identify whether they are positive materials at the seedling stage, extract the DNA of maize leaves, and then perform PCR amplification with the primers in Table 5. After the amplification products are electrophoresed on a gel, check whether the size of the target band is correct.

[0101] Table 5 Identification primers

[0102]

[0103] GUS staining:

[0104] Preparation of staining solution: Add 20 μL of X-GluC reagent to 1 mL of GUS buffer, and prepare it immediately before use.

[0105] Staining: Add 1 mL of the prepared GUS staining working solution to a 1.5 mL EP tube, immerse the tissues such as roots, stems, leaves, tassels, anthers, filaments and seeds of positive transgenic plants into the GUS staining solution, and place them in a 37 °C incubator for staining for 8 h.

[0106] Rinsing: Rinse the samples successively with 50%, 75% and absolute ethanol, soaking for 5 min each time.

[0107] Decolorization: For tissues with more chlorophyll, decolorize the tissues successively with 75% and 95% ethanol.

[0108] Recording: Take pictures and record under a stereomicroscope.

[0109] II. Experimental Results

[0110] RT-qPCR results: To verify the gene expression pattern of GRMZM2G151755, RT-qPCR experiments were used for verification. In this experiment, the CDS sequence of the target gene was obtained from MaizeGDB (https: / / www.MaizeGDB.org / ). Primers capable of specifically amplifying the target gene were designed using Primer5 software. Sixty-two maize samples were classified according to 14 parts such as primary roots, crown roots, brace roots, young leaves, old leaves, meristems, stems, tassels, anthers, filaments, ear axes, embryos, endosperms, and seeds.

[0111] This experiment selected maize Actin gene as the internal reference gene. Actin The gene is a highly conserved gene widely present in eukaryotes, and its expression level is relatively stable. Therefore, it is suitable to be used as an internal reference gene to correct errors in the experiment. According to the above experimental method, the expression pattern of GRMZM2G151755 gene in maize was detected. Simplified annotation was carried out using the last four digits of the ID ( Figure 1 ).

[0112] Expression vector construction: For subsequent maize stable transformation experiments, the promoter was ligated to the GUS reporter gene to construct a new expression vector. First, use restriction enzymes Xba I and Nco I to digest the vector pBDRS-SP. The purpose of this step is to obtain a linearized vector backbone. Through agarose gel electrophoresis, the target fragment was successfully separated and recovered. Use homologous recombinase to ligate the cloned promoter region to the GUS reporter gene. And the vector was named p1755:: GUS . GUS The reporter gene is a commonly used biological marker gene, which can show blue through specific chemical reactions, thus facilitating the observation and determination of the promoter expression. To ensure the successful construction of the expression vector, the recombinant was transformed into DH5α competent cells. Through colony PCR, positive clones were verified and sequenced. After the sequencing results were confirmed to be correct, plasmids were extracted and transformed into EHA105 Agrobacterium competent cells according to the above experimental method ( Figure 2)

[0113] Molecular identification of transgenic maize materials: To verify whether the target fragment was integrated into the genome of transgenic maize materials, PCR amplification was performed on the obtained transgenic maize plants. First, the DNA of transgenic maize plants was extracted, and then primers were designed for specific PCR amplification. The presence of the GUS gene was detected in the T0 generation transgenic materials. The primer pair JX-GUS-F / R was used to detect the presence of the GUS reporter gene. As can be seen from the result graph, all transgenic maize materials were positive and could be used for subsequent experiments ( Figure 3 、 4 )

[0114] Identification of spatiotemporal specificity and expression level of the promoter: To deeply explore the spatiotemporal specificity of promoter expression, GUS staining experiments were performed on the obtained transgenic maize materials. Samples were taken from the roots, stems, leaves, silk, tassels, and anthers at different growth stages, and GUS staining was carried out according to the above experimental method, and the results are as Figure 5 shown.

[0115] Figure 5 The results showed that the p1755:: GUS transgenic lines were only expressed in the anthers and not in other tissues such as roots, stems, leaves, tassels, and silk. By further observing the anthers and pollen grains at different magnifications, it can be seen from Figure 6 that the p1755:: GUS transgenic plants were only expressed in the pollen grains, and no blue color was found in the anther glumes. The pollen of the p1755:: GUS transgenic plants was partially stained blue, and the degree of blue color was different, deep or shallow. Although samples were taken at the same time, there were differences in the maturity of different pollen grains. It is speculated that this may be related to the pollen maturity.

[0116] RT-qPCR was used to prove that the 1755 gene was a high-expression gene specific to anthers, and its promoter was cloned and ligated to the 5' end of GUS to construct a vector. Transgenic maize lines were obtained through stable transformation, and GUS staining experiments were used to prove that the p1755 promoter was a high-expression gene specific to pollen, providing a molecular tool of pollen-specific promoter for maize biological breeding and having great application potential in the subsequent creation of male sterile lines.

[0117] The above-described embodiments only represent several implementation modes of the present invention. The description is relatively specific and detailed, but it should not be construed as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can still be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the present invention patent shall be subject to the appended claims.

Claims

1. A pollen-specific expression promoter p1755, characterized in that, The nucleotide sequence of the promoter p1755 is shown in SEQ ID NO: 1 in the sequence listing.

2. A recombinant vector, expression cassette or recombinant bacterium containing the pollen-specific expression promoter p1755 described in claim 1.

3. A method for creating transgenic maize, characterized in that, Transfer the recombinant vector, expression cassette or recombinant bacterium described in claim 2 into maize.

4. Use of a recombinant vector, expression cassette or recombinant bacterium of the pollen-specific expression promoter p1755 described in claim 2 for initiating the expression of a target gene in maize.

5. The application according to claim 4, characterized in that, The target gene is specifically expressed in maize pollen.

6. Use of a recombinant vector, expression cassette or recombinant bacterium of the pollen-specific expression promoter p1755 described in claim 2 for maize genetic improvement.

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