Maize nutritional tissue efficient expression promoter and application thereof

Abstract

The invention discloses a corn nutrition tissue specific expression promoter p7098. The nucleotide sequence of the promoter p7098 is as shown in a sequence table SEQ ID NO: 1. According to the present invention, RT-qPCR results prove that the 7098 gene is the nutritional tissue high expression gene, the promoter of the 7098 gene is cloned, and the promoter is connected with the GUS 5'end to construct the vector, the transgenic corn strain is obtained through stable transformation, the GUS dyeing experiment results prove that the p7098 promoter is the nutritional tissue high expression promoter, and the molecular tool of the nutritional tissue high expression promoter is provided for corn biological breeding;

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and particularly relates to a promoter with high expression in maize vegetative tissues and its application. Background Art

[0002] Green tissues are one of the most basic structures in plants. They are the most important sites for photosynthesis in plants, providing energy and nutrients required for plant growth, and also protecting plants from environmental stress (Liu et al. 2018). Researchers discovered a rice green tissue-specific promoter (DX1) through microarray and RT-PCR methods. By using the DX1 promoter to drive GUS gene expression, it was proven that it was highly expressed in green tissues (Rongjian et al. 2012). Green tissue-specific promoters have great potential in crop breeding. It has been found that green tissue-specific promoters can drive foreign genes to increase the resistance of crops. For example, using the rice green tissue-specific promoter PD540 to drive the expression of the Osoxo4 gene, compared with the wild type, the transgenic lines had higher oxalate oxidase enzyme activity and showed higher resistance to sheath blight (Molla et al. 2013). Not only can it increase the disease resistance of crops, but also the green tissue-specific promoter can be used to drive the expression of insect-resistant genes to enhance their insect resistance. For example, the GSX7R promoter found in japonica rice is expressed in green tissues except for the endosperm. Using this promoter to drive the expression of the insect-resistant gene cry1Ab, experiments have proven that the transgenic lines are not affected by pests and grow well (Lin et al. 2022). It has also been applied not only in rice but also in cotton. Using the green tissue-specific promoter PNZIP in cotton to drive the expression of the gene Cry9C, the PNZIP::Cry9C cotton plants showed strong resistance to both cotton bollworms and pink bollworms. The PNZIP promoter can effectively drive the expression of Bt toxin in cotton green tissues and reduce the accumulation level of Bt protein in seeds (Wang et al. 2016).

[0003] The use of non-seed-specific promoters is of great significance in the field of agricultural biotechnology. First of all, it makes the insect resistance of transgenic crops more precise and efficient. Since the promoter is only not expressed in seeds and is expressed in other parts, Bt toxin can act more accurately on pests and reduce the impact on non-target organisms. Secondly, reducing the accumulation level of Bt protein in seeds helps to alleviate the public's concerns about the safety of transgenic crops. This improvement makes the promotion and application of transgenic crops smoother and provides strong support for the sustainable development of agricultural production. Summary of the Invention

[0004] The object of the present invention is to provide a promoter with high expression in maize vegetative tissues and its application.

[0005] A maize vegetative tissue-specific expression promoter p7098, wherein the nucleotide sequence of the promoter p7098 is as shown in SEQ ID NO: 1 in the sequence listing.

[0006] A recombinant vector, expression cassette or recombinant bacterium containing the maize vegetative tissue-specific expression promoter p7098.

[0007] A method for creating a transgenic plant, comprising transferring the recombinant vector, expression cassette or recombinant bacterium into a plant.

[0008] The plant is maize.

[0009] Application of the recombinant vector, expression cassette or recombinant bacterium of the maize vegetative tissue-specific expression promoter p7098 in initiating the expression of a target gene in a plant.

[0010] The target gene is specifically expressed in the vegetative tissue of the plant.

[0011] The plant is maize.

[0012] Application of the recombinant vector, expression cassette or recombinant bacterium of the maize vegetative tissue-specific expression promoter p7098 in plant genetic improvement.

[0013] Advantages of the present invention: The present invention proves through RT-qPCR that the 7098 gene is a gene highly expressed in maize vegetative tissue, clones its promoter, and connects it to the 5' end of GUS to construct a vector. Transgenic maize lines are obtained through stable transformation, and the GUS staining experiment proves that the p7098 promoter is a promoter highly expressed in vegetative tissue, providing a molecular tool of a promoter highly expressed in vegetative tissue for maize biological breeding. Description of the Drawings

[0014] Figure 1 It is the RT-qPCR result of the 7098 gene.

[0015] Figure 2 It is the transgenic plant GUS Gene detection.

[0016] Figure 3 It is the Bar gene detection of the transgenic plant.

[0017] Figure 4 It is the GUS staining result of the transgenic maize plant. Detailed Embodiments

[0018] 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, these embodiments are provided to make the understanding of the disclosure of the present invention more thorough and comprehensive.

[0019] Example 1 RT-qPCR verification of the gene expression pattern of GRMZM2G027098 Obtain the CDS sequence of the target gene from MaizeGDB (https: / / www.MaizeGDB.org / ). Use Primer5 software to design primers that can specifically amplify the target gene. Classify 62 maize samples according to 14 parts, namely primary root, crown root, brace root, young leaf, old leaf, meristem, stem, tassel, anther, silk, ear axis, embryo, endosperm, and seed.

[0020] Extraction of maize tissue RNA: (1) Take out the mortar sterilized by high temperature and high pressure, rinse it with liquid nitrogen, and place 80 mg of tissues such as the root, stem, and leaf of B73 maize after quick-freezing in liquid nitrogen into the mortar for grinding.

[0021] (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 completely separate the nucleoprotein complex.

[0022] (3) Add 600 µL of chloroform, cover it, vortex for 15 sec, 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 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.

[0023] (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, white RNA precipitates can be seen on the side wall and bottom of the tube.

[0024] (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, the rotation speed is 12,000 r / min, and centrifuge for 5 min to completely wash away the organic solvent.

[0025] (6) Discard the supernatant, place it in the air for 8 min to dry the RNA precipitate, and do not completely dry the RNA precipitate, as this will greatly reduce its solubility.

[0026] (7) Resuspend the RNA pellet in 50 μL of DEPC water, pipette up and down several times, place 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 the RNA using the cDNA synthesis SuperMix kit, and perform PCR amplification with the Actin200 primer to check the quality of the cDNA.

[0027] Reverse transcription of RNA into cDNA: After the RNA extracted in the above experiment was qualified by electrophoresis, 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 is 20 μL each time, and the reverse transcription program is carried out using a PCR instrument. The reverse transcription system is configured as follows (Table 1).

[0028] Table 1 Reverse transcription system

[0029] Reverse transcription program: Step 1: Incubate at 42 °C for 30 min; 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.

[0030] RT-qPCR: The qualified cDNA is detected using an RT-qPCR kit to detect the expression location and level of the candidate gene. Use the MaizeGDB (https: / / www.MaizeGDB.org / ) and Gramene (https: / / www.gramene.org) websites to find the gene sequence information respectively, and use the software primer 5 for primer design. In this example, the maize Actin gene is used as the internal reference gene, and an ABI 7500 Real-Time PCR instrument is used. The system configuration is shown in Table 2.

[0031] Table 2 RT-qPCR system

[0032] RT-qPCR program: Step 1: Pre-denature at 94 °C for 30 sec; Step 2: Denature at 94 °C for 5 sec; Step 3: Anneal at 60 °C for 15 sec; Repeat steps 1 - 3 for 40 cycles.

[0033] According to the above experimental method, the expression pattern of the GRMZM2G027098 gene in maize was detected; the last four digits of the ID were used for simplified annotation ( Figure 1 ).

[0034] Example 2 Construction of expression vector Promoter cloning: The candidate gene sequence was retrieved through the MaizeGDB (https: / / www.MaizeGDB.org / ) website, and primer design was performed using primer 5 software. In this study, a 50 μL system was used for promoter cloning. Using the B73 maize genome as a template, the primer listed in Table 3 was used to configure the system, which was placed in a PCR instrument and amplified by the high-fidelity enzyme KOD three-step method. The target fragment was recovered through gel electrophoresis. The system configuration is as follows: Table 3 Promoter cloning system

[0035] For subsequent maize stable transformation experiments, the promoter was ligated to GUS the reporter gene to construct a new expression vector. The vector pBDRS-SP was digested with restriction enzymes Xba I and Nco I. The purpose of this step was to obtain a linearized vector backbone. The operating steps are as follows: (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 10× restriction enzyme reaction buffer to each tube, then add ddH2O to make the total volume 19 μL. Mix the solution in the tube and then add 1 μL of enzyme solution. Gently flick the tube wall to mix, and then use a tabletop centrifuge to centrifuge the solution to the bottom of the tube. When using restriction enzymes, try to minimize the time they are out of the refrigerator to avoid loss of activity.

[0036] (2) After mixing the reaction system, place the EP tube on a metal bath and incubate at 37°C for 30 min to complete the digestion reaction.

[0037] (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%.

[0038] Homologous recombination: The linearized vector and PCR product were subjected to gel electrophoresis and recovered and purified. The All-in-One Biotech Basic Seamless Cloning and Assembly Kit was used for homologous recombination ligation. The specific reaction system is shown below: Table 4 Configuration of homologous recombination system

[0039] In a 10-µL reaction system, it is recommended that the addition amounts of the vector and each insert fragment be both 0.01 - 0.25 pms, and the optimal molar ratio of the vector to each insert fragment is 1:2; pmols = mass ng / (fragment length bp × 0.65 kDa).

[0040] 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.

[0041] Transformation of Escherichia coli: Use the Trelief® 5α competent cells from Tsingke Biotechnology Co., Ltd. The specific operation is as follows: (1) Take 100 µL of competent cells and melt them in an ice bath.

[0042] (2) After the competent cells are melted, add 1 ng of the recombinant to the competent cell suspension, gently flick to mix, and let it stand in the ice bath for 30 min.

[0043] (3) Heat shock at 42 °C for 2 min, then quickly transfer the centrifuge tube to the ice bath and let it stand for 4 min without shaking the centrifuge tube.

[0044] (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 and shake at 200 r / min for 45 min to resuscitate the bacteria.

[0045] (5) Take 200 µL of the transformed competent cells and add them to the LB solid agar medium containing the corresponding antibiotic. Use a sterile spreader to spread the cells evenly. Place the plate at 37 °C until the liquid is absorbed, then invert and culture at 37 °C for 14 h.

[0046] Small-scale plasmid DNA extraction: Use the TransGen Biotech EasyPure ® Plasmid MiniPrep Kit. The specific experimental steps are as follows: (1) Pipette 2 mL from the overnight culture into a centrifuge tube, centrifuge at 10,000 r / min for 1 min, discard the supernatant, and invert the tube on filter paper to remove the remaining liquid. If the amount of the bacterial liquid is too large, it can be collected by centrifugation in multiple batches.

[0047] (2) Add 250 µL of colorless RB solution (containing RNaseA) to the centrifuge tube, shake to suspend the bacterial pellet, and there should be no small bacterial clumps left, otherwise it will affect the subsequent lysis effect.

[0048] (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.

[0049] (4) Continue to add 350 µL of yellow NB solution to the centrifuge tube, gently mix 5 times until a firm yellow flocculent precipitate is formed, and let it stand at room temperature for 2 min.

[0050] (5) Centrifuge at 12,000 r / min for 5 min, carefully aspirate the supernatant with a pipette and add it to the centrifugal column; centrifuge at 12,000 r / min for 1 min, and discard the waste liquid in the collection tube.

[0051] (6) Add 650 µL of solution WB, centrifuge at 12,000 r / min for 1 min, and discard the waste liquid in the collection tube; repeat the operation twice.

[0052] (7) Centrifuge at 12,000 r / min for 1 - 2 min to thoroughly remove the residual WB.

[0053] (8) Place the centrifugal 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.

[0054] (9) Centrifuge at 10,000 r / min for 1 min to elute the DNA. The eluted DNA product can be stored short-term at 4℃ or long-term at -20℃.

[0055] Transformation of Agrobacterium: Use the competent cells of EHA105 from Tsingke Biological Co., Ltd. The specific steps are as follows: (1) Place the Agrobacterium competent cells stored at -80℃ at room temperature or hold them between your fingertips for a moment. After partial thawing, insert them on ice.

[0056] (2) Add 1 µg of the target plasmid, gently mix, and let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and in an ice bath for 5 min in sequence.

[0057] (3) Add 700 µL of sterile medium without antibiotics to the centrifuge tube, mix well, and incubate with shaking at 28℃ and 200 r / min for 3 h to resuscitate the bacteria.

[0058] (4) Aspirate 400 µL of the resuscitation solution and spread it evenly on the YEB plate containing the corresponding antibiotic. Invert the plate and place it in an incubator at 28℃ for 3 days.

[0059] Verify the positive clones by colony PCR and perform sequencing. After the sequencing results are confirmed to be correct, extract the plasmid and transform it into the competent cells of Agrobacterium tumefaciens EHA105 according to the above experimental method ( Figure 2 ).

[0060] In this example, the cloned promoter region was ligated to the GUS reporter gene using a homologous recombinase. And the vector was named p7098::GUS. The GUS reporter gene is a commonly used biomarker gene, which can show blue through a specific chemical reaction, 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. Verify the positive clones by colony PCR and perform sequencing. After the sequencing results are confirmed to be correct, extract the plasmid and transform it into the competent cells of Agrobacterium tumefaciens EHA105 according to the above experimental method.

[0061] Example 3 Creation and Molecular Identification of Transgenic Maize Materials Agrobacterium-mediated Stable Transformation of Maize: (1) Spread the revived Agrobacterium evenly on a solid YEB medium containing double resistance to Kan and Rif, and culture it at 28 °C for use in maize stable transformation. Generally, use the plates within two weeks.

[0062] (2) Immerse the maize ears at 11 DAP in 5% NaClO + 0.02% Tween 20 for 30 min for disinfection, and then rinse them 3 times with sterile water to remove the mixture on the surface of the ears.

[0063] (3) Carefully remove the maize embryos and place them in a well-separated 2 mL EP tube (containing liquid infection medium), with 90 embryos in each tube.

[0064] (4) Agrobacterium infection and co-culture: Pick Agrobacterium from the solid medium with an inoculation loop and put it into a 5 mL EP tube (containing liquid infection medium), place it on a shaker, and incubate it at 180 r / min for 3 h until its OD 600 reaches 0.55.

[0065] (5) Wash the embryos in the 2.0 mL EP tube twice with the liquid infection medium, then suck out the liquid with a pipette, add 1.5 - 2.0 mL of the Agrobacterium infection solution, gently mix the liquid about 20 times, and incubate it at room temperature in the dark for 5 min.

[0066] (6) Then pour it onto the sterilized filter paper to absorb the excess infection solution, place the scutellum facing up in the co-culture medium, and culture it in the dark at 22°C for 4 days; Recovery culture: The recovery medium is cultured in the dark at 28°C for 7 days; Screening for transformation events: The screening medium is cultured in the dark at 28°C, once every 2 weeks, about 4 times; Seedling light culture: Screen with the differentiation medium for 2 weeks; Transfer the seedlings to the rooting medium, and after normal and strong roots grow, transfer them to pots; Select the seedlings with better development and culture them in the greenhouse until normal growth and development.

[0067] PCR identification of transgenic maize materials: 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 the table. After the amplification products are electrophoresed on a gel, detect whether the size of the target band is correct.

[0068] Table 5 Identification primers

[0069] The JX-GUS-F / R primer pair is used to detect the presence of the reporter gene. As can be seen from Figure 2 it, all the transgenic maize materials are positive and can be used for subsequent experiments.

[0070] The JX-Bar-F / R primer pair is used to detect the presence of the selection marker gene. As can be seen from Figure 3 it, all the transgenic maize materials are positive and can be used for subsequent experiments.

[0071] Example 3 Identification of promoter spatiotemporal specificity and expression level GUS staining: Preparation of staining solution: Add 20 µL of X-GlnC reagent to 1 mL of GUS buffer, and prepare it for immediate use.

[0072] 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 about 8 h.

[0073] Rinsing: Rinse the samples successively with 50%, 75%, and absolute ethanol, and soak for 5 min each time.

[0074] Decolorization: For tissues with more chlorophyll, decolorize the tissues successively with 75% and 95% ethanol.

[0075] Recording: Take pictures and record under a stereomicroscope.

[0076] To deeply explore the spatiotemporal specificity of promoter expression, a GUS staining experiment was conducted on the obtained transgenic maize materials. Samples were taken from roots, stems, leaves, silk, tassels, and anthers at different growth stages, and GUS staining was performed according to the above experimental method. The results are as follows Figure 4 as shown

[0077] Figure 4 The results showed that other tissues such as roots, stems, leaves, tassels, and silk of the p7098::GUS transgenic lines were stained blue, and there was no color in the seeds. Therefore, it can be seen that the p7098 promoter drives the expression of the GUS gene in roots, stems, leaves, tassels, and silk, but not in seeds and anthers.

[0078] The above-described embodiments only represent several implementation manners 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 maize vegetative tissue-specific expression promoter p7098, characterized in that, The nucleotide sequence of the promoter p7098 is shown in Sequence Listing SEQ ID NO:

1.

2. A recombinant vector, expression cassette or recombinant bacterium containing the maize vegetative tissue-specific expression promoter p7098 described in claim 1.

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

4. A method for creating a transgenic plant according to claim 3, characterized in that, The plant is maize.

5. Use of a recombinant vector, expression cassette or recombinant bacterium containing the maize vegetative tissue-specific expression promoter p7098 described in claim 2 for initiating the expression of a target gene in a plant.

6. The application according to claim 5, wherein The target gene is specifically expressed in the vegetative tissue of the plant.

7. The application according to claim 5, wherein The plant is maize.

8. Use of a recombinant vector, expression cassette or recombinant bacterium containing the maize vegetative tissue-specific expression promoter p7098 described in claim 2 for plant genetic improvement.

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