Sugar beet root-specific promoter BvTSP1 and application thereof

Abstract

The present application relates to a sugar beet root-specific expression promoter BvTSP1 and its application, the nucleotide sequence of the promoter is shown as SEQ ID NO. 1. The application of the promoter is the application of regulating the specific expression of the target gene in the sugar beet root. The present application screens the root-specific, high-expression, constitutive promoter under stress conditions in the sugar beet gene through transcriptome data, which has root-specific transcriptional activity in sugar beet. Based on the promoter, the target gene is transferred into the sugar beet genome through transgenic means, which can make the target gene only expressed in the sugar beet root and root part, and not expressed or weakly expressed in the stem, leaf and other tissues. Meanwhile, the expression level of the target gene is not affected by the external environmental changes such as low temperature, drought, salt stress, alkali stress, etc. The overexpression vector designed by the promoter can be used for transgenic sugar beet root improvement.

Description

Technical Field

[0001] This invention relates to the beet root-specific expression promoter BvTSP1 and its applications, belonging to the field of plant genetic engineering technology. Background Technology

[0002] Sugar beets are one of the major sugar crops, with the enlarged tuberous roots being the primary sugar-producing part. Tuber enlargement mainly occurs through the three-stage growth of the taproot, involving the active division of multiple cambium cells. The tuber is mainly divided into the root head, root neck, root body, and root tail. The root head is where the petioles attach; the root neck is the transitional area between the stem and root; the root body is the main site of tuber enlargement, and the vacuoles of its parenchyma cells are the main storage sites for sugar; and the root tail is the un-enlarged taproot portion. In transgenic sugar beet improvement, the specific expression of exogenous genes targeting the tuberous root is of great significance for increasing tuber yield and sugar content.

[0003] Using tissue-specific promoters enables more precise and effective transgenic improvement by allowing the expression of introduced genes in specific tissues. In plant transgenics, constitutive promoters such as CaMV35S, Ubiquitin, and MAS are commonly used. These promoters result in high expression of downstream genes in all tissues throughout the plant, including roots, stems, and leaves. When using transgenics to improve root traits, the high expression of introduced genes in tissues other than roots not only leads to additional energy loss but may also disrupt the function of the introduced gene in the roots due to expression in other tissues. Currently, some root-specific expression promoters have been developed for plants such as tomatoes and soybeans. However, the roots of these plants do not undergo tertiary growth processes and do not have enlarged tuberous root structures. Therefore, the developed root-specific expression promoters cannot guarantee specific expression function in beet tubers, and these promoters cannot guarantee root specificity and high transcriptional activity under different stress conditions.

[0004] Currently, no constitutive promoters have been reported for root-specific expression in sugar beets (especially for root head, root body, and root tail of sugar beet tubers). Summary of the Invention

[0005] To overcome the above-mentioned deficiencies of the prior art, the present invention provides the sugar beet tuber-specific expression promoter BvTSP1 and its application, which enables the target gene to be specifically expressed only in the young roots and tubers of sugar beets.

[0006] The technical solution adopted in this invention is: In a first aspect, the present invention provides a beet root-specific expression promoter BvTSP1, wherein the nucleotide sequence of the promoter is any one of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.1 that has been substituted, deleted and / or added with one or more nucleotides and exhibits promoter activity in a stationary phase-specific manner; (3) A nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO.1 under stringent conditions and exhibits promoter activity in a stationary phase-specific manner; (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequences of (1), (2) or (3) and exhibits promoter activity in a stationary phase-specific manner.

[0007] Secondly, the present invention provides an expression box containing the promoter.

[0008] Thirdly, the present invention provides a recombinant expression vector containing the promoter or the expression cassette.

[0009] Preferably, the carrier is pCAMBIA1300.

[0010] Fourthly, the present invention provides engineered bacteria containing the promoter, the expression cassette, or the recombinant expression vector.

[0011] Preferably, the method for obtaining the engineered bacteria is to transfer the promoter, the expression cassette, or the recombinant expression vector into Agrobacterium.

[0012] Fifthly, the present invention provides the application of the promoter in regulating the specific expression of the target gene in beet tubers.

[0013] The beneficial effects of this invention are: (1) This invention screened a root-specific, highly expressed, constitutive promoter in sugar beet genes through transcriptome data. This promoter has root-specific transcriptional activity in sugar beet and its transcriptional activity in the root is not affected by low temperature, drought, salt stress, or alkali stress. The overexpression vector designed based on this sugar beet root-specific highly expressed constitutive promoter can be used for transgenic modification of sugar beet roots. (2) Based on the constitutive promoter that is highly expressed in beet tubers, the present invention transfers the target gene into the beet genome through transgenic means, which enables the transferred target gene to be expressed only in the beet young roots and tubers, while not expressed or expressed very weakly in the stems, leaves and other tissues. At the same time, the expression level of the transferred target gene is not affected by changes in the external environment such as low temperature, drought, salt stress, and alkali stress. Attached Figure Description

[0014] Figure 1 This is a fluorescence image of BvTSP1::eGFP transformed into beet roots in an experimental example of the present invention. Figure 2This is a fluorescence image of BvTSP1::eGFP transformation expressed in beet leaves in an experimental example of the present invention. Detailed Implementation

[0015] This invention discloses a beet root-specific expression promoter BvTSP1, wherein the nucleotide sequence of the promoter is any one of the following: (1) The nucleotide sequence as shown in SEQ ID NO.1; (2) A nucleotide sequence of the nucleotide sequence shown in SEQ ID NO.1 that has been substituted, deleted and / or added with one or more nucleotides and exhibits promoter activity in a stationary phase-specific manner; (3) A nucleotide sequence that hybridizes to the nucleotide sequence shown in SEQ ID NO.1 under stringent conditions and exhibits promoter activity in a stationary phase-specific manner; (4) A nucleotide sequence that has more than 90% homology with the nucleotide sequences of (1), (2) or (3) and exhibits promoter activity in a stationary phase-specific manner.

[0016] The present invention also discloses an expression box containing any of the promoters disclosed herein.

[0017] The present invention also discloses a recombinant expression vector containing any of the promoters or expression cassettes disclosed herein, preferably pCAMBIA1300.

[0018] The present invention also discloses engineered bacteria containing any of the promoters, expression cassettes, or recombinant expression vectors disclosed herein.

[0019] The preferred method for obtaining the engineered bacteria is to transfer the promoter, the expression cassette, or the recombinant expression vector into Agrobacterium.

[0020] This invention also discloses the application of any of the promoters disclosed herein in regulating the specific expression of a target gene in sugar beet tubers. The application includes linking the promoter upstream of the target gene sequence to be expressed in a vector to construct a recombinant expression vector, and transforming the recombinant expression vector into sugar beets for cultivation.

[0021] Example: Step 1: Obtaining the promoter sequence and constructing the expression plasmid Primers were designed based on the sugar beet root-specific promoter (BvTSP1) sequence and the vector homologous arm sequence (BvTSP1-F: TAAGCTCGCGGATCCTAGCCTCTGAAGCTCCCTCT; BvTSP1-R: CAGAAGCTTTCTAGATCGACTTCACCAGATGAGGC). The promoter was amplified from the sugar beet genome. The pCAMBIA1300 vector was double-digested with restriction endonucleases. The digested vector and the amplified target gene were analyzed by 1% agarose gel electrophoresis to determine the sequence length and content of the target gene and the digested vector. The promoter can also be obtained chemically based on the BvTSP1 sequence. The BvTSP1 promoter and the digested vector, after verification, were purified by agarose gel electrophoresis recovery kit. The concentration and mass of the recovered and purified BvTSP1 promoter and digested vector were determined using a Nano Drop 2000 UV spectrophotometer. The BvTSP1 promoter and digested vector were ligated using the ClonExpress II One Step Cloning Kit. The BvTSP1 promoter and digested vector were added at a 1:2 molar ratio (20 ng BvTSP1 promoter and 134 ng digested vector), followed by 2 μL of 5×CEII Buffer and 1 μL of Exnase II. After mixing, homologous recombination ligation was performed. 1 µg of the ligation product was added to 25 µL of *E. coli* TOP10 competent cells and incubated on ice for 30 min. After incubation, the cells were heat-shocked in a metal bath at 42 °C for 45 s, and then incubated on ice again for 2 min. Subsequently, 700 µL of antibiotic-free LB liquid medium was added, mixed, and the cells were incubated at 37 °C and 200 rpm for 1 h on a shaker. The revived bacterial culture was centrifuged at 5000 rpm for 1 min, and 900 µL of supernatant was discarded. The resuspended bacterial culture was spread onto LB solid medium containing the antibiotic Kan and incubated upside down at 37°C for 12 h. Single colonies growing well on LB solid medium were picked and transferred to LB liquid medium and incubated on a shaker at 37°C for 3 h. Colony PCR was performed using universal primers or primers designed based on BvTSP1 (M13-F: GTTGTAAAACGACGGCCAG or BvTSP1-R: CAGAAGCTTTCTAGATCGACTTCACCAGATGAGGC). Positive clones were selected based on the colony PCR gel electrophoresis results and Sanger sequencing was performed to confirm successful construction of the BvTSP1 promoter into the vector. The sequenced bacterial culture was incubated overnight on a shaker at 37°C and 200 rpm. Finally, the pCAMBIA1300-BvTSP1 plasmid was extracted using a plasmid miniprep kit.Using the same method as for constructing BvTSP1, the eGFP gene (target gene) was constructed into the pCAMBIA1300-BvTSP1 plasmid.

[0022] The nucleotide sequence (SEQ ID NO.1) of promoter BvTSP1 is as follows:

[0023] Step 2: Transformation with Agrobacterium The constructed pCAMBIA1300-BvTSP1::eGFP vector was transformed into competent Agrobacterium GV1301 cells using a freeze-thaw method. 25 µL of competent Agrobacterium GV1301 cells were thawed and incubated on ice, and 1 µg of the pCAMBIA1300-BvTSP1::eGFP vector was added. Subsequently, the cells were incubated on ice for 5 min, flash-frozen in liquid nitrogen for 5 min, thawed in a metal bath at 37°C for 5 min, and incubated on ice for 5 min. 700 µL of antibiotic-free LB broth was added and mixed thoroughly. The cells were then incubated at 200 rpm for 3 h at 28°C on a shaker. After centrifugation at 5000 rpm for 1 min, 900 µL of supernatant was discarded. The resuspended and mixed cells were spread onto LB solid medium containing 50 μg / mL of antibiotic Rif and 50 μg / mL of antibiotic Kan, and incubated upside down at 28°C for 72 h. Single colonies that grew well on solid culture medium were picked and transferred to LB liquid medium. After incubation at 37°C for 3 hours on a shaker, bacterial culture was identified by PCR using universal primers and designed primers (M13-F: GTTGTAAAACGACGGCCAG and BvTSP1-R: CAGAAGCTTTCTAGATCGACTTCACCAGATGAGGC). The plasmid was successfully transformed based on the results of 1% agarose gel electrophoresis.

[0024] Step 3: Genetically modifying sugar beet plants Wash beet seeds 3-5 times with sterile water and shake in a conical flask for 20 minutes. Sterilize the washed beet seeds with 0.1% mercuric chloride for 8 minutes under sterile conditions, then rinse with sterile water at least 5 times, each rinse lasting 1 minute. Sow the seeds in tissue culture bottles containing sterile vermiculite at a 1:1 (v / v) ratio with Hogrange nutrient solution. 10-14 days after sowing, cut sterile beet plants from the hypocotyl and insert them into pH 5.8 solid MS medium containing 1 mg / L 6-BA and 30 g / L sucrose. After 14-21 days of culture, cut leaf and petiole midrib tissues into 1 cm segments and place them in a pre-culture medium. The pre-culture medium was a pH 5.8 solid MS medium containing 1 mg / L 6-BA, 2 μM trichostatin A (TSA), 0.2 mM acetylsuccinone (AS), 30 g / L sucrose, and 0.5 g / L 2-(N-morpholine)ethanesulfonic acid (MES). After 7 days of pre-culture, the pre-cultured perivascular tissue was placed in a GV3101 resuspension with an OD600 of 0.8 and co-cultured with shaking at 125 rpm for 10 min. The GV3101 resuspension consisted of Agrobacterium GV3101 containing the pCAMBIA1300-BvTSP1::eGFP vector cultured overnight in LB medium until an OD600 of 0.6–0.8 was reached. The bacterial pellet was collected by centrifugation at 5000 rpm and resuspended in the infection medium until an OD600 of 0.8 was reached. The infection culture medium was a pH 5.8 liquid MS medium containing 30 g / L sucrose, 5.0 g / L MES, and 0.2 mM AS. After co-culturing, the beet tissue was placed on a sterile filter and rinsed five times with 50 mL of sterile water, followed by regeneration medium. The regeneration medium was a pH 5.8 solid MS medium containing 30 g / L sucrose, 1 mg / L 6-BA, 1 mg / L triiodobenzoic acid (TIBA), 0.5 g / L MES, and 400 mg / L termethin. Positive transgenic shoots were selected by adding antibiotics such as hygromycin to the regeneration medium based on the vector's resistance gene. After 21–28 days of culture, the induced shoots were transferred to a rooting medium. The rooting medium was a pH 5.8 solid MS medium containing 1 mg / L indolebutyric acid (IBA), 30 g / L sucrose, and 0.5 g / L MES. After rooting, the expression of GFP in the roots was detected using a fluorescent light source, and positive plants were selected. Selected positive plants were cultured to obtain more mature tubers. The culture conditions were 26℃, 8000 Lux light intensity, 16h / 8h photoperiod, and 60% humidity.

[0025] Experimental example: This experiment was conducted to verify the expression of the target gene introduced via BvTSP1 in beet seedlings, tubers, stems, and leaves.

[0026] 1. The recombinant plasmid pCAMBIA1300-BvTSP1 containing BvTSP1 was transformed using Agrobacterium rhizogenes K599. Adventitious roots (hairy roots) were induced by infecting the hypocotyl of sugar beet seedlings. The expression specificity of BvTSP1 in sugar beet adventitious roots was analyzed by combining eGFP fluorescence detection. The specific procedures are as follows: Take 10 μL of bacterial suspension containing pCAMBIA1300-BvTSP1 of the K599 engineered strain, inoculate it into 50 mL of TY liquid medium containing 50 μg / mL kanamycin, and incubate at 28℃ and 200 rpm for 12-16 h to complete liquid activation. Measure the OD600 value of the bacterial suspension using a spectrophotometer. When the OD600 value reaches 0.8-1.0, take 200 μL of the bacterial suspension and spread it onto a TY solid medium plate containing 50 μg / mL kanamycin. Incubate at 28℃ with the plate inverted for 16-20 h until a uniform bacterial growth forms on the plate surface. At the same time, take 20 μL of the activated bacterial suspension and inoculate it into 50 mL of TY liquid medium containing 50 μg / mL kanamycin. Incubate at 28℃ and 200 rpm until the OD600 value reaches 0.8-1.0. Prepare the bacterial suspension for infection and store it temporarily on ice. Healthy sugar beet seedlings that have grown for 10 days and have developed cotyledons and a few true leaves were selected. In a clean bench, the seedlings were cut approximately 1 cm below the growing point using a sterile scalpel to obtain rootless explants, preserving the hypocotyl cut. A viscous K599 mycelium was scraped from a TY solid plate using a sterile inoculation loop and evenly applied to the cut surface of the sugar beet seedling explants, ensuring the wound area was completely covered. A clean culture container was prepared, and a 3-4 cm layer of vermiculite sterilized at 150°C for 2 hours was placed inside. The vermiculite was moistened with sterile water and gently compacted. The explants coated with the mycelium were inserted into the vermiculite to a depth of 1-2 cm, burying the cut in the substrate. The vermiculite around the explants was gently compacted to secure them. Multiple explants were planted in each container, maintaining appropriate spacing. Evenly pour 2.5 mL of the bacterial inoculum solution around the base of each explant into the vermiculite, ensuring the solution penetrates the area around the cut without splashing onto the leaves. If the substrate is too wet, tilt the container to drain excess liquid. Cover the inoculated culture containers and place them in a 25±2℃ incubator in the dark for 24 hours. Then remove the shading material and transfer the container to a light incubator with a 16-hour light / 8-hour dark photocycle and a light intensity of 100–150 μmol·m⁻¹. -2 ·s -1Maintain a temperature of 25±2℃ and keep the substrate moist but not waterlogged. Begin hardening off the seedlings gradually on day 5: loosen the container lid or open it 1 / 4 of the way on day 5; open 1 / 2 of the lid on day 6; open 3 / 4 of the lid on day 7; and completely open the lid on days 8-9. During hardening off, maintain stable temperature and humidity. If seedlings wilt, spray water as needed. Begin observing the growth of adventitious roots around the cut at the base of the explant on day 10 post-infection. Once hairy roots have emerged, remove the explant from the vermiculite, gently rinse the roots with water to remove vermiculite particles and impurities, and observe the fluorescence signal of the adventitious roots under a fluorescence stereomicroscope using an eGFP-specific filter (excitation wavelength 488nm, emission wavelength 509nm). The fluorescence image is shown below. Figure 1 As shown in the image, the left image is a fluorescence image of a sugar beet plant transformed with BvTSP1::eGFP, and the right image is an image of a sugar beet plant without the target gene. The expression specificity of BvTSP1 in adventitious roots of sugar beets was determined based on the presence and intensity of green fluorescence.

[0027] 2. Agrobacterium tumefaciens GV3101 was transformed with the recombinant plasmid pCAMBIA1300-BvTSP1 containing the beet root-specific expression promoter BvTSP1 to prepare an engineered bacterial culture that mediated transient expression in beet cotyledons. The expression characteristics of BvTSP1 in beet cotyledon tissues were detected by the fluorescence signal of the eGFP reporter gene. The specific procedure is as follows: Take 10 μL of bacterial culture containing pCAMBIA1300-BvTSP1 of the GV3101 engineered strain, inoculate it into 50 mL of LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampin, and culture at 28℃ and 200 rpm for 16-18 h to complete the activation of the strain; prepare 80 mL of infection buffer, and add 100 μL of 1 mol / L MgCl2 stock solution, 500 μL of 0.2 mol / L MES stock solution, and 20 μL of 100 mmol / L acetylsyl syringone stock solution activated by 37℃ water bath for 5-10 min in sequence, and add sterile deionized water to make up to 80 mL, so that the final concentrations of each component are 10 mmol / L, 10 mmol / L, and 200 μmol / L, respectively. After expanding the bacterial culture, the bacterial suspension was centrifuged at 6000 rpm for 10 min to collect the bacterial cells. The supernatant was discarded, and the bacterial cells were resuspended in 1 mL of fresh infection buffer. The resuspended solutions were combined, and the OD600 value of the bacterial suspension was adjusted to 0.8-1.0 with infection buffer to prepare the working infection solution. Healthy beet seedlings that had grown for 10 days, with fully expanded cotyledons and no true leaves, were selected. Using a sterile syringe needle, 1-2 small holes were made in the mesophyll area on both sides of the midrib of the cotyledon to penetrate the upper epidermis. A 1 mL sterile syringe without a needle was aimed at the small holes, and the working infection solution was slowly injected until the leaf area appeared water-soaked. 1-2 sites were injected on each cotyledon, and at least 3-5 seedlings were injected for each treatment and marked. The infected seedlings were placed in a culture environment of 25±2℃, with a photocycle of 16h light / 8h dark. The soil was kept moist, and the seedlings were appropriately shaded for 24 hours to avoid direct sunlight. Ventilation was maintained during the culture period, and the growth status of the seedlings was observed in a timely manner. On the 7th day after infection, a 3mm × 3mm piece of cotyledon lower epidermis was torn from the injection area, laid flat on a glass slide with sterile water, covered with a coverslip, and placed under a fluorescence microscope. The fluorescence signal was observed using an eGFP-specific filter (excitation wavelength 488nm, emission wavelength 509nm). The fluorescence image is shown below. Figure 2 As shown. Fluorescence intensity was recorded and transformation efficiency was statistically analyzed to determine the transient expression of BvTSP1 in beet cotyledons.

[0028] The above experiments and results show that BvTSP1 has root-specific transcriptional activity and can make the introduced target gene expressed only in the young roots and tubers of beet, while it is not expressed or is expressed very weakly in leaf tissues.

Claims

1. The beet root-specific expression promoter BvTSP1, characterized in that... Its nucleotide sequence is shown in SEQ ID NO.

1.

2. An expression cassette containing the beet root-specific expression promoter BvTSP1 as described in claim 1.

3. A recombinant expression vector containing the beet root-specific expression promoter BvTSP1 as described in claim 1 or the expression cassette as described in claim 2.

4. Engineered bacteria containing the beet root-specific expression promoter BvTSP1 as described in claim 1, the expression cassette as described in claim 2, or the recombinant expression vector as described in claim 3.

5. The application of the beet root-specific expression promoter BvTSP1 as described in claim 1 in regulating the specific expression of the target gene in beet roots.

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