Tissue-specific rcsdh2-3 gene promoter and use thereof

Abstract

Provided in the present invention is a tissue-specific RcSDH2-3 gene promoter from Ricinus communis, which can drive gene expression in seeds and cotyledon-stage seedlings of plants, or in roots, stems, leaves, flowers, seeds and cotyledon-stage seedlings of plants.

Description

A tissue-specific RcSDH2-3 gene promoter and its applications Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a tissue-specific RcSDH2-3 gene promoter and its applications. Background Technology

[0002] In plant technology, gene expression regulation is a core component in gene function research, metabolic regulation pathway research, and trait improvement. Promoters, as key elements in gene expression regulation, specifically bind to RNA polymerases and other transcription factors, thereby initiating gene transcription. However, widely used constitutive promoters, such as the CaMV 35S promoter and the Ubiquitin promoter, while capable of driving stable gene expression in various tissues and cells, lack tissue-specific regulatory capabilities, leading to unnecessary expression of exogenous genes in non-target tissues. This can not only waste biological resources and increase metabolic burden but also trigger unexpected phenotypic changes and even negatively impact the normal physiological functions of the organism.

[0003] On the other hand, existing tissue-specific promoters also have limitations in application. Many known tissue-specific promoters only exhibit certain specificity in specific species or developmental stages, and the strength and stability of their driving gene expression are insufficient to meet complex application requirements. For example, in plant genetic engineering, root-specific promoters may exhibit non-specific activation under certain environmental stresses. Furthermore, for some rare tissues or cell types, there is currently a lack of effective specific promoter resources, which greatly restricts the research and application development in related fields. Therefore, developing efficient and widely applicable tissue-specific gene promoters to achieve precise and controllable specific expression of exogenous genes in different tissues is of great significance for promoting the development of genetic engineering technology in basic research and practical applications. Summary of the Invention

[0004] The purpose of this invention is to provide a tissue-specific RcSDH2-3 gene promoter and its applications to address the problems existing in the prior art. This invention provides a promoter fragment capable of expression in specific plant tissues. Compared to traditional constitutive promoters, it effectively avoids unnecessary expression of exogenous genes in non-target tissues, reduces waste of biological resources and metabolic burden, and can initiate the expression of target genes in specific tissues at appropriate times according to different developmental stages of the plant. This provides technical support for research on plant gene function, metabolic regulation pathways, and trait improvement.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] The present invention provides a tissue-specific RcSDH2-3 gene promoter, wherein the nucleotide sequence of the RcSDH2-3 gene promoter is shown in any one of SEQ ID NO.19-23.

[0007] Furthermore, when the nucleotide sequence of the RcSDH2-3 gene promoter is as shown in SEQ ID NO.19, SEQ ID NO.22 or SEQ ID NO.23, it can drive the expression of the gene in plant seeds and cotyledon seedlings;

[0008] When the nucleotide sequence of the RcSDH2-3 gene promoter is as shown in SEQ ID NO.20 or SEQ ID NO.21, it can drive gene expression in the roots, stems, leaves, flowers, seeds, and cotyledon seedlings of plants.

[0009] This invention also provides the application of the above-mentioned RcSDH2-3 gene promoter in the preparation of exogenous gene expression vectors.

[0010] The present invention also provides a recombinant expression vector containing the above-mentioned RcSDH2-3 gene promoter.

[0011] The present invention also provides a recombinant microorganism comprising the above-described recombinant expression vector.

[0012] The present invention also provides the application of the above-mentioned RcSDH2-3 gene promoter, the above-mentioned recombinant expression vector, or the above-mentioned recombinant microorganism in regulating tissue-specific gene expression in plants.

[0013] Furthermore, the tissues include roots, stems, leaves, flowers, seeds, and cotyledonary seedlings.

[0014] Optionally, the plants include castor beans and Arabidopsis thaliana.

[0015] The present invention discloses the following technical effects:

[0016] This invention found that the RcSDH2-3 gene promoter can drive high expression of the gene in the cell nucleus and low expression in the cell membrane. Through further analysis of the RcSDH2-3 gene promoter region and design and synthesis of promoter fragments of different lengths, including full-length promoters and stepwise deletion promoter fragments, it was found that the full-length promoter and stepwise deletion promoter fragments have different expression patterns in different plant tissues. The SDH2-3(1) and SDH2-3(2) deletion promoter fragments drive gene expression in tissues and organs such as roots, stems, leaves, and flowers; SDH2-3(3), SDH2-3(4) and the full-length promoter SDH2-3 drive gene expression in cotyledon seedlings and seeds.

[0017] This invention provides promoter fragments that can be expressed in specific plant tissues. Compared with traditional constitutive promoters, it can effectively avoid unnecessary expression of exogenous genes in non-target tissues, reduce waste of biological resources and metabolic burden, and can initiate the expression of target genes in specific tissues at the appropriate time according to different developmental stages of plants, providing technical support for research on plant gene function, metabolic regulation pathways and trait improvement. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0019] Figure 1 shows the expression of four seed-specific genes in castor bean tissues at different developmental stages;

[0020] Figure 2 is an electrophoresis diagram of PCR products from E. coli transformed with pCAMBIA1303-SDH2-3; where M: DL2000 Maker; lane 1: PCR products;

[0021] Figure 3 shows the electrophoresis diagram of Agrobacterium plaque PCR products transformed by pCAMBIA1303-SDH2-3; where M: DL2000Maker; lane 1: PCR products;

[0022] Figure 4 shows the promoter activity verification results; where A: pCAMBIA1303 fluorescence; B: pCAMBIA1303 bright field; C: pCAMBIA1303-SDH2-3 fluorescence; D: pCAMBIA1303-SDH2-3 bright field;

[0023] Figure 5 is a schematic diagram of the construction process of different promoter vectors;

[0024] Figure 6 shows the electrophoresis diagram of the amplification products of the SDH2-3 promoter deletion; where M: DL2000Maker; lane 1: SDH2-3(1); lane 2: SDH2-3(2); lane 3: SDH2-3(3); lane 4: SDH2-3(4);

[0025] Figure 7 shows the electrophoresis diagram of E. coli plaque PCR products transformed with the promoter-deficient expression vector; where M: DL2000Maker; lanes 1 and 2: pCAMBIA1303-SDH2-3(1); lanes 3 and 4: pCAMBIA1303-SDH2-3(2); lanes 5 and 6: pCAMBIA1303-SDH2-3(3); lanes 7 and 8: pCAMBIA1303-SDH2-3(4);

[0026] Figure 8 shows the electrophoresis diagram of Agrobacterium plaque PCR products transformed with the promoter-deficient expression vector; where M: DL2000Maker; lanes 1 and 2: pCAMBIA1303-SDH2-3(1); lanes 3 and 4: pCAMBIA1303-SDH2-3(2); lanes 5 and 6: pCAMBIA1303-SDH2-3(3); lanes 7 and 8: pCAMBIA1303-SDH2-3(4);

[0027] Figure 9 shows the PCR identification results of transgenic Arabidopsis thaliana; where M: DL2000Maker; lanes 1 and 2: pCAMBIA1303-SDH2-3(1); lanes 3 and 4: pCAMBIA1303-SDH2-3(2); lanes 5 and 6: pCAMBIA1303-SDH2-3(3); lanes 7 and 8: pCAMBIA1303-SDH2-3(4); lanes 9 and 10: pCAMBIA1303-SDH2-3; lanes 11 and 12: pCAMBIA1303;

[0028] Figure 10 shows the GUS staining results of various tissues of transgenic Arabidopsis thaliana;

[0029] Figure 11 shows the BSA standard curve;

[0030] Figure 12 shows the results of GUS enzyme activity assay in various tissues of transgenic Arabidopsis thaliana;

[0031] Figure 13 shows the results of GUS gene expression level detection in various tissues of transgenic Arabidopsis thaliana. Detailed Implementation

[0032] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0033] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0034] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0035] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0036] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0037] Example 1

[0038] 1. Test materials

[0039] 1.1 Plant materials

[0040] The seeds of the Colombian wild-type (WT) Arabidopsis thaliana are preserved by the Key Laboratory of Castor Bean Breeding and Comprehensive Utilization of Inner Mongolia Autonomous Region. The castor bean material of the Tongbi 5 strain was provided by the Tongliao Municipal Institute of Agricultural Sciences, Inner Mongolia.

[0041] 1.2 Experimental Reagents

[0042] Pst I, Nco I, Maker2000, 10× Loading Buffer, T4 DNA Ligase, Plant RNA Extraction Kit, TaKaRa MiniBEST Plant RNA Extraction Kit HD Cloning Kit Seamless Cloning Reagent, PrimeScript TMRT Master Mix (Perfect Real Time) Reverse Transcription Kit, TB Premix Ex Taq TM The Tli RNaseH Plus fluorescence quantitative reagent kit was purchased from Takara Biotech Ltd.; the 2×Taq PCR MasterMix, plasmid miniprep kit, agarose gel DNA recovery kit, and hygromycin were purchased from Solarbio Biotech Ltd. FastPfu Fly PCR Super Mix The Plant Tissue PCR Kit was purchased from Beijing TransGen Biotech Co., Ltd.; X-Gluc was purchased from Beijing Cooler Master Technology Co., Ltd.; other reagents were domestically produced analytical grade.

[0043] 1.3 Preparation of Culture Media and Reagents

[0044] LB medium, YEB medium, MS medium.

[0045] The formula for GUS staining solution is shown in Table 1:

[0046] Table 1 GUS dye solution formulation

[0047] 1.4 Primer sequences are shown in Table 2

[0048] Table 2 Primer sequences

[0049] 2. Test methods

[0050] 2.1 Validation of the expression of seed-specific genes in castor beans

[0051] Based on publicly available transcriptome data of various castor bean tissues and the castor bean transcriptome data preserved in our laboratory, several seed-specific expressed genes were screened. Four genes, RcICL, RcMS1, RcOLE1, and RcSDH2-3, were selected, and specific quantitative fluorescent primers (SEQ ID NO.11-18) were designed. Using cDNA obtained from reverse transcription of genomic RNA from castor bean 'Tongbi 5' at different growth stages as templates, the expression levels of the four genes were detected.

[0052] The detection results are shown in Figure 1. The expression patterns of the four genes, RcICL, RcMS1, RcOLE1, and RcSDH2-3, were consistent with the transcriptome sequencing results and exhibited seed specificity. Among them, the expression level of the RcSDH2-3 gene was higher than that of the other genes, and it showed an increasing trend during seed development. RcSDH2-3 is a succinate dehydrogenase, which participates not only in the tricarboxylic acid cycle but also in the electron transport chain, playing an important role in seed material accumulation. Therefore, the promoter of this gene was selected as the research object.

[0053] Analysis of the cis-regulatory elements of the RcSDH2-3 gene promoter using the online software PlantCARE revealed that the promoter region contains multiple abscisic acid response elements (ABRE), cis-regulatory elements essential for anaerobic induction (ARE), cis-regulatory elements involved in light responsiveness (G-Box), cis-regulatory elements involved in salicylic acid responsiveness (TCA-element), cis-regulatory elements involved in MeJA responsiveness (TGACG-motif), and drought-induced elements at the MYB binding site (MBS).

[0054] The SDH2-3 promoter sequence of the RcSDH2-3 gene is shown in SEQ ID NO.19:

[0055] 2.2 Expression vector construction and activity analysis

[0056] Castor bean seeds were flash-frozen in liquid nitrogen and ground in a mortar. Genomic DNA was extracted according to the instructions provided with the plant genomic DNA extraction kit. The RcSDH2-3 gene promoter was cloned using primers SDH2-3R and SDH2-3F, which contain restriction enzyme sites. The PCR reaction mixture consisted of 2 μL of castor bean genomic DNA template, 1 μL each of forward and reverse primers, 25 μL of 2×Fly Mix, and 21 μL of ddH2O. The PCR program was as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 57℃ annealing for 30 s, 72℃ extension for 3 min, 30 cycles; 72℃ for 3 min; storage at 4℃.

[0057] After confirming the electrophoresis results were correct, 50 μL of the PCR product was electrophoresed for 30 min (the 1×TAE electrophoresis buffer was replaced beforehand). The SDH2-3 promoter was then recovered by agarose gel DNA recovery kit using agarose gel electrophoresis. The large fragment of pCAMBIA1303, recovered by Pst I / Nco I restriction enzyme digestion, was ligated into the fragment. The ligation system consisted of: 1 μL of pCAMBIA1303 linearized vector, 3 μL of SDH2-3 promoter, 1 μL of T4 DNA Ligase, 1 μL of 10×T4 DNA Ligase Buffer, 4 μL of ddH2O, for a total of 10 μL. The pCAMBIA1303-SDH2-3 recombinant plasmid was thus constructed.

[0058] The ligation product was transferred into *E. coli* DH5α competent cells as follows: First, thaw 50 μL of competent cells on ice for 5 min, then add 10 μL of the ligation product to the competent cells. Incubate on ice for 30 min, then in a 42°C water bath for 90 s, followed by 5 min on ice. Next, in a clean bench, add 800 μL of antibiotic-free LB medium to the competent cells and incubate *E. coli* on a 37°C shaker at 220 rpm for 1 h. Aspirate all the revived bacterial culture and transfer it to a pre-pollinated LB agar plate containing Kans antibiotic. Gently invert the plate to spread the culture evenly, and seal it after the culture has dried. Incubate overnight in a 37°C *E. coli* incubator, inverted.

[0059] After round, single colonies grew on LB solid medium, two colonies were picked in a clean bench and dissolved in 10 μL of ultrapure water. 2 μL of each colony was used as a template for colony PCR using the vector primers (SEQ ID NO. 7-8). The colony PCR reaction system was as follows: 5 μL of *E. coli* bacterial culture, 25 μL of 2×Master PCR Mix, 1 μL of 1303F, 1 μL of 1303R, and 18 μL of ddH2O. The reaction program was: 94℃ for 3 min; 94℃ for 30 s, 59℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ for 5 min, 4℃ at ∞.

[0060] The results are shown in Figure 2. The band size is 1793 bp, which is consistent with expectations.

[0061] Plasmids were extracted from the correctly validated *E. coli* culture by shaking. The recombinant plasmid pCAMBIA1303-SDH2-3 and the pCAMBIA1303 control were transformed into GV3101 *Agrobacterium* competent cells and incubated upside down at 18°C ​​for 2 days. Once single colonies appeared on the plates, they were picked for *Agrobacterium* plaque PCR. The reaction system and procedure were the same as above. The results are shown in Figure 3, displaying bands of the same size as in Figure 2. The correctly validated *Agrobacterium* culture was preserved and stored at -80°C for subsequent experiments.

[0062] To verify promoter activity, transient expression of onion was performed. The method is as follows:

[0063] In a clean bench, take 3-4 layers of onion tissue and sterilize them in a large beaker containing 75% alcohol for 10 minutes, then rinse three times with ultrapure water. After sterilization, cut the inner epidermis of the onion into 1cm pieces using a scalpel. 2 Slowly peel off the onion skin to form a square shape, and lay the wounded side flat on MS solid medium for pre-incubation at 25°C for 24 hours (16 hours light / 8 hours dark). Inoculate the two identified Agrobacterium species into 250 μL Kan and 500 μL Rif liquid YEB medium, and incubate at 28°C with shaking at 180 rpm for 12 hours. After centrifugation at 4500 rpm for 5 minutes, discard the supernatant. Resuspend the cells in MS medium supplemented with 10 mmol / L MgCl2 and 100 μmol / L acetylsyringone, until OD... 600 =0.6-1.0. Place the pre-cultured onion epidermis into the resuspended bacterial cells, shake to infect for 10 minutes, blot dry with absorbent paper, and incubate at 25℃ for 2 days (16h light / 8h dark). After incubation, prepare temporary slides, observe the slides under an inverted fluorescence microscope, and photograph for record-keeping.

[0064] The results are shown in Figure 4. The promoter was successfully expressed after Agrobacterium-mediated permeation treatment. Confocal microscopy revealed that the RcSDH2-3 gene promoter enabled high expression of the GFP fluorescent tag in the cell nucleus and low expression in the cell membrane, although its initiation strength was weaker compared to the 35S promoter in pCAMBIA1303.

[0065] 2.3 Cloning of promoters and construction of expression vectors

[0066] To explore the core functional region of the RcSDH2-3 promoter, a promoter deletion mutation was cloned at its 5' end. A schematic diagram of the promoter vector construction is shown in Figure 5. Using the pCAMBIA1303-SDH2-3 recombinant plasmid as a template, amplification was performed using primers (SEQ ID NO.1, 3-6) listed in Table 3, resulting in 5'-terminal deleted promoters SDH2-3(1), SDH2-3(2), SDH2-3(3), and SDH2-3(4), respectively. The amplification system consisted of: 2 μL of pCAMBIA1303-SDH2-3 plasmid, 1 μL of SDH2-3(1 / 2 / 3 / 4)F, 1 μL of SDH2-3 R, 25 μL of 2×Fly Mix, 20 μL of ddH2O, and a total of 50 μL. The amplification program was as follows: 98℃ for 1 min; 98℃ for 10 s, 58℃ for 5 s, 72℃ for 20 s, 30 cycles; 72℃ for 1 min, 4℃ at ∞. The electrophoresis diagram of the amplification product is shown in Figure 6. The amplification band is bright and uniform, with no extraneous bands, indicating that the cloned promoter was successfully cloned.

[0067] The missing promoter sequences are as follows:

[0068] SDH2-3(1) sequence, SEQ ID NO.20:

[0069] SDH2-3(2) sequence, SEQ ID NO.21:

[0070] SDH2-3(3) sequence, SEQ ID NO.22:

[0071] SDH2-3(4) sequence, SEQ ID NO.23:

[0072] The amplification products of each promoter deletion were gel-recovered and ligated into the pCAMBIA1303 linearized vector digested with Pst I and Nco I restriction endonucleases, respectively. The recombinant vectors were named pCAMBIA1303-SDH2-3(1), pCAMBIA1303-SDH2-3(2), pCAMBIA1303-SDH2-3(3), and pCAMBIA1303-SDH2-3(4). After transformation of the recombinant vectors into E. coli, amplification was performed using the vector primers. The results are shown in Figure 7. The amplification product sizes of pCAMBIA1303-SDH2-3(1), pCAMBIA1303-SDH2-3(2), pCAMBIA1303-SDH2-3(3), and pCAMBIA1303-SDH2-3(4) were 697bp, 940bp, 1258bp, and 1538bp, respectively. The amplified bands were consistent with the expectations. Positive plaques were shaken and sent to the company for sequencing. After comparison, it was confirmed that four promoter fragments were successfully ligated to the expression vector, and plasmids were extracted.

[0073] The correctly verified plasmid was transformed into GV3101 Agrobacterium competent cells, and the transformation was verified by colony PCR using the vector primers. The results are shown in Figure 8. The correct bacterial culture was then shaken and preserved for subsequent Arabidopsis genetic transformation.

[0074] 2.4 Arabidopsis genetic transformation

[0075] Wild-type (WT) Arabidopsis thaliana seeds were sown in pots filled with a 1:1 mixture of potting soil and vermiculite. A layer of plastic wrap was placed over the soil, and the pots were cultured in a climate chamber with 16 hours of light and 8 hours of darkness. The plastic wrap was removed after three days. When the Arabidopsis thaliana reached the 4-leaf stage, the wild-type plants were transplanted into the pots, ensuring one plant per pot. At 7-8 weeks of growth, the main stem of the Arabidopsis thaliana was pruned to encourage more inflorescences. Agrobacterium, containing pCAMBIA1303-SDH2-3 recombinant plasmid, pCAMBIA1303-SDH2-3(1), pCAMBIA1303-SDH2-3(2), pCAMBIA1303-SDH2-3(3), pCAMBIA1303-SDH2-3(4), and pCAMBIA1303 vector (positive control), were used to infect Arabidopsis thaliana using the dip-flower method.

[0076] Positive transgenic Arabidopsis thaliana were screened by adding 50 mg / mL of hygromycin to MS solid medium. First, seeds of the T0 generation were collected, dried, and vernalized at 4°C for two days. They were then washed with 75% alcohol for 10 minutes, followed by three washes with sterile water. These seeds were sown in MS medium supplemented with hygromycin. Once the Arabidopsis thaliana reached the 4-leaf stage, they were transplanted into flowerpots. They were cultured in an artificial climate chamber (under the same conditions as the wild type: 16 h light / 8 h dark). After the seeds matured, seeds of the T1 generation Arabidopsis thaliana were harvested from individual plants.

[0077] T1 generation transgenic Arabidopsis seeds were transplanted onto solid MS medium supplemented with 50 mg / mL hygromycin to culture positive-positive Arabidopsis plants. Using a gold-based direct PCR kit, leaves of the transgenic Arabidopsis were used to amplify the GUS gene on the vector using qGUS F / qGUS R primers (SEQ ID NO. 9-10). Leaves from each transgenic plant were lysed, and approximately 5 mg of leaf tissue was minced and placed in a 200 μL centrifuge tube. 40 μL of PD1 buffer was added, and the mixture was vortexed. The tube was incubated at 95°C for 10 min. 40 μL of PD2 buffer was added to the centrifuge tube and mixed to serve as the PCR template. The PCR reaction mixture consisted of: 3 μL lysed template, 1 μL qGUS F, 1 μL qGUS R, and 2×... PCR Mix 10 μL, ddH2O 5 μL, total 20 μL. PCR reaction program: 94℃ for 10 min; 94℃ for 30 s, 55℃ for 30 s, 72℃ for 15 s, 35 cycles; 72℃ for 10 min, 4℃ to infinity. After amplification, 1% agarose gel electrophoresis was performed for detection. The results are shown in Figure 9. A 156 bp band was amplified in each transgenic Arabidopsis thaliana, and the band size was correct, indicating successful acquisition of transgenic Arabidopsis thaliana.

[0078] 2.5 GUS Expression Analysis in Transgenic Arabidopsis

[0079] 2.5.1 GUS staining of transgenic Arabidopsis thaliana

[0080] Take different tissue materials from WT and various transgenic Arabidopsis thaliana species, place them in 0.2 mL centrifuge tubes, and add 150 μL of GUS staining solution (protect from light). Wrap the tubes with aluminum foil and place them in a shaking incubator at 220 rpm and 37°C for 10 h. After staining, destain with 75% ethanol for 1 h, repeating the destaining process three times. Take photographs for observation.

[0081] The results are shown in Figure 10. WT represents wild-type Arabidopsis thaliana, and no GUS gene was expressed in any tissue of this plant; the staining results serve as the negative control. The positive control is pCAMBIA1303 transgenic Arabidopsis thaliana, whose CaMV35S promoter is a constitutive promoter. In transgenic Arabidopsis thaliana, it can drive high expression of the GUS gene in all tissues, resulting in a darker GUS staining color. Notably, regardless of promoter length, cotyledonary seedlings of all transgenic Arabidopsis thaliana can be stained, all showing GUS gene expression. Driven by promoter SDH2-3(1), the GUS gene is expressed in all tissues, especially in cotyledonary seedlings, rosette leaves, embryos, and seeds, where the color is very dark, similar to the color of GUS gene expression driven by the CaMV35S promoter. Expression is weaker in roots, stems, cauline leaves, and flowers. Root staining results showed that, except for the SDH2-3(1) promoter, no GUS gene expression was observed in SDH2-3(2), SDH2-3(3), SDH2-3(4), and SDH2-3. Staining results of transgenic Arabidopsis stems and stem leaves showed that only the stems and stem leaves of the SDH2-3(1) transgenic plants showed low expression of the GUS gene. Rosette leaf staining results showed that the SDH2-3(1) promoter resulted in high expression of the GUS gene in rosette leaves, the SDH2-3(2) promoter showed low expression, and other promoters did not show color, indicating no GUS gene expression. In flower staining, the results were similar to those of rosette leaves, with only the SDH2-3(1) and SDH2-3(2) promoters staining. The SDH2-3(1) promoter showed low expression, and the SDH2-3(2) promoter showed trace amounts of GUS gene expression. The staining results of seeds and embryos show that the staining color is relatively dark, and all transgenic plants have high expression of GUS in seeds and embryos. Among them, SDH2-3(2) stains lighter than other seeds and embryos. There is no obvious difference between other plants. The difference in GUS gene expression in seeds needs to be verified by fluorescence quantitative and GUS enzyme activity experiments.

[0082] 2.5.2 GUS enzyme activity assay

[0083] Proteins were extracted from various tissues of each transgenic plant, and a BSA standard curve was plotted, following the instructions of the Bradford Protein Assay Kit (Sole Protein). The extracted protein samples were diluted, and 20 μL was added to each well of a 96-well plate. 200 μL of 1×G250 staining solution was added to each well, and the A595 value was measured using a microplate reader. The protein content of each sample was calculated by subtracting the values ​​from the BSA standard curve. The BSA standard curve is shown in Figure 11. The regression equation for protein concentration and absorbance was y = 1.8217x + 0.0156, R0. 2 =0.9953.

[0084] A standard curve was prepared using 4-MU standard: First, 1 mM 4-MU standard was serially diluted with 0.2 mol / L Na2CO3. After 12 dilutions, 5 samples with low concentrations were taken and their fluorescence values ​​were measured under 365 nm excitation light and 455 nm emission light. The standard curve was plotted, and the regression equation between 4-MU content and fluorescence intensity was obtained: y = 0.4836x + 53.326, R2 = 0.9952.

[0085] Take 20 μL of total protein from each sample and 200 μL of GUS extraction buffer containing 1 mM 4-MUG, preheated to 37°C, and react in a 37°C water bath. Add 160 μL of 0.2 mol / L Na2CO3 to each well of a black 96-well plate, and add reaction solutions with reaction times of 5 min, 15 min, 25 min, and 35 min to the wells of the 96-well plate, respectively. Measure the fluorescence value, calculate the 4-MU content according to the standard curve, and obtain the enzyme activity of each protein.

[0086] The results of GUS enzyme activity assays in various tissues of transgenic Arabidopsis thaliana are shown in Figure 12. The enzyme activity in all WT tissues was close to 0, and no GUS enzyme activity was detected. The results of the positive control (1303) showed that, under the influence of the CaMV 35S promoter, GUS enzyme activity was very high in the roots, stems, stem leaves, rosette leaves, flowers, seeds, and cotyledon seedlings of transgenic Arabidopsis thaliana 1303, reaching 1325 in seeds. Driven by the SDH2-3(1) and SDH2-3(2) promoters, GUS enzyme activity was present in all tissues, with higher activity in cotyledon seedlings and seeds. The SDH2-3(3), SDH2-3(4), and SDH2-3 promoters showed high GUS enzyme activity in cotyledon seedlings and seeds, while almost no GUS enzyme activity was detected in the roots, stems, stem leaves, rosette leaves, and flowers. The results of the GUS enzyme activity assays were basically consistent with the GUS staining results.

[0087] 2.5.3 Quantitative fluorescence analysis of the transgenic Arabidopsis thaliana GUS gene

[0088] Following the instructions of the Takara Bio RNA Extraction Kit, RNA was extracted from various tissues of transgenic Arabidopsis thaliana, reverse transcribed into cDNA, and then used as a template. Actin-2 was used as an internal control gene in Arabidopsis thaliana. -ΔΔCt The expression level of the gene was calculated, and the expression of the GUS gene in different transgenic Arabidopsis and different tissues was analyzed (the experiment was set up with three biological replicates and three technical replicates).

[0089] The results are shown in Figure 13. Analysis revealed that, compared with wild-type Arabidopsis, transgenic Arabidopsis with SDH2-3(1) and SDH2-3(2) promoters expressed the GUS gene in all tissues, but the expression level in seeds and seedlings at the cotyledon stage was significantly higher than that in other tissues. The promoters SDH2-3, SDH2-3(3), and SDH2-3(4) still showed seed-specific expression in seeds.

[0090] In summary, this invention utilizes transient expression in onion, and the results show that the RcSDH2-3 gene promoter enables high expression of the GFP fluorescent tag in the cell nucleus and low expression in the cell membrane. Its promoter strength is weaker than that of the 35S promoter in pCAMBIA1303. Cis-acting element analysis of the RcSDH2-3 gene promoter region revealed multiple cis-acting elements. Based on the distribution of cis-acting elements, promoter deletion fragments of different lengths were designed and synthesized, including full-length promoters and stepwise deletion fragments, and expression vectors with GUS reporter genes were constructed. The constructed expression vectors were introduced into Arabidopsis thaliana using Agrobacterium-mediated genetic transformation. The expression patterns of the full-length promoter and different promoter fragments in Arabidopsis thaliana were analyzed by GUS histochemical staining. The results showed that genes driven by the deletion of SDH2-3(1) and SDH2-3(2) promoter fragments were expressed in roots, stems, leaves, flowers, and other tissues and organs. Genes driven by SDH2-3(3), SDH2-3(4), and the full-length promoter SDH2-3 were expressed in cotyledonary seedlings and seeds of Arabidopsis thaliana, exhibiting significant tissue-specific or expression intensity changes. It is speculated that the seed-specific expression core region of the seed-specific gene RcSDH2-3 promoter is located between -920 bp and -602 bp. These results indicate that specific cis-regulatory elements in the promoter play an important regulatory role in its expression pattern.

[0091] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A tissue-specific RcSDH2-3 gene promoter, characterized in that, The nucleotide sequence of the RcSDH2-3 gene promoter is shown in any one of SEQ ID NO.19-23.

2. The RcSDH2-3 gene promoter according to claim 1, characterized in that, When the nucleotide sequence of the RcSDH2-3 gene promoter is as shown in SEQ ID NO.19, SEQ ID NO.22 or SEQ ID NO.23, it can drive the expression of the gene in the seeds and cotyledon seedlings of plants; When the nucleotide sequence of the RcSDH2-3 gene promoter is as shown in SEQ ID NO.20 or SEQ ID NO.21, it can drive gene expression in the roots, stems, leaves, flowers, seeds, and cotyledon seedlings of plants.

3. The application of the RcSDH2-3 gene promoter as described in claim 1 or 2 in the preparation of exogenous gene expression vectors.

4. A recombinant expression vector comprising the RcSDH2-3 gene promoter as described in claim 1 or 2.

5. A recombinant microorganism comprising the recombinant expression vector of claim 4.

6. The application of the RcSDH2-3 gene promoter of claim 1 or 2, the recombinant expression vector of claim 4, or the recombinant microorganism of claim 5 in regulating tissue-specific gene expression in plants.

7. The application according to claim 6, characterized in that, The tissues include roots, stems, leaves, flowers, seeds, and seedlings in the cotyledon stage.

8. The application according to claim 6, characterized in that, The plants mentioned include castor bean and Arabidopsis thaliana.

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