A soybean bidirectional promoter and application thereof

Abstract

The application discloses a soybean bidirectional promoter and application thereof, and belongs to the technical field of bidirectional promoter separation and application.The nucleotide sequence of the soybean bidirectional promoter is shown as SEQ ID NO.2.It is verified by experiments that the soybean bidirectional promoter can simultaneously drive the expression of target genes in a bidirectional mode, and the soybean bidirectional promoter can be applied to improve plant properties, cultivate transgenic plants or cultivate new plant varieties, and is favorable for promoting the biological breeding process.

Description

Technical Field

[0001] This invention belongs to the field of separation and application technology of bidirectional promoters, specifically relating to a soybean bidirectional promoter and its application. Background Technology

[0002] In crop molecular breeding and functional genomics research, the development of multi-gene synergistic transformation and rapid trait aggregation technologies is a core direction for overcoming the bottlenecks of traditional genetic improvement techniques. Traditional single-gene transformation systems struggle to cope with the complex agronomic traits (such as yield, stress resistance, and quality) of crops through multi-gene regulatory networks, failing to achieve the simultaneous introduction and synergistic expression of multiple functional genes. Currently, the cauliflower mosaic virus CaMV 35S promoter and the maize UBI promoter, as stable and efficient exogenous gene expression regulatory elements, remain commonly used tools in plant genetic engineering. However, during transgenic operations, the presence of repetitive DNA sequences often leads to gene silencing, especially during multi-gene transformation, where this gene expression inhibition effect is significantly enhanced, severely impacting the successful acquisition of transgenic plants and the genetic stability of their offspring. When introducing two or more exogenous genes, these genes typically need to maintain consistency in expression levels and spatiotemporal expression patterns to ensure the introduced genes can perform their biological functions normally. However, existing research indicates that the CaMV35S promoter or the maize UBI promoter cannot meet these regulatory requirements, and these technical challenges have become key bottlenecks that urgently need to be overcome in the field of plant genetic engineering.

[0003] With the development of genomics, the sequencing of genomes of many organisms has been completed. Bioinformatics analysis has revealed the existence of bidirectional promoters, making it possible to utilize plant-derived bidirectional promoters for transgenic operations, thus avoiding gene silencing caused by multiple gene insertions. Bidirectional promoters require only one promoter to drive the transcription of two genes in opposite directions, significantly reducing repetitive sequences in vectors and lowering the risk of gene silencing caused by sequence duplication. Their shared cis-regulatory elements ensure high synchronization of expression levels and spatiotemporal patterns between the two genes, meeting the needs of multi-gene co-expression in metabolic or signaling pathways. In terms of vector construction, bidirectional promoters can shorten vector length and improve vector stability and Agrobacterium-mediated transformation efficiency. Furthermore, some bidirectional promoters possess tissue-specific or inducible response characteristics, enabling co-expression of two genes under specific conditions. Soybean is an important dual-purpose crop for both food and oil; exploring endogenous bidirectional promoters in soybean and using them for the co-expression of multiple genes is beneficial for advancing the process of biobreeding. Summary of the Invention

[0004] In view of the above-mentioned prior art, the present invention provides a soybean bidirectional promoter and its application, and develops a new bidirectional promoter to achieve the synergistic expression of two genes in plants.

[0005] To achieve the above objectives, the technical solution adopted by the present invention is to provide a soybean bidirectional promoter, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0006] Based on the above technical solution, the present invention can be further improved as follows.

[0007] Furthermore, an expression cassette containing the aforementioned soybean bidirectional promoter.

[0008] Furthermore, recombinant vectors containing the aforementioned soybean bidirectional promoter.

[0009] Furthermore, transgenic cell lines containing the aforementioned soybean bidirectional promoter.

[0010] Furthermore, recombinant bacteria containing the aforementioned soybean bidirectional promoter.

[0011] Furthermore, the above-mentioned soybean bidirectional promoters, expression cassettes, recombinant vectors, transgenic cell lines, or recombinant bacteria are used to drive the expression of target genes in plants.

[0012] Furthermore, the plant is either soybean or tobacco.

[0013] Furthermore, the above-mentioned soybean bidirectional promoters, expression cassettes, recombinant vectors, transgenic cell lines, or recombinant bacteria are used in improving plant traits, cultivating transgenic plants, or cultivating new plant varieties.

[0014] Furthermore, the plant is either soybean or tobacco.

[0015] The beneficial effects of this invention are: the soybean bidirectional promoter of this invention can simultaneously drive the expression of its upstream and downstream target genes. By introducing the soybean bidirectional promoter sequence into a vector, the recombinant vector pTF101-GFP-SXPRO08-RUBY is obtained. This recombinant vector can effectively express its upstream and downstream GFP and RUBY genes in both the tobacco transient expression system and the soybean hairy root transformation system. The soybean bidirectional promoter provided by this invention can be applied to the construction of transgenic soybeans, the improvement of soybean agronomic traits, and the cultivation of new soybean germplasm with superior traits. Attached Figure Description

[0016] Figure 1 The vector map for pTF101-GFP-SXPRO08-RUBY;

[0017] Figure 2 To verify the function of the soybean bidirectional promoter using a tobacco transient expression system; where A is the RUBY colorimetric result under white light conditions, and B is the result of GFP fluorescence signal detected by a plant in vivo imaging system;

[0018] Figure 3To verify the function of the soybean bidirectional promoter using the soybean hairy root transformation system; where A is the RUBY color development result under white light, B and C are the results of RUBY and GFP fluorescence signals observed under a plant stereomicroscope, CK represents the control group, and the scale bar is 500 μm. Detailed Implementation

[0019] The specific embodiments of the present invention will be described in detail below with reference to examples.

[0020] Example 1

[0021] Cloning and construction of recombinant vectors for soybean bidirectional promoters

[0022] 1.1 Construction of the intermediate carrier

[0023] (1) The synthesized GFP-RUBY sequence (SEQ ID NO.1) was ligated into the pUC57 vector (Beijing Qingke Biotechnology Co., Ltd.) to obtain the pUC57-GFP-RUBY vector.

[0024] (2) Using restriction endonucleases Xba I and Pme I. The pTF101.1 vector was double-digested in a 50 μL system at 37°C. After 4 hours of digestion, the digested vector was detected by 1% agarose gel electrophoresis and recovered from the gel.

[0025] (3) Using the pUC57-GFP-RUBY vector plasmid as a template, amplification was performed using the following primers:

[0026] Primer GFP-RUBY-F:

[0027] 5'-TGCATGCCTGCAGGTCGACTCTAGACTTATCTTTAATCATATTCCATAGT-3' (SEQ ID NO. 3);

[0028] Primer GFP-RUBY-R:

[0029] 5'-CCTGTCAAACACTGATAGTTTAAACGGCGCGCCTGAAGGCGGGAAACGAC-3' (SEQ ID NO. 4).

[0030] The amplification products were separated by 1% agarose gel electrophoresis, and the target band (5564 bp) was recovered from the gel. Subsequently, the target sequence was ligated into a gel using the ClonExpress Ultra One Step Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number C115-01). Xba I and PmeThe PTF101.1 vector, which was double-digested with enzyme I, was transformed into E. coli Fast-T1 (Nanjing Novizan Biotechnology Co., Ltd., catalog number C505-02). After sequencing, the target sequence was confirmed to be correct, and the intermediate vector pTF101-GFP-RUBY was obtained.

[0031] 1.2 Cloning of the Soybean Bidirectional Promoter

[0032] (1) Based on the results of bioinformatics analysis, some potential bidirectional promoter sequences exist in the soybean genome. In this study, the bidirectional promoter of soybean (named SXPRO08) was amplified using DNA from the soybean variety Zhonghuang 13 (from the Institute of Crop Science, Chinese Academy of Agricultural Sciences) as a template. Its nucleotide sequence is shown in SEQ ID NO.2. PCR amplification was performed using the primers described below, with restriction enzyme sites introduced at both ends of the SXPRO08 sequence. Nco I and Spe I:

[0033] Primer SXPRO08-F: 5'-CATGCCATGGTGTGAGATAATAATGCTTGA-3' (SEQ ID NO.5);

[0034] Primer SXPRO08-R: 5'-GACTAGTTCTTGGTGATGGTTATAGTCTAT-3' (SEQ ID NO.6).

[0035] The amplification products were separated by 1% agarose gel electrophoresis, and the target band (1079 bp in size) was recovered from the gel and ligated into the cloning vector of the TA / Blunt-Zero Cloning Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number C601-01). The vector was transformed into E. coli Fast-T1, and the ligated target sequence was confirmed to be correct after sequencing, thus obtaining the Blunt-Zero-SXPRO08 vector.

[0036] 1.3 Construction of recombinant expression vectors

[0037] (1) Using restriction endonucleases Spe I and Nco I. The intermediate vector pTF101-GFP-RUBY was double-digested in a 50 μL system at 37 °C for 3 hours. The target fragment to be recovered by 1% agarose gel electrophoresis was approximately 14412 bp.

[0038] (2) Using restriction endonucleases Spe I and NcoI. The vector Blunt-Zero-SXPRO08 was double-digested in a 50 μL system at 37°C for 3 hours. The target fragment required for gel recovery was approximately 1079 bp, as detected by 1% agarose gel electrophoresis.

[0039] (3) The target fragments obtained in steps (1) and (2) were ligated using T4 DNA ligase (Beijing Bailinke Biotechnology Co., Ltd., catalog number M0202T), and transformed into E. coli Fast-T1 to obtain the recombinant expression vector pTF101-GFP-SXPRO08-RUBY, the pattern of which is shown below. Figure 1 As shown in the figure, this vector contains two marker genes, GFP and RUBY, located on either side of the soybean bidirectional promoter SXPRO08, for the purpose of verifying the function of the bidirectional promoter.

[0040] Example 2

[0041] Verification of the function of the soybean bidirectional promoter using a tobacco transient expression system

[0042] (1) The recombinant expression vector pTF101-GFP-SXPRO08-RUBY was transformed into Agrobacterium tumefaciens GV3101 (PSoup-P19, Shanghai Weidi Biotechnology Co., Ltd., catalog number AC1003L) competent cells by freeze-thaw method to obtain recombinant Agrobacterium, which was named GV3101-pTF101-GFP-SXPRO08-RUBY.

[0043] (2) Agrobacterium injection infection of tobacco: The first step is to activate the bacterial culture. For the first activation of Agrobacterium, 100 μL of the preserved GV3101 bacterial culture is added to 5 mL of LB+Rif+Spe liquid medium and the culture is shaken at 220 rpm for 12 h at 28℃. Then, the bacterial culture is activated a second time by transferring 200 μL of the first-activated bacterial culture to 200 mL of LB+Rif+Spe liquid medium and incubating at 220 rpm for 14 h at 28℃ until the bacterial culture reaches OD. 600 At approximately 1.0, the bacterial culture was enriched by centrifugation (6000 rpm, 10 min), the supernatant was discarded, and the bacterial cells were retained. Then, an infection buffer (100 mL) was prepared by weighing 0.4 g of MgCl₂·6H₂O, 150 μL of 0.1 M acetylsalicylic acid, and 0.4 g of MES monohydrate, and bringing the volume to 100 mL with water. The bacterial cells were then resuspended in the infection buffer to OD₀. 600 = 0.5. The bacterial culture was left to stand at room temperature for 3 h, then injected into tobacco leaves of about 5 weeks old using a 1 mL syringe. After 24 h of dark treatment, the leaves were transferred to a greenhouse at 25°C for about 4 days of cultivation before subsequent observation.

[0044] like Figure 2 As shown in Figure A, tobacco leaves injected with recombinant Agrobacterium GV3101-pTF101-GFP-SXPRO08-RUBY exhibited a magenta color from betalains, indicating that SXPRO08 drove the expression of the RUBY gene. Subsequently, this invention used a plant in vivo imaging system to detect the GFP fluorescence signal, and the results are as follows... Figure 2 As shown in Figure B, the GFP gene was also driven to express. These results demonstrate that the soybean bidirectional promoter SXPRO08 can simultaneously drive the expression of both the RUBY and GFP genes in tobacco.

[0045] Example 3

[0046] Verification of the function of the soybean bidirectional promoter using a soybean hairy root transformation system

[0047] (1) The recombinant expression vector pTF101-GFP-SXPRO08-RUBY was transformed into competent Agrobacterium rhizogenes K599 (from the Institute of Crop Science, Chinese Academy of Agricultural Sciences) cells by electroporation to obtain recombinant Agrobacterium, which was named K599-pTF101-GFP-SXPRO08-RUBY.

[0048] (2) Induction of hairy roots by puncture method: First, take healthy, plump, and uniform soybean seeds of variety Jack (from the Institute of Crop Science, Chinese Academy of Agricultural Sciences) that are free from pests and diseases and spots, plant them in nutrient soil, and cultivate them for 7 days at 28°C under 16 hours of light / 8 hours of darkness.

[0049] (3) Preparation of bacterial culture: Take a small amount of the preserved hairy rhizome culture carrying the target vector and add it to 20 mL of YEP liquid medium containing the corresponding antibiotic. Incubate overnight at 28°C with shaking to complete the first activation of the bacterial culture. Add the first activated bacterial culture to fresh medium containing the corresponding antibiotic at a ratio of 1 / 1000 and incubate at 28°C with shaking until OD. 600 The secondary activation of the bacterial solution is completed at approximately 0.8.

[0050] (4) Using a 1 mL syringe, inject K599-pTF101-GFP-SXPRO08-RUBY into the hypocotyl. Cover the flowerpot with a transparent plastic bag to keep it moist. Culture at 28℃ for 15 days under 16 hours of light / 8 hours of darkness to produce roots. Then test the results.

[0051] The results are as follows Figure 3As shown, untransformed soybean hair roots are white, while positive hair roots transformed with *Agrobacterium tumefaciens* pTF101-GFP-SXPRO08-RUBY exhibit a magenta color resembling betaine, indicating that SXPRO08 drives the expression of the RUBY gene. Subsequently, this invention used a stereomicroscope equipped with a fluorescence excitation module (Nikon INTENSILIGHT C-HGFI) to identify the GFP signal. The results showed that hair roots expressing the RUBY gene emitted green fluorescence under excitation light, indicating that the GFP gene was also driven to express. These results demonstrate that the soybean bidirectional promoter SXPRO08 can simultaneously drive the expression of both the RUBY and GFP genes in soybean.

[0052] The nucleotide sequence of GFP-RUBY is shown below (SEQ ID NO.1):

[0053]

[0054] The nucleotide sequence of the soybean bidirectional promoter SXPRO08 is shown below (SEQ ID NO.2):

[0055]

[0056] Although specific embodiments of the present invention have been described in detail with reference to examples, they should not be construed as limiting the scope of protection of this patent. Various modifications and variations that can be made by those skilled in the art without inventive effort within the scope described in the claims are still within the scope of protection of this patent.

Claims

1. A soybean bidirectional promoter, characterized in that: The nucleotide sequence of the soybean bidirectional promoter is shown in SEQ ID NO.

2.

2. An expression cassette containing the soybean bidirectional promoter as described in claim 1.

3. A recombinant vector containing the soybean bidirectional promoter as described in claim 1.

4. A transgenic cell line containing the soybean bidirectional promoter as described in claim 1.

5. Recombinant bacteria containing the soybean bidirectional promoter as described in claim 1.

6. The application of the soybean bidirectional promoter of claim 1, the expression cassette of claim 2, the recombinant vector of claim 3, the transgenic cell line of claim 4, or the recombinant bacteria of claim 5 in driving the expression of the target gene in plants.

7. The application according to claim 6, characterized in that: The plant in question is either soybean or tobacco.

8. The application of the soybean bidirectional promoter of claim 1, the expression cassette of claim 2, the recombinant vector of claim 3, the transgenic cell line of claim 4, or the recombinant bacteria of claim 5 in improving plant traits, cultivating transgenic plants, or cultivating new plant varieties.

9. The application according to claim 8, characterized in that: The plant in question is either soybean or tobacco.

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