Soybean transcription enhancer element eefb and uses thereof

Abstract

The application provides a soybean transcription enhancer element EEFB and an application thereof, and belongs to the technical field of plant genetic engineering. The transcription enhancer element EEFB overcomes the limitation that an efficient enhancer element in existing plant genetic engineering depends on a virus source, provides an endogenous element with higher safety, simultaneously realizes accurate positioning of a key transcription activation region of soybean, and obtains a transcription enhancement level that is significantly better than that of an existing commonly used endogenous enhancer.

Description

Technical Field

[0001] This application belongs to the field of plant genetic engineering technology, and more specifically, relates to the soybean transcription enhancer element EEFB and its application. Background Technology

[0002] In plant genetic engineering, promoters and transcriptional enhancers are key factors determining the expression level and pattern of exogenous genes. The discovery and utilization of efficient transcriptional enhancers are of great significance for improving the expression level of target genes, reducing expression heterogeneity, and achieving precise gene regulation in transgenic plants.

[0003] Currently, constitutive promoters widely used in plant genetic engineering are mainly derived from the 35S promoter of cauliflower leaf virus (CaMV), the maize UBI promoter, and the Arabidopsis UBQ10 promoter. Among these, the CaMV 35S promoter is the most commonly used constitutive promoter in dicotyledonous plants. Its core region (positions -46 to +8, i.e., mini35S) only has basic transcription initiation function and its ability to drive gene expression alone is very weak. To improve expression levels, transcriptional enhancer elements are usually added to the mini35S promoter. Although the CaMV 35S promoter itself contains a transcriptional enhancer region, it is derived from a virus, and its safety assessment in transgenic plants is controversial. The maize UBI promoter and the Arabidopsis UBQ10 promoter have relatively long sequences, and the transcriptional enhancer elements within them are unclear.

[0004] In existing technologies, non-viral endogenous transcriptional enhancer elements derived from plant genomes are scarce and have limited enhancement folds, making it difficult to reach or surpass the transcriptional enhancement levels of viral-derived transcriptional enhancers. Ning Zhang et al. identified soybean... eEF1A Gene( GmScreamM8The promoter of a Soybean Elongation Factor 1A (eEF1A) and its 5'UTR leader intron were analyzed, and the results showed that the 222bp intronic sequence contributed to high expression (A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters, PLOS ONE, pp. 1-20, 20161102). Its enhancer elements are derived from introns, and the fold increase in gene expression level is limited to no more than 7-fold, resulting in a relatively low enhancement level. Furthermore, existing transcriptional enhancers are usually used only as a whole of their natural promoter fragments, lacking independently modularly operable minimal transcriptional enhancer elements, which limits their flexible combination and application in expression vectors and makes it difficult to meet the modular assembly requirements of different expression levels. Summary of the Invention

[0005] The purpose of this application is to provide a soybean transcription enhancer element EEFB and its application, which aims to overcome the limitation of existing plant genetic engineering's efficient enhancer elements relying on viral sources, provide a safer endogenous element, and achieve precise localization of key transcriptional activation regions in soybeans, thereby obtaining a transcription enhancement level that is significantly better than that of commonly used endogenous enhancers.

[0006] A first aspect of the embodiments of this application provides a soybean transcription enhancer element EEFB having a nucleotide sequence as shown in SEQ ID NO:5.

[0007] A second aspect of this application provides a transcriptional activation element, including the transcriptional enhancer element EEFB, wherein the transcriptional activation element is a transcriptional activation element composed of a nucleotide sequence as shown in SEQ ID NO:3.

[0008] A third aspect of this application provides a promoter comprising the transcription enhancer element EEFB or the transcription activation element, the promoter having a nucleotide sequence as shown in SEQ ID NO:1.

[0009] A fourth aspect of this application provides a gene expression cassette including the aforementioned transcription enhancer element EEFB.

[0010] In one embodiment, the gene expression cassette further includes a mini35S minimal promoter operatively linked to the transcription enhancer element EEFB.

[0011] In one embodiment, the gene expression cassette further includes a target gene and a plant endogenous promoter operatively linked to the transcription enhancer element EEFB, wherein the transcription enhancer element EEFB is positioned upstream of the 5' end of the plant endogenous promoter or downstream of the 3' end of the target gene, and the plant is soybean or tobacco.

[0012] In one embodiment, the target gene includes at least one of a reporter gene, a screening marker gene, and a gene associated with the target trait.

[0013] A fifth aspect of the embodiments of this application provides an expression vector, including the transcription enhancer element EEFB, the transcription activation element, the promoter, or the gene expression cassette.

[0014] A sixth aspect of the embodiments of this application provides a transformant comprising the transcription enhancer element EEFB, the transcription activation element, the promoter, the gene expression cassette, or the expression vector.

[0015] A seventh aspect of this application provides a plant having a foreign nucleic acid molecule incorporated into the genome or the transformant; when having a foreign nucleic acid molecule incorporated into the genome, the nucleic acid molecule includes the transcription enhancer element EEFB or the transcription activation element or the promoter or the gene expression cassette or the expression vector. The plant in question is either soybean or tobacco.

[0016] An eighth aspect of the embodiments of this application provides the application of the transcriptional enhancer element EEFB, the transcriptional activation element, the promoter, the gene expression cassette, the expression vector, the transformant, or the plant in the following C1-C4: C1. Enhances gene transcription and expression; C2. Plant genetic improvement; C3, modular transcriptional regulation; C4. Plant breeding; The plant in question is either soybean or tobacco.

[0017] A ninth aspect of this application provides a method for enhancing the expression of exogenous genes, comprising the step of introducing the transcriptional enhancer element EEFB into plant cells; The plant in question is either soybean or tobacco.

[0018] The beneficial effects of the soybean endogenous transcription enhancer element provided in this application are as follows: This application is the first to isolate the transcriptional enhancer element EEFB from the promoter of a soybean endogenous high-expression gene, providing a well-known and safe endogenous enhancer resource for plant genetic engineering, effectively overcoming the limitation of existing technologies where high-efficiency transcriptional enhancer elements mainly rely on viral sources. Validated using a dual-luciferase reporter system, the EEFB transcriptional enhancer element of this application can increase the expression level of genes driven by the mini35S minimal promoter by 23-fold, significantly better than the enhancement level of existing endogenous transcriptional enhancer elements, effectively solving the problems of weak promoter-driven ability and limited enhancement fold of existing endogenous transcriptional enhancers. Furthermore, the transcriptional enhancer element of this application originates from a key transcriptional activation region of the soybean endogenous promoter, providing important element resources and theoretical basis for the fine analysis of soybean promoter functional elements and the modular design of artificial promoters. Attached Figure Description

[0019] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0020] Figure 1 The sequencing confirmation results for the full-length pGmEF1a promoter and pGmTCTP promoter are shown in the figure. Figure 2 Figure showing the sequencing confirmation results of the sequence deletion variant of promoter pGmEF1a; Figure 3 A schematic diagram illustrating the identification and detection results of the transcriptional enhancer element EEFB in tobacco leaves; In the figure: A is a schematic diagram of the dual-luciferase reporter system used to test enhancer activity; B is a graph showing the transient expression detection results in the first round of truncated tobacco leaves; C is a graph showing the transient expression detection results in the second round of truncated tobacco leaves. Figure 4 Figure showing the sequencing confirmation results of the sequence deletion variant of the promoter pGmTCTP; Figure 5 A schematic diagram illustrating the identification and detection results of transcriptional enhancer elements ETFD and ETFE in tobacco leaves; In the figure: A is a schematic diagram of the dual-luciferase reporter system used to test enhancer activity; B is a graph showing the transient expression detection results in the first round of truncated tobacco leaves; C is a graph showing the transient expression detection results in the second round of truncated tobacco leaves. Figure 6 A schematic diagram showing the identification results of transcriptional enhancer elements EEFB, ETFD, and ETFE in soybean hairy roots; In the figure: A shows the results of the activity test of the pGmEF1a promoter and the fragment deletion version of the enhancer in soybean hairy roots; B shows the results of the activity test of the pGmTCTP promoter and the fragment deletion version of the enhancer in soybean hairy roots. Detailed Implementation

[0021] The present application will now be described in detail with reference to preferred embodiments and comparative examples. The preferred embodiments described below according to the present application can be modified in various ways, and therefore the scope of the present application should not be construed as limited to the preferred embodiments described in detail below. Preferred embodiments are provided to help those skilled in the art to more readily understand the present application.

[0022] To make the technical solution of the present invention clearer, the terminology will be explained below.

[0023] Transcription enhancer element: refers to a specific DNA sequence that can significantly increase the transcriptional level of downstream genes by binding to transcription factors and their linked promoters.

[0024] Strict conditions: These refer to the highly stringent conditions used in nucleic acid hybridization experiments. They typically include specific temperature and salt concentration conditions (such as 6×SSC, 0.5% SDS, 65℃ hybridization). Under these conditions, only highly complementary sequences can bind stably, and these sequences are used to screen for functionally equivalent variant sequences.

[0025] Complementary sequence: refers to the reverse sequence between two nucleotide single strands according to the base pairing rule (AT, GC). In this application, it is used to define nucleic acid molecules capable of hybridizing with a target sequence under stringent conditions.

[0026] Combinatorial elements: Fusion polynucleotides consisting of at least two transcriptional enhancer elements linked together by head-to-tail tandem or other means, which can achieve superposition or synergistic enhancement of activity.

[0027] Transcriptional activation elements: These are long DNA fragments containing transcriptional enhancer elements and their natural upstream and downstream sequences, which can independently drive or synergistically enhance high-level transcription of genes.

[0028] Promoter: A DNA sequence located upstream of the 5' end of a gene that can be recognized and bound by RNA polymerase, thereby initiating the transcription of downstream genes.

[0029] Operative linkages refer to the functional interrelation of two or more nucleic acid elements, enabling one element to regulate the function of another (such as an enhancer enhancing the transcriptional activity of a promoter), thereby achieving the desired regulatory effect.

[0030] Gene expression cassettes are linear DNA molecules that contain complete expression regulatory elements such as promoters, transcriptional enhancers, target genes, and terminators. Once assembled, they can be directly used for plant transformation to achieve efficient expression of exogenous genes.

[0031] Reporter genes are genes whose encoded products have easily detectable activity. They do not confer new traits to plants themselves but are used to verify the activity intensity of promoters or enhancers. In this application, reporter genes include, but are not limited to, luciferase gene LUC, green fluorescent protein GFP, β-glucuronidase GUS, red fluorescent protein RFP, and betaine biosynthesis gene RUBY.

[0032] Selection marker genes: These are genes that confer specific resistance to transformed cells, used to distinguish successfully transformed cells from untransformed cells during the transformation process. In this application, selection marker genes include, but are not limited to, antibiotic selection genes and herbicide resistance genes.

[0033] Target trait-related genes: These are genes that impart specific desirable traits to plants. In this application, target trait-related genes include, but are not limited to, genes related to agronomic traits (yield, plant type), genes related to stress resistance (drought and disease resistance), and genes related to quality improvement (oil content, protein content).

[0034] Expression vector: refers to DNA capable of autonomously replicating and expressing exogenous genes in host cells. In this application, expression vectors include, but are not limited to, plant expression vectors such as the pCAMBIA series vectors, pBI121 series vectors, pGreen series vectors, and pPZP series vectors. Expression vectors can be used for transformation by plant transformation methods such as Agrobacterium-mediated transformation, gene gun transformation, PEG-mediated transformation, or electroporation transformation.

[0035] Transformed organisms: These are cells and their derived populations that have successfully received exogenous genetic material through transformation techniques (such as heat shock, electroporation, and Agrobacterium-mediated transformation) in genetic engineering operations, and that stably maintain or express specific traits within the transformed organism.

[0036] Plants: In this application, plants do not specifically refer to any particular plant species, and these plants are not required to have the same or highly similar genetic background.

[0037] Modular transcriptional regulation refers to treating transcriptional regulatory elements with different functions as standardized components, which can be flexibly assembled, replaced, or combined as needed to achieve precise artificial regulation of gene expression intensity and patterns.

[0038] Dicotyledons: A group of plants whose seeds have two cotyledons, characterized by reticulate leaf venation and taproot systems. Soybeans, tobacco, tomatoes, and cotton are all dicotyledons. In this application, the transcriptional enhancer element exhibits good functional transferability among species in the same family or closely related species.

[0039] Soybeans Glycine max Soybean is one of the world's most important oilseed and economic crops, and its genome contains abundant resources of endogenous transcriptional regulatory elements. Promoters of highly expressed genes in soybean (such as GmEF1a encoding elongation factor 1-alpha and GmTCTP encoding proteins that are mainly involved in cell proliferation regulation and stress response in plants and are highly conserved with translational regulation of tumor proteins in animals) often contain enhancer elements capable of driving high-level transcription. These elements have potential applications in plant genetic engineering. However, the systematic identification and functional analysis of key transcriptional enhancer elements in soybean endogenous promoters remain insufficient.

[0040] Sequence deletion analysis is a classic method for identifying key enhancer elements in promoters. By progressively truncating the promoter sequence and comparing the differences in the expression activities of reporter genes driven by different deletion variants, the key regions in the promoter that contribute the most to transcriptional activity can be located. The dual-luciferase reporter system is a commonly used method for quantitative analysis of promoter and enhancer activity. Using firefly luciferase (LUC) as the experimental reporter gene and Renal luciferase (REN) as an internal control, and normalizing the difference in transformation efficiency by the LUC / REN ratio, quantitative assessment of transcriptional enhancer element activity under in vivo conditions can be achieved in plant tissues such as soybean hairy roots and tobacco leaves.

[0041] Based on this, this application proposes a soybean endogenous transcription enhancer element, a gene expression cassette containing the above-mentioned transcription enhancer element and an expression vector, as well as a method for enhancing the expression of exogenous genes in plants using the above-mentioned transcription enhancer element, providing a modular, combinable and efficient expression tool for the genetic improvement of soybean and other dicotyledonous crops.

[0042] The technical solution of this application is as follows: Starting from the soybean endogenous highly expressed promoters pGmEF1a (GmEF1a gene promoter, 2288bp, nucleotide sequence as shown in SEQ ID NO:1) and pGmTCTP (GmTCTP gene promoter, 2229bp, nucleotide sequence as shown in SEQ ID NO:2), key transcriptional activating elements and minimal transcriptional enhancer elements were systematically identified through two rounds of truncation segmentation using sequence deletion analysis.

[0043] The first round of truncation and segmentation involved progressively truncating the 5' ends of the full-length pGmEF1a and pGmTCTP promoters to construct a series of deletion variants. For pGmEF1a, in addition to testing the full-length pGmEF1a-Full promoter, we also tested pGmEF1a-T1, pGmEF1a-T2, and pGmEF1a-T3 deletion variants with varying degrees of N-terminal deletion; pGmEF1a-S1, pGmEF1a-S2, and pGmEF1a-S3 deletion variants with segment selection and truncation; and pGmEF1a-Fni, pGmEF1a-T1ni, and pGmEF1a-T2ni deletion variants with introns removed from the 5'UTR. For pGmTCTP, in addition to testing the full-length pGmTCTP promoter pGmTCTP-Full, we also tested pGmTCTP-T1, pGmTCTP-T2, and pGmTCTP-T3 variants with varying degrees of N-terminal deletion; and pGmTCTP-S1, pGmTCTP-S2, pGmTCTP-S3, and pGmTCTP-S4 variants with selected and truncated segments. The activity of each variant was detected in soybean hairy roots and tobacco leaves using a dual-luciferase reporter system, identifying the key transcriptional activation regions pGmEF1a-S3 (811 bp, nucleotide sequence as shown in SEQ ID NO:3, corresponding to positions 912 to 1722 of the full-length pGmEF1a promoter) and pGmTCTP-T3 (691 bp, nucleotide sequence as shown in SEQ ID NO:4, corresponding to positions 1539 to 2229 of the full-length pGmTCTP promoter).

[0044] Second-round segmentation: pGmEF1a-S3 and pGmTCTP-T3 were further segmented, and each subfraction was placed upstream of the mini35S minimal promoter. Minimal transcription enhancer elements were identified using a dual-luciferase reporter system: EEFB (243 bp, nucleotide sequence as shown in SEQ ID NO:5, located at positions 212 to 454 of pGmEF1a-S3, corresponding to positions 1124 to 1366 of the full-length pGmEF1a promoter), ETFD (142 bp, nucleotide sequence as shown in SEQ ID NO:6, located at positions 458 to 599 of pGmTCTP-T3, corresponding to positions 1995 to 2136 of pGmTCTP), and ETFE (107 bp, nucleotide sequence as shown in SEQ ID NO:5). As shown in NO:7, it is located at positions 584 to 690 of pGmTCTP-T3 (corresponding to positions 2121 to 2227 of pGmTCTP) and the ETFD+ETFE merging region (233bp, nucleotide sequence as shown in SEQ ID NO:8).

[0045] The identified transcription enhancer elements were combined with mini35S or plant endogenous promoters to construct gene expression cassettes and expression vectors, enabling efficient expression of exogenous genes in plant cells.

[0046] The present invention will now be described in detail with reference to preferred embodiments. The preferred embodiments of the invention described below can be modified in various ways, and therefore the scope of the invention should not be construed as limited to the preferred embodiments described in detail below. Preferred embodiments are provided to help those skilled in the art to more readily understand the invention.

[0047] Experimental methods in the following examples, unless otherwise specified, are generally performed under standard conditions or as recommended by the manufacturer. All materials and reagents used are commercially available unless otherwise specified.

[0048] Example 1: Promoter Cloning Based on the annotation information of the GmEF1a and GmTCTP genes in the soybean genome database, primers were designed to amplify the pGmEF1a and pGmTCTP promoter regions, respectively. The PCR amplification products were cloned into the pDual-LUC / REN vector and sequenced for verification.

[0049] Primer sequences (lowercase letters indicate recombinant arms cloned into the pDual-LUC / REN vector): Forward primer pGmEF1a-F: 5'-tatagggcgaattgggtaccCAGGCGAGAAGAAATCAGTTGC-3', as shown in SEQ ID NO:9; Reverse primer pGmEF1a-R: 5'-gaggaagggtcttggctagcCTTCCTTAAATCTGCAAAAATCC-3', as shown in SEQ ID NO:10; Forward primer pGmTCTP-F: 5'-tatagggcgaattgggtaccCTTCATGCAGAAAAATATACTCCG-3', as shown in SEQ ID NO:11; The reverse primer pGmTCTP-R: 5'-gaggaagggtcttggctagcGTTTACAGAGTTTTTCTCGGAT-3', as shown in SEQ ID NO:12.

[0050] PCR reaction system (20 μL): 2× PCR Mix (containing enzyme, buffer, dNTP, Mg²⁺) +Add 10 μL of forward primer (0.5 μM), 0.5 μL of reverse primer (0.5 μM), 2.0 μL of template DNA (100 ng genomic DNA), and ddH2O (sterile deionized water) to a total volume of 20 μL.

[0051] PCR amplification procedure: Stage 1: Pre-denaturation: 95℃, 2 min; Phase Two: Touchdown Loop (15 loops); Denaturation: 95℃, 10 s; Annealing: 65℃→58℃ (decreasing by 0.5℃ per cycle), 15 s; Extension: 72℃, 2 min (adjusted in increments of 1 kb / min); Phase 3: Regular loop (20 loops); Denaturation: 95℃, 10 s; Annealing: 58℃ (fixed), 15 s; Extension: 72℃, 2 min (adjusted in increments of 1 kb / min); Phase 4: Final extension: 72℃, 3 min.

[0052] Sequencing validation results as follows Figure 1 As shown. Sequence comparison revealed that the full length of the promoter is consistent with the reference sequence.

[0053] Example 2: First-round truncation and segmentation of the pGmEF1a promoter and identification of pGmEF1a-S3 The full-length pGmEF1a promoter was truncated at the 5' end stepwise, and deletion variants pGmEF1a-T1, pGmEF1a-T2, and pGmEF1a-T3 with different degrees of N-terminal deletion were constructed by PCR amplification. The pGmEF1a-S1, pGmEF1a-S2, and pGmEF1a-S3 deletion variants were selected and the introns in the 5' UTR (located at positions 1508 to 2277 of pGmEF1a) were removed to form pGmEF1a-Fni, pGmEF1a-T1ni, and pGmEF1a-T2ni deletion variants.

[0054] Primer sequences (lowercase letters indicate recombinant arms cloned into the pDual-LUC / REN vector): Forward primer pGmEF1a-T1-F: 5'-tatagggcgaattgggtaccCACTTTTTGAATCAATCAGTGTACT-3', as shown in SEQ ID NO: 13; Reverse primer pGmEF1a-T1-R: 5'-gaggaagggtcttggctagcCTTCCTTAAATCTGCAAAAATCC-3', as shown in SEQ ID NO: 14; Forward primer pGmEF1a-T2-F: 5'-tatagggcgaattgggtaccCACGCACTCAACTGCACTGTACG-3', as shown in SEQ ID NO: 15; Reverse primer pGmEF1a-T2-R: 5'-gaggaagggtcttggctagcCTTCCTTAAATCTGCAAAAATCC-3', as shown in SEQ ID NO: 16; Forward primer pGmEF1a-T3-F: 5'-tatagggcgaattgggtaccCTGAATCTGTCGTCGTTGTCTTC-3', as shown in SEQ ID NO:17; Reverse primer pGmEF1a-T3-R: 5'-gaggaagggtcttggctagcCTTCCTTAAATCTGCAAAAATCC-3', as shown in SEQ ID NO: 18; Forward primer pGmEF1a-S1-F: 5'-tatagggcgaattgggtaccCAGGCGAGAAGAAATCAGTTGC-3', as shown in SEQ ID NO:19; Reverse primer pGmEF1a-S1-R: 5'-gaggaagggtcttggctagcGTCTCACAGCACACGTAAAAGG-3', as shown in SEQ ID NO:20; Forward primer pGmEF1a-S2-F: 5'-tatagggcgaattgggtaccCACTTTTTGAATCAATCAGTGTACT-3', as shown in SEQ ID NO: 21; Reverse primer pGmEF1a-S2-R: 5'-gaggaagggtcttggctagcGCGGCTTCTATTATGCGGCTGTGAA-3', as shown in SEQ ID NO: 22; Forward primer pGmEF1a-S3-F: 5'-tatagggcgaattgggtaccATCTATTATTTGTGAGAGCCAAC-3', as shown in SEQ ID NO:23; Reverse primer pGmEF1a-S3-R: 5'-gaggaagggtcttggctagcCACTTTTGAAAACACCATAGCACC-3', as shown in SEQ ID NO: 24; Forward primer pGmEF1a-Fni-F: 5'-tatagggcgaattgggtaccCAGGCGAGAAGAAATCAGTTGC-3', as shown in SEQ ID NO:25; Reverse primer pGmEF1a-Fni-R: 5'-gaggaagggtcttggctagcCTTCCTTAAATCTAGAAGAAGCTGCGCTAAAAC-3', as shown in SEQ IDNO: 26; Forward primer pGmEF1a-T1ni-F: 5'-tatagggcgaattgggtaccCACTTTTTGAATCAATCAGTGTACT-3', as shown in SEQ ID NO: 27; Reverse primer pGmEF1a-T1ni-R: 5'-gaggaagggtcttggctagcCTTCCTTAAATCTAGAAGAAGCTGCGCTAAAAC-3', as shown in SEQ IDNO: 28; Forward primer pGmEF1a-T2ni-F: 5'-tatagggcgaattgggtaccCACGCACTCAACTGCACTGTACG-3', as shown in SEQ ID NO: 29; Reverse primer pGmEF1a-T2ni-R: 5'-gaggaagggtcttggctagcCTTCCTTAAAATCTAGAAGAAGCTGCGCTAAAAC-3', as shown in SEQ ID NO:30.

[0055] PCR reaction system (20 μL): 2× PCR Mix (containing enzyme, buffer, dNTP, Mg²⁺) +10 μL of forward primer (0.5 μM), 0.5 μL of reverse primer (0.5 μM), 2.0 μL of template DNA (100 ng genomic DNA), and ddH2O (sterile deionized water) to bring the total volume to 20 μL.

[0056] PCR amplification procedure: Stage 1: Pre-denaturation: 95℃, 2 min; Phase Two: Touchdown Loop (15 loops); Denaturation: 95℃, 10 s; Annealing: 65℃→58℃ (decreasing by 0.5℃ per cycle), 15 s; Extension: 72℃, 2 min (adjusted in increments of 1 kb / min); Phase 3: Regular loop (20 loops); Denaturation: 95℃, 10 s; Annealing: 58℃ (fixed), 15 s; Extension: 72℃, 2 min (adjusted in increments of 1 kb / min); Phase 4: Final extension: 72℃, 3 min.

[0057] Sequencing validation results as follows Figure 2 As shown. Sequence comparison revealed that the sequence deletions were identical to the reference sequence.

[0058] All deletion variants shared the mini35S minimal promoter as the minimum transcriptional baseline. Each deletion variant was cloned into a dual-luciferase reporter vector to drive the expression of the firefly luciferase (LUC) reporter gene, with Renilla luciferase (REN) used as an internal control. For activity assays, each deletion variant was placed upstream of the mini35S minimal promoter (containing the CaMV 35S promoter region from -46 to +8), and transcriptional enhancement activity was detected in soybean hairy roots and tobacco leaves, respectively. Specifically, this was achieved using Agrobacterium rhizogenes (… Agrobacterium rhizogenes Agrobacterium-mediated transformation of soybean hairy roots was performed by obliquely cutting the hypocotyl of 7-day-old soybean seedlings, removing the roots, applying Agrobacterium-mediated transformation to the cut surfaces, and inserting vermiculite. The seedlings were cultured at 25°C for 2 weeks under a controlled photoperiod of 16 h light and 8 h dark. Positive hairy roots were selected using the visual screening marker RUBY, and tissue proteins were extracted. Reaction substrates were added, and chemiluminescence was detected using a microplate reader. Dual-luciferase activity was then assessed. The Agrobacterium-mediated transformation of *Nicotiana benthamiana* (a type of soybean) was then performed. Nicotiana benthamianaLeaves were used. Agrobacterium suspension was adjusted to an OD of 0.8, and the leaves were subjected to two vacuum permeation incubation cycles at 5 kPa for 60 s. After 48 h of culture, leaf proteins were extracted, reaction substrates were added, and chemiluminescence was detected using a microplate reader for dual-luciferase activity assay. The mini35S minimal promoter driving LUC alone served as a negative control. Transcriptional enhancement activity was quantified using the LUC / REN ratio.

[0059] The results showed that pGmEF1a-S3 retained significant transcriptional activation activity; corresponding truncated deletions excluding the S3 region showed significantly decreased transcriptional activation. This indicates that pGmEF1a-S3 is a key region contributing to transcriptional activity in the pGmEF1a promoter. pGmEF1a-S3 is located in the middle region of the pGmEF1a promoter, as shown in the detection results. Figure 3 As shown. Figure 3 Figure A shows a schematic diagram of a dual-luciferase reporter system used to test enhancer activity, containing the reporter gene Fluc and the internal reference gene Rluc. The internal reference gene Rluc is constitutively stably expressed by a 35S promoter, while the reporter gene Fluc is expressed by an artificial promoter composed of a potential enhancer derived from the pGmEF1a promoter and a TATA-Box. The enhancing effect of the potential enhancer on gene expression can be determined by detecting and calculating the ratio of the signal intensity of the reporter gene Fluc to that of the internal reference gene Rluc. Figure 3 B in the figure represents the transient expression detection results in the first round of truncated tobacco leaf segments. The activity test of the pGmEF1a promoter and the enhancer of the fragment deletion version showed that the S3 fragment and the full-length promoter F containing the S3 region, the truncated versions T1 and T2, and the intron-removed Fni, T1ni, and T2ni all had good enhancement effects, suggesting that the core enhancer exists in the S3 region.

[0060] Example 3: Second-round truncation and segmentation of pGmEF1a-S3 and identification of the transcriptional enhancer element EEFB pGmEF1a-S3 was further segmented to construct subfractions AB, BC, CD, A, B, C, D, and E. Each subfraction was placed upstream of the mini35S minimal promoter. With the mini35S minimal promoter driving LUC alone as a control (activity set at 1-fold), transcriptional enhancement activity was detected in soybean hairy roots and tobacco leaves, respectively, following the transformation and detection methods in Example 2.

[0061] The results showed that fragment B, namely EEFB, had the strongest transcriptional enhancement activity: EEFB increased gene expression levels by approximately 23-fold based on the mini35S minimal promoter and determined approximately 50% of the expression level of the pGmEF1a promoter. A schematic diagram of the detection results is shown below. Figure 3 C in Figure 6As shown.

[0062] However, in existing technologies, the promoter activity of an intron-derived enhancer (A Leader Intron of a Soybean Elongation Factor 1A (eEF1A) Gene Interacts with Proximal Promoter Elements to Regulate Gene Expression in Synthetic Promoters, PLOS ONE, pp. 1-20, 20161102) only decreases to about 60% of its original level after removal, meaning the upper limit of this enhancer's contribution to expression is approximately 40%. In contrast, the enhancement effect contributed by the transcriptional enhancer element EEFB from the soybean endogenous promoter in this application is significantly higher.

[0063] Example 4: First-round truncation and segmentation of the pGmTCTP promoter and identification of pGmTCTP-T3 The full-length pGmTCTP promoter was truncated at the 5' end stepwise, and deletion variants pGmTCTP-T1, pGmTCTP-T2, and pGmTCTP-T3 with different degrees of N-terminal deletion were constructed by PCR amplification; and deletion variants pGmTCTP-S1, pGmTCTP-S2, pGmTCTP-S3, and pGmTCTP-S4 were selected and truncated.

[0064] Primer sequences (lowercase letters indicate recombinant arms cloned into the pDual-LUC / REN vector): Forward primer pGmTCTP-T1-F: 5'-tatagggcgaattgggtaccCTCCGATAGAGCTGTAATTGCCG-3', as shown in SEQ ID NO:31; Reverse primer pGmTCTP-T1-R: 5'-gaggaagggtcttggctagcGTTTACAGAGTTTTTCTCGGAT-3', as shown in SEQ ID NO:32; Forward primer pGmTCTP-T2-F: 5'-tatagggcgaattgggtaccCACTTTAACAACCTCCATGATGGA-3', as shown in SEQ ID NO: 33; Reverse primer pGmTCTP-T2-R: 5'-gaggaagggtcttggctagcGTTTACAGAGTTTTTCTCGGAT-3', as shown in SEQ ID NO:34; Forward primer pGmTCTP-T3-F: 5'-tatagggcgaattgggtaccTTCGTTTGCCATTCTGCG-3', as shown in SEQ ID NO:35; Reverse primer pGmTCTP-T3-R: 5'-gaggaagggtcttggctagcGTTTACAGAGTTTTTCTCGGAT-3', as shown in SEQ ID NO:36; Forward primer pGmTCTP-S1-F: 5'-tatagggcgaattgggtaccCTTCATGCAGAAAAATATACTCCG-3', as shown in SEQ ID NO:37; Reverse primer pGmTCTP-S1-R: 5'-gaggaagggtcttggctagcTACTAACTTGACTTCTCTTCTTGGA-3', as shown in SEQ ID NO: 38; Forward primer pGmTCTP-S2-F: 5'-tatagggcgaattgggtaccCTCCGATAGAGCTGTAATTGCCG-3', as shown in SEQ ID NO:39; Reverse primer pGmTCTP-S2-R: 5'-gaggaagggtcttggctagcCAGGCGAGGACTTTTGCGCTCG-3', as shown in SEQ ID NO: 40; Forward primer pGmTCTP-S3-F: 5'-tatagggcgaattgggtaccCACTTTAACAACCTCCATGATGGA-3', as shown in SEQ ID NO: 41; Reverse primer pGmTCTP-S3-R: 5'-gaggaagggtcttggctagcCCCGTTTTTACACTCTTTACACCA-3', as shown in SEQ ID NO:42; Forward primer pGmTCTP-S4-F: 5'-tatagggcgaattgggtaccCTTCATGCAGAAAAATATACTCCG-3', as shown in SEQ ID NO:43; Reverse primer pGmTCTP-S4-R: 5'-gaggaagggtcttggctagcCCCGTTTTTACACTCTTTACACCA-3', as shown in SEQ ID NO:44.

[0065] PCR reaction system (20 μL): 2× PCR Mix (containing enzyme, buffer, dNTP, Mg²⁺) + 10 μL of forward primer (0.5 μM), 0.5 μL of reverse primer (0.5 μM), 2.0 μL of template DNA (100 ng genomic DNA), and ddH2O (sterile deionized water) to bring the total volume to 20 μL.

[0066] PCR amplification procedure: Stage 1: Pre-denaturation: 95℃, 2 min; Phase Two: Touchdown Loop (15 loops); Denaturation: 95℃, 10 s; Annealing: 65℃→58℃ (decreasing by 0.5℃ per cycle), 15 s; Extension: 72℃, 2 min (adjusted in increments of 1 kb / min); Phase 3: Regular loop (20 loops); Denaturation: 95℃, 10 s; Annealing: 58℃ (fixed), 15 s; Extension: 72℃, 2 min (adjusted in increments of 1 kb / min); Phase 4: Final extension: 72℃, 3 min.

[0067] Sequencing validation results as follows Figure 4 As shown. Sequence comparison revealed that the sequence deletions were identical to the reference sequence.

[0068] All deletion variants shared the mini35S minimal promoter as the minimum transcriptional baseline. Each deletion variant was cloned into a dual-luciferase reporter vector to drive the expression of the firefly luciferase (LUC) reporter gene, with Renilla luciferase (REN) used as an internal control. For activity assays, each deletion variant was placed upstream of the mini35S minimal promoter (containing the CaMV 35S promoter region from -46 to +8), and transcriptional enhancement activity was detected in soybean hairy roots and tobacco leaves, respectively. Specifically, this was achieved using Agrobacterium rhizogenes (… Agrobacterium rhizogenes Agrobacterium-mediated transformation of soybean hairy roots was performed by obliquely cutting the hypocotyl of 7-day-old soybean seedlings, removing the roots, applying Agrobacterium-mediated transformation to the cut surfaces, and inserting vermiculite. The seedlings were cultured at 25°C for 2 weeks under a controlled photoperiod of 16 h light and 8 h dark. Positive hairy roots were selected using the visual screening marker RUBY, and tissue proteins were extracted. Reaction substrates were added, and chemiluminescence was detected using a microplate reader. Dual-luciferase activity was then assessed. The Agrobacterium-mediated transformation of *Nicotiana benthamiana* (a type of soybean) was then performed. Nicotiana benthamiana Leaves were used. Agrobacterium suspension was adjusted to an OD of 0.8, and the leaves were subjected to two vacuum permeation incubation cycles at 5 kPa for 60 s. After 48 h of culture, leaf proteins were extracted, reaction substrates were added, and chemiluminescence was detected using a microplate reader for dual-luciferase activity assay. The mini35S minimal promoter driving LUC alone served as a negative control. Transcriptional enhancement activity was quantified using the LUC / REN ratio.

[0069] The results showed that pGmTCTP-T3 retained significant transcriptional activation activity; corresponding truncated deletions without the T3 region showed significantly decreased transcriptional activation. This indicates that pGmTCTP-T3 is a key region contributing to transcriptional activity in the pGmTCTP promoter. pGmTCTP-T3 is located in the 3' end region of the pGmTCTP promoter, as shown in the detection results below. Figure 5 As shown. Figure 5 Figure A shows a schematic diagram of a dual-luciferase reporter system used to test enhancer activity, containing the reporter gene Fluc and the internal reference gene Rluc. The internal reference gene Rluc is constitutively stably expressed by a 35S promoter, while the reporter gene Fluc is expressed by an artificial promoter composed of a potential enhancer derived from the pGmEF1a promoter and a TATA-Box. The enhancing effect of the potential enhancer on gene expression can be determined by detecting and calculating the ratio of the signal intensity of the reporter gene Fluc to that of the internal reference gene Rluc. Figure 5 B in the figure represents the transient expression detection results in the first round of truncated tobacco leaf segments. The activity test of the pGmTCTP promoter and the enhancer of the fragment deletion version showed that the T3 fragment and the full-length promoter F containing the T3 region, as well as the truncated versions T1 and T2, all had good enhancement effects, suggesting that the core enhancer exists in the T3 region.

[0070] Example 5: pGmTCTP-T3 second-round truncation and segmentation and identification of transcriptional enhancer elements ETFD and ETFE pGmTCTP-T3 was further segmented to construct subfractions AB, BC, DE, A, B, C, D, E, F, and G. Each subfraction was placed upstream of the mini35S minimal promoter. Using the mini35S minimal promoter driving LUC alone as a control (activity set at 1-fold), transcriptional enhancement activity was detected in soybean hairy roots and tobacco leaves according to the transformation and detection methods in Example 4.

[0071] The results showed that fragment D (ETFD) and fragment E (ETFE) possessed the strongest transcriptional enhancement activity: ETFD increased gene expression levels by approximately 12-fold based on the mini35S minimal promoter and determined approximately 70% of the expression level of the pGmTCTP promoter; ETFE increased gene expression levels by approximately 8-fold based on the mini35S minimal promoter, with slightly weaker transcriptional enhancement activity than ETFD. A 16bp overlap region existed between ETFD and ETFE, located at the 3' end of ETFD and the 5' end of ETFE. The merged ETFD+ETFE region encompassed the complete sequences of both transcriptional enhancer elements. A schematic diagram of the detection results is shown below. Figure 5 C in Figure 6 As shown. Figure 6 In the figure, A represents the results of the enhancer activity test of the pGmEF1a promoter and fragment deletion version in soybean hairy roots, which showed that the S3 fragment and fragment B (S3-B) had a strong transcriptional enhancement effect. Figure 6 B in the figure represents the results of the activity test of the pGmTCTP promoter and the fragment deletion version of the enhancer in soybean hairy roots. It was found that the T3 fragment and the split structures of the T3 fragment DE (A2-DE), fragment D (A2-D), and fragment E (A2-E) are the core enhancer elements.

[0072] The above experimental results demonstrate that, through two rounds of sequence deletion analysis, this application successfully identified three transcriptional enhancer elements (EEFB, ETFD, and ETFE) with significant transcriptional enhancement activity from the soybean endogenously highly expressed promoter. Among them, EEFB showed the most significant enhancement effect (approximately 23-fold), while ETFD and ETFE also achieved enhancement effects of approximately 12-fold and 8-fold, respectively. All transcriptional enhancer elements were validated in two systems using a dual-luciferase reporter system: soybean hairy roots and tobacco leaves, with consistent results.

[0073] The above are merely specific embodiments of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A soybean transcriptional enhancer element EEFB, characterized in that, It has a nucleotide sequence as shown in SEQ ID NO:

5.

2. A transcriptional activation element, characterized in that, Includes the transcription enhancer element EEFB as described in claim 1, wherein the transcription activation element is a transcription activation element composed of a nucleotide sequence as shown in SEQ ID NO:

3.

3. A promoter, characterized in that, The promoter includes the transcription enhancer element EEFB of claim 1 or the transcription activation element of claim 2, wherein the promoter has a nucleotide sequence as shown in SEQ ID NO:

1.

4. A gene expression cassette, characterized in that, Includes the transcription enhancer element EEFB as described in claim 1.

5. A gene expression cassette according to claim 4, characterized in that, It also includes the mini35S minimal promoter, which is operatively linked to the transcription enhancer element EEFB.

6. A gene expression cassette according to claim 4, characterized in that, It also includes a target gene and a plant endogenous promoter operatively linked to the transcription enhancer element EEFB, wherein the transcription enhancer element EEFB is located upstream of the 5' end of the plant endogenous promoter or downstream of the 3' end of the target gene, and the plant is soybean or tobacco.

7. A gene expression cassette according to claim 4, characterized in that, The target gene includes at least one of a reporter gene, a screening marker gene, and a gene related to the target trait.

8. An expression carrier, characterized in that, It includes the transcription enhancer element EEFB as described in claim 1, the transcription activator element as described in claim 2, the promoter as described in claim 3, or the gene expression cassette as described in any one of claims 4 to 7.

9. A transformant, characterized in that, It includes the transcription enhancer element EEFB as described in claim 1, or the transcription activation element as described in claim 2, or the promoter as described in claim 3, or the gene expression cassette as described in any one of claims 4 to 7, or the expression vector as described in claim 8.

10. A plant, characterized in that, The nucleic acid molecule is a foreign nucleic acid molecule incorporated into the genome or the transformant of claim 9; when it is a foreign nucleic acid molecule incorporated into the genome, the nucleic acid molecule includes the transcription enhancer element EEFB of claim 1 or the transcription activation element of claim 2 or the promoter of claim 3 or the gene expression cassette of any one of claims 4 to 7 or the expression vector of claim 8. The plant in question is either soybean or tobacco.

11. The use of the transcription enhancer element EEFB according to claim 1, or the transcription activator element according to claim 2, or the promoter according to claim 3, or the gene expression cassette according to any one of claims 4-7, or the expression vector according to claim 8, or the transformant according to claim 9, or the plant according to claim 10 in the following C1-C4: C1. Enhances gene transcription and expression; C2. Plant genetic improvement; C3, modular transcriptional regulation; C4. Plant breeding; The plant in question is either soybean or tobacco.

12. A method for enhancing the expression of exogenous genes, characterized in that, Includes the step of introducing the transcription enhancer element EEFB of claim 1 into plant cells; The plant in question is either soybean or tobacco.

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