Carp u6 promoter that is functionally conserved across species and drives long fragment sequence expression

Abstract

This invention discloses a carp U6 promoter with cross-species functional conservation that drives the expression of long sequence fragments and its applications, belonging to the fields of genetic engineering and molecular biology. Existing U6 promoters exhibit strong species specificity, low activity in non-mammals such as fish, and are mainly suitable for short RNA transcription, making it difficult to drive the expression of long target genes, thus limiting complex gene editing and the creation of transgenic aquatic animals. This invention provides a carp-derived U6 promoter, the nucleotide sequence of which is shown in SEQ ID NO:1. This promoter exhibits cross-species functional conservation and can drive the transcription of long target genes. Expression cassettes and recombinant vectors constructed based on this promoter can achieve efficient expression of long target genes in fish and mammalian cells. The promoter and related biomaterials provided by this invention can provide an efficient, cross-species applicable key technical tool for fish gene function research and editing breeding.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering and molecular biology, and relates to the carp U6 promoter, which is functionally conserved across species and drives the expression of long sequence fragments, and its applications. Background Technology

[0002] As a core cis-regulatory element of gene transcription, the promoter's activity, species adaptability, and transcription fragment adaptability directly determine the expression efficiency of exogenous genes in recipient cells, making it a key element in the construction of gene engineering vectors. The U6 promoter, a typical representative of type III RNA polymerase promoters, is widely used in molecular operations such as gene editing (e.g., CRISPR / Cas9 system), miRNA, siRNA, and other small RNA expression regulation due to its precise transcription initiation site and stable transcription efficiency. It is extensively applied in gene function research and variety improvement in plants, animals, and microorganisms. These small RNAs are characterized by a length of less than 100 nucleotides. However, existing U6 promoters have the following key problems: (1) Species-specific limitations: Currently used U6 promoters (such as mouse U6 and human U6) show significant differences in activity across different species. For example, their activation efficiency is relatively low in non-mammals such as fish, making it difficult to meet the needs of cross-species gene editing applications. The application of U6 promoters has obvious species limitations: although their core functional elements are somewhat conserved in closely related species, the overall sequence conservation across distant species is significantly reduced. Furthermore, the transcriptional activity of U6 promoters from model organisms such as mammals and rodents is greatly reduced in aquatic animal cells due to the lack of species-specific auxiliary regulatory elements, which cannot meet the needs of aquatic animal genetic engineering research. At the same time, research on U6 promoters in aquatic animals is mostly concentrated on a few species such as zebrafish, grouper, and grass carp (patent number: CN202510574889.2).

[0003] (2) Insufficient transcriptional capacity for long fragments: The existing U6 promoter is mainly suitable for driving short RNA fragments (such as 20-30 base sgRNAs), and has low transcriptional efficiency for long RNA fragments (such as long non-coding RNAs and polycistronic sgRNAs), which limits the realization of complex gene editing strategies (such as the expression of multiple gRNAs).

[0004] In summary, the limitations of short-fragment transcription and species-specific adaptability of existing fish U6 promoters have become one of the major technical bottlenecks restricting research on fish functional genes and the creation of gene-edited / transgenic fish. Summary of the Invention

[0005] One object of the present invention is to solve at least the aforementioned problems and / or defects, and to provide at least the advantages described below. The present invention provides a U6 promoter derived from carp and its applications, which exhibits functional conservation across multiple species, can drive the expression of long sequence fragments, and is particularly suitable for scenarios such as gene editing and gene expression regulation in aquatic animals.

[0006] This study addresses the technical challenges of existing U6 promoters, such as their high species specificity, low transcriptional activity in non-mammalian cells like fish, and the lack of endogenous U6 promoters from aquatic animals that can stably drive long RNA transcription across species simultaneously.

[0007] This study addresses the problem that the known U6 promoter is generally only applicable to transcription of short RNA fragments (such as sgRNA), and cannot effectively drive the transcription of long target genes with a length of not less than 300 bp, thus limiting the research and application of polycistronic sgRNA expression.

[0008] Therefore, the present invention provides the following technical solution: The carp U6 promoter, which is functionally conserved across species and drives the expression of long sequence fragments, has its nucleotide sequence shown in SEQ ID NO:1.

[0009] Preferably, in the carp U6 promoter, which is functionally conserved across species and drives the expression of long sequence fragments, the carp U6 promoter can drive the transcription of a target gene with a length of not less than 300 bp.

[0010] A recombinant expression vector containing the aforementioned cyprinid U6 promoter, or containing the aforementioned expression cassette.

[0011] An animal cell for non-diagnostic purposes, comprising the recombinant expression vector or the expression cassette.

[0012] A method for expressing a long target gene in fish or mammalian cells, comprising introducing the recombinant expression vector into an animal cell, wherein the recombinant expression vector contains the carp U6 promoter and a long target gene operatively linked downstream of the carp U6 promoter.

[0013] Preferably, in the method for expressing a long target gene in fish cells or mammalian cells, the long target gene is one or more tandem sgRNA coding sequences or a long gene sequence.

[0014] Preferably, in the method for expressing a long target gene fragment in fish cells or mammalian cells, the fish cells are carp cells or grass carp cells, and the mammalian cells are human cells.

[0015] The use of the carp U6 promoter, the expression cassette, the recombinant expression vector, or the non-diagnostic animal cells in the preparation of a kit for genetic manipulation, wherein the genetic manipulation includes the expression of a long target gene fragment in fish or mammalian cells.

[0016] The application of the described carp U6 promoter, the described expression cassette, the described recombinant expression vector, or the described non-diagnostic animal cells in the preparation of transgenic fish or gene-edited fish.

[0017] The present invention has at least the following beneficial effects: This invention yielded a carp U6 promoter, cloned and constructed a fusion expression vector driving either green fluorescent protein (GFP) or red fluorescent protein (RFP) using the U6 promoter, and expressed GFP or RFP in human cells and grass carp cell lines. GFP is 238 amino acids long, with a coding region of 714 bases, longer than small RNA. RFP is 236 amino acid residues long, with a coding region of 708 bases. The U6 promoter screened in this invention not only exhibits cross-species functional conservation but also demonstrates the ability to transcribe long gene sequences. This research fills a gap in the application of endogenous U6 promoters in carp, providing a functionally conserved tool suitable for expressing long gene sequences. This invention is expected to significantly improve gene editing efficiency in fish and expand its application scope, providing key technical support for new germplasm creation, fish gene function research, and molecular breeding, and has broad market application prospects.

[0018] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0019] Figure 1 This is an analysis diagram of the carp U6 promoter sequence SEQ ID NO:1 in one embodiment of the present invention.

[0020] Figure 2 This is a comparison diagram of the U6 promoter sequences of carp and grass carp in one embodiment of the present invention.

[0021] Figure 3 This is a schematic diagram of the pcDNA3.1-carpU6-mcherry plasmid containing the carp U6 promoter in one embodiment of the present invention.

[0022] Figure 4 This is a diagram showing the expression of green fluorescent protein in human HEK293T cells using the pcDNA3.1-carpU6-EGFP plasmid in one embodiment of the present invention.

[0023] Figure 5 This image shows the expression of red fluorescent protein in human HEK293T cells using the pcDNA3.1-carpU6-mcherry plasmid in one embodiment of the present invention.

[0024] Figure 6 This image shows the expression of green fluorescent protein in grass carp L8824 cells using the pcDNA3.1-carpU6-EGFP plasmid in one embodiment of the present invention.

[0025] Figure 7 This image shows the expression of red fluorescent protein in grass carp L8824 cells using the pcDNA3.1-carpU6-mcherry plasmid in one embodiment of the present invention. Detailed Implementation

[0026] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0027] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0028] It should be noted that, unless otherwise specified, the experimental methods in the following implementation schemes are all conventional methods, and the reagents and materials mentioned are all commercially available unless otherwise specified.

[0029] carp( Cyprinus carpio As one of the most widely distributed and economically valuable freshwater aquaculture fish in the world, the discovery, identification, and application of endogenous U6 promoters in carp are still in a state of flux. No endogenous U6 promoters that can be efficiently transcribed in carp cells have been cloned and verified, and there are no reports on U6 promoters in aquatic animals that combine multi-species conservation and long-fragment transcriptional driving capabilities. This has limited research on gene editing and functional verification in carp due to the lack of suitable promoter elements.

[0030] Currently, fish gene editing is transitioning from single-gene editing to multi-gene editing, thus requiring the simultaneous expression of multiple gRNAs. The main current method for simultaneous expression of multiple gRNAs is to add a U6 promoter to each gRNA. However, while this strategy can transcribe multiple gRNAs simultaneously, it significantly increases the total plasmid length. Furthermore, transfection efficiency typically decreases with increasing plasmid size. Therefore, if a U6 promoter capable of transcribing long sequences can be discovered and combined with a polycistronic sequence structure, it is hoped that a single U6 promoter can simultaneously transcribe multiple gRNAs, thereby achieving simultaneous multi-gene editing. Therefore, discovering and identifying a carp-endogenous U6 promoter that possesses both multi-species functional conservation and long-sequence transcriptional driving capabilities is of great significance for conducting in vivo expression studies of small RNAs such as gRNAs in carp and other species, as well as expanding the application of the U6 promoter in long-sequence transcription.

[0031] The purpose of this invention is to address the above-mentioned problems by providing a carp endogenous U6 gene promoter and its application.

[0032] The first aspect of the present invention provides a carp U6 gene promoter, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0033] A second aspect of the present invention provides an expression cassette containing the aforementioned promoter. Preferably, it includes the described carp U6 promoter and a target gene driven by the carp U6 promoter.

[0034] A third aspect of the present invention provides a recombinant vector containing the aforementioned promoter or expression cassette. Preferably, the recombinant vector is a recombinant expression vector.

[0035] A fourth aspect of the present invention provides an Escherichia coli containing the above-described promoter, expression cassette, or recombinant vector.

[0036] The application of the described carp U6 promoter, the described gene expression cassette, or the described expression plasmid in genetic manipulation of cells, embryos, or living organisms. Preferably, the genetic manipulation is gene expression. Preferably, the cell is an animal cell. Preferably, the animal cell is a fish-derived cell or a mammal-derived cell. More preferably, the mammal-derived cell is a human cell.

[0037] The fifth aspect of the present invention provides the use of the above-described carp U6 gene promoter in any of the following: (1) Application in the construction of transgenic fish; (2) Application in conducting fish functional research; (3) Application in the construction of gene-edited fish.

[0038] A sixth aspect of the present invention provides a method for expressing a target nucleic acid molecule in fish, the method comprising: introducing a nucleic acid construct into fish, the nucleic acid construct containing the aforementioned promoter and a target nucleic acid molecule operatively linked to the promoter.

[0039] According to one embodiment of the present invention, a carp U6 promoter that is functionally conserved across species and drives the expression of long sequence fragments has the nucleotide sequence shown in SEQ ID NO:1. This promoter overcomes the limitation of existing U6 promoters that are only effective in a limited number of species, and can maintain stable transcription initiation function in both human and fish cells. It provides a universal promoter tool for cross-species gene manipulation in aquatic animals and mammals, significantly expanding the applicability of gene editing and functional genomics research.

[0040] According to one embodiment of the present invention, the carp U6 promoter is a cross-species functionally conserved promoter that drives the expression of long sequence fragments. This promoter can stably transcribe fluorescent protein coding sequences longer than 700 bp, providing a key transcriptional driving element for the functional study of polycistronic sgRNA tandem expression, long non-coding RNAs, and protein-coding genes, greatly enriching the toolbox of genetic manipulation.

[0041] According to one embodiment of the present invention, an expression cassette containing the carp U6 promoter is provided. The expression cassette includes the carp U6 promoter and a target gene operably linked downstream of the carp U6 promoter. The target gene is at least 300 bp in length and is preferably a gene encoding a fluorescent protein. This expression cassette can be directly used as a modular component for constructing various recombinant vectors, and can be used to verify whether the carp U6 promoter identified in this patent is applicable to other mammals or fish, significantly improving the efficiency of U6 promoter function verification and providing a convenient and reliable tool for aquatic animal functional gene research and mammalian cell gene manipulation.

[0042] According to one embodiment of the present invention, a method for expressing a long-fragment target gene in fish cells or mammalian cells includes introducing the recombinant expression vector into animal cells, wherein the recombinant expression vector contains the carp U6 promoter and a long-fragment target gene operably linked downstream of the carp U6 promoter. This method is simple and standardized, eliminating the need for promoter replacement and condition optimization for different species or target genes of different lengths. It significantly improves the success rate and reproducibility of cross-species gene expression experiments, providing a reliable operational procedure for gene editing, RNA interference, and functional genomics research.

[0043] According to one embodiment of the present invention, in the method for expressing a long target gene in fish cells or mammalian cells, the long target gene may encode one or more tandem sgRNAs. This method can simultaneously generate multiple functional small RNAs through a single promoter, thereby greatly simplifying the vector construction process, lowering the experimental threshold for multi-site editing, and providing an efficient and convenient technical means for fish genome function research and precision molecular breeding.

[0044] According to one embodiment of the present invention, the method for expressing a long target gene fragment in fish cells or mammalian cells, wherein the fish cells are carp cells or grass carp cells, and the mammalian cells are human cells, enables the promoter to be directly applied to gene function studies of grass carp and carp, two important economically farmed fish species, as well as high-throughput screening studies in human cell models, possessing clear species-specificity and industrial applicability.

[0045] According to one embodiment of the present invention, the carp U6 promoter, the expression cassette containing the carp U6 promoter, the recombinant expression vector, or the non-diagnostic animal cells are used in the preparation of transgenic fish or gene-edited fish. This application can significantly improve the success rate of multi-site gene editing in fish and the stability of transgenic expression, accelerating the creation of new germplasm with faster growth, stronger disease resistance, and superior quality. It has significant application value and industrial prospects for promoting the sustainable development of aquaculture.

[0046] To enable those skilled in the art to better understand the technical solution of the present invention, the following embodiments are provided for further explanation: Example 1: Obtaining the Carp U6 Promoter 1.1 Screening of the Carp U6 promoter By analyzing the carp genome sequence, we searched for U6 spliceosomal RNA gene clusters and calculated the number of U6 nucleomicroRNAs (U6 snRNAs) in each gene cluster. The candidate gene clusters were required to meet the following requirements: (1) There must be a TTTTT transcription termination signal downstream of the nucleomicroRNA to conform to the transcription characteristics of the U6 promoter; (2) The number of nucleomicroRNA genes must be at least 10.

[0047] A total of 81 U6 microRNA sites were identified in the carp genome. Among them, 34 sites contained only one microRNA, 3 sites contained two microRNAs, 2 sites contained four microRNAs, and 1 site contained 33 microRNAs (located at NC_056586.1:1375674-1417692). The gene cluster with the most microRNAs was selected, and the genomic sequence between its microRNA sites was used as the carp U6 promoter sequence, named the carp U6 promoter.

[0048] 1.2 Prediction of key cis-components in the U6 promoter Using the online tool JASPAR, with human POU2F1 as a reference, the OCT binding site of carp U6 was predicted. Simultaneously, based on the conserved sequence of the proximal sequence element (PSE) "[AG]TCCCACATCG" and the conserved sequence of the TATA-like box element, respectively, they were aligned to the carp U6 sequence to predict the PSE and TATA-like box element sites of carp U6.

[0049] 1.3 Sequence Feature Analysis of the Carp U6 Promoter The U6 promoter base sequence of the carp is shown in SEQ ID NO:1. Figure 1 The sequence, 860 bp in length, contains TATA-frame elements, PSE elements, and OCT elements that bind to PolIII RNA polymerase. The positions of each element are labeled. Figure 1 middle.

[0050] The nucleotide sequence of the U6 promoter of carp is SEQ ID NO:1: CGTGATTGCGATATTTGTGCTCCTCTACTCTATCAGCCCCTCGTCTGGTGAATAGACACAGGTCTCAGCGAGGGAGCGTTTTTTGTTGTAAAATCACTCACCGCCTCCTTTGTCTGAGAGATGCTCAGCTGCGCGCCCCCGCTCCATCACACCATGTGATGAACTCATGAGAACTACATGGCCATCGTCAGACTCGGAGCTTGAGAGCAGGGAGAGGAGACCTGGGTCACCTGCGTATTTGACAACTCGAAAATGAGCCAGAGAACTTCAACGCTCATTTGCAGAATGTGAGAAGAGGATCCATGATAGGACACCGTCCTGGCCCCTTCGCTGCCCACCACTTCTTTACCGCCACCTCTTCTTTCGATAAAAGGACAAGGCCACGCCCCCTTCAGCGCCAGAACTTCAGCGCTCATCTGTAGAAAGTAAGAAGAGAATCAATGACAAGACATCAGCACGTGGAGATCCAAAGTTCGTCCGGTAAGACCCCCTTCATCCTGACCCTTCGCTGCCCACCACTTCTTCACTGCCACCTCTTTTTTGAAAAAAAAAAAGGACAAGGCCAAGCCCCCTTCAGCGCAGTACTCCGGTACCATCGGCAAGTGCGTCCACCCGCTTCCTCATGTTCAGCAGATGCTCCTGTACATCGCTGTGAAATCTGGCACATGTGCAACACGAGGCGCTACCGTCCCCTGTCCCCCGAACTCGGTGAACGATTGCAACAGTCAGGGTGCGTCCGGCCTCAGAACCGAATGGTACGGCATGCGAAGTGGTGAGATGGCTCACTGGGCCCTACCACCTCTGAGAGGGCTCGCGCGGCCCCGAGGGGTACACATACCTCGAGCGGGACGCATCTCTTG Example 2: Sequence comparison between the carp U6 promoter and the grass carp U6 promoter Using the BLAST tool in the NCBI database, the carp U6 promoter sequence obtained in this invention was compared with the grass carp U6 promoter sequence (see Chinese patent application number: CN202510574889.2) using BLAST. The comparison results showed that the two had low homology. The specific comparison results are as follows: Figure 2 As shown.

[0051] Furthermore, the obtained carp U6 promoter sequence was compared with the grass carp genome using BLAST analysis in the NCBI database. The results showed that the maximum alignment score of the sequence with the grass carp genome was only 46, and no statistically significant homologous match was detected.

[0052] In summary, the U6 promoter sequence obtained in this invention does not exhibit sequence conservation and is a novel, previously unreported promoter.

[0053] Example 3: Construction of a recombinant vector plasmid carrying the carp U6 promoter and a long RNA fragment for the EGFP reporter gene Because this region contains highly similar repetitive sequences, it was impossible to design specific primers for amplification. Therefore, the fragment was synthesized de novo based on the obtained carp U6 promoter sequence. To facilitate subsequent ligation into the pcDNA3.1 vector via enzyme digestion, MluI and NheI restriction sites were synthesized at both ends of the fragment.

[0054] Furthermore, the universal vector pcDNA3.1 was linearized by double digestion with MluI and NheI to remove the original CMV promoter. Subsequently, the carp U6 promoter sequence containing MluI and NheI restriction sites was ligated into the linearized pcDNA3.1 plasmid using a ligase. The resulting recombinant plasmid served as the template plasmid for subsequent PCR amplification. This plasmid contained the coding sequence for green fluorescent protein (EGFP). EGFP consists of 238 amino acid residues, and its coding region has a nucleic acid sequence length of 714 bases (bp).

[0055] The recombinant product was transformed into *E. coli* DH10b competent cells, and single colonies were picked and inoculated into liquid LB medium for culture. The recombinant plasmid was then extracted. Whole-genome resequencing was performed on the extracted recombinant plasmid, and the sequence reads were assembled to obtain the full-length nucleotide sequence of the recombinant plasmid. This full-length sequence was compared with the obtained *Carp U6* promoter sequence. The alignment results showed that the obtained *Carp U6* promoter was accurately ligated into the pcDNA3.1 vector without mutations or deletions. Therefore, a successfully constructed recombinant plasmid carrying the *Carp U6* promoter was obtained and named pcDNA3.1-*Carp U6*-EGFP plasmid.

[0056] In this recombinant expression vector, the carp U6 promoter was cloned into the original CMV promoter region of the expression vector pcDNA3.1, replacing the vector's native CMV promoter. Figure 3 As shown, in the constructed recombinant plasmid pcDNA3.1-carpU6-EGFP, the carpU6 promoter is inserted at the position of the vector's original CMV promoter, located before EGFP. Since this vector replaces the CMV promoter, if green fluorescence is observed after transfection into cells, it indicates that the carpU6 promoter can transcribe EGFP.

[0057] Example 4: Construction and functional verification of the mCherry reporter gene recombinant vector plasmid carrying the carp U6 promoter and long RNA fragment. To verify whether the carp U6 promoter can drive the expression of other long RNA fragments, the EGFP coding sequence in the plasmid (pcDNA3.1-carpU6-EGFP) carrying the carp U6 promoter was replaced with the mcherry red fluorescent protein coding sequence.

[0058] The mcherry red fluorescent protein consists of 236 amino acids, and its coding region has a nucleic acid sequence length of 708 bp, as shown in SEQ ID NO:2. This sequence references the sequence annotation information of mcherry in the pmRi-mCherry plasmid. The mcherry nucleic acid sequence was obtained through de novo chemical synthesis and replaced the EGFP sequence in the pcDNA3.1-carpU6-EGFP plasmid, thereby obtaining a recombinant plasmid carrying the carp U6 promoter and the encoding sequence of the mcherry fluorescent protein, named pcDNA3.1-carpU6-mcherry plasmid.

[0059] Since the CMV promoter has been replaced in this vector, if red fluorescence can be observed after transfecting the above recombinant plasmid into cells, it indicates that the carp U6 promoter has the ability to transcribe the mcherry sequence.

[0060] The base sequence of mcherry red fluorescent protein SEQ ID NO:2: atggtgagcaagggcgaagaggacaacatggccatcatcaaagagttcatgcgcttcaaggtgcacatggaaggcagcgtgaacggccacgagttcgagatcgaaggcgaaggcgagggtcgtccgtacgagggcacccagaccgccaagctgaaggtgaccaaaggcggtccgctg ccgttcgcctgggacatcctgtcgccacagttcatgtacggcagcaaggcctacgtgaagcacccagcggacatcccggactacctgaagctgagcttcccggaaggcttcaagtggggagcgcgtgatgaacttcgaggacggtggcgtggtgaccgtgacccaggacagcagcctgc aggacggcgagttcatctacaaggtgaagctgcgtggcaccaacttcccgagcgcggtccggtgatgcagaaaaagaccatgggctgggaagccagcagcgagcgcatgtacccggaagatggtgccctgaagggcgagatcaagcagcgcctgaaactgaaggatggcggtcacta cgacgccgaggtcaagaccacctacaaggccaagaagccggtccagctgccaggtgcctacaacgtgaacatcaagctggacatcaccagccacaacgaggactacaccatcgtggaacagtacgagcgtgccgaaggccgtcacagcaccggtggcatggacgagctgtacaagtga Example 5: Activity detection of carp U6 promoter in human HEK293T cells Based on the successful acquisition of the carp U6 promoter sequence and the completion of the recombinant vector plasmid construction, in order to verify whether it has the basic function of driving downstream gene expression and its cross-species functional conservation, an activity detection experiment was conducted in human HEK293T cells to detect the expression of green fluorescent protein and red fluorescent protein.

[0061] The HEK293T cell line in good growth condition was used at a density of 2 × 10⁶ cells per well. 5The cells were seeded at a density of 1 / 2 wells into 12-well plates and cultured in complete medium (90% DMEM + 10% fetal bovine serum + 1% triple antibody) at 37°C in a 5% CO2 incubator. When the cell lines reached 60%–80% confluency, the pcDNA3.1-carp U6-EGFP plasmid was transfected into the cell lines. The intensity of green fluorescence was observed under a microscope periodically to determine the activity of the carp U6 promoter. The results showed that 17 hours after transfection, a significant fluorescence signal was observed for the first time in HEK293T cells containing the carp U6 promoter plasmid, demonstrating that the obtained carp U6 promoter has transcriptional activity in human cells. Figure 4 ).

[0062] Similarly, under the same culture and transfection conditions, the pcDNA3.1-carp U6-mcherry recombinant plasmid was transfected into the HEK293T cell line. The intensity of red fluorescence was observed under a microscope at regular intervals to determine the activity of the carp U6 promoter. The results showed that 23 hours after transfection, a significant red fluorescence signal was observed for the first time in HEK293T cells containing the carp U6 promoter plasmid, further demonstrating that the obtained carp U6 promoter has transcriptional activity in human cells. Figure 5 ).

[0063] Example 6: Activity detection of the carp U6 promoter in grass carp L8824 cells Based on the successful acquisition of the carp U6 promoter sequence and the completion of the recombinant vector plasmid construction, in order to verify whether it has the basic function of driving downstream gene expression and its cross-species functional conservation, and to evaluate the transcriptional activity of the above promoter in other fish cells, this invention further detected the activity of the promoter in the grass carp liver cell line L8824.

[0064] Grass carp liver cell lines were selected, using 2×10⁻⁶ cells per cell line. 5 The cells were seeded at a density of 3 wells into 12-well plates and cultured in complete medium (90% M199 + 10% fetal bovine serum + 1% trivalent antibiotics) at 28°C in a 5% CO2 incubator. Once the cells reached 60%–80% confluency, they were transfected with the carp U6 promoter plasmid. The results showed that 48 hours after transfection, a distinct green fluorescence signal was observed for the first time in L8824 cells containing the carp U6 promoter plasmid, demonstrating that the obtained carp U6 promoter has transcriptional activity in grass carp cells. Figure 6 ).

[0065] Similarly, under the same culture and transfection conditions, pcDNA3.1-carp U6-mcherry was transfected into the cell line. The intensity of red fluorescence was observed under a microscope at regular intervals to determine the activity of the carp U6 promoter. The results showed that 48 hours after transfection, a significant red fluorescence signal was observed for the first time in L8824 cells containing the carp U6 promoter plasmid, further demonstrating that the obtained carp U6 promoter has transcriptional activity in grass carp cells. Figure 7 ).

[0066] In summary, the carp U6 promoter disclosed in this invention is a novel, previously unreported promoter that exhibits transcriptional activity in both human cells and grass carp cell lines. This demonstrates that while this promoter lacks sequence conservation, it possesses functional conservation, enabling it to perform transcriptional activity in different species.

[0067] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A carp U6 promoter that is functionally conserved across species and drives the expression of long sequence fragments, characterized in that, Its nucleotide sequence is shown in SEQ ID NO:

1.

2. The carp U6 promoter as described in claim 1, which is functionally conserved across species and drives the expression of long sequence segments, characterized in that... The carp U6 promoter can drive the transcription of target genes with a length of not less than 300 bp.

3. An expression box, characterized in that, The expression cassette includes the carp U6 promoter as described in claim 1, the carp U6 promoter, and a target gene operatively linked downstream of the carp U6 promoter, wherein the target gene is not less than 300 bp in length and is a protein-coding gene or a long non-coding RNA gene.

4. A recombinant expression vector, characterized in that, The recombinant expression vector contains the carp U6 promoter as described in claim 1 or the expression cassette as described in claim 3.

5. An animal cell for non-diagnostic purposes, characterized in that, Includes the recombinant expression vector as described in claim 4 or the expression cassette as described in claim 3.

6. A method for expressing long-fragment target genes in fish cells or mammalian cells, characterized in that, This includes introducing the recombinant expression vector of claim 4 into animal cells, wherein the recombinant expression vector contains the carp U6 promoter of claim 1 and a long target gene operatively linked downstream of the carp U6 promoter.

7. The method for expressing a long target gene in fish cells or mammalian cells as described in claim 6, characterized in that, The sequence of the long target gene fragment is one or more tandem sgRNA sequences, or a long gene sequence.

8. The method for expressing a long target gene in fish cells or mammalian cells as described in claim 6, characterized in that, The fish cells are carp cells or grass carp cells, and the mammalian cells are human-derived cells.

9. The use of the carp U6 promoter as described in claim 1, the expression cassette as described in claim 3, the recombinant expression vector as described in claim 4, or the non-diagnostic animal cells as described in claim 5 in the preparation of a kit for genetic manipulation, characterized in that, The genetic manipulation involves expressing long segments of the target gene in fish or mammalian cells.

10. The use of the cross-species functionally conserved carp U6 promoter that drives the expression of long fragment sequences as described in claim 1, or the expression cassette as described in claim 3, or the recombinant expression vector as described in claim 4, or the non-diagnostic animal cells as described in claim 5, in the preparation of transgenic fish or gene-edited fish.

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