Promoter element derived from asmt gene and use of promoter element

Abstract

Provided are a promoter element and the use thereof. The promoter element comprises the nucleotide sequence represented by SEQ ID NO: 17 or one or more sequences having 85% sequence identity thereto. The promoter element can be used as a cis-acting element for expressing a target gene, enhancing the expression of a target protein gene; the promoter element has broad application prospects and high application potential.

Description

A promoter element derived from ASMT gene and its application Technical Field

[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a promoter element and application thereof. Background Art

[0002] Melatonin is an indole signaling substance secreted by the pineal gland in the brain. It is stored in the pineal gland and the sympathetic nerves excite the pineal cells to release melatonin. Melatonin synthesis occurs in two steps: acetylation and methylation. 5-hydroxytryptamine (5-HT) is acetylated by the key enzyme arylalkylamine N-acetyl transferase (AANAT) to form N-acetylserotonin (NAS). NAS is then methylated to melatonin by N-acetylserotonin O-methyltransferase (ASMT). AANAT and ASMT are key genes regulating melatonin levels in vivo. Animal studies have shown that AANAT gene expression levels are closely associated with the onset of human depression. Mice carrying specific SNPs in the AANAT gene exhibit increased immobility time and depressive-like behaviors in a forced swim test. AANAT expression and enzyme activity further increase at night, while melatonin synthesis levels remain stable. It is speculated that the rate of melatonin synthesis at night is limited by ASMT gene expression. The reduced plasma melatonin concentration in patients with depression is associated with immune cell infiltration of the blood-brain barrier, leading to brain inflammatory response and increased levels of soluble pro-inflammatory proteins, which may be related to the genetic polymorphism of ASMT.

[0003] At present, the sequence of goat ASMT gene in NCBI and ensmbl databases is incomplete, especially the core promoter region is still unknown, which in turn affects the development of research on melatonin secretion in goats.

[0004] Summary of the Invention

[0005] In order to solve at least one of the above problems, the present disclosure provides a promoter element and application thereof.

[0006] According to a first aspect of the present disclosure, a promoter element is provided, comprising the nucleotide sequence shown in SEQ ID NO: 17, or at least one or more sequences having 85% sequence identity thereto.

[0007] In some embodiments, the promoter element is of mammalian origin.

[0008] In some specific embodiments, the mammal is an artiodactyl.

[0009] In some specific embodiments, the mammal is a goat.

[0010] In some embodiments, the promoter element is derived from -778 to -658 bp upstream of the transcription start site of the goat ASMT gene.

[0011] According to a second aspect of the present disclosure, a transcription expression cassette is provided, comprising the promoter element of the first aspect and a target protein coding sequence, wherein the promoter element is operably linked to the target protein coding sequence.

[0012] In some embodiments, the target protein comprises arylalkylamine-N-acetyltransferase, N-acetylserotonin methyltransferase, or firefly luciferase.

[0013] In some embodiments, the target protein is N-acetylserotonin methyltransferase.

[0014] In some embodiments, the target protein is goat N-acetylserotonin methyltransferase.

[0015] According to a third aspect of the present disclosure, an expression vector is provided, wherein the expression vector comprises the promoter element according to the first aspect or the transcription expression cassette according to the second aspect.

[0016] In some embodiments, the expression vector is selected from one or more of a prokaryotic expression vector, a eukaryotic expression vector, or a viral expression vector.

[0017] According to a fourth aspect of the present disclosure, a host cell is provided, which carries one or more of the promoter element described in the first aspect, the transcription expression cassette described in the second aspect, or the expression vector described in the third aspect.

[0018] In some embodiments, the host cell is selected from a prokaryotic cell and a eukaryotic cell.

[0019] In some embodiments, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces.

[0020] In some embodiments, the eukaryotic cells include yeast cells, mammalian cells, and rodent cells.

[0021] In some specific embodiments, the mammalian cells include human, monkey, mouse, rat, hamster, goat, sheep, cow, pig, dog, and cat.

[0022] In some embodiments, the eukaryotic cells include human cells, mouse cells, rat cells, hamster cells, goat cells, sheep cells, or any other host cells.

[0023] In some embodiments, the mammalian cells include A549, WEHI, 3T3, 10T1 / 2, BHK, MDCK, COS1, COS7, BSC1, BSC40, BMT10, VERO, WI38, HeLa, 293 cells (which can express functional adenovirus E1), Saos, C2C12, L cells, HT1080, and HepG2.

[0024] In some embodiments, the mammalian cells include fibroblasts, hepatocytes, and myoblasts.

[0025] In some embodiments, the fibroblasts are selected from embryonic fibroblasts.

[0026] In some embodiments, the fibroblasts are selected from goat embryonic fibroblasts.

[0027] According to a fifth aspect of the present disclosure, there is provided use of the promoter element described in the first aspect or the transcription expression cassette described in the second aspect in regulating gene expression.

[0028] The present invention successfully uses a dual-luciferase reporter gene system to identify the core region of the goat ASMT promoter and screens a promoter element. The promoter element disclosed in the present invention can serve as a cis-acting element for expressing the target gene, thereby increasing the expression effect of the target protein gene and having a broad application prospect and application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] FIG1 shows the first part of the Sanger sequencing peak diagram of the 3′ RACE fragment of the ASMT gene.

[0030] FIG. 2 shows the second part of the Sanger sequencing peak diagram of the 3′ RACE fragment of the ASMT gene.

[0031] FIG3 shows the first part of the Sanger sequencing peak diagram of the 5′ RACE fragment of the ASMT gene.

[0032] FIG4 shows the second part of the Sanger sequencing peak diagram of the 5′ RACE fragment of the ASMT gene.

[0033] Figure 5 shows the full-length sequence of the ASMT gene, where the sequence order is from 5' to 3'; the yellow part is the 5' and 3' UTR; and the green part is the protein coding region.

[0034] FIG6 shows the electrophoresis diagram of each fragment of the ASMT promoter.

[0035] FIG7 shows the results of luciferase activity detection. DETAILED DESCRIPTION

[0036] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0037] The method for identifying a gene core promoter sequence using a dual-luciferase reporter gene comprises the following steps:

[0038] Step 1: Design RACE primers based on the known sequence fragments of the gene in the database, extract the total RNA of the gene, and clone the full-length cDNA. The cloned fragment is connected to the vector plasmid and transformed into competent cells. After a period of culture, sequencing is performed. The vector sequence is removed from the sequenced sequence and spliced ​​to obtain the full-length genomic sequence of the gene.

[0039] Step 2: The base on the DNA chain corresponding to the first nucleotide of the mRNA chain is used as the transcription start site, and the upstream sequence of the gene transcription start site is searched in the database and multiple pairs of primers are designed, wherein the primers have specific enzyme cutting sites.

[0040] Step 3: Use the primers in step 2 to perform PCR amplification on the whole genomic DNA to obtain amplification products of different lengths.

[0041] Step 4: Load the various amplification products from Step 3 into plasmids and transfect them into competent cells for expression. Luciferase activity is then measured using a dual-luciferase reporter gene. Luciferase activity is used to determine the transcriptional activity of the promoter and to screen for the gene's core promoter sequence.

[0042] definition

[0043] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0044] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0045] As used herein, the expression "about" is understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If the use of the term is not understood by one of ordinary skill in the art based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0046] The term "promoter" or "promoter element" as used herein is defined herein as a DNA sequence that binds RNA polymerase and directs the polymerase to the correct downstream transcription start site of a polynucleotide to initiate transcription, wherein the polynucleotide encodes a biologically active polypeptide. RNA polymerase effectively catalyzes the assembly of messenger RNA complementary to the appropriate DNA strand of the coding region. The term "promoter" or "promoter element" should also be understood to include a 5' non-coding region (between the promoter and the translation start point) for translation after transcription into mRNA, cis-acting transcriptional regulatory elements such as enhancers, and / or other nucleotide sequences that can interact with transcription factors. A promoter or promoter element can be a wild-type promoter, a variant promoter, a hybrid promoter body, or a consensus promoter.

[0047] The term "core promoter" as used herein refers to the nucleic acid sequence contained in a promoter. A core promoter is typically the smallest portion of a promoter required to correctly initiate transcription. A core promoter typically includes a transcription start site and a binding site for RNA polymerase.

[0048] As used herein, the term "sequence identity" refers to the "percentage of sequence identity" or "percentage of identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within the comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide, a gap present in the target sequence is not counted. Similarly, since the target sequence nucleotides are counted and the nucleotides from the reference sequence are not counted, the gap present in the reference sequence is not counted. At least 85% sequence identity includes a continuous segment having at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity over the full length of the sequence.

[0049] As used herein, the term "expression vector" refers to the process of linking the isolated nucleic acid molecule to a vector, where the nucleic acid sequence can be directly or indirectly linked to regulatory elements on the vector, as long as these regulatory elements are capable of regulating the translation and expression of the nucleic acid molecule. These regulatory elements can be derived directly from the vector itself or exogenously, i.e., not from the vector itself. In other words, the nucleic acid molecule and the regulatory elements are operably linked. Commonly used expression vectors include prokaryotic expression vectors, eukaryotic expression vectors, viral expression vectors, and the like.

[0050] The term "operably linked" as used herein refers to linking an exogenous gene to a vector so that regulatory elements within the vector, such as transcriptional regulatory sequences and translational regulatory sequences, can play their intended function of regulating the transcription and translation of the exogenous gene.

[0051] The following examples and accompanying drawings are provided to facilitate understanding of the present invention. However, it should be understood that these examples and accompanying drawings are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0052] Example

[0053] Example 1. RACE cloning of goat ASMT gene

[0054] RACE primers were designed based on the known sequence fragment of goat ASMT gene in the Ensembl database (Gene No: ENSCHIG00000006740). The primer sequences are shown in Table 1.

[0055] Table 1. RACE primer sequences

[0056] Total RNA was extracted from goat pineal cells according to the instructions of the FastPure Cell / Tissue Total RNA Isolation Kit, and the concentration and purity of the extracted RNA were measured using a Nanodrop one ultramicro spectrophotometer.

[0057] The RNA obtained in the previous step was reverse transcribed and cloned by RACE using the HiScript-TS 5' / 3' RACE Kit and the two primer pairs described in Table 1, respectively. The procedures were carried out according to the kit instructions. The cloned product was purified using the FastPure Gel DNA Extraction Mini Kit and ligated into the pCE2-TA-Blunt-Zero vector (Novagen). The purified product was transformed into DH5α-competent Escherichia coli (TAKARA) and cultured overnight before being sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The sequencing results were spliced ​​using SnapGene software. The sequencing results of the ASMT gene 3'UTR sequence are shown in Figures 1 and 2, and the sequencing results of the ASMT gene 5'UTR sequence are shown in Figures 3 and 4.

[0058] As shown in Figure 5, the full-length sequence of the ASMT gene is as shown in SEQ ID NO: 11, wherein the 5'UTR region (marked in yellow at the upper end of Figure 5) is as shown in SEQ ID NO: 12, the protein coding region (marked in green in Figure 5) is as shown in SEQ ID NO: 13, and wherein the 3'UTR region (marked in yellow at the lower end of Figure 5) is as shown in SEQ ID NO: 14.

[0059] Example 2. Construction of dual luciferase reporter gene vector

[0060] The ASMT gene 3'UTR and 5'UTR spliced ​​together in Example 1 constituted the full-length ASMT gene cDNA. The first nucleotide corresponding to the base on the DNA chain was used as the transcription start site. The upstream 2000 bp sequence (LWLT01000043.1: 126,459-128,459) was searched in the Ensembl database and primers were designed, with the underlined region being the restriction enzyme cleavage site. The primers are shown in Table 2.

[0061] Table 2. Promoter search primer sequences

[0062] Three amplification products of different lengths were amplified using the primers shown in Table 2: ASMT-p0 (SEQ ID NO: 15), ASMT-p1 (SEQ ID NO: 16), and ASMT-p2 (SEQ ID NO: 17). The amplification products were subjected to electrophoresis to identify the product band lengths. The electrophoretogram is shown in Figure 6.

[0063] A portion of the skin tissue of a 4-week-old goat fetus was minced and digested with 0.25% trypsin to form a single-cell suspension. The cells were cultured in DMEM high-glucose medium containing 10% fetal bovine serum. Fresh medium was replaced every 3 days until the confluence of the cultured cells reached 60% to 70%. Goat embryonic fibroblasts were obtained and cryopreserved.

[0064] Frozen goat embryonic fibroblasts were thawed in a 37°C water bath. After centrifugation to remove the cell cryopreservative, the cells were resuspended and cultured in DMEM high-glucose medium containing 5% fetal bovine serum. The cells were cultured in 6-well plates and passaged when the cell confluence reached 80%. The cell passage ratio was 3:1, and at least 1×10 6 Genomic DNA was extracted from each cell using the FastPure Cell / Tissue DNA Isolation Mini Kit, and PCR amplification was performed using the primers in step 2. The amplification procedure was as follows: pre-denaturation at 98°C for 30 s, denaturation at 98°C for 10 s, annealing at 65°C for 5 s, and extension at 72°C for 5 s for a total of 35 cycles, and extension at 72°C for 60 s. The reaction system was as follows: 2×Phanta Flash Master Mix (Dye Plus) 25 μL, ddH2O 20 μL, 2 μL each of upstream and downstream primers, and 1 μL of DNA template.

[0065] pGL3-basic and the product amplified in the previous step were digested with KpnI and HindIII restriction enzymes (TAKARA), respectively. The digestion system was as follows: 1 μg of pGL3-basic and the product amplified in the previous step, 1 μL of KpnI, 1 μL of HindIII, 2 μL of 10x digestion buffer, and embryonic water to a total of 20 μL. The system was thoroughly mixed and incubated at 37°C for 1 hour.

[0066] The digestion products obtained in the previous step were purified by gel recovery and then ligated to pGL3-basic using T4 ligase (TAKARA). The ligation system was as follows: 1 μL of pGL3-basic vector digestion product, 16 μL of PCR fragment digestion product, 1 μL of T4 ligase, and 2 μL of T4 buffer. The T4 ligase reaction conditions were 16°C for 2 hours. The ligation products were transformed into DH5α competent Escherichia coli (TAKARA), shaken overnight, and sent to Shanghai Sangon Biotechnology Co., Ltd. for sequencing. The recombinant protein expression vectors with correct sequencing were named pGL3-basic-ASMT-p0, pGL3-basic-ASMT-p1, and pGL3-basic-ASMT-p2, respectively.

[0067] Example 3. Identification of the ASMT gene core promoter

[0068] pGL3-basic-ASMT-p0 and pRL-TK, pGL3-basic-ASMT-p1 and pRL-TK, and pGL3-basic-ASMT-p2 and pRL-TK plasmids were co-transfected into goat embryonic fibroblasts using Lipofectamine 3000. When the confluence of goat embryonic fibroblasts reached 70%, 3.75 μL of Lipofectamine 3000 was diluted in 250 μL of OPTI-MEM medium in tube A and mixed thoroughly. 5 μg of plasmid was diluted in 250 μL of OPTI-MEM medium in tube B to prepare a DNA premix. Then, 10 μL of P3000 reagent was added and mixed thoroughly. Tubes A and B were mixed and incubated at room temperature for 10 minutes. The mixture was then added to the cells in a 6-well plate and incubated at 37°C for 2 days before the cells were analyzed.

[0069] Example 4. Luciferase activity identification

[0070] Transfected cells were lysed and reacted with Firefly luciferase using the Dual Luciferase Reporter Assay Kit (Novagen). Detection was performed using a microplate reader. The fluorescence produced by firefly luciferase is represented by "Firefly," and the fluorescence produced by Renilla luciferase is represented by "Renilla."

[0071] Each constructed pGL3-basic-ASMT plasmid and pRL-TK plasmid were transfected into goat embryonic fibroblasts. The blank pGL3-basic vector served as a control (i.e., the control Firefly and control Renilla) and untransfected cells served as blank controls (i.e., background Firefly and background Renilla). Luciferase activity was assayed two days after transfection. The results are shown in Figure 7.

[0072] The experimental results show that the luciferase activities of pGL3-basic-ASMT-p0, pGL3-basic-ASMT-p1, and pGL3-basic-ASMT-p2 are 1.5376±1.21, 5.3413±1.43, and 15.32±2.12, respectively; the luciferase activity of the blank vector pGL3-basic is 0.19314±0.73; compared with the blank vector pGL3-basic, the luciferase activity of the experimental group pGL3-basic-ASMT-p2 was significantly higher than that of the control group (P<0.05). As the ASMT promoter fragment in the experimental group was reduced, the relative luciferase activity of the vector gradually increased. The luciferase activity of the identified core promoter region was higher than that of the two promoter fragments pGL3-basic-ASMT-p0 and pGL3-basic-ASMT-p1. The ASMT core promoter region sequence ranges from -338 bp to -778 bp, as shown in SEQ ID NO:17.

[0073] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A promoter element, characterized in that: The promoter element includes the nucleotide sequence shown in SEQ ID NO: 17, or at least one or more of the sequences having at least 85% sequence identity thereto.

2. The promoter element according to claim 1, characterized in that The promoter element is derived from mammals.

3. The promoter element according to claim 1, characterized in that The mammal is an artiodactyl, preferably a goat.

4. The promoter element according to claim 1, characterized in that The promoter element is derived from -778 to -658 bp upstream of the transcription start site of goat ASMT gene.

5. A transcription expression cassette, characterized in that: The transcription expression cassette comprises the promoter element of claim 1 and a target protein coding sequence, wherein the promoter element is operably linked to the target protein coding sequence.

6. An expression vector, characterized in that: The expression vector comprises the promoter element according to any one of claims 1 to 4 or the transcription expression cassette according to claim 5.

7. The expression vector according to claim 6, characterized in that The expression vector is selected from one or more of a prokaryotic expression vector, a eukaryotic expression vector or a viral expression vector.

8. A host cell, characterized in that The host cell carries one or more of the promoter element according to any one of claims 1 to 4 or the transcription expression cassette according to claim 5 or the expression vector according to any one of claims 6 or 7.

9. The host cell according to claim 8, characterized in that The host cell is selected from prokaryotic cells and eukaryotic cells; Preferably, the prokaryotic cells include bacterial cells, Escherichia coli, and Streptomyces; Preferably, the eukaryotic cells include yeast cells, mammalian cells, and insect cells; more preferably, the mammals are selected from mice, rats, hamsters, goats, sheep, cattle, pigs, dogs, and cats.

10. Use of the promoter element according to any one of claims 1 to 4 or the transcription expression cassette according to claim 5 in regulating gene expression.

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