CN117384906B - Promoter element derived from AANAT gene and application thereof - Google Patents
Promoter element derived from AANAT gene and application thereof Download PDFInfo
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- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
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- C12N2800/10—Plasmid DNA
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/80—Vector systems having a special element relevant for transcription from vertebrates
- C12N2830/85—Vector systems having a special element relevant for transcription from vertebrates mammalian
Abstract
The present disclosure provides a promoter element comprising at least one or more of the nucleotide sequence shown as SEQ ID NO. 19, or a sequence having 85% sequence identity thereto, and uses thereof. The promoter element disclosed by the disclosure can be used as a cis-acting element for expressing a target gene, so that the expression effect of the target protein gene is improved, and the promoter element has wider application prospect and application potential.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a promoter element and application thereof.
Background
In mammals, the AANAT gene encodes an arylalkylamine-N-acetyltransferase, an important cytoplasmic enzyme, and also a rate-limiting enzyme for melatonin synthesis. In-vivo synthesis of melatonin comprises multiple reaction steps, wherein serotonin is catalyzed by a synthesis rate-limiting enzyme AANAT to generate N-acetylserotonin, and then is catalyzed by another rate-limiting enzyme HIOMT to generate methylation, so that melatonin is finally generated. Another approach is that serotonin is first subjected to HIOMT catalysis to produce 5-methoxy tryptamine (methoxytyrptamine), and then AANAT catalysis to synthesize melatonin. While the pathways synthesized in different species may not be identical. In previous studies, it was found that AANAT gene in the pine cone of flatfish can affect thyroid hormone secretion, and has a certain relationship with development. The activity of AANAT is consistent with the secretion of MLT in rhythm, and AANAT gene participates in regulating the circadian rhythm and reproductive performance of mammals by regulating the synthesis and secretion amount of melatonin. The study shows that the G619A mutation of AANAT gene is related to human sleep phase delay syndrome.
At present, the AANAT gene of the Hainan black goat is incomplete in sequence in NCBI and ensmbl databases, and especially the core promoter region is unknown, so that the development of melatonin secretion research of the Hainan black goat is affected.
Disclosure of Invention
To solve at least one of the problems described above, the present disclosure provides a promoter element and applications thereof.
According to a first aspect of the present disclosure there is provided a promoter element comprising at least one or more of the nucleotide sequence shown as SEQ ID NO. 19, or a sequence having 85% sequence identity thereto.
In some embodiments, the promoter element is derived from a mammal.
In some specific embodiments, the mammal is a artiodactyla.
In some specific embodiments, the mammal is a black goat.
In some specific embodiments, the mammal is a Hainan black goat.
In some embodiments, the promoter element is derived from-540 bp to-958 bp upstream of the Hainan black goat AANAT gene transcription initiation site.
According to a second aspect of the present disclosure there is provided a transcriptional expression cassette 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.
In some embodiments, the target protein comprises an arylalkylamine-N-acetyltransferase, a hydroxyindole-oxo-methyltransferase, or a firefly luciferase.
In some embodiments, the target protein is an arylalkylamine-N-acetyltransferase.
In some embodiments, the target protein is an arylalkylamine-N-acetyltransferase of a black goat in Hainan.
According to a third aspect of the present disclosure there is provided an expression vector comprising the promoter element of the first aspect or the transcriptional expression cassette of the second aspect.
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.
According to a fourth aspect of the present disclosure there is provided a host cell carrying one or more of the promoter element of the first aspect or the transcription expression cassette of the second aspect or the expression vector of the third aspect.
In some embodiments, the host cell is selected from the group consisting of a prokaryotic cell and a eukaryotic cell.
In some specific embodiments, the prokaryotic cell comprises a bacterial cell, e.coli, streptomyces.
In some specific embodiments, the eukaryotic cell comprises a yeast cell, a mammalian cell, an insect cell.
In some specific embodiments, the mammalian cells comprise humans, monkeys, mice, rats, hamsters, goats, sheep, cows, pigs, dogs, cats.
In some specific embodiments, the eukaryotic cell comprises a human cell, a mouse cell, a rat cell, a hamster cell, a goat cell, a sheep cell, or any other host cell.
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 may express functional adenovirus E1), saos, C2C12, L cells, HT1080, hepG2.
In some embodiments, the mammalian cells include fibroblasts, hepatocytes, and myoblasts.
In some embodiments, the fibroblast is selected from embryonic fibroblast.
In some embodiments, the fibroblast is selected from a goat embryo fibroblast.
According to a fifth aspect of the present disclosure there is provided the use of the promoter element of the first aspect or the transcriptional expression cassette of the second aspect for regulating gene expression.
The method successfully utilizes the dual-luciferase reporter gene system to identify the AANAT promoter core region of the Hainan black goat, screens to obtain a promoter element, can be used as a cis-acting element for expressing a target gene, increases the expression effect of a target protein gene, and has wider application prospect and application potential.
Drawings
FIG. 1 shows the first part of the peak diagram of the Sanger sequencing sequence of the 5' RACE fragment of the AANAT gene.
FIG. 2 shows the second part of the peak diagram of the sequence of Sanger sequencing of the 5' RACE fragment of the AANAT gene.
FIG. 3 shows the first part of the peak diagram of the Sanger sequencing sequence of the 3' RACE fragment of the AANAT gene.
FIG. 4 shows the second part of the peak diagram of the Sanger sequencing sequence of the 3' RACE fragment of the AANAT gene.
FIG. 5 shows the nucleotide sequence of the AANAT gene from the transcription initiation site to the transcription termination site. Wherein the upper red portion represents the 5 'non-coding region, the lower red portion represents the 3' non-coding region, and the middle yellow portion represents the protein coding region.
Fig. 6 shows AANAT promoter region electrophoresis.
FIG. 7 shows the results of luciferase activity assay.
Detailed Description
The present invention will be described in further detail with reference to the following examples in order to make the objects, technical solutions and advantages of the present invention more apparent. The specific embodiments described herein are for purposes of illustration only and are not to be construed as limiting the invention in any way. In addition, in the following description, descriptions of well-known structures and techniques are omitted so as not to unnecessarily obscure the concepts of the present disclosure. Such structures and techniques are also described in a number of publications.
Definition of the definition
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly used in the art to which this invention belongs. For the purposes of explaining the present specification, the following definitions will apply, and terms used in the singular will also include the plural and vice versa, as appropriate.
The terms "a" and "an" as used herein include plural referents unless the context clearly dictates otherwise.
The term "promoter" or "promoter element" as used herein is defined as a DNA sequence that binds to an RNA polymerase and directs the polymerase to the correct downstream transcription initiation site of a polynucleotide encoding a polypeptide having biological activity to initiate transcription. 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" is also understood to include the 5' non-coding region (between the promoter and the translation origin) for translation after transcription into mRNA, cis-acting transcriptional regulatory elements such as enhancers, and/or other nucleotide sequences capable of interacting with transcription factors. The promoter or promoter element may be a wild-type promoter, a variant promoter, a hybrid promoter or a consensus promoter (consensus promoter).
The term "core promoter" as used herein refers to a nucleic acid sequence comprised by a promoter. The core promoter is typically the smallest part of the promoter required to properly initiate transcription. The core promoter typically includes a transcription initiation site and a binding site for RNA polymerase.
The term "sequence identity" as used herein refers to a "percentage of sequence identity" or "percentage of identity" between two polynucleotides, i.e., the number of identical matching positions shared by sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. The matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since the gaps are not nucleotides, the gaps present in the target sequence are not accounted for. Also, since the target sequence nucleotides are counted, and the nucleotides from the reference sequence are not counted, gaps present in the reference sequence are not counted. At least 85% sequence identity includes a contiguous 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 entire length of the sequence.
As used herein, the term "expression vector" when used in connection with an isolated nucleic acid molecule as described above, may be used to directly or indirectly link the nucleic acid sequence to regulatory elements on the vector, so long as such regulatory elements are capable of regulating translation and expression of the nucleic acid molecule, and the like. These regulatory elements may of course be derived directly from the vector itself or may be exogenous, i.e.not derived from the vector itself. That is, the nucleic acid molecule is operably linked to a regulatory element. Common expression vectors such as prokaryotic expression vectors, eukaryotic expression vectors or viral expression vectors, and the like.
The term "operably linked" as used herein refers to the linkage of a foreign gene to a vector such that regulatory elements within the vector, such as transcription and translation regulatory sequences and the like, are capable of performing their intended functions of regulating transcription and translation of the foreign gene.
Examples and figures are provided below to aid in the understanding of the invention. It is to be understood that these examples and drawings are for illustrative purposes only and are not to be construed as limiting the invention in any way. The actual scope of the invention is set forth in the following claims. It will be understood that any modifications and variations may be made without departing from the spirit of the invention.
Examples
EXAMPLE 1 RACE cloning of Hainan Black goat AANAT Gene
Step 1: the AANAT amino acid sequences of different species of cattle (Genbank No: NM-177509.2), sheep (Genbank No: NM-001009461.1), mouse (Genbank No: NM-001009461.1) and goat (Genbank No: XM-018064062.1) were aligned, and 2 pairs of primers were designed for the conserved sequences in the protein coding region, the primer sequences are shown in Table 1.
Step 2: the total RNA extracted from the pine cone tissue of Hainan black goat is used as a template, the first strand of cDNA is reversely transcribed into a primer pair 2 shown in table 1 to be used as the template, RACE cloning is carried out, and 3 'and 5' end sequence fragments of the AANAT gene are obtained after PCR amplification.
The resulting fragment was ligated into pCE2-TA/Blunt-Zero vector (Northenzan) and subjected to first generation sequencing (Sanger sequencing). The peak pattern of the 5'RACE fragment Sanger of AANAT gene is shown in FIGS. 1 and 2, and the peak pattern of the 3' RACE fragment Sanger of AANAT gene is shown in FIGS. 3 and 4.
And (3) respectively removing carrier sequences from the obtained 3 '-end RACE fragment and the obtained 5' -end RACE fragment, and then splicing to obtain the full-length sequence (SEQ ID NO: 13) of the Hainan black goat AANAT gene. FIG. 5 shows the full length sequence of goat AANAT gene, wherein the 5'UTR region (red at the upper end of FIG. 5) is shown as SEQ ID NO. 14, the protein coding region (yellow color in FIG. 5) is shown as SEQ ID NO. 15, and the 3' UTR region (red at the lower end of FIG. 5) is shown as SEQ ID NO. 16.
Example 2 use of a double luciferase reporter to identify goat AANAT promoter Activity
1. Construction of double luciferase reporter vector
The AANAT gene 5' UTR sequence obtained by sequencing in example 1 was aligned with the sequences in Ensembl database (chr 19: 54512968-chr19: 54512546), the base on the DNA strand corresponding to the first nucleotide of the mRNA strand was used as the transcription initiation site, and the region within 2000bp upstream of the transcription initiation site was used as the promoter region to design the primer, the primer sequences are shown in Table 2.
Four unequal length amplification products amplified using the primers shown in table 2: AANAT-P0 (SEQ ID NO: 17), AANAT-P1 (SEQ ID NO: 18), AANAT-P2 (SEQ ID NO: 19) and AANAT-P3 (SEQ ID NO: 20), the amplified products were subjected to electrophoresis to identify the product band length, and the electrophoresis pattern is shown in FIG. 6.
Taking a 4-week-old large goat fetus, cutting part of skin tissues, then digesting the skin tissues into single cell suspension by using 0.25% trypsin, culturing the single cell suspension by using a DMEM high-sugar culture medium containing 10% fetal bovine serum, and replacing fresh culture medium every 3 days until the confluency of cultured cells is 60-70%, thereby obtaining goat embryo fibroblasts. The goat embryo fibroblasts obtained by the above method were subjected to DNA extraction using a magnetic bead extraction DNA kit (VAMNE Magnetic Pathogen DNA Kit) of Norfluzan, and the above AANAT promoter fragment was amplified in a PCR amplification system containing 25. Mu.L of 2X Phanta Flash Master Mix (Dye Plus) per 50. Mu.L, 2. Mu.L of 10. Mu.M upstream primer, 2. Mu.L of 10. Mu.M downstream primer, 1. Mu.L of 200 ng/. Mu.L template and 20. Mu.L of embryo water. The PCR amplification procedure was: pre-denaturation at 98 ℃ for 30 seconds; denaturation at 98℃for 10 sec; annealing at 65 ℃ for 5 seconds; extending at 72 ℃ for 10 seconds, 30 cycles; final extension at 72℃for 1 min. And was subjected to product recovery using a norvazan gum recovery DNA kit (FastPure Gel DNA Extraction Mini Kit).
pGL3-Basic plasmid and amplified AANAT promoter fragment were treated by double cleavage with KpnI and Hind III restriction enzymes (TAKARA), respectively, and ligated using T4 ligase (TAKARA), the ligated system contained 7. Mu.L of double-digested AANAT gene promoter, 1. Mu.L of double-digested pGL3-Basic plasmid 1. Mu. L, T4 ligase buffer and 1. Mu.L of T4 ligase per 10. Mu.L; the conditions of the connection include: 16℃for 4 hours. The ligation product is transformed into E.coli DH5 alpha competent cells (TAKARA), the transformation steps are carried out according to the specification of the E.coli DH5 alpha competent cells, positive clone colonies are picked up after transformation and sent to Shanghai biological engineering company for first generation sequencing (Sanger sequencing), and recombinant protein expression vectors with correct sequencing are named pGL3-AANAT-P0, pGL3-AANAT-P1, pGL3-AANAT-P2 and pGL3-AANAT-P3 respectively.
Identification of AANAT Gene core promoter
The correct sequencing plasmid bacterial fluid was subjected to endotoxin removal by using a nonipran plasmid endotoxin removal kit (FastPure EndoFree Plasmid Mini Kit).
Thawing and resuscitating goat embryo fibroblasts at 37 ℃, culturing the goat embryo fibroblasts by using a DMEM high-sugar culture medium containing 10% of fetal bovine serum, converging the cells to 70% to 80% for passaging, discarding the original culture medium, flushing the goat embryo fibroblasts twice by using a sterile PBS buffer, placing the goat embryo fibroblasts in a 37 ℃ incubator, digesting the goat embryo fibroblasts by using trypsin of 0.25% EDTA for about 2 minutes, taking out the goat embryo fibroblasts, adding 20% of fetal bovine serum DMEM culture medium to neutralize the trypsin, blowing the goat embryo cells by using a suction tube, and removing the goat embryo fibroblasts. Inoculating into 6-well plate of cell culture plate at a ratio of 1:3, adding 5mL of 10% fetal calf serum DMEM medium, 37deg.C, and 5% CO 2 Culturing in an incubator. Cell passaging can be performed every 3 days, at a ratio of 1:3.
And (3) when the fibroblasts grow to 70-90% confluence, carrying out transfection, diluting a Lipofectamine ™ reagent (Sieimer) by using an Opti-MEM culture medium, fully mixing, then diluting endotoxin plasmids by using the Opti-MEM culture medium, preparing a plasmid premix, carrying out cotransfection on four plasmids of pGL3-AANAT-P0, pGL3-AANAT-P1, pGL3-AANAT-P2 and pGL3-AANAT-P3 respectively with pRL-TK plasmids, carrying out cotransfection on negative control of pGL3-Basic and pRL-TK, adding the P3000 reagent into a tube, and fully mixing. Diluted DNA (1:1 ratio) was added to each tube of diluted Lipofectamine ™ 3000 reagent and incubated in a chamber for 5 minutes, DNA-liposome complexes were added to the cells, cells were incubated at 37 ℃ for 2 to 4 days, and transfected cells were analyzed.
3. Luciferase Activity assay
Transfected cells were collected and luciferase assay was performed on transfected cells using the double luciferase reporter assay kit for norpran (Duo-Lite Luciferase Assay System). Wherein the fluorescence value generated by firefly luciferase catalysis is represented by F, and the fluorescence value generated by Renilla luciferase catalysis is represented by R. Untransfected cells were used as blank controls (i.e., background F and background R) for background subtraction; transfected cells were treated with the test compound as test group cells (i.e., test group F and test group R), transfected cells were untreated to normalize the results (i.e., control group F and control group R),
the relative activity of the pGL3-AANAT-P0 of the experimental group is 12.5376 +/-1.21 according to the experimental result measured by an enzyme-labeling instrument; pGL3-AANAT-P1 has relative activity of 15.34+ -1.432; pGL3-AANAT-P2 has relative activity of 35.1233 +/-2.331; pGL3-AANAT-P3 has relative activity of 17.322 + -1.306, and luciferase of control group has relative activity of 0.12147 + -1.342. The results are shown in FIG. 7. From the results of FIG. 7, it can be seen that the relative activities of luciferases in the experimental group pGL3-AANAT-P2 are significantly higher than those in the control group (P < 0.05), so that 540bp to 958bp upstream of the AANAT promoter can be judged as the promoter core region, and the effect of transcription initiation is obvious.
The technical scheme of the invention is not limited to the specific embodiment, and all technical modifications made according to the technical scheme of the invention fall within the protection scope of the invention.
Claims (6)
1. A promoter element, characterized in that:
the nucleotide sequence of the promoter element is shown as SEQ ID NO. 19, and the promoter element is derived from Hainan black goats.
2. The promoter element according to claim 1, wherein the promoter element is derived from-540 bp to-958 bp upstream of the transcription initiation site of the Hainan black goat AANAT gene.
3. A transcriptional expression cassette comprising 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.
4. An expression vector comprising the promoter element of claim 1 or 2 or the transcriptional expression cassette of claim 3.
5. A host cell carrying one or more of the promoter element of claim 1 or 2 or the transcriptional expression cassette of claim 3 or the expression vector of claim 4, said host cell being a mammalian cell.
6. Use of the promoter element of claim 1 or 2 or the transcriptional expression cassette of claim 3 for regulating gene expression in mammalian cells.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990042573A (en) * | 1997-11-27 | 1999-06-15 | 이황희 | Insulin-like growth factor 1 promoter 2 of black goat |
| WO2001087909A2 (en) * | 2000-05-18 | 2001-11-22 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the aanat gene |
| CN106852157A (en) * | 2014-06-16 | 2017-06-13 | 约翰斯·霍普金斯大学 | Composition and method for expressing guide CRISPR RNA using H1 promoters |
| CN113278709A (en) * | 2021-05-27 | 2021-08-20 | 贵州省种畜禽种质测定中心 | Guizhou black goat multi-lamb major gene application, primer pair and kit |
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| US11519005B2 (en) * | 2017-09-28 | 2022-12-06 | The Governors Of The University Of Alberta | Retinoic acid-inducible gene I promoter and compositions and methods relating to same |
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- 2023-12-08 CN CN202311676378.9A patent/CN117384906B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR19990042573A (en) * | 1997-11-27 | 1999-06-15 | 이황희 | Insulin-like growth factor 1 promoter 2 of black goat |
| WO2001087909A2 (en) * | 2000-05-18 | 2001-11-22 | Genaissance Pharmaceuticals, Inc. | Haplotypes of the aanat gene |
| CN106852157A (en) * | 2014-06-16 | 2017-06-13 | 约翰斯·霍普金斯大学 | Composition and method for expressing guide CRISPR RNA using H1 promoters |
| CN113278709A (en) * | 2021-05-27 | 2021-08-20 | 贵州省种畜禽种质测定中心 | Guizhou black goat multi-lamb major gene application, primer pair and kit |
Non-Patent Citations (2)
| Title |
|---|
| Overexpression of ovine AANAT and HIOMT genes in switchgrass leads to improved growth performance and salt-tolerance;Yan-Hua Huang et al.;Sci Rep .;第7卷(第1期);文献号：12212，第1-13页 * |
| Peng,O.K. et al..GenBank: KF220307.1.GenBank.2013,ORIGIN. * |
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