CN113151355A - Dual-luciferase reporter gene vector of chicken STRN3 gene 3' UTR and construction method and application thereof - Google Patents
Dual-luciferase reporter gene vector of chicken STRN3 gene 3' UTR and construction method and application thereof Download PDFInfo
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Abstract
The invention discloses a dual-luciferase reporter gene vector of a chicken STRN3 gene 3' untranslated region, and a construction method and application thereof, belonging to the technical field of genetic engineering. The dual-luciferase reporter gene vector of the 3' untranslated region of the chicken STRN3 gene is pmiR-Glo, the dual-luciferase reporter gene vector comprises a sequence of the 3' untranslated region of the chicken STRN3 gene, and the recombinant vector can stably express the sequence gene of the 3' untranslated region of the chicken STRN 3. The dual-luciferase reporter gene vector of the 3' untranslated region of the chicken STRN3 gene can be used for detecting the influence of the SNP site on the 3' untranslated region of the STRN3 gene on the expression of the STRN3 gene, and can be used for detecting the targeting relation between the SNP site on the 3' untranslated region of the STRN3 gene and miR-383-5p and the negative regulation and control of miR-383-5p to inhibit the expression of the STRN3 gene.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a dual-luciferase reporter gene vector of a chicken STRN3 gene 3' untranslated region, and a construction method and application thereof.
Background
STRN, STRN3(SG2NA) and STRN4(Zinedin) are members of the family of proteins, comprising a variety of protein binding regions, such as notch binding regions, coiled coil regions, calmodulin binding regions and WD repeat regions, which modulate calmodulin family dimerization, in close association with a variety of proteins, including calmodulin and notch. The protein family of the Striatin is highly expressed in central and peripheral nervous systems, partial knocking-down of calmodulin in mouse brain and mouse neuron culture can induce reduction of night activity and dendritic cell growth defect respectively, and the Striatin protein is also expressed in other tissues and cell lines and can regulate and control the orientation of zon 1 and estrogen-induced activation of endothelial NO synthase. The family of Striatin proteins can also form complexes of protein phosphatases and protein kinases. Research shows that the striatin protein family can be used as a regulation subunit of protein phosphate 2A (PP2A) to regulate and control signal paths related to cell proliferation, differentiation, apoptosis and transformation. Recent studies have shown that the holoenzyme PP2A contains striated proteins which are the core components of a number of protein complexes known as striated protein cross-linked phosphatases and kinases (STRIPAKs). The STRIPAK complex is conserved in most species and is thought to play a crucial role in fundamental biological processes.
miRNA is a 19-23nt endogenous non-coding single-stranded RNA molecule, can degrade target mRNA or inhibit translation, thereby completing transcriptional control of target mRNA, participating in embryonic development, organ formation, cell proliferation, differentiation, apoptosis, metabolism, virus infection, tumorigenesis and a series of physiological or pathological processes. mirnas act by interacting with target mrnas through incomplete base pairing with the 3' UTR of the target mRNA, either triggering target mRNA degradation or inhibiting its translation. Studies have shown that hermaphroditic mirnas in the gonads of mammals and chickens may play a key role in gonad development, including germ cell differentiation, gametogenesis, and steroidogenesis. In animals, miRNAs are thought to act primarily through translational inhibition rather than mRNA degradation, transcriptome sequencing analysis of differentially expressed miRNAs during differentiation of embryonic stem cells into male germ cells revealed important clues for miRNA function and suggested that some miRNAs may down-regulate large amounts of mRNA.
miR-383 is a miRNA which is popular in the field of cancer research in recent years, and the expression level of the miRNA is considered to be significantly and negatively correlated with the malignancy degree of various cancers. miR-383-5p is a main mature body of miR-383, so that the research on the influence of the miR-383 on apoptosis and proliferation has important reference value for the related research on tumors. A plurality of research results show that miR-383-5p has the phenomenon of abnormal down-regulation of expression in medulloblastoma, glioma, liver cancer cells and colon cancer cells. In addition, researches show that the expression of miR-383-5p also has the abnormal down-regulation phenomenon in testis tissues of a mature stasis type male sterile patient and a male sterile animal, and meanwhile, spermatogenic cells are in a hyperproliferation state.
In conclusion, miR-383-5p and STRN3 play a crucial role in the body regulation process, however, the related researches of miR-383-5p and STRN3 on the gene and signal path regulation mechanism are not reported.
Disclosure of Invention
In view of the above, the invention aims to provide a dual-luciferase reporter gene vector of chicken STRN3 gene 3' UTR, and a construction method and application thereof.
The purpose of the invention is realized by the following modes:
the invention provides a chicken STRN3 gene 3'UTR dual-luciferase reporter gene vector, which contains a dual-luciferase reporter gene vector and a nucleotide fragment of the 3' UTR region of a STRN3 gene.
Further, the dual-luciferase reporter gene vector is pmiR-Glo.
Further, the nucleotide sequence of the nucleotide fragment of the 3' UTR region of the STRN3 gene is shown as follows:
gagggaacgcgtcactgccatcgataaggttaccaataagacactacgtctgatctgcctgagtgaaggctcttaagtggggcaggtttcagttggtgaagcgtatgtctgttctttaggtgtctttatgcaaacgtggagtctgatcttacaaaagacttttattttggactctgtgtgctgccttagggttaggaatgaatacaataaagtattttttctagtcttccacaaaacttttctgatcagtttgcgagttttgatgagttttgtaaggtttctgttttacaaactatgaat。
further, the nucleotide fragment of the 3' UTR region of the STRN3 gene can be combined with miR-383-5p through base complementary pairing.
The invention also provides a construction method of the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector, which mainly comprises the following steps: and (2) connecting the nucleotide fragment of the 3'UTR region of the STRN3 gene cut by the restriction endonuclease to a dual-luciferase reporter gene vector cut by the same restriction endonuclease through a ligase, and screening to obtain the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector.
Further, a nucleotide fragment of the 3' UTR region of the STRN3 gene is obtained by PCR amplification, wherein a primer oligo used for the PCR amplification comprises:
STRN3-3'UTR—oligo-1:5’-TCTAGTTGTTTAAACGAGCTCGAG-3’；
STRN3-3'UTR—oligo-10:5’-CAGGTCGACTCTAGACTCGAGATTCATAGTT
TGTAAAACAGAAACCTTACAAAACTCA-3’。
further, the STRN3-3' UTR-oligo-1 contains the restriction enzyme cutting site of SacI restriction enzyme; the STRN3-3' UTR-oligo-10 contains the restriction site of XhoI restriction endonuclease.
Further, the dual-luciferase reporter gene vector is pmiR-Glo subjected to double digestion by SacI and XhoI restriction enzymes.
Further, the ligase was T4DNA ligase, and the ligation was performed at 22 ℃ for 2 hours.
The invention also provides application of the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector in detection of chicken STRN3 gene expression activity.
The invention also provides application of the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector in screening miRNA target genes.
The invention also provides application of the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector in detecting a regulation mechanism of miRNA regulation chicken STRN3 gene.
Further, the miRNA is miRNA-383-5 p.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention recycles enzyme digestion products after double enzyme digestion pmiR-Glo plasmid of SacI and XhoI, clones nucleotide fragment of the 3' UTR region of the STRN3 gene to the downstream of luciferase luc gene of firefly, constructs 3' UTR sequence which can be used for analyzing the STRN3 gene in cells from different tissue sources, the vector can stably express the chicken STRN3 gene, and meanwhile, the dual-luciferase reporter gene can be used as a detection index to reflect the expression quantity of the chicken STRN3 gene, and can be used for researching the specific mechanism of the 3' UTR cell of the STRN3 gene.
2. The chicken STRN3 gene dual-luciferase report vector provided by the invention can be used for detecting the influence of SNP sites on the 3 'untranslated region of the STRN3 gene on the expression of the STRN3 gene, and can be used for detecting the targeting relationship between the SNP sites on the 3' untranslated region of the STRN3 gene and miR-383-5p, and miR-383-5p negatively regulates and inhibits the expression of the STRN3 gene.
3. The chicken STRN3 gene dual-luciferase report vector and the preparation method thereof provided by the invention can also be used for screening candidate miRNA prediction target genes and researching a regulation mechanism for regulating and predicting the target genes by miRNA at a cellular level.
Drawings
In order to more clearly illustrate the embodiments of the present invention, the drawings to which the embodiments relate will be briefly described below.
FIG. 1: miR-383-5p and a stem-loop structure diagram of the prediction target gene STRN3 gene 3' UTR region base complementation.
FIG. 2: PCR electrophoresis result of STRN3-3' UTR region fragment, wherein, 1: mutant, 398 bp; 2: wild type, 378 bp.
FIG. 3: schematic structure of the pmiR-Glo vector.
FIG. 4: the result of double enzyme cutting electrophoresis of the chicken STRN3 gene double luciferase report vector.
FIG. 5: sequencing result graph of the wild chicken STRN3 gene dual-luciferase reporter vector.
FIG. 6: sequencing result graph of mutant chicken STRN3 gene dual-luciferase reporter vector.
FIG. 7: influence of miR-383-5p mimics on 293T cell luciferase expression.
Detailed Description
The present invention is described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto, and it is obvious that the examples in the following description are only some examples of the present invention, and it is obvious for those skilled in the art to obtain other similar examples without inventive exercise and falling into the scope of the present invention.
Example 1: miR-383-5p target gene prediction
The miR-383-5p target gene is predicted by using three websites of mirDB, targetScan and miRanda, intersection of the target genes is obtained, the STRN3 gene is obtained by screening miR-383-5p according to the base complementary pairing principle, score and research focus of the target gene and 3' UTR, and the binding site of the gene and miR-383-5p is AGACUAGA. On-line prediction results show that the target genes are highly conserved and stable with the binding sites of the differential expression miRNA gga-miR-383-5 p. And predicting the binding site and the base complementary stem-loop structure of the miR-383-5p and the 3' -UTR of the target gene by using Targetscan and RNAhybrid software.
Example 2: obtaining the 3' UTR sequence fragment of the STRN3 gene by a PCR method
1. According to a 3' UTR region in a chicken STRN3 gene sequence (XM015287636.1) in GenBank, 100bp near a miR-383-5p and STRN3 gene 3' UTR binding site is intercepted, a primer oligo sequence is synthesized, SacI and XhoI enzyme cutting sites and protective bases are respectively added to STRN3-gga383 wild-type gene upstream and downstream primers, and the primer oligo sequence amplified by the STRN3 gene 3' UTR is as follows:
TABLE 1 primer oligo sequences for STRN3 Gene 3' UTR amplification
| oligo-1 | TCTAGTTGTTTAAACGAGCTCGAG |
| oligo-2 | TTATTGGTAACCTTATCGATGGCAGTGACGCGTTCCCTCGAGCTCGTTTAAACAACTAG |
| oligo-3 | GCCATCGATAAGGTTACCAATAAGACACTACGTCTGATCTGCCTGAGTGAAGGCTCTTA |
| oligo-4 | GAACAGACATACGCTTCACCAACTGAAACCTGCCCCACTTAAGAGCCTTCACTCAGGCA |
| oligo-5 | TGGTGAAGCGTATGTCTGTTCTTTAGGTGTCTTTATGCAAACGTGGAGTCTGATCTTAC |
| oligo-6 | CTAAGGCAGCACACAGAGTCCAAAATAAAAGTCTTTTGTAAGATCAGACTCCACGTTTG |
| oligo-7 | GACTCTGTGTGCTGCCTTAGGGTTAGGAATGAATACAATAAAGTATTTTTTCTAGTCTT |
| oligo-8 | GCAAACTGATCAGAAAAGTTTTGTGGAAGACTAGAAAAAATACTTTATTGTATTCATTC |
| oligo-9 | ACAAAACTTTTCTGATCAGTTTGCGAGTTTTGATGAGTTTTGTAAGGTTTCTGTTTTAC |
| oligo-10 | CAGGTCGACTCTAGACTCGAGATTCATAGTTTGTAAAACAGAAACCTTACAAAACTCA |
The primer is synthesized by Suzhou Jima gene GmbH;
2. dissolving the primer oligos in Table 1 to prepare 50. mu.M primer oligo solution, respectively taking the primer oligo solution with the same volume to a 1.5ml centrifuge tube, and mixing uniformly to prepare oligo mix;
3. the prepared oligo mix is used as a template to carry out a first round of PCR reaction, and the PCR system is as follows:
| oligo mix | 2μl |
| 10×Pfu Buffer(+Mg2+) | 3μl |
| dNTP | 0.6μl |
| DMSO | 1.2μl |
| ddH2O | 23μl |
| Pfu DNA polymerase | 0.2μl |
circulation conditions are as follows:
4. taking the product of the first round of PCR reaction as a template, and carrying out a second round of PCR reaction by using oligo-1 and oligo-10;
the PCR system was as follows:
| first round PCR product | 1μl |
| 10×Pfu Buffer(+Mg2+) | 5μl |
| dNTP | 1μl |
| DMSO | 2μl |
| oligo-1 | 1μl |
| oligo-10 | 1μl |
| ddH2O | 39μl |
| Pfu DNA polymerase | 0.3μl |
Circulation conditions are as follows:
obtaining a non-single-band PCR product mixture mixed with a target gene band by the first round of PCR, and obtaining a single target gene band by the second round of PCR by using the PCR product of the first round as a template;
5. after the PCR reaction was completed, the STRN3-gga383 wild-type gene fragment was recovered by Agarose electrophoresis and gel cutting.
Example 3: construction of chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector
1. The wild-type gene fragment of the STRN3-gga383 is subjected to enzyme digestion by SacI and XhoI at 37 ℃ for 2 hours, wherein the enzyme digestion system is as follows:
| 10×Buffer | 5μl |
| STRN3-gga383 wild-type gene | 42μl |
| SacI | 1.5μl |
| XhoI | 1.5μl |
2. The gene vector P-mirGLO (see FIG. 3) was digested with SacI and XhoI at 37 ℃ for 2 hours as follows:
| 10×Buffer | 5μl |
| P-miRGLO | 5ug |
| SacI | 1.5μl |
| XhoI | 1.5μl |
| ddH2O | make up to 50. mu.l |
3. Electrophoresis, and recovering the STRN3-gga383 wild type gene fragment and the vector P-mirGlLO by using a DNA gel recovery kit;
4. t4DNA ligase is used for connecting the STRN3-gga383 wild-type gene fragment obtained by double enzyme digestion and a linearized gene vector P-mirGlLO, and the wild-type gene fragment and the linearized gene vector P-mirGlLO are connected for 2 hours at the temperature of 22 ℃, and the connection system is as follows:
| T4DNA ligase buffer | 2μl |
| P-miRGLO | 2μl |
| STRN3-gga383 wild-type gene fragment | 5μl |
| T4 DNA ligase | 1μl |
| ddH2O | 10μl |
5. Transforming the ligation product into a competent cell;
6. selecting clone colonies from the plate, extracting plasmids and identifying to select positive clones;
7. the sequencing of the recombinant plasmid is verified, and the wild-type recombinant vector with correct sequencing is named as STRN 3-WT.
Example 4: construction of mutant chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector
1. Designing primer oligo, adding SacI, XhoI and protective basic groups to the upstream and downstream primers of the STRN3-gga383 mutant gene respectively, using the primers for cloning and designing mutation of a target gene, mutating TCTGATC in a target sequence into AGACATG, and using other primer oligo sequences as wild primers. The chicken STRN3-gga383 mutant dual-luciferase reporter gene vector is constructed according to the steps of example 2 and 3, the sequencing verification is carried out on the recombinant vector, the result is shown in figure 5 and figure 6, the target sequence TCTGATC is successfully mutated into AGACATG, and the mutant recombinant vector with correct sequencing is named as STRN 3-MT.
Example 5: cell transfection assay
293T cells with good growth state are divided into 2X 105The dual-luciferase reporter gene vector prepared in example 3 and miR-383-5p mix (20 mu M) or negative control (20 mu M) are co-transfected into 293T cells according to the instructions of transfection reagents by inoculating the dual-luciferase reporter gene vector into a 24-well plate at a density of 500 mu L. The method comprises the following specific steps: proliferating 293T cell to 80%, washing with PBS for 2 times, adding pancreatin, digesting for 1min, discarding pancreatin, adding complete culture medium, blowing, and diluting to 5 × 105Inoculating each cell/mL into a 24-well plate, wherein each well is 0.5mL, and continuously culturing for 24 h; diluting miR-383-5p mimics to 20 mu M, adding 50 mu L of opti-DMEM into a 1.5mL centrifuge tube, adding 0.8ug of dual-luciferase reporter gene vector, adding 50 mu L of opti-DMEM and 2 mu L of lip2000 into another new 1.5mL centrifuge tube, respectively mixing uniformly, standing for 5min, mixing the two components after standing, and standing for 20min at room temperature; removing the old culture medium in the pore plate, adding a new non-antibiotic culture medium, slowly and gently dripping the well-standing solution into the pore plate with the cells, gently shaking up, and replacing with a complete culture medium after 4 hours.
Each test group was set with 3 parallel wells and each test was repeated 3 times. Transfected cells were grouped as follows: transfection of pmirGLO (empty plasmid group); ② cotransfection pmirGLO (empty plasmid) + NC (negative control); ③ cotransfecting the STRN3-WT + NC; fourthly, co-transfecting STRN3-WT + miR-383-5p mimics; cotransfecting STRN3-MT + NC; sixthly, transfecting the STRN3-MT + miR-383-5p mimics together.
Example 6: dual luciferase Activity assay and statistical analysis
And after the cells are transfected for 48 hours, collecting the cells, and detecting the dual-luciferase reporter activity in the cells. Each test group was set with 3 parallel wells and each test was repeated 3 times. The method comprises the following specific steps: removing the old culture medium, washing with PBS for 2 times, adding 250 μ L of diluted 1 XPLB (passive lysis buffer), placing in a horizontal shaking table, shaking for 15min at room temperature to passively lyse cells to obtain a lysate, adding 100uL LARII (luciferase detection Reagent II) into an EP tube, adding 20 μ L of the lysate, mixing uniformly, detecting the activity of firefly luciferase, after detection, rapidly adding 100 μ L of Stop & Glo Reagent into a primary tube, mixing uniformly to detect the activity of renilla luciferase, and calculating the ratio of the two.
All data are expressed as mean ± standard deviation. P <0.05 was significant for all statistical analyses. One-way analysis of variance and t-test were performed for all statistical significance using GraphPad Prism software. Each test group was set with 3 parallel wells and each test was repeated 3 times.
The dual-luciferase activity detection result shows that the luciferase activity of the 293T cell group of the STRN3-WT + miR-383-5P mimics group is remarkably reduced by about 46 percent (P <0.01) compared with that of other groups, the statistical difference is very remarkable, and the other 5 groups have no remarkable difference.
The results show that miR-383-5p can inhibit the activity of the STRN3-3' UTR luciferase reporter gene vector, mainly because miR-383-5p and STRN3-3' UTR in the STRN3-3' UTR luciferase reporter gene vector have interaction; and after site-directed mutagenesis is carried out on the STRN3-3' UTR in the STRN3-3' UTR luciferase reporter gene vector, the interaction of miR-383-5p and the mutated STRN3-3' UTR luciferase reporter vector disappears, and meanwhile, the regulation and control effect of miR-383-5p on the STRN3-3' UTR is further proved, meanwhile, the STRN3-3' UTR luciferase reporter gene vector can be used for analyzing and detecting miR-383-5p, and the miR-383-5p and the STRN3 are closely related to the differentiation process of chicken embryonic stem cells to male germ cells on the basis of that the miR-383-5p and the STRN3 are closely related to the differentiation process of the chicken embryonic stem cells to the male germ cells, so that a foundation is laid for researching a gene and signal path regulation and control mechanism in the process.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
SEQUENCE LISTING
<110> Jilin province academy of agricultural sciences
Double-luciferase reporter gene vector of <120> chicken STRN3 gene 3' UTR and construction method and application thereof
<130> 20210316
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 300
<212> DNA
<213> Artificial sequence
<400> 1
gagggaacgc gtcactgcca tcgataaggt taccaataag acactacgtc tgatctgcct 60
gagtgaaggc tcttaagtgg ggcaggtttc agttggtgaa gcgtatgtct gttctttagg 120
tgtctttatg caaacgtgga gtctgatctt acaaaagact tttattttgg actctgtgtg 180
ctgccttagg gttaggaatg aatacaataa agtatttttt ctagtcttcc acaaaacttt 240
tctgatcagt ttgcgagttt tgatgagttt tgtaaggttt ctgttttaca aactatgaat 300
Claims (10)
1. The chicken STRN3 gene 3' UTR dual-luciferase reporter vector is characterized by comprising a dual-luciferase reporter vector and a STRN3 gene 3' UTR region nucleotide fragment, wherein the nucleotide sequence of the STRN3 gene 3' UTR region nucleotide fragment is shown in SEQ ID NO: 1 is shown.
2. The chicken STRN3 gene 3' UTR dual-luciferase reporter vector of claim 1, wherein the dual-luciferase reporter vector is pmiR-Glo.
3. The method for constructing a dual-luciferase reporter vector with 3' UTR of chicken STRN3 gene according to claim 1 or 2, which is characterized by mainly comprising the following steps: and (2) connecting the nucleotide fragment of the 3'UTR region of the STRN3 gene cut by the restriction endonuclease to a dual-luciferase reporter gene vector cut by the same restriction endonuclease through a ligase, and screening to obtain the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector.
4. The method for constructing according to claim 3, wherein the nucleotide fragment in the 3' UTR region of the STRN3 gene is obtained by PCR amplification using primer oligos comprising:
STRN3-3'UTR—oligo-1:5’-TCTAGTTGTTTAAACGAGCTCGAG-3’；
STRN3-3'UTR—oligo-10:5’-CAGGTCGACTCTAGACTCGAGATTCATAGTT
TGTAAAACAGAAACCTTACAAAACTCA-3’。
5. the method for constructing a recombinant plasmid according to claim 4, wherein the STRN3-3' UTR-oligo-1 comprises a cleavage site of SacI restriction endonuclease; the STRN3-3' UTR-oligo-10 contains the restriction site for XhoI restriction endonuclease.
6. The method of claim 3, wherein the dual-luciferase reporter gene vector is subjected to double restriction with SacI and XhoI restriction endonucleases.
7. The use of the chicken STRN3 gene 3' UTR dual-luciferase reporter vector of claim 1 or 2 in detecting chicken STRN3 gene expression activity.
8. The use of the chicken STRN3 gene 3' UTR dual-luciferase reporter gene vector of claim 1 or 2 in screening miRNA-383-5p target genes.
9. Use of the chicken STRN3 gene 3' UTR dual-luciferase reporter vector of claim 1 or 2 in the mechanism of detecting miRNA-regulated chicken STRN3 gene.
10. The use of claim 9, wherein the miRNA is miRNA-383-5 p.
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