CN114480496A - Dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application - Google Patents
Dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application Download PDFInfo
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Abstract
The invention belongs to the field of reporter gene vectors, and particularly relates to a dual-luciferase reporter gene vector for insect cells, a construction method, a recombinant vector and application. The dual-luciferase reporter gene vector for the insect cells is obtained by modifying a psiCHECK-2 vector; the modification comprises replacing the original SV40 promoter with the OpIE2 promoter and inserting a Synthetic DNA fragment between HSV-TK promoter and Synthetic poly (A); the hRluc gene downstream of the psiCHECK-2 vector is used for inserting a target gene of an interested insect; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1 is shown. The dual-luciferase reporter gene vector for the insect cells ensures strong gene transcription and mRNA processing in the insect cells, and is suitable for the research of the insect gene function.
Description
Technical Field
The invention belongs to the field of reporter gene vectors, and particularly relates to a dual-luciferase reporter gene vector for insect cells, a construction method, a recombinant vector and application.
Background
Insects are the most huge organisms in the world, are numerous and have strong environmental adaptability. Because of the advantages of easily available materials, simple gene operation, obvious genetic phenotype and the like, the gene chip is favored by researchers and plays an important role in various aspects such as biomedicine, insect insecticides, basic research and the like. With the continuous development of molecular biology technology, more than 800 insect cell lines have been established and are commonly used for research on immunofluorescence, protein interaction, regulation of small-molecule RNA on target genes and the like.
MiRNAs are non-coding RNA molecules of about 22 nucleotides (nt) that either degrade the target mRNA or suppress translation, and regulate gene expression at the post-transcriptional level in almost all biological processes. The Untranslated Region (UTR) refers to a segment of mRNA that is not translated into protein at both ends of the coding Region of the mRNA chain, and a segment of mRNA located upstream of the coding Region is called the 5' -Untranslated Region (5' -UTR) and downstream of the coding Region is called the 3' -Untranslated Region. Numerous studies have shown that miRNAs induce mRNA degradation or translational silencing by sequence complementarity to the 3'-UTR, 5' -UTR or genome of target mrnas and are demonstrated to be evolutionarily conserved during the major evolution of eukaryotes. In recent years, a large number of mirnas have been identified and annotated from eukaryotes including insects. They are considered regulators of many biological processes, including cell proliferation differentiation, metabolism, viral infection, cell cycle progression, stress resistance, and longevity prolongation. Potential miRNA or target genes are typically screened by a dual luciferase reporter system.
The luciferase reporter gene system is a method for indirectly screening potential regulatory factors influencing the expression of target genes and quantifying the regulatory activity of the potential regulatory factors by quantifying bioluminescence emitted by the oxidation of luciferin to luciferin under the catalysis of luciferase. In insect cells, the commonly used luciferase reporter assay mainly utilizes a cotransfection method, i.e., a method of transfecting a recombinant luciferase reporter gene plasmid and an internal reference reporter gene to achieve research purposes. If the target promoter sequence is cloned into a PGL3-Basic vector, the activation effect of the endogenous transcription factor on the gene promoter is explored through cotransformation of an internal reference plasmid TK. Similarly, when the targeting relationship between the 3'/5' -UTR and the miRNA is researched, three vectors, namely miRNA over-expression vectors (mics), a recombinant luciferase reporter gene vector containing the 3'/5' -UTR gene sequence and an internal reference vector, need to be co-transformed. The co-rotation experiment has the following defects: first, the length of the full-length 3' -UTR of most target genes varies from several hundred to several thousand bases, and a recombinant reporter vector is constructed. In the co-transfection system, the size and the addition ratio of each vector can have a serious influence on the transfection efficiency of each vector, and the accuracy of the experimental result is probably influenced. Second, co-transfection with multiple vectors increases the amount of transfection reagent used, increases cellular stress and toxicity, and often detects parameters that are not numerically reflected in the normal physiological state of the cell.
The Chinese patent with the publication number of CN111440823B researches the regulation and control mechanism of the brown planthopper Ccdc124 gene and miRNA thereof by constructing a recombinant vector comprising the brown planthopper Ccdc124 gene dual-luciferase reporter gene. The dual-luciferase reporter gene vector used in the method is a Psicheck-2 vector, and the cell line used in the method is a Drosophila S2 cell (embryonic cell). Strictly speaking, the drosophila S2 cell is a model species cell, has strong compatibility, and the physicochemical property, common carrier and expression element are closer to the use of higher animal cells; in addition, most technical approaches of the S2 cell line are not applicable to the conventional insect cell line.
In the prior art, although there is a Psicheck-2 vector design, the change of gene expression can be monitored by fusing firefly and sea cucumber luciferase reporter genes, the vector has almost no activity in insect cells due to the specificity of promoter species, and can not normally start the luciferase gene expression on the vector, so the use of the vector in insect cells is limited.
Disclosure of Invention
The invention aims to provide a dual-luciferase reporter gene vector for insect cells, which is designed aiming at the insect cells, ensures the transcription and mRNA processing of a target gene and is suitable for detecting the targeting relation between a 3' untranslated region of the insect gene and miRNA.
The second purpose of the invention is to provide a construction method of the dual-luciferase reporter gene vector.
The third purpose of the invention is to provide a recombinant vector based on the dual-luciferase reporter gene vector.
The fourth purpose of the invention is to provide the application of the recombinant vector.
In order to achieve the above purpose, the technical scheme adopted by the invention is as follows:
a dual-luciferase reporter gene vector for insect cells is obtained by modifying a psiCHECK-2 vector; the modification comprises replacing the original SV40 promoter with the OpIE2 promoter and inserting a Synthetic DNA fragment between HSV-TK promoter and Synthetic poly (A); the hRluc gene downstream of the psiCHECK-2 vector is used for inserting a target gene of an interested insect; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1 is shown.
The dual-luciferase reporter gene vector for insect cells comprises an OpIE2 promoter, a transcription terminator SV40 polyA late signal, a synthetic DNA element sequence, a holothurian and firefly luciferase reporter gene and a resistance marker ampicillin. Wherein the combination of OpIE2 and SV40 poly A ensures that the target gene is transcribed while ensuring that transcription termination is strictly regulated; the synthetic DNA element can weaken the influence of the OpIE2 promoter on the firefly luciferase, and ensure the powerful gene transcription and mRNA processing in insect cells.
Preferably, the nucleotide sequence of the OpIE2 promoter is as set forth in SEQ ID NO: 2, respectively. Further preferably, the OpIE2 promoter fragment was obtained by PCR amplification of the pIZ/V5-His vector. Experiments prove that the OpIE2 promoter is matched with an SV40 polyA late signal to ensure that a target gene is transcribed in insect cells and transcription termination is strictly regulated.
Still more preferably, the upstream primer and the downstream primer of the PCR amplification are respectively shown in SEQ ID NO: 3. SEQ ID NO: 4, respectively.
The technical scheme of the construction method of the dual-luciferase reporter gene vector is as follows:
a method for constructing the dual-luciferase reporter gene vector for the insect cells comprises the following steps: after the plasmid psiCHECK-2 is subjected to double enzyme digestion by Kpn I and Nhe I, an enzyme digestion product is recovered, an OpIE2 gene fragment is connected with the enzyme digestion product, and then the synthesized DNA fragment is connected between HSV-TK promoter and Synthetic poly (A).
The construction method of the dual-luciferase reporter gene vector for the insect cells is simple and convenient to operate and high in stability, and can be used for researching a specific regulation mechanism of the insect miRNA on the 3' UTR of the target gene in the cells.
The technical scheme of the insect cell dual-luciferase reporter gene recombinant vector is as follows:
an insect cell dual-luciferase reporter gene recombinant vector is characterized in that an insect target gene is inserted into the downstream of the hRluc gene of the dual-luciferase reporter gene vector.
The dual-luciferase reporter gene recombinant vector provided by the invention is used for detecting the targeting relation between the 3' untranslated region of a target gene and miRNA in insect cells; the whole detection process is short in time consumption, good in repeatability and high in accuracy.
Preferably, the insect target gene is an insect gene 3' UTR sequence.
Further preferably, the insect gene 3' UTR sequence is selected from the group consisting of a trehalase gene 3' UTR sequence and a glucose dehydrogenase gene 3' UTR sequence. Experiments prove that the dual-luciferase reporter gene vector can be used for detecting the targeting relationship between miRNA and the target genes, so that the dual-luciferase reporter gene vector is used for researching related regulation and control mechanisms.
The insect cell dual-luciferase reporter gene recombinant vector is applied to the aspect of detecting the targeting relationship between the 3' UTR of an insect gene and miRNA.
The dual-luciferase reporter gene vector can be used for researching a specific regulation mechanism of insect miRNA on the 3' UTR of the target gene in cells; the miRNA gene expression vector can also be used for screening candidate miRNA prediction target genes and researching the regulation mechanism of miRNA in regulating and controlling the predicted target genes on the cellular level.
Preferably, the 3' UTR of the insect gene is a trehalase gene 3' UTR sequence or a glucose dehydrogenase gene 3' UTR sequence, and the miRNA is miRNA-317-5p or miRNA-34-5 p-2.
Drawings
FIG. 1 is a prior art plasmid map of vector psiCHECK-2;
FIG. 2 is a prior art map of the vector pIZ/V5-His plasmid;
FIG. 3 is a plasmid map of the vector pPIZ/3UTR constructed in the present invention;
FIG. 4 is a diagram showing an activity analysis of a pPIZ/3UTR bifluorescent reporter gene in an example of the present invention; (A) the histogram sequentially shows No Mimic, control, miRNA-317-5P 100nM and miRNA-317-5P 200nM from left to right; (B) the histogram sequentially comprises No Mimic, control, miRNA-34-5P-2100 nM and miRNA-317-5P 200nM from left to right; represents p < 0.01; n.s. represents p > 0.05.
Detailed Description
The design of the Psicheck-2 vector can be used for quickly and quantitatively detecting the regulation and control effect of miRNA on the target gene. In the vector, the change of gene expression is monitored by fusing firefly and holothurian luciferase reporter genes, the holothurian luciferase is the main reporter gene, and the foreign gene of interest is cloned to the downstream. When miRNA is combined with target gene, the regulation and control function is started, and the luciferase activity of sea cucumber is correspondingly changed. The vector realizes the expression of the exogenous fragment and the reference gene on the same vector, and is widely applied to researching RNAi, the target relation between the target gene 3' -UTR and miRNA in mammalian cells. However, the psicheck-2 vector does not function in insect cells due to the species specificity of the promoter, limiting the use of this vector.
In the invention, by designing primers OpIE2primer1 and OpIE2primer2, a complete OpIE2 promoter fragment is obtained through amplification, and is connected into a psiCheck-2 vector through double enzyme digestion to replace a partial sequence of the original vector, and meanwhile, a synthetic DNA original sequence is inserted, so that a dual-luciferase reporter gene vector pPIZ/3UTR is constructed.
The main function of the synthetic DNA fragment is to isolate the influence of the promoter on the hluc reporter gene. If the fragment is not inserted, the value of the hluc reporter gene is too high. The higher value directly affects the evaluation of whether miRNA has inhibitory effect on target site. Especially when the binding of miRNA to the target site is weak, it is masked, which affects the result.
The dual-luciferase report vector obtained by the method reduces the number of vectors during co-transfection and improves the transfection efficiency, the experimental repeatability is good, the obtained result is closer to the real situation in vivo than the traditional luciferase report plasmid, the dual-luciferase report vector can be used for detecting the targeting relation between a 3' untranslated region and miRNA, and the research process of miRNA on the target gene regulation mechanism in insect cells can be greatly accelerated.
Specifically, the restriction endonuclease cut OpIE2 promoter gene fragment was ligated into the same restriction endonuclease cut dual-luciferase reporter gene vector by a ligase to screen for the OpIE2/3 UTR. Further, the dual-luciferase reporter gene vector of the 3' untranslated region of the insect gene is inserted with a synthetic DNA sequence, such as SEQ ID NO: 1 is shown. Further, the fragment is obtained by connecting the Synthetic DNA fragment sequence between HSV-TK promoter and Synthetic poly (A) in the vector by a seamless cloning method. The vector was named pPIZ/3 UTR.
Further, the OpIE2 promoter gene fragment was obtained by PCR amplification using primers including: the primers were an OpIE2primer (1/2) containing restriction sites for Kpn I and Nhe I restriction enzymes, respectively.
Further, the dual-luciferase reporter gene vector is psiCHECK-2 subjected to double enzyme digestion by Kpn I and Nhe I restriction enzymes.
Further, the ligase is T4 DNA ligase, and the ligation is performed at 16 ℃ for more than 3 hours.
The targeting relationship between insect miRNAs and the 3'-UTR of the gene can be detected by utilizing the insect gene 3' UTR dual-luciferase reporter gene vector. In the following examples, the regulation mechanism of miRNA (miRNA-317-5p and miRNA-34-5p-2) on insect trehalase and glucose dehydrogenase genes was verified by using the dual-luciferase reporter gene vector described above.
The following describes the practice of the present invention in detail with reference to specific examples.
Example 1 Dual luciferase reporter Gene vectors and Process for constructing the same
The dual-luciferase reporter gene vector of the insect gene 3' UTR is obtained by modifying a psiCHECK-2 vector, and specifically comprises replacing an original SV40 promoter with an OpIE2 promoter and inserting a synthetic DNA fragment. The Synthetic DNA fragment sequence was ligated between HSV-TK promoter and Synthetic poly (A) of the psiCHECK-2 vector; inserting an insect gene 3' UTR sequence into the hRluc downstream of the psiCHECK-2 vector; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1 is shown. The OpIE2 promoter sequence is shown in SEQ ID NO: 2, respectively.
The dual-luciferase reporter gene vector (pPIZ/3UTR) is constructed by using a vector pIZ/V5-His and a vector psiCHECK-2, and the construction method comprises the following steps:
(1) plasmid maps of the vectors psiCHECK-2 and pIZ/V5-His are shown in FIGS. 1 and 2. Based on the sequence analysis of pIZ/V5-His vector, primers OpIE2 fragment, OpIE2primer1 and OpIE2primer2, were synthesized, the upstream and downstream primers of the gene were added with Kpn I and Nhe I cleavage sites and protective bases, respectively, and the sequences of primer oligos amplified from the OpIE2 promoter gene are shown in Table 1 below.
TABLE 1 OpIE2 promoter Gene amplification primer sequences
| OpIE2primer1(SEQ ID NO：3) | GGGGTACCTCATGATGATAAACAATGTATGGTGCTAATG |
| OpIE2primer2(SEQ ID NO：4) | CTAGCTAGC TAAATTCGAACAGATGCTGTTCAACGTT |
The primer is synthesized by Suzhou Hongxn Biotechnology Limited; the primer dry powder synthesized in table 1 was prepared into 10 μ M working solution for PCR amplification, and the reaction system is shown in table 2 below:
TABLE 2 PCR amplification reaction System
| PCR Mix | 10μL |
| OpIE2primer1 | 1μL |
| OpIE2primer2 | 1μL |
| DNA template | 1μL |
| ddH2O | 7μL |
The amplification procedure is shown in table 3 below:
TABLE 3 PCR amplification procedure
After the PCR amplification reaction was completed, the OpIE2 fragment was recovered by Agarose electrophoresis and gel cutting. The OpIE2 gene fragment and the vector were digested with KpnI and Nhe I, respectively, at 37 ℃ for 3 hours, and the double digestion system is shown in the following Table 4:
TABLE 4 double enzyme digestion System
| 10xBuffer | 5μL |
| DNA fragment/vector | 25μL/1μg |
| KpnI | 2μL |
| Nhe I | 2μL |
| ddH2O | Make up to 50. mu.L |
Electrophoresis, recovering the OpIE2 gene fragment and the vector psiCHECK-2 by using a DNA gel recovery kit; the OpIE2 gene fragment obtained by double digestion and the linearized vector psiCHECK-2 were ligated with T4 DNA ligase for more than 3 hours at 16 ℃ as follows:
TABLE 5 connection System
| 10xBuffer | 2μL |
| psiCHECK-2 | 2μL |
| DNA fragment | 8μL |
| T4 DNA ligase | 0.5μL |
| ddH2O | Make up to 20. mu.L |
Transforming the ligation product into DH5 alpha competent cells; selecting clone colonies from the plate, extracting plasmids and identifying to select positive clones; the vector was named OpIE2/3UTR for sequencing validation.
(2) The Synthetic DNA fragment (SEQ ID NO: 2) was ligated into the OpIE2/3UTR vector between HSV-TK promoter and Synthetic poly (A) by means of seamless cloning, and a positive monoclonal colony was picked and verified by sequencing, this vector was designated pPIZ/3UTR, and its vector map is shown in FIG. 3.
Example 2 recombinant vectors and application experiments
The insect cell dual-luciferase reporter gene recombinant vector and the application thereof are specifically described as follows:
2.1 construction of recombinant vectors
The pPIZ/3UTR vector is respectively connected with the trehalase gene of the insect and the 3' UTR sequence of the glucose dehydrogenase gene to construct recombinant plasmids pPIZ/3UTR-Tre and pPIZ/3 UTR-GDH.
(1) 3' UTR sequence:
the sequence of the 3' UTR of the trehalase gene is shown as SEQ ID NO: 5, respectively.
The 3' UTR sequence of the glucose dehydrogenase gene is shown as SEQ ID NO: and 6.
(2) Vector construction:
the recombinant plasmids pPIZ/3UTR-Tre and pPIZ/3UTR-GDH were constructed in a manner similar to that described in example 1, and the trehalase gene and the glucose dehydrogenase gene 3' UTR fragments were obtained by PCR amplification. The above fragments were then cleaved and ligated into the pPIZ/3UTR vector, respectively, with the restriction enzymes Pme I (F) and Not I (R).
2.2 cell transfection experiments
Sf9 cells with good growth state are divided into 5X 10 cells4The prepared recombinant dual-luciferase reporter gene vector is co-transfected with miRNA mimics (100/200nM) or negative control (200nM) according to the instructions of the transfection reagent.
Artificially synthesizing miR-317-5 p: 5-CGGGUGCCACGCUGUGCUCUCU-3(SEQ ID NO: 7); miR-34-5 p: 5-UGGCAGUGUGGUUAGCUGGUUGUG-3(SEQ ID NO: 8) and an NC control, the nucleic acid sequence being as follows, the NC control: 5-GCGACGAUCUGCCUAAGAUTT-3(SEQ ID NO: 9), and miRNA mimics was synthesized by Shanghai Jima pharmaceutical technology, Inc.
The method comprises the following specific steps:
(1) the Sf9 cells are proliferated to 80-90%, washed for 3 times by PBS, added with complete culture medium, blown and beaten uniformly, inoculated into a 96-well plate with 0.1mL per well, and continuously cultured for 24 hours; miR-317-5p or miRNA-34-5p-2 mices is diluted to 10 mu M.
(2) mu.L Grace medium and 10ng pPIZ/3UTR-Tre or pPIZ/3UTR-GDH vector were added to a 1.5mL centrifuge tube. Then, adding negative control, 100nM (final concentration) single-chain miR-317-5p/miRNA-34-5p-2mimics, and adding 20 ul Grace culture medium and 2 ul new 1.5mL centrifuge tube
Respectively mixing HD transfection reagents, standing for 5min, mixing the two components, and standing at room temperature for 15 min; and (3) replacing the old culture medium in the plate hole with a new antibiotic-free culture medium, slightly dropping the well-standing transfection system into the plate hole, slightly shaking uniformly, and replacing with a complete culture medium after 5 hours.
Each set of experiments was repeated 3 times. Transfected cells were grouped as follows: no mic group: transfecting pPIZ/3UTR-Tre or pPIZ/3 UTR-GDH; ② (-) Control group: cotransfection with pPIZ/3UTR-Tre + NC or pPIZ/3UTR-GDH + NC (negative control); ③ 100 groups: co-transfecting pPIZ/3UTR-Tre +100nM mimics or pPIZ/3UTR-GDH +100nM mimics; 200 groups: and (3) co-transfecting pPIZ/3UTR-Tre +200nM mimics or pPIZ/3UTR-GDH +200nM mimics.
2.2 measurement and statistical analysis of Dual luciferase Activity
Cells were harvested 48h after transfection and intracellular dual luciferase reporter activity was detected. Each set of experiments was repeated 3 times.
The method comprises the following specific steps: removing old cell culture medium, washing with PBS for 2 times, adding 100 μ L of 1 XPLB (lysis buffer), and shaking in horizontal shaker at room temperature for 30min to fully lyse cells; adding 50 mul of the lysis solution and 100uL of LARII (luciferase detection Reagent II) into an enzyme label plate, uniformly mixing, detecting the activity of firefly luciferase, quickly adding 100 mul of Stop and Glo Reagent into an original tube after detection, uniformly mixing, detecting the activity of the sea cucumber luciferase, and calculating the ratio of the two.
P <0.05 was significant for all statistical analyses. A one-way ANOVA (one-way ANOVA) was performed for all statistical significance using SPSS software, and each set of experiments was repeated 3 times. Represents p < 0.01; n.s. represents p > 0.05.
As can be seen from the dual-luciferase activity detection results in fig. 4, the luciferase activity of the Sf9 cell group of the co-transformed miRNA mimics group is significantly reduced by about 50% (P <0.01) compared with that of the other groups, and the two groups have a statistically significant difference, and no significant difference exists between the control groups.
<110> Xinxiang medical college
<120> dual-luciferase reporter gene vector for insect cells, construction method, recombinant vector and application
<160> 9
<170> PatentIn version 3.5
<210> 1
<211> 368
<212> DNA
<213> Artificial sequence
<221> synthetic DNA fragment
<400> 1
ctcccctgtg aggaactact gtcttcacgc agaaagcgtc tagccatggc gttagtatga 60
gtgtcgtgca gcctccaggc ccccccctcc cgggagagcc atagtggtct gcggaaccgg 120
tgagtacacc ggaattgcca ggacgaccgg gtcctttctt ggatcaatcc cgctcaatgc 180
ctggagattt gggcgtgccc ccgcgagact gctagccgag tagtgttggg tcgcgaaagg 240
ccttgtggta ctgcctgata gggtgcttgc gagtgccccg ggaggtctcg tagaccgtgc 300
accatgagca caaatcctaa acctcaaaga aaaaccaaac gtaacaccaa ccgccgccca 360
caggacgt 368
<210> 2
<211> 559
<212> DNA
<213> moth nuclear polyhedrosis virus (Orgyia pseudomegata multiple nucleopoliohydrovirus)
<221> OpIE2 promoter
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ggatcatgat gataaacaat gtatggtgct aatgttgctt caacaacaat tctgttgaac 60
tgtgttttca tgtttgccaa caagcacctt tatactcggt ggcctcccca ccaccaactt 120
ttttgcactg caaaaaaaca gcttttgcac gcgggcccat acatagtaca aactcacgtt 180
tcgtagacta ttttacataa atagtctaca ccgttgtata cgctccaaat acactaccac 240
acattgaacc tttttgcagt gcaaaaaagt acgtgtcggc agtcacgtag gccggcctta 300
tcgggtcgcg tcctgtcacg tacgaatcac attatcggac cggacgagtg ttgtcttatc 360
gtgacaggac gccagcttcc tgtgttgcta accgcagccg ggacgcaact ccttatcgga 420
acaggacgcg cctccatatc agccgcgcgt tatctcatgc gcgtgaccgg acacgaggcg 480
cccgtcccgc ttatcgcgcc tataaataca gcccgcaacg atctggtaaa caacgttgaa 540
cagcatctgt tcgaattta 559
<210> 3
<211> 39
<212> DNA
<213> Artificial sequence
<221> OpIE2 primer1
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<213> Artificial sequence
<221> OpIE2 primer2
<400> 4
ctagctagct aaattcgaac agatgctgtt caacgtt 37
<210> 5
<211> 419
<212> DNA
<213> Cotton bollworm (Helicoverpa armigera)
<221> trehalase gene 3' UTR sequence
<400> 5
aattcttcca tttactgcgg cggggatctc ctccggactg tgcagctcgc tagaatattt 60
cccgactcga agactttcgt tgatctgaga cttgcaaaca gcgaaattga aactttggcc 120
aattttacat attttatgaa gaaaacagac aataaaccat ctcgagaaca aattaggtca 180
ttcgtagatg ctaattttat cgatggagat gagcttttta gctggcaccc gcctgatttt 240
gaccctaacc caccagttct taatgagatt gctgatccga aactgagaca attcgcgaag 300
gatatcatca gtatatggcc aggactcggc aggaaagtgt cgccagatgt tcatcgtaac 360
tctgaacaat acagttttat ctacgtgcct aacggattca ttgttcctgg cgggaggtt 419
<210> 6
<211> 322
<212> DNA
<213> Helicoverpa armigera (Helicoverpa armigera)
<221> glucose dehydrogenase Gene 3' UTR
<400> 6
ctgtggaagt caggaacagc tttggtggaa ctgagaccat ttttgctaac aatgagatta 60
ttttgagcgc cggagcagta gcgtctcctc aaatcctgca actaagcggg gtgggtgaac 120
cgtccctgct gacccgcgtc ggagtgcggc cggtgcacgc actgccgggc gtggggcgca 180
acctgcacaa ccacgtggcg cacttcctca acttcagagt tagtgacaat aacaccacgc 240
cgctcaactg ggccacggcc atggagtacc tgctgttcag ggatggactt atgtcgggga 300
ctggcatatc agaagtgacg gg 322
<210> 7
<211> 22
<212> RNA
<213> Artificial sequence
<221> miR-317-5p
<400> 7
cgggugccac gcugugcucu cu 22
<210> 8
<211> 22
<212> RNA
<213> Artificial sequence
<221> miRNA-34-5p-2
<400> 8
uggcagugug guuagcuggu ugug 24
<210> 9
<211> 21
<212> RNA
<213> Artificial sequence
<221> NC comparison
<400> 9
gcgacgaucu gccuaagaut t 21
Claims (10)
1. A dual-luciferase reporter gene vector for insect cells is characterized in that the vector is obtained by modifying a psiCHECK-2 vector; the modification comprises replacing the original SV40 promoter with the OpIE2 promoter and inserting a Synthetic DNA fragment between HSV-TK promoter and Synthetic poly (A); the hRluc gene downstream of the psiCHECK-2 vector is used for inserting a target gene of an interested insect; the nucleotide sequence of the synthesized DNA fragment is shown as SEQ ID NO: 1 is shown.
2. The dual-luciferase reporter vector for insect cells of claim 1, wherein the nucleotide sequence of the OpIE2 promoter is as set forth in SEQ ID NO: 2, respectively.
3. The dual-luciferase reporter vector for insect cells as claimed in claim 2, wherein the OpIE2 promoter fragment is obtained by PCR amplification of pIZ/V5-His vector.
4. The dual-luciferase reporter vector for insect cells as claimed in claim 3, wherein the upstream primer and the downstream primer of the PCR amplification are respectively shown in SEQ ID NO: 3. SEQ ID NO: 4, respectively.
5. A method for constructing a dual-luciferase reporter vector for insect cells according to any one of claims 1 to 4, comprising the steps of: after the plasmid psiCHECK-2 is subjected to double enzyme digestion by Kpn I and Nhe I, an enzyme digestion product is recovered, an OpIE2 gene fragment is connected with the enzyme digestion product, and then the synthesized DNA fragment is connected between HSV-TK promoter and Synthetic poly (A).
6. An insect cell dual-luciferase reporter gene recombinant vector, which is characterized in that an insect target gene is inserted into the downstream of the hRluc gene of the dual-luciferase reporter gene vector as claimed in any one of claims 1 to 4.
7. The insect cell dual-luciferase reporter recombinant vector of claim 6, wherein the insect target gene is an insect gene 3' UTR sequence.
8. The insect cell dual-luciferase reporter recombinant vector according to claim 7, wherein the insect gene 3' UTR sequence is selected from a trehalase gene 3' UTR sequence or a glucose dehydrogenase gene 3' UTR sequence.
9. The application of the insect cell dual-luciferase reporter gene recombinant vector as defined in any one of claims 6-8 in detecting the targeting relationship between the 3' UTR of an insect gene and miRNA.
10. The use of claim 9, wherein the insect gene 3' UTR is a trehalase gene 3' UTR sequence or a glucose dehydrogenase gene 3' UTR sequence and the miRNA is miRNA-317-5p or miRNA-34-5 p-2.
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