CN109467595B - Application of transcription factor MyoD in regulation and control of pig RTL1 gene expression - Google Patents
Application of transcription factor MyoD in regulation and control of pig RTL1 gene expression Download PDFInfo
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Abstract
The invention provides application of a transcription factor MyoD in regulation and control of pig RTL1 gene expression. With RTL1 as a research object, a core promoter region of RTL1 is found by constructing a porcine RTL1 gene promoter dual-luciferase reporter gene recombinant plasmid; then verifying the interaction between the transcription factor MyoD and the core promoter region of RTL 1; then constructing a MyoD overexpression vector and synthesizing small interfering RNA, and detecting the influence of MyoD on RTL 1; and constructing a mutant fluorescent expression vector MyoD-mut of the transcription factor MyoD binding site to transfect PK15 and C2C12 cells and then detecting luciferase activity. The invention proves the influence of the transcription factor MyoD on the RTL1 gene transcription regulation for the first time, brings deeper cognition for the expression regulation of RTL1, and has better application prospect when being applied to the improvement of livestock meat quality traits and the research of a muscle development molecular regulation mechanism.
Description
Technical Field
The invention relates to the technical field of genetic engineering, in particular to application of a transcription factor MyoD in regulation and control of pig RTL1 gene expression.
Background
The RTL1 gene (retrotransposon 1 gene, retrotransposon-like-1), also known as paternally expressed gene 11(paternally expressed gene 11, Peg11), the imprinted gene expressed paternally in humans and mice, has no intron sequences. The expression level of the RTL1 gene in embryo and placenta tissues is very high, which plays an important role in nutrition transmission of maternal placenta and fetus, and mice with the gene knocked out have the symptoms of high fetal death rate, newborn growth retardation and small placenta size; it is also closely related to human fetal development and neonatal growth. The gene is positioned in a DLK1-DIO3 imprinting area, the gene in the area has certain influence on muscle development, and the ectopic expression of the RTL1 gene in a transgenic mouse can cause muscle hypertrophy; furthermore, the gene was associated with muscle hypertrophy in callyphge sheep, and the expression level of RTL1 gene was 12-fold higher in the double-gluteal sheep than in the normal sheep.
The research on gene expression regulation is one of the hot spots in current molecular biology research, the regulation of transcription level is the most important first step in the gene expression process, and transcription factors are protein molecules which can be specifically combined with specific sequences of genes so as to regulate the expression of target genes. The transcription factor is directly or indirectly recognized and combined on the core sequence of the cis-acting element in the process of transcriptional regulation and is involved in regulating the transcription of target genes. Therefore, screening and identifying the transcription factor of the gene becomes the most important step for researching the regulation and control of gene expression, and lays a foundation for researching the regulation and control of gene expression. However, no report is found on the regulation of the RTL1 gene.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides the application of the transcription factor MyoD in regulation and control of the expression of the RTL1 gene of a pig, and firstly proves the influence of the transcription factor MyoD on the transcription and control of the RTL1 gene: a core promoter region of the RTL1 gene is found through a dual-luciferase report system, further, the interaction between the transcription factor MyoD and the core promoter region of the RTL1 gene is verified by utilizing a chromatin immunoprecipitation technology (ChIP), a MyoD overexpression vector and a synthetic small interfering RNA (MyoD-siRNA) are constructed to detect the influence of MyoD on RTL1, and luciferase activity is detected after a transcription factor MyoD binding site mutant type fluorescence expression vector MyoD-mut is constructed to transfect PK15 cells and C2C12 cells.
To achieve the above object, the present invention is realized by:
one of the purposes of the invention is to provide the application of the transcription factor MyoD in regulation and control of the expression of the RTL1 gene of the pig. The transcription factor MyoD has an inhibiting effect on the promoter activity of the RTL1 gene, so that the expression of the RTL1 gene is down-regulated.
The invention also aims to provide siRNA for inhibiting expression of a transcription factor MyoD, which is characterized by comprising at least one of the following three siRNAs:
siRNA1, wherein the nucleotide sequence of a sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 2;
siRNA2, wherein the nucleotide sequence of a sense strand is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 4;
the nucleotide sequence of the sense strand of the siRNA3 is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense strand thereof is shown as SEQ ID NO. 6.
Preferably, the siRNA is siRNA 2.
Specifically, dTdT is suspended at the 3' end of each of the siRNA1, siRNA2 and siRNA3 sequences.
The invention also aims to provide application of the siRNA for inhibiting expression of the transcription factor MyoD in promotion of expression of the RTL1 gene of the pig.
The fourth purpose of the invention is to provide a binding site mutant vector of the transcription factor MyoD and RTL1 genes, which comprises a nucleotide sequence shown as SEQ ID NO. 7.
The fifth purpose of the invention is to provide a super expression vector of the transcription factor MyoD, and the super expression vector comprises a CDS region of the transcription factor MyoD.
Specifically, the CDS region of MyoD is connected into a pcDNA3.1 eukaryotic expression vector to construct a transcription factor overexpression vector pc-MyoD.
The sixth purpose of the invention is to provide the application of the overexpression vector in inhibiting the expression of the porcine RTL1 gene.
The invention has the beneficial effects that:
1. in the research of the regulation mechanism of the porcine RTL1 gene, the inventor finds that the transcription factor MyoD can interact with a promoter region at the upstream of the RTL1 gene so as to down-regulate the expression of the RTL1 gene. The invention firstly proves the influence of the transcription factor MyoD on the transcription regulation of RTL1 gene: a core promoter region of the RTL1 gene is found through a dual-luciferase report system, further, the interaction between the transcription factor MyoD and the core promoter region of the RTL1 gene is verified by utilizing a chromatin immunoprecipitation technology (ChIP), a MyoD overexpression vector and a synthetic small interfering RNA (MyoD-siRNA) are constructed to detect the influence of MyoD on RTL1, and luciferase activity is detected after a transcription factor MyoD binding site mutant type fluorescence expression vector MyoD-mut is constructed to transfect PK15 cells and C2C12 cells. Brings deeper cognition for the expression regulation of RTL1, is applied to the improvement of livestock meat quality traits and the research of a muscle development molecular regulation mechanism, and has better application prospect.
2. The technical scheme of the invention is thoroughly designed and has reliable results. In order to confirm the effect of the transcription factor MyoD on the transcription regulation effect of the RTL1 gene and the function of cells, the invention is verified from multiple levels and angles and verified on the transcription activity, the mRNA level and the protein level.
Drawings
FIG. 1 shows the results of measurement of relative luciferase activity of pGL3-P1, pGL3-P2, pGL3-P3, pGL3-P4, pGL3-P5, and pGL3-Basic transfected C2C12 cells in example 1 for 24 hours, respectively; in the figure: the ordinate of the relative luciferase activity represents the relative luciferase activity value, and Basic is a blank control;
FIG. 2 is a PCR electrophoretogram of chromatin co-immunoprecipitation (ChIP) after binding of transcription factor MyoD to core promoter of RTL1 gene in example 2;
FIG. 3 shows the results of detecting relative luciferase activities of the transcription factor MyoD binding site mutant fluorescent expression vectors MyoD-mut and pGL3-P2 wild-type vector and pGL3Basic transfected PK15 cells and C2C12 cells for 24h, respectively, in example 3; in the figure: the ordinate relative luciferase activity represents the relative luciferase activity value, P2 is the control group,. P < 0.01;
FIG. 4 shows the results of luciferase activity assay of pGL3-P2 co-transfected in PK15 cells for 24h with overexpression vector pc-MyoD of transcription factor MyoD and pcDNA3.1 blank control, respectively, in example 4; in the figure: the ordinate Related Luciferase Activity represents relative Luciferase Activity value, pcDNA3.1 is blank control, P is less than 0.01;
FIG. 5 shows the effect of overexpression vector pc-MyoD of transcription factor MyoD on the expression level of RTL1 gene in example 4;
FIG. 6 is a graph showing the effect of fluorescent quantitative PCR detection on the interference of MyoD gene mRNA levels after siRNA transfection in example 5;
FIG. 7 is a graph showing the effect of fluorescent quantitative PCR detection on the promoter activity of RTL1 gene after transfection of siRNA in example 5;
FIG. 8 is a graph showing the effect of western-blot assay on the interference of RTL1 gene protein level after transfection of siRNA in example 5.
Detailed Description
Example 1 determination of the core promoter region of the porcine RTL1 Gene
1. Biological information analysis of pig RTL1 gene promoter region
Primers RPF1 (shown as SEQ ID NO: 9) and RPR1 (shown as SEQ ID NO: 10) are designed according to the sequence of the porcine RTL1 gene in NCBI, and the nucleotide sequence (shown as SEQ ID NO: 8) of 1475bp (-1394/+81) including a part of the first exon of the RTL1 gene is cloned.
The online website (http:// www.fruitfly.org/seq _ tools/promoter. html) is used to predict that the segment of-1027 bp to-978 bp may be the core promoter region of the gene.
2. Primer design
For further verification, 5 upstream deletion fragment primers (P1F-P5F) for amplifying and obtaining 5 deletion fragments (P1-P5) of the promoter region and 1 common downstream Primer PR are designed by using the Primer5 software, 5 'end of each upstream Primer is added with Mlu I (a ↓) cgcgcgt cleavage site and protection base CG, 5' end of the downstream Primer is added with Xho I (C ↓) TCGAG cleavage site and protection base CC (underlined part represents cleavage site), and the sequences are as following table 1:
TABLE 1
3. Amplification of promoter deletion fragments and PCR product recovery
The designed primers P1F to P5F are respectively used for forming a primer pair with PR, the nucleotide sequence (shown as SEQ ID NO: 8) obtained by cloning is used as a template, Touch-down PCR strategy amplification is adopted for PCR amplification, 5 deletion fragments with different lengths of the promoter are obtained, the PCR products (the product lengths are 1394bp, 1046bp, 940bp, 529bp and 205bp respectively) are cut by a gel recovery kit of Omega Bio-tek company for recovery, and the specific operation steps are carried out according to the kit specification.
4. Construction of a promoter-deleted fragment luciferase reporter vector
And (3) recovering the 5 segments by agarose gel, sequentially carrying out Mlu I and Xho I double enzyme digestion on the 5 segments and pGL3-Basic vectors, connecting the deleted segments into the pGL3-Basic vectors by using T4 ligase, cloning, extracting plasmids, sequencing to determine the correctness of the plasmids, and naming the plasmids as P1-P5. The P1-P5 recombinant plasmid is extracted by using an endotoxin-removing plasmid miniprep extraction kit (Omega E.Z.N.A.TM. Endo-Free plasmid Mini KitISpin), and the specific steps are shown in the kit specification.
5. Transfection of C2C12 cells with luciferase reporter vectors
Sequentially transferring the constructed vector P1-P5 into a C2C12 cell, taking pGL3-Basic as a negative control, determining the activity of dual-luciferase after transfection for 24h, and determining a core promoter region, wherein the specific steps are as follows:
(1) 1 day before transfection, at 1X105C2C12 cells are inoculated in a 24-hole plate at the density, and the culture is continued until the cell density reaches 80%;
(2) adding 0.8. mu.g plasmid transfected into each well into 50. mu.L OPTI-MEM culture medium, mixing, adding 2.0. mu.L Lipofectamine 2000 and 1/20. mu.g pRL-TK into 50. mu.L OPTI-MEM culture medium, mixing, and standing at room temperature for 5 min;
(3) and (3) uniformly mixing the two mixed solutions in the step (2), and standing at room temperature for 20 min.
(4) During this time, the original cell culture medium in each well was aspirated and washed twice with OPTI-MEM.
(5) The mixture in (3) was added to 100. mu.L of each well of cells, and the mixture was made up to 500. mu.L with OPTI-MEM.
(6) Culturing at 37 deg.C in 5% CO2 cell culture box for 24h, collecting cells, lysing the cells with 1 XPLB (passive lysis buffer), storing the obtained cell lysate in-70 deg.C refrigerator, and detecting.
6. Luciferase Activity detection
Using Promega corporation
The reporters assay System kit detects the relative fluorescence activity of the fluorescent carrier in a functional microplate reader. Luciferase assay buffer II (LARII) and Stop were performed prior to the dual luciferase assay&The Glo Reagent is balanced to the room temperature, 10 mu L of cell lysate is absorbed and added into an enzyme label plate, 50 mu L of LARII is added to read the activity value A of the firefly luciferase in the sample, and 50 mu L of Stop is added&Glo Reagent reads the activity value B of renilla luciferase in the sample. The A/B ratio represents the activity of the fluorescent carrier, and each group is provided with 3 repeats.
The single-factor variance analysis is carried out on the obtained result by adopting SAS8.0 software, the difference is obvious when P is less than 0.05, and the difference is extremely obvious when P is less than 0.01. The results of measuring the relative fluorescence activity of the 5 promoter-deleted fragments are shown in FIG. 1. The activity of P2(-1084/+81, as shown in SEQ ID NO: 17) was the highest, indicating that this region has a core promoter region, consistent with the results predicted by the web site NNPP.
Example 2 determination of the binding of the transcription factor MyoD to the core promoter of the RTL1 Gene
1. Biological information analysis
Potential transcription factor binding sites of a promoter region are predicted by using a TESS website (http:// www.cbil.upenn.edu/cgi-bin/TESS/TESS) and a TFSEARCH (http:// www.cbrc.jp/research/db/TFSEARCH. html) website, and a transcription factor MyoD binding site with a higher score is found to be (-458bp/-453 bp).
2. Co-immunoprecipitation
The binding of the transcription factor MyoD to the RTL1 promoter in an in vivo environment was tested by chromatin co-immunoprecipitation (ChIP) assay of PK15 cells. The EZ-ChIPTM kit of Millipore company in the United states is adopted, and the specific operation process refers to the kit use instruction. The method mainly comprises the following steps:
(1) culturing and collecting PK15 cells for later use, treating PK15 cell genome by ultrasonic disruption, disrupting the cells on ice to make chromatin DNA become 200-and 1000-bp fragments, centrifuging at 12000r/min at 4 ℃ for 10min, and removing insoluble substances;
(2) chromatin immunoprecipitation: each 100ul of the lysate contains 1x106900uL of ChIP dilution buffer containing protease inhibitor and 60uL of protein G-agarose are added into each cell, the cells are subjected to rotary incubation at 4 ℃ for 60min, the cells are centrifuged at 5000r/min for 1min to obtain agarose particles (to remove non-specifically bound molecules), the supernatant is transferred to a new centrifuge tube, and 10uL (1%) is taken out to serve as an input control. Adding corresponding antibodies into the remaining supernatant, wherein an IgG antibody group is added as a negative control group, and PK15 cell DNA (Input group) which is not treated by the antibodies after being crushed is used as a positive control group; the MyoD antibody group was added as an experimental group, incubated overnight at 4 ℃ with rotation, then 60uL of protein G-Sepharose was added, incubated at 4 ℃ with rotation for 60min (to collect antibody/transcription factor complexes), 5000r/min, and the supernatant (containing unbound and non-specific DNA) was carefully removed. And finally washing the protein G/antibody/transcription factor/DNA complex by using a low-salt washing buffer solution, a high-salt washing buffer solution, a LiCl washing buffer solution and a TE buffer solution.
(3) And (3) performing crosslinking release and DNA purification: adding 200uL of elution buffer solution into the compound, flicking the tube wall, uniformly mixing, standing at room temperature for 15min, centrifuging at 5000r/min for 1min, collecting supernatant, adding 5mol/L NaCl, carrying out water bath at 65 ℃ for 5h to obtain free DNA through crosslinking, adding RNaseA, incubating at 37 ℃ for 30min, adding 0.5mol/L EDTA, 1mol/L Tris-HCl (pH 6.5) and proteinase K, and incubating at 45 ℃ for 60 min; and (3) purifying by using a kit and a centrifugal column to obtain DNA.
(4) PCR identification
The PCR primers used to detect the MyoD binding site were:
TABLE 2
A20 uL reaction system was used. The reaction conditions are as follows: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s, for 32 cycles; extension at 72 ℃ for 10 min. The amplification product is sent for sequencing.
3. Results of the experiment
Analyzing the sequence of the RTL1 gene promoter region, selecting a conserved region and a sequence containing a predicted MyoD transcription factor binding site to design a PCR primer, and taking an immunoprecipitated DNA fragment as a template, wherein the target fragment is 120 bp. As shown in FIG. 2, the results show that the experimental group and the Input control both have the target amplified band, and the negative control group does not detect the target amplified band, which indicates that the experimental results are correct and reliable. And (3) performing PCR amplification by using DNA of MyoD antibody immunoprecipitation as a template to obtain a target amplification band, and sequencing and identifying the sequence of an amplification product to be correct, thereby confirming that the MyoD transcription factor in the PK15 cell can be combined with the sequence of the RTL1 gene promoter region.
The experimental results prove that: in an in vivo environment, the transcription factor MyoD can specifically bind to the RTL1 promoter. Indicating that the transcription factor MyoD is involved in the expression regulation of the RTL1 gene.
Example 3 detection of Activity of transcription factor MyoD binding site mutant luciferase reporter vectors
Firstly, construction of mutant vector MyoD-mut
A wild-type P2 vector is used as a template, and a recombination PCR rapid gene site-directed mutation method is used for constructing a transcription factor MyoD binding site mutation vector MyoD-mut.
1. Design of binding site mutation primers
A wild-type P2 vector is used as a template, mutant primers are designed aiming at a MyoD binding site CACCTG as follows, base groups after mutation are underlined, an upstream primer and a downstream primer are reversely complementary, and 47bp sequences are overlapped. The upstream primer (MyoD-mut-F) and the downstream primer (MyoD-mut-R) are as follows:
TABLE 3
2. Amplification of overlapping fragments
And amplifying a fragment F1 by using the primer P2F as an upstream primer and MyoD-mut-R as a downstream primer, amplifying a fragment F2 by using MyoD-mut-F as an upstream primer and PR as a downstream primer, wherein the overlapping part of the fragment F1 and the fragment F2 is the overlapping part of the primer MyoD-mut-F and the MyoD-mut-R. The sequence of the wild-type P2 carrier after mutation is shown in SEQ ID NO. 7.
3. Construction of binding site mutation vector
And connecting the fragments F1 and F2 by fusion PCR, and connecting the fragments into a whole to complete the construction of the transcription factor MyoD binding site mutation vector.
Two, two luciferase activity detection
The wild-type vector and the mutant-type vector MyoD-mut are transfected into PK15 cells and C2C12 cells respectively in a transient transfection mode, and dual-luciferase activity detection is carried out after 24 hours.
Three, result in
As shown in FIG. 3, in both cells, MyoD binding site mutation significantly enhanced promoter activity (P < 0.01), and MyoD inhibited promoter activity.
Experimental example 4 construction of transcription factor MyoD overexpression vector and regulation and control effect on RTL1
Construction of transcription factor MyoD overexpression vector and regulation and control effect on fluorescence activity of RTL1 core promoter
According to CDS sequence of MyoD gene (GeneBank accession No.: GU249575.1) of pig, a primer is designed: corresponding restriction enzyme cutting sites and protective bases are respectively added at the 5' ends of the upstream and downstream primers, and meanwhile, in order to ensure the expression efficiency of the vector, a Kozaka sequence GCCACC is added in front of the translation initiation site. The construction of the MyoD overexpression vector uses KpnI and EcoR I as endonucleases. The cDNA of the large white pig muscle tissue is taken as a template, the CDS region of MyoD is connected into a pcDNA3.1 eukaryotic expression vector, and an over-expression vector pc-MyoD of the transcription factor MyoD is constructed. The overexpression vector construction primers are as follows:
TABLE 4
The pc-MyoD and the core promoter reporter gene vector P2 were co-transfected, and 24h after transfection with P2 as a control, the change in promoter activity was detected using a dual-luciferase reporter system. As a result, as shown in FIG. 4, the promoter activity of RTL1 gene was significantly decreased (P < 0.01) after MyoD overexpression, indicating that MyoD can inhibit the promoter activity of RTL1 gene.
Secondly, the influence of the transcription factor MyoD overexpression vector on the RTL1 gene expression level
The constructed transcription factor over-expression vector pc-MyoD is transferred into a skeletal muscle satellite cell of a pig through transient transfection, a cell transferred into an empty vector pcDNA3.1 is used as a negative control, and the cell is collected, protein is extracted and the expression quantity of the RTL1 gene is detected 48 hours after the transfection. The results are shown in FIG. 5, and show that the expression level of RTL1 gene is significantly reduced (P < 0.01) after Myod is overexpressed, which indicates that Myod has an inhibitory effect on the expression of RTL1 gene.
Example 5 MyoD-siRNA
1. Design and synthesis of MyoD-siRNA
According to the sequence of the transcription factor MyoD in Genbank, 3 pieces of siRNA (siRNA-1, siRNA-2 and siRNA-3) are designed aiming at different target sites of the transcription factor MyoD gene by using BLOCK-iT RNAi Designer software (http:// rnaidesigner. thermofisher. com/rnainexpress/sort. do), and siRNA Negative Control (NC) is designed, wherein the sequence is synthesized by Sharp Biotech, Inc. of Guangzhou.
TABLE 5
2. Transfection of siRNA
Separately transfecting porcine PK15 cells with riboFECTTM CP from Ruibo; one day before transfection, cells were plated at 1X105The density of individual cells/well was seeded in 6-well cell culture plates; mu.l of 20. mu.M siRNA was diluted with 120. mu.l of 1 XriboFECTTM CP Buffer and gently mixed; adding 12 μ l riboFECTTM CP Reagent, gently blowing, mixing, and standing at room temperature for 15 min; cells in 6-well plates were washed 3 times with PBS, PBS was removed, the mixture was seeded onto cell plates, and complete medium was added to make up to a final volume of 2ml per well.
3. Fluorescent quantitative PCR detection of interference efficiency of siRNA to MyoD mRNA level
Will interfere with the group(siRNA-1, siRNA-2, siRNA-3) and negative control group (NC) transfect PK15 cells respectively, collect cells after 24h of transfection, extract total RNA by Trizo1, reverse transcribe by TaKaRa Primer Script RT reverse transcription kit, perform fluorescent quantitative PCR experiment by adopting SYBR Green fluorescent dye of Bio-Rad company, LightCycler480 fluorescent quantitative PCR instrument of Roche company and taking beta-actin as internal reference, and perform 2 steps of fluorescence quantitative PCR experiment by using 2 steps of-ΔΔCtThe method calculates the relative mRNA expression of each gene, 3 replicates for each experiment, three replicates for each experiment: the reaction system is 20 mu 1; reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, denaturation at 95 ℃ for 15sec, annealing at 60 ℃ for 15sec, and annealing at 72 ℃ for 15sec for 40 cycles.
The results show that: as shown in FIG. 6, compared with negative control group (NC) siRNA-1 to siRNA-3, siRNA-2 has the best interference effect, so that siRNA-2 is selected for subsequent experiments in the embodiment of the invention.
4. After siRNA transfection, fluorescent quantitative PCR detection on the effect of RTL1 gene promoter activity
The siRNA-2 and the core promoter reporter gene vector P2 were co-transfected into PK15 cells by transient transfection, and the siRNA-2 was not transfected as a negative control, and the change in promoter activity was detected by using a dual-luciferase reporter system. After 24h, dual-luciferase activity detection is carried out, and the result is shown in figure 7, wherein siRNA-2 has promotion effect on promoter activity.
5. WesternBlot test for inhibition of siRNA to RTL1 protein expression
Respectively transfecting C2C12 cells to an interference group (siRNA-1) and a negative control group (NC), collecting the cells after transfection for 48 hours, extracting protein by RIPA lysate (P0013B) of Biyunstian biotechnology company, and measuring the protein concentration by using a BCA protein concentration measuring kit, wherein the specific method refers to the instruction of the kit. Adding the protein solution into 5 Xprotein loading buffer solution according to the ratio of 4:1, performing boiling water bath denaturation for 15min, leveling the loading amount, and running gel by SDS-polyacrylamide gel electrophoresis. The electrophoresis is stopped when bromophenol blue just runs out, the membrane is rotated for 1 hour at 200mA, then the antibody is incubated, the primary antibody is incubated for 3 hours, the secondary antibody is incubated for 30 minutes, and the coloration exposure is carried out. The results are shown in FIG. 8, which indicate that siRNA-2 is able to promote the expression of RTL1 protein. The siRNA interferes the expression of MyoD, and the transcription factor MyoD can down-regulate the expression of RTL1 gene, so that the expression of RTL1 gene is up-regulated after MyoD is interfered.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> institute of zootechnics of academy of agricultural sciences of Hubei province
Application of <120> transcription factor MyoD in regulation and control of pig RTL1 gene expression
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<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
uugacuuugc ucaagcggc 19
<210> 5
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gcaaacgcaa gaccacuaa 19
<210> 6
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
uuaguggucu ugcguuugc 19
<210> 7
<211> 1046
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
cccaagaaca aggggggcag tctgagcaac gcggagcttt taggaggggt ggggaactga 60
tgccaggatc tcctcgacag ctcagagcgg gggagaacgc tgcctggaag cggcccagag 120
aggggaaagg gaaaagtgag caaaaattcc gatgtggaat cgagtcatgt atgcgcatgt 180
ctctggcaca cagtaggtgt ttttatcaag gtttaaaggg gaaaaaaaga aaagccagaa 240
tccagattcc ctacatcagc agtccctcaa tttgaatcca ggagaggaaa caagcgctat 300
ttggggcggc tgacccgacc gacgggcgag cggtcaaagg cccattttct cttcttgccc 360
tgagcctgtc gaagggcagg catccgggct agagattccc acctccgacc agggaggccc 420
ggcctgccag gggtggcttt ggacggcacc gagaggcgac ctctgtgact tgcggctgac 480
cagagaaggg agggcagatt gaccgggcac gtgtgccagg gttgaaacga agccaccagt 540
gcatcctgcc aggtacccgc ctgccctgga gaaggcactg ggcccatcgc gccacagaat 600
ttatgggctg tgaatttctc acagccacac gggaagtagg ctggccagac aggcagaggg 660
taagcccgag tgagccggca ggaaggtcag aggctgacag gttctctctc cccccggggg 720
ggcgggggcc ctgagttgga aacgggggca gggggtgggg gggtgctttc ctcagtttct 780
ctctgttctg gtgccttggg catctgattt gtcccagaga ccctgacctc gccagaccct 840
caacggctgc ctttacttat tcccaccccc acccccccag gttctgaccg ctgctgtccc 900
agtcgcagac cggacgcacc gcgaccttac ccgtcttcag agggcactcc tttccatccg 960
acgacatgat agaaccctct gaagactcat ttgagacgat gatggagcgt aagaatccat 1020
catcaaaaca aatggagtcc tccgag 1046
<210> 8
<211> 1475
<212> DNA
<213> pig (Sus scrofa)
<400> 8
agccagtgag gagttttacg catgggattt tttttttttt tttaatagct tggcccagtg 60
gctaaaaaga aaactgagaa ggaacccaaa gggctacatg cgtgcagagc aggatgtgca 120
gagctcagag tggatgagca gaagcagcac gcaaataacc ccaaccaaaa cagctctgtg 180
aatcctaccc acgaacgcca cccatgccaa ggcagtcaga ggtcaccgtc acctcctgcc 240
ctctgcacac cctctccccc tactgaacat cccagctcta cccatgctgg cgggggaatc 300
aaaccagctc cagagaaggg gtgtccacta aagaaccaag agatcgggcc caccttggaa 360
agactgcagg ggcgggtaaa catgcctgca caggcggaac taaggccaaa tgtctcaagt 420
tcctgaaccc ccaagaacaa ggggggcagt ctgagcaacg cggagctttt aggaggggtg 480
gggaactgat gccaggatct cctcgacagc tcagagcggg ggagaacgct gcctggaagc 540
ggcccagaga ggggaaaggg aaaagtgagc aaaaattccg atgtggaatc gagtcatgta 600
tgcgcatgtc tctggcacac agtaggtgtt tttatcaagg tttaaagggg aaaaaaagaa 660
aagccagaat ccagattccc tacatccacc tgccctcaat ttgaatccag gagaggaaac 720
aagcgctatt tggggcggct gacccgaccg acgggcgagc ggtcaaaggc ccattttctc 780
ttcttgccct gagcctgtcg aagggcaggc atccgggcta gagattccca cctccgacca 840
gggaggcccg gcctgccagg ggtggctttg gacggcaccg agaggcgacc tctgtgactt 900
gcggctgacc agagaaggga gggcagattg accgggcacg tgtgccaggg ttgaaacgaa 960
gccaccagtg catcctgcca ggtacccgcc tgccctggag aaggcactgg gcccatcgcg 1020
ccacagaatt tatgggctgt gaatttctca cagccacacg ggaagtaggc tggccagaca 1080
ggcagagggt aagcccgagt gagccggcag gaaggtcaga ggctgacagg ttctctctcc 1140
ccccgggggg gcgggggccc tgagttggaa acgggggcag ggggtggggg ggtgctttcc 1200
tcagtttctc tctgttctgg tgccttgggc atctgatttg tcccagagac cctgacctcg 1260
ccagaccctc aacggctgcc tttacttatt cccaccccca cccccccagg ttctgaccgc 1320
tgctgtccca gtcgcagacc ggacgcaccg cgaccttacc cgtcttcaga gggcactcct 1380
ttccatccga cgacatgata gaaccctctg aagactcatt tgagacgatg atggagcgta 1440
agaatccatc atcaaaacaa atggagtcct ccgag 1475
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
<210> 11
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
cgacgcgtag ccagtgagga gttttac 27
<210> 12
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
cgacgcgtcc caccttggaa agactgc 27
<210> 13
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
cgacgcgtgc aacgcggagc ttttagg 27
<210> 14
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
cgacgcgtct ttggacggca ccgaga 26
<210> 15
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgacgcgtgg gtgctttcct cagtttc 27
<210> 16
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
ccctcgaggt cgtcggatgg aaaggag 27
<210> 17
<211> 1046
<212> DNA
<213> pig (Sus scrofa)
<400> 17
cccaagaaca aggggggcag tctgagcaac gcggagcttt taggaggggt ggggaactga 60
tgccaggatc tcctcgacag ctcagagcgg gggagaacgc tgcctggaag cggcccagag 120
aggggaaagg gaaaagtgag caaaaattcc gatgtggaat cgagtcatgt atgcgcatgt 180
ctctggcaca cagtaggtgt ttttatcaag gtttaaaggg gaaaaaaaga aaagccagaa 240
tccagattcc ctacatccac ctgccctcaa tttgaatcca ggagaggaaa caagcgctat 300
ttggggcggc tgacccgacc gacgggcgag cggtcaaagg cccattttct cttcttgccc 360
tgagcctgtc gaagggcagg catccgggct agagattccc acctccgacc agggaggccc 420
ggcctgccag gggtggcttt ggacggcacc gagaggcgac ctctgtgact tgcggctgac 480
cagagaaggg agggcagatt gaccgggcac gtgtgccagg gttgaaacga agccaccagt 540
gcatcctgcc aggtacccgc ctgccctgga gaaggcactg ggcccatcgc gccacagaat 600
ttatgggctg tgaatttctc acagccacac gggaagtagg ctggccagac aggcagaggg 660
taagcccgag tgagccggca ggaaggtcag aggctgacag gttctctctc cccccggggg 720
ggcgggggcc ctgagttgga aacgggggca gggggtgggg gggtgctttc ctcagtttct 780
ctctgttctg gtgccttggg catctgattt gtcccagaga ccctgacctc gccagaccct 840
caacggctgc ctttacttat tcccaccccc acccccccag gttctgaccg ctgctgtccc 900
agtcgcagac cggacgcacc gcgaccttac ccgtcttcag agggcactcc tttccatccg 960
acgacatgat agaaccctct gaagactcat ttgagacgat gatggagcgt aagaatccat 1020
catcaaaaca aatggagtcc tccgag 1046
<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
<210> 19
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
<210> 20
<211> 46
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
tccagattcc ctacatcagc agtccctcaa tttgaatcca ggagag 46
<210> 21
<211> 47
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
ctcctggatt caaattgagg gactgctgat gtagggaatc tggattc 47
<210> 22
<211> 33
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
ggggtaccgc caccatggag ctgctgtcgc cac 33
<210> 23
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ggaattctca gagcacctgg tagatag 27
Claims (10)
1. Application of a porcine transcription factor MyoD in inhibiting expression of a porcine RTL1 gene.
2. The use of claim 1, wherein the transcription factor MyoD inhibits the expression of the RTL1 gene by inhibiting the promoter activity of the RTL1 gene.
3. The use according to claim 1, wherein the expression of the porcine RTL1 gene is promoted by inhibiting the expression of the transcription factor MyoD.
4. The use of claim 3, wherein the expression of the transcription factor MyoD is inhibited by an siRNA selected from at least one of the following three siRNAs:
siRNA1, wherein the nucleotide sequence of a sense strand is shown as SEQ ID NO.1, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 2;
siRNA2, wherein the nucleotide sequence of a sense strand is shown as SEQ ID NO.3, and the nucleotide sequence of an antisense strand is shown as SEQ ID NO. 4;
the nucleotide sequence of the sense strand of the siRNA3 is shown as SEQ ID NO.5, and the nucleotide sequence of the antisense strand thereof is shown as SEQ ID NO. 6.
5. The use of claim 4, wherein said siRNA is siRNA 2.
6. The use of claim 4 wherein the 3' ends of the siRNA1, siRNA2, siRNA3 sequences are each suspended with dTdT.
7. The use according to claim 1, wherein the expression of the porcine RTL1 gene is inhibited by promoting the expression of the transcription factor MyoD.
8. The use according to claim 7, wherein the expression of the transcription factor MyoD is promoted by constructing a overexpression vector for the transcription factor MyoD.
9. The use of claim 8, wherein the overexpression vector comprises the CDS region of the transcription factor MyoD.
10. The use of claim 9, characterized in that the CDS region of MyoD is ligated into pcDNA3.1 eukaryotic expression vector to construct transcription factor overexpression vector pc-MyoD, the upstream primer nucleotide sequence being shown as SEQ ID No.22 and the downstream primer nucleotide sequence being shown as SEQ ID No. 23.
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| CN103849618A (en) * | 2014-03-10 | 2014-06-11 | 湖北省农业科学院畜牧兽医研究所 | SNP (single nucleotide polymorphism) molecular marker related to pig carcass and meat quality trait and application thereof |
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