CN117448334A - siRNA targeting chicken demethylase gene FTO and application thereof - Google Patents
siRNA targeting chicken demethylase gene FTO and application thereof Download PDFInfo
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Abstract
The invention discloses an siRNA targeting chicken demethylase gene FTO and application thereof, wherein the sense strand of the siRNA has the sequence of 5'-CAAGAGGAACAUUGGAAUA-3' (SEQ NO. 1) and the antisense strand has the sequence of 5'-UAUUCCAAUGUUCCUCUUG-3' (SEQ NO. 2). The invention provides siRNA capable of targeting chicken demethylase gene FTO, which can effectively reduce the expression of chicken demethylase gene FTO after being transferred into chicken myoblasts, and has inhibition efficiency of more than 50 percent, thus being high in knocking-down efficiency. The siRNA of the invention can identify the gene function by specifically knocking down the expression of the chicken demethylase gene FTO, can be applied to genetic breeding of high-quality chickens and provide reference data for human disease research, and has great economic value and scientific research value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to siRNA for inhibiting chicken demethylase FTO gene expression and application thereof.
Background
Muscle fibers are the basic building blocks of muscle, and different muscle fiber type compositions give specific physiological properties and functions to different muscle groups. The muscle fiber type is closely related to exercise and muscle metabolism diseases (such as obesity, type 2 diabetes, muscular atrophy and the like) and the quality of livestock and poultry meat. Overall, the muscle quality with a high content of slow muscle fibers is better than the muscle with a high content of fast white muscle fibers.
Research shows that m6A methylation is the most common chemical modification of eukaryotic RNA, and is widely involved in the regulation of biological processes such as disease occurrence, embryo development, cell proliferation and differentiation and the like by regulating activities such as RNA shearing, mRNA transfer, mRNA stability, translation, miRNA processing and the like. Has important roles in various physiological functions such as cell differentiation, development, stress response and the like. Fat mass and obesity-related proteins (FTOs) are the first identified demethylases of m6A that can forward regulate mTOR-PGC-1 a pathway-mediated mitochondrial biosynthesis through demethylating of m6A, promoting skeletal muscle differentiation. Previous studies have shown that FTO may play a role in the growth and development of chicken skeletal muscle by modulating m6A modifications.
Chickens have been extremely valuable model animals in life sciences research. The development of molecular genetics basic research on the formation and transformation of muscle fiber types in chicken skeletal muscles has important significance for improving the market competitiveness of domestic poultry products, and has good reference value for revealing the molecular mechanism of muscle related diseases caused by abnormal human skeletal muscle metabolism. However, there are no reports in the prior art on the use of siRNA to interfere with the expression of the chicken demethylase FTO gene.
Disclosure of Invention
Aiming at the problem that siRNA is not utilized to interfere the expression of chicken demethylase FTO genes at present, the invention provides the siRNA targeting chicken demethylase genes FTO and the application thereof, which can be applied to genetic breeding of high-quality chickens and provide reference data for human disease research, and have great economic value and scientific research value.
To achieve the above object, the first aspect of the present invention provides an siRNA targeting chicken demethylase gene FTO, the sense strand of the siRNA having a sequence of 5'-CAAGAGGAACAUUGGAAUA-3' (SEQ No. 1) and the antisense strand having a sequence of 5'-UAUUCCAAUGUUCCUCUUG-3' (SEQ No. 2).
Further, two dangling bases dT are added to the 3' -end of the sense strand and the antisense strand.
In a second aspect, the invention provides a recombinant expression vector, transgenic cell line or recombinant virus comprising a nucleotide sequence encoding the siRNA targeting chicken demethylase gene FTO of the first aspect.
In a third aspect, the invention also provides a kit containing the siRNA targeting chicken demethylase gene FTO according to the first aspect.
Further, the kit also comprises a primer group for detecting the expression of the chicken demethylase gene FTO.
In a fourth aspect, the invention also provides the siRNA of the first aspect, the recombinant expression vector, the transgenic cell line or the recombinant virus of the second aspect, and the application of the kit of the third aspect in identifying the FTO function of the chicken demethylase gene.
Through the technical scheme, the invention has the following beneficial effects:
the invention provides siRNA capable of targeting chicken demethylase gene FTO, which can effectively reduce the expression of chicken demethylase gene FTO after being transferred into chicken myoblasts, and has inhibition efficiency of more than 50 percent, thus being high in knocking-down efficiency. The siRNA of the invention can identify the gene function by specifically knocking down the expression of the chicken demethylase gene FTO, can be applied to genetic breeding of high-quality chickens and provide reference data for human disease research, and has great economic value and scientific research value.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments will be briefly described below.
FIG. 1 shows the Western blot for the detection of cytoplasmic and nuclear expression of FTO proteins in chicken muscle tissue and myoblasts, lane 1 is 11 embryonic-year-old chicken breast muscle tissue cytoplasmic protein, lane 2 is 11 embryonic-year-old chicken breast muscle tissue cytoplasmic protein, lane 3 is adult chicken breast muscle tissue cytoplasmic protein, lane 4 is adult chicken breast muscle tissue cytoplasmic protein, lane 5 is differentiated chicken myoblast cytoplasmic protein, lane 6 is 11 embryonic-year-old chicken breast muscle tissue nuclear protein, lane 7 is 11 embryonic-year-old chicken breast muscle tissue nuclear protein, lane 8 is adult chicken breast muscle tissue nuclear protein, lane 9 is adult chicken breast muscle tissue nuclear protein, and lane 10 is differentiated chicken myoblast nuclear protein;
FIG. 2 is the efficiency of different siRNAs in interfering with chicken FTO gene expression during primary myoblast proliferation and differentiation, where ns indicates that the interference group is not significantly different (P > 0.05) compared to the NC control group; * Indicating that the interference group was very significant compared to NC control group (P < 0.01);
FIG. 3 is a graph showing the efficacy of Western blot to detect siRNA2 interfering with chicken FTO protein expression during primary myoblast differentiation, where the interference group is very different (P < 0.01) compared to NC control;
FIG. 4 is a graph showing the effect of knockdown of FTO gene expression in chicken myoblasts on gene expression associated with cell proliferation and differentiation, wherein the interference group showed a very significant difference (P < 0.01) compared to the NC control group;
FIG. 5 is a cellular immunofluorescence graph;
fig. 6 is the effect on myotube area after immunofluorescence detection of chicken FTO gene expression knockdown, where x represents significant differences (P < 0.05) in the interference group compared to NC control group;
FIG. 7 is a homology comparison of FTO gene in chicken and human;
FIG. 8 is a homology comparison of FTO gene in chicken and mouse;
FIG. 9 is a homology comparison of FTO gene in chicken and rat;
FIG. 10 is a homology comparison of FTO gene in chicken and duck;
FIG. 11 is a homology comparison of FTO gene in chicken and goose.
Detailed Description
Further advantages and effects of the present invention will become apparent to those skilled in the art from the disclosure of the present invention, which is described by the following specific examples. It will be apparent that the described embodiments are some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. The following examples were carried out by conventional methods, and the reagents used were all commercially available ones unless otherwise specified.
EXAMPLE 1Western blot detection of cytoplasmic Nuclear expression location of the chicken demethylase Gene FTO Gene
Extraction of chicken muscle tissue-like and chicken myoblast-like cytoplasmic and nuclear proteins, proStatain, using Nuclear Extract Kit (Active Motiff, 40010) kit TM Protein quantitative reagent kit (Active Mot)iff, 15001) protein concentration was determined, FTO antibody (NOVUS, NB100-60935SS, 1:1000) was used to detect FTO protein expression in cytoplasm and nucleus, and β -action antibody (ERWAN, ER001, 1:10000) and history H3 (ERWAN, EAB02004, 1:1000) antibody were used to detect cytoplasmic and nuclear internal reference proteins β -action H3, respectively. As can be seen from FIG. 1, the nuclear internal reference protein Histone H3 is expressed only in chicken muscle tissue and myoblast nuclei, the cytoplasmic internal reference protein beta-action is expressed in both the cytoplasm and nuclei of chicken muscle tissue and myoblast cells, and the FTO protein is expressed in both the cytoplasm and nuclei of chicken muscle tissue and myoblast cells, and is relatively highly expressed in the cytoplasm. It is suggested that the expression of FTO in chicken muscle tissue and myoblasts is mainly localized to cytoplasm, and that expression of chicken FTO can be regulated by siRNA which exerts regulation of target gene expression at posttranscriptional level.
Example 2 selection of optimal sequence for targeting chicken FTO Gene siRNA
2.1 SiRNA design
According to NCBI online database, obtaining chicken FTO gene mRNA sequence information (accession number: NM_ 001185147.1), adopting optimized siPatch (TM) siRNA design software to design 4 pairs of siRNA targeting chicken FTO gene CDS region. When designing, the ideal siRNA is searched from 100bp nucleotide downstream of the target gene initiation codon AUG, and the boundary region between exons is avoided. To enhance the stability of the siRNA duplex, 2 overhanging bases dT were added to the 3' ends of the sense and antisense strands of each pair of sirnas, synthesized by the guangzhou sharp biotechnology limited. The sequences of the 4 pairs of siRNAs obtained by design are shown in Table 1, the targeting sequences of the 4 pairs of siRNAs are shown in Table 2, and targets start at 1370, 1078, 557 and 235 respectively.
TABLE 14 sequences for siRNA
TABLE 24 targeting sequence to siRNA
2.2 siRNA transfection of primary chicken myoblasts
The primary cells isolated and extracted from animal tissues maintain a number of important biological characteristics and functions of the cells in vivo, so that the primary cells are widely applied to basic research of molecules, cell biology and biomedicine and have non-replaceable effects in the field of biomedicine. However, primary cells are difficult to culture and the transfection efficiency of foreign genes is low compared with cell lines. In this example, primary chicken myoblasts isolated from leg muscles of 11-embryo-old chickens were used as transfected cells. The primary chicken myoblasts isolated and extracted are treated with 3X 10 5 The density of each cell/hole is inoculated into a 24-hole culture plate, after the cell density grows to 70-90%, induced differentiation is carried out for 1 day, 4 pairs of siRNA transfection targeting chicken FTO genes and negative control siRNA NC are respectively transfected into chicken myoblasts by means of a transfection reagent Lipofectamine 3000, 4 repeated holes are arranged in each group, and the transfection concentration is 100 n mol/L.
2.3 RNA extraction and cDNA preparation
After 48h of transfection, each well was washed 2 times with PBS, and total RNA of transfected cells was collected and extracted by selecting RNA isolater Total RNA Extraction Reagent reagent for extracting total RNA of cells from Nanjinouzan Biotechnology Co., ltd. The nucleic acid quantitative instrument measures the RNA concentration. cDNA synthesis was performed according to HiScript III RT SuperMix for qPCR, inc. of Nanjinovone Biotech Co.
2.4 Real-time fluorescence quantitative PCR detection of siRNA interference efficiency
The efficiency of siRNA specific interference on chicken FTO gene expression was detected by performing real-time fluorescent quantitative PCR using SYBR Green I method with HiScript III RT SuperMix for qPCR (+gDNA wind) reagent from Nanjinouzan Biotechnology Co., ltd. The system of the real-time fluorescent quantitative PCR reaction is shown in Table 3. Each sample was set up with 3 replicates.
TABLE 3 real-time fluorescent quantitative PCR reaction System
The primer pair nucleic acid sequence for detecting chicken FTO gene expression is as follows:
an upstream primer: 5'-CCAGCTGATGCTGATTTTCA-3' (SEQ ID NO. 13)
A downstream primer: 5'-GCATCCTGGCTTTCCAATTA-3' (SEQ ID NO. 14)
The primer pair nucleic acid sequence for detecting the expression of the chicken internal reference ACTB gene is as follows:
an upstream primer: 5'-TGCTGTGTTCCCATCTATCG-3' (SEQ ID NO. 15)
A downstream primer: 5'-TTGGTGACAATACCGTGTTCA-3' (SEQ ID NO. 16)
The results of 4-pair siRNA specific to the efficiency of interfering with chicken FTO gene expression are shown in FIG. 2. As can be seen from fig. 2, compared with the control group siRNA NC, 4 pairs of sirnas, only siRNA2 and siRNA 4 can significantly (P < 0.01) inhibit expression of FTO gene in cells, the interference efficiency of siRNA2 is best, the inhibition efficiency is up to more than 50%, and the subsequent experiment selects siRNA2 for researching chicken FTO gene function. The sense strand of siRNA2 is the base sequence 5'-CAAGAGGAACAUUGGAAUA-3' shown in SEQ ID NO.1, the antisense strand is the base sequence 5'-UAUUCCAAUGUUCCUCUUG-3' shown in SEQ ID NO.2, and two overhang bases dT are added at the 3' ends of the sense strand and the antisense strand.
2.5 Western blot detection siRNA interference efficiency
After 72h transfection, each well was washed 2 times with PBS, the total cell proteins were extracted from the cell lysates of the Siemens RIPA, and after the BCA method was used to determine the concentration of the extracted proteins, western blot experiments were performed using FTO (NOVUS, NB110-60935SS, 1:1000) and beta-Actin (ERWAN, ER001, 1:10000) as primary antibodies, and sheep anti-rabbit IgG (Invitrogen, A32732, 1:5000) and sheep anti-mouse IgG (Invitrogen, A32723, 1:5000) as secondary antibodies, respectively. As can be seen from fig. 3, siRNA2 was able to inhibit FTO protein expression very significantly (P < 0.01) compared to NC control.
Example 3 Effect of FTO Gene expression on cell proliferation and differentiation-related Gene expression after knockdown
The effect of knockdown expression of chicken FTO gene on expression of chicken myoblast proliferation related gene PAX7 and differentiation related genes MYOD1 and MYOG was detected by a 2.4 real-time fluorescent quantitative PCR method in reference example 2, and the result is shown in FIG. 4. As can be seen from fig. 4, expression of PAX7, MYOD1 and MYOG genes was significantly inhibited after knock-down expression of FTO genes in chicken myoblasts.
The primer pair nucleic acid sequence for detecting chicken PAX7 gene expression is as follows:
an upstream primer: 5'-TCAGCAACCGACGAGCAAG-3' (SEQ ID NO. 17)
A downstream primer: 5'-ATGGTGGATGGTGGCAAGG-3' (SEQ ID NO. 18)
The primer pair nucleic acid sequence for detecting chicken MYOD1 gene expression is as follows:
an upstream primer: 5'-CAACGCCATCCGCTACAT-3' (SEQ ID NO. 19)
A downstream primer: 5'-GTCGAGGCTGGAAACAAC-3' (SEQ ID NO. 20)
The primer pair nucleic acid sequence for detecting chicken MYOG gene expression is as follows:
an upstream primer: 5'-AGCGCCATCCAGTACATCG-3' (SEQ ID NO. 21)
A downstream primer: 5'-ATCCTTCCAGCATCACCATC-3' (SEQ ID NO. 22)
Example 4 Effect of chicken FTO Gene on myotube area after knockdown expression
In induced differentiated chicken myoblasts, siRNA2 and siRNA NC were transfected, fixed with 4% paraformaldehyde for 10 min after 72H, PBS-rinsed, 0.5% Triton-X100 permeabilized for 15 min, PBST-rinsed, 1% BSA blocked for 30 min, myotube marker protein MYHC antibody (DSHB, B103, 1:50) diluted with 1% BSA, incubated for 2H at 37℃and PBS-rinsed, FITC-labeled goat anti-mouse IgG (H+G) secondary antibody diluted with 1% BSA was added, incubated for 1H at 37℃and PBS-rinsed, distilled water-rinsed, cells incubated with 1ug/ml DAPI for 1 min after PBS-rinsing, and the results were examined in fluorescence microscopy after sealing as shown in FIGS. 5 and 6. As can be seen from fig. 5 and 6, the myotube area was significantly reduced (P < 0.05) after FTO gene knockdown expression in chicken myoblasts relative to NC control.
EXAMPLE 5 analysis of mRNA sequence of chicken FTO Gene homology with mRNA sequence of other species FTO Gene
Homology analysis was performed on chicken FTO gene mRNA sequences with FTO gene mRNA sequences in human, mouse, rat, duck, and goose species by NCBI online alignment software blastn. As can be seen from FIG. 7, the full length of chicken FTO gene mRNA sequence NM_001185147.1 is 2787bp, the full length of human FTO gene mRNA sequence NM_001080432.3 is 11573bp, only 48% of the region can be used for alignment, and the homology of the alignment is 71.60%. As can be seen from FIG. 8, the mRNA sequence NM-011936.2 of the mouse FTO gene has a total length of 3586bp, and only 44% of the region can be used for alignment, and the homology of the alignment is 70.11%. As can be seen from FIG. 9, the mRNA sequence NM-001039713.1 of the rat FTO gene has a total length of 1571bp, and only 40% of the region can be used for alignment, with an alignment homology of 71.15%. As can be seen from FIG. 10, the whole length 5634bp of the mRNA sequence XM_027467172.2 of the duck FTO gene can be used for comparison, and the homology of the comparison is 87.82%. As can be seen from FIG. 11, the whole length of the mRNA sequence XM_048075344.1 of the goose FTO gene is 2787bp, and only 64% of the region can be used for alignment, and the homology of the alignment is 85.74%. It can be seen that the FTO gene mRNA sequences of the above species are of no reference value in selecting targeting sites for chicken FTO gene mRNA and in siRNA design.
The preferred embodiments of the present invention have been described in detail above with reference to the examples, but the present invention is not limited to the specific details of the above embodiments, and various simple modifications can be made to the technical solutions of the present invention within the scope of the technical concept of the present invention, and all the simple modifications belong to the protection scope of the present invention.
In addition, the specific features described in the above embodiments may be combined in any suitable manner, and in order to avoid unnecessary repetition, various possible combinations are not described further.
Moreover, any combination of the various embodiments of the invention can be made without departing from the spirit of the invention, which should also be considered as disclosed herein.
Claims (6)
1. An siRNA targeting chicken demethylase gene FTO, wherein the sense strand of the siRNA has the sequence 5'-CAAGAGGAACAUUGGAAUA-3' (SEQ No. 1) and the antisense strand has the sequence 5'-UAUUCCAAUGUUCCUCUUG-3' (SEQ No. 2).
2. The siRNA of claim 1, wherein the 3' ends of the sense strand and the antisense strand are further added with two overhanging bases dT.
3. A recombinant expression vector, transgenic cell line or recombinant virus comprising a nucleotide sequence encoding the siRNA targeting chicken demethylase gene FTO of claim 1 or 2.
4. A kit comprising the siRNA targeting chicken demethylase gene FTO of claim 1 or 2.
5. The kit of claim 4, further comprising a primer set for detecting expression of chicken demethylase gene FTO.
6. The siRNA of claim 1 or 2, the recombinant expression vector, the transgenic cell line or the recombinant virus of claim 3, the kit of claim 4 or 5 for use in identifying chicken demethylase gene FTO function.
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| CN115820640A (en) * | 2022-10-24 | 2023-03-21 | 江苏省家禽科学研究所 | siRNA for inhibiting chicken demethylase gene ALKBH5 and application thereof |
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| CN115948391A (en) * | 2022-07-20 | 2023-04-11 | 江苏省家禽科学研究所 | SiRNA (small interfering ribonucleic acid) of targeted chicken methylation transferase gene METTL16, kit and application of siRNA and kit |
| CN115820640A (en) * | 2022-10-24 | 2023-03-21 | 江苏省家禽科学研究所 | siRNA for inhibiting chicken demethylase gene ALKBH5 and application thereof |
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