CN113817845A - miRNA (micro ribonucleic acid) molecule related to porcine luminal ovarian follicle atresia and application thereof - Google Patents
miRNA (micro ribonucleic acid) molecule related to porcine luminal ovarian follicle atresia and application thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of livestock molecular biology, and particularly relates to microRNA (miRNA) related to antral follicular atresia of pigs and application thereof; the miRNA is miR-9820-5p, and the linear nucleotide sequence of the miRNA is shown in SEQ ID NO 1; the invention designs a specific hybridization probe aiming at miR-9820-5p, and the real existence of the miR-9820-5p is verified through fluorescence in situ hybridization; specific amplification primers are designed aiming at miR-9820-5p, the expression of miR-9820-5p in healthy luminal follicle tissues in pig ovaries is lower than that of atretic follicles detected by fluorescent quantitative PCR, and the miR-9820-5p is treated by corresponding analogues and inhibitors to prove that the miR-9820-5p has a remarkable promoting effect on granular cell apoptosis; meanwhile, the invention designs nucleotide sequences respectively providing miR-9820-5 analogue and inhibitor; the miR-9820-5p disclosed by the invention is expected to be used as a molecular marker related to sow reproduction, so that a new theoretical basis and a potential molecular target are provided for evaluation of pig reproduction traits and improvement of reproductive capacity, and occurrence and prevention of diseases related to human follicular abnormality.
Description
Technical Field
The invention belongs to the technical field of livestock molecular biology, and particularly relates to miRNA related to antral follicular atresia of pigs and application thereof.
Background
Reproductive performance, particularly litter size, of female animals is closely related to their number of ovulations. Atresia folliculorum is a natural physiological phenomenon in mammalian follicles. Around the birth of the sow, the primordial follicle lumen develops and the follicle reserve contains approximately 500 ten thousand primordial follicles. However, during the growth and development and oestrus cycle, a large number of follicles become atretic and obliterate before reaching 6 mm in diameter, resulting in a final achievable ovulation rate of less than 14%. Granulosa cells function as a major component of the follicle by interacting intimately with oocytes and theca cells and producing paracrine substances. Studies have shown that granulosa cell proliferation and function are critical in follicular development and maturation, while the rate of granulosa cell apoptosis increases significantly with the progression of follicular atresia, and therefore the level of granulosa cell apoptosis is one of the major hallmarks of follicular atresia.
Micrornas (mirnas) are a class of non-coding single-stranded RNA molecules of about 20-24 nucleotides in length encoded by endogenous genes and are generally involved in the regulation of gene expression through transcriptional inhibition or post-transcriptional degradation. Research shows that 15-30% of animal genomes are directly regulated by miRNA, and 1/3 genes in human are all regulated by miRNA. miRNA is widely distributed and participates in biological processes of cell growth, differentiation, apoptosis, proliferation and the like. In 2007, the involvement of miRNA in the regulation of oocyte development and maturation was first confirmed in a Dicer enzyme gene knockout mouse model. In 2010, miR-21 becomes the first miRNA which is proved to play a role in apoptosis of follicular granular cells of mammals, and in recent years, important regulation and control roles of the miRNA in oocytes, follicular membrane cells and luteal cells are found, so that the regulation and control roles and application potentials of the miRNA in the follicular atresia process are suggested.
Disclosure of Invention
In order to achieve the aim, the invention provides a miRNA related to sow breeding, wherein the miRNA is pig miR-9820-5p, and the nucleotide sequence of the miRNA is shown as SEQ ID NO 1.
The invention provides a reagent for detecting miR-9820-5p expression level in a sample, wherein the nucleotide sequence of miR-9820-5p is shown in SEQ ID NO:1, and the reagent contains a reverse transcription stem-loop primer for specifically amplifying miR-9820-5p and an upstream primer for fluorescent quantitative PCR detection, or a fluorescent in-situ hybridization probe for specifically marking miR-9820-5 p; the sequence of the stem-loop primer of the specific reverse transcription miR-9820-5p is shown in SEQ ID NO. 2;
the invention provides an upstream primer for specific amplification miR-9820-5p and can be used for fluorescent quantitative PCR detection, wherein the sequence of the upstream primer is shown as SEQ ID NO.3, the upstream primer is used by being matched with a miRNA amplification universal downstream primer, and the sequence of the universal downstream primer is shown as SEQ ID NO. 10; provides an in-situ hybridization probe of a specific marker miR-9820-5p, and the sequence of the probe is shown in SEQ ID NO. 4.
The invention provides a specific mics double-chain sequence of miR-9820-5p, which is shown in SEQ ID NO. 5 and SEQ ID NO. 6.
The invention provides a specific inhibitor sequence of miR-9820-5p, which is shown in SEQ ID NO. 7.
The miRNA provided by the invention is applied to the detection of the expression quantity of animal cells and tissues.
The miRNA provided by the invention is applied to selection evaluation, hybridization, genetic improvement and ovarian pathology detection of sows.
The reagent provided by the invention is applied to the detection of the expression quantity of animal cells and tissues.
The reagent provided by the invention is applied to selection evaluation, hybridization, genetic improvement and ovarian pathology detection of sows.
The application of the mimics sequence provided by the invention in the treatment for enhancing the miRNA; the specific mimics sequence of the miR-9820-5p can be used for the functional up-regulation of the miR-9820-5p in cells, tissues and living bodies.
The inhibitor sequence provided by the invention is applied to the treatment for inhibiting the miRNA; the specific inhibitor sequence of the miR-9820-5p can be used for the functional down-regulation of the miR-9820-5p in cells, tissues and living bodies.
Compared with the prior art, the invention has the following advantages and beneficial effects:
the invention provides microRNA (miRNA) related to atresia of pig antral follicles and application thereof; the miRNA is miR-9820-5p, and the linear nucleotide sequence of the miRNA is shown in SEQ ID NO 1; the invention designs a specific hybridization probe aiming at miR-9820-5p, and the real existence of the miR-9820-5p is verified through fluorescence in situ hybridization; specific amplification primers are designed aiming at miR-9820-5p, the expression of miR-9820-5p in healthy luminal follicle tissues in pig ovaries is lower than that of atretic follicles detected by fluorescent quantitative PCR, and the miR-9820-5p is treated by corresponding analogues and inhibitors to prove that the miR-9820-5p has a remarkable promoting effect on granular cell apoptosis; meanwhile, the invention designs nucleotide sequences respectively providing miR-9820-5 analogue and inhibitor; the miR-9820-5p disclosed by the invention is expected to be used as a molecular marker related to sow reproduction, so that a new theoretical basis and a potential molecular target are provided for evaluation of pig reproduction traits and improvement of reproductive capacity, and occurrence and prevention of diseases related to human follicular abnormality.
The miRNA provided by the invention has the advantages of short and short sequence, fast transcription and high correlation with swine follicular atresia, and can be used as one of direct indications or judgment bases for identifying follicular health or atresia status. Since miRNA can directly act through modes of intraperitoneal injection or tissue injection, etc., the miRNA can also be used as a tool for artificially interfering with follicular development or atresia process. The reverse transcription stem-loop primer in the related detection reagent provided by the invention, and the matched quantitative primer and the fluorescent probe thereof have strong specificity, and can definitely reflect the expression level and the subcellular distribution condition of the miRNA. The mimics and the inhibitor provided by the invention have remarkable promoting and inhibiting effects on the biological action of miRNA and strong specificity.
Drawings
FIG. 1 shows that expression of miR-9820-5p in healthy and antral follicles of pigs is detected by qRT-PCR;
FIG. 2 is a distribution diagram of fluorescence in situ hybridization detection of miR-9820-5p in porcine granulosa cells, wherein the left diagram is the positioning of a green fluorescence labeled miR-9820-5p probe, the middle diagram is DAPI stained cell nuclei, and the right diagram is an overlay diagram of the left and middle diagrams;
FIG. 3 shows the expression level of miR-9820-5p in pig granular cells transfected with miR-9820-5pmimics detected by qRT-PCR;
FIG. 4 shows the expression level of miR-9820-5p in miR-9820-5p inhibitor transfected porcine granulosa cells;
FIG. 5 is a graph showing the change of the apoptosis rate of pig granule cells before and after the detection of miR-9820-5p mimics by flow cytometry, wherein the left graph is an original graph of the detection result of a flow cytometer; the right graph is the result graph after the statistical analysis processing. FIG. 6 is a graph showing the change of the apoptosis rate of the porcine granule cells before and after the detection of miR-9820-5p inhibitor by flow cytometry, wherein the left graph is an original graph of the detection result of the flow cytometer; the right graph is the result graph after the statistical analysis processing.
Detailed Description
The technical solutions provided by the present invention will be described in detail below with reference to specific examples, and it should be understood that the following specific embodiments are only illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedures, in which specific conditions are not specified in the examples, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers.
Description of sequence listing:
SEQ ID NO 1 is a linear nucleotide sequence of pig miR-9820-5 p;
SEQ ID NO.2 is a reverse transcription stem-loop primer sequence for specific amplification of miR-9820-5 p;
SEQ ID NO.3 is an upstream primer sequence of a primer pair for specifically amplifying miR-9820-5 p;
SEQ ID NO. 4 is an in situ hybridization probe sequence of a specific marker miR-9820-5 p;
SEQ ID NO. 5 is a specific mimics double-stranded positive chain sequence of miR-9820-5 p;
SEQ ID NO. 6 is a specific mimics double-stranded negative strand sequence of miR-9820-5 p;
SEQ ID NO. 7 is a specific inhibitor sequence of miR-9820-5 p;
SEQ ID NO 9 is the downstream primer sequence of the primer pair for specific amplification of U6;
The invention relates to a method for effectively detecting the relative expression quantity of miRNA in animal cells or tissues, namely a method for carrying out real-time quantitative PCR by taking miRNA specific primers and U6 as reference genes.
The miRNA reverse transcription primer, the upstream amplification primer and the U6 primer have sequences shown as SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.8 and SEQ ID NO. 9.
The invention provides a method for effectively detecting the qualitative distribution of miRNA in animal cells or tissues, namely a method for carrying out fluorescence in situ hybridization by using miRNA specific probes; the sequence of the miRNA specific probe is shown in SEQ ID NO. 4.
The invention provides a method for effectively up-regulating miRNA expression in animal cells, namely a method for transfection of liposome-mediated miRNAmimics.
The invention provides a method for effectively down-regulating the expression of miRNA in animal cells, namely a liposome-mediated mirnAinhibitor transfection method.
The miRNA specific mimics double-chain sequences are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6.
The sequence of the miRNA specific inhibitor single strand is shown in SEQ ID NO. 7.
The invention relates to a method for detecting apoptosis, namely Annexin V-PI staining and a flow cytometry detection method.
Example 1
Expression difference of pig miR-9820-5p in healthy and atretic luminal follicles
(1) Design of pig miR-9820-5p primer
Designing a primer according to the sequence (NR _128505.1) of the sus-miR-9820-5 p; the specific sequence is as follows:
reverse transcription primer SEQ ID No.2: CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGAGCCTTTC;
specific forward primer sequence of miR-9820-5p is SEQ ID No. 3: GCCGAGGAGGAGGAGGGAA, respectively;
forward primer sequence of U6 SEQ ID No. 8: CGCTTCGGCAGCACATATAC, respectively;
reverse primer sequence of U6 SEQ ID No. 9: TTCACGAATTTGCGTGTCAT, respectively;
reverse universal primer sequence SEQ ID No.10 for miRNA amplification: CTCAACTGGTGTCGTGGA, respectively;
the above primers were synthesized by primer synthesis, in which U6 is an internal reference gene.
(2) Extracting pig healthy antral follicle RNA (conventional TRIZOL method):
healthy and atretic antral follicles with a diameter of about 5mm were isolated from healthy swine ovaries before estrus by using tweezers and scissors, and 1ml of TRIZOL reagent (purchased from Vazyme Nanjing, Ltd.) was repeatedly aspirated for each follicle tissue, and the homogenate was transferred to a 1.5ml centrifuge tube (RNase free) and left at room temperature for 5 minutes to completely separate the nucleic acid-protein complex. 0.2ml of chloroform was added thereto, followed by vigorous shaking for 15 seconds and standing at room temperature for 3 min. Centrifuging at 12000 Xg for 15min at 4 deg.C, separating the sample into three layers, collecting RNA mainly in the uppermost layer of water phase, transferring 600ul of supernatant to 1.5ml centrifuge tube, adding 500ul of isopropanol, mixing by inversion, and ice-cooling for 10 min. Then, the mixture was centrifuged at 12000 Xg at 2-8 ℃ for 10min, and the supernatant was discarded. Adding 1ml of 75% ethanol, shaking, mixing, centrifuging at 4 deg.C and 7500 Xg for 5min, and removing supernatant. Drying RNA at room temperature for 5-10min, adding 20ul RNase-free water, mixing, sucking 1ul, measuring concentration and purity with Thermo NANO Drop2000 ultraviolet spectrophotometer, and storing qualified RNA at-80 deg.C.
(3) Reverse transcription of RNA
RNA reverse transcription was performed according to the protocol described in the Vazyme reverse transcription kit (R223-01), and the procedure was as follows: 1) genomic DNA removal: 1ug of RNA, 4ul of 4 XgDNA wiper Mix, RNase free ddH2O to 16ul, mixed well and incubated at 42 ℃ for 2 min. 2) Reverse transcription reaction: adding 5 XHiScript II qRTSupermix II4ul and RT primer SEQ ID NO:2 into the reaction solution in the step 1), mixing uniformly, and keeping the temperature at 50 ℃ for 15min and the temperature at 85 ℃ for 5 s.
(4)qPCR
qPCR reaction system: SYBR Green I real-time PCR Mix (from Takara Bio Inc., Dalian China) 5. mu.L, upstream and downstream primers (SEQ ID NO:3, SEQ ID NO:10 or SEQ ID NO:8, SEQ ID NO: 9) 0.15. mu.L, 200ng cDNA, water to 10. mu.L. Reaction procedure: pre-denaturation at 95 ℃ for 2 min; 40 cycles (denaturation at 95 ℃ for 15 s; annealing at 60 ℃ for 15 s; extension at 72 ℃ for 15 s). Biologically and technically repeating 3 times, and calculating relative gene expression amount by using 2-delta-Ct method, wherein the delta-Ct is the maximum value of (Ct target gene-Ct internal reference) - (Ct target gene-Ct internal reference), and the internal reference is U6. T-test for significance analysis. P <0.05 indicates significant difference, and P <0.01 indicates very significant difference.
As a result:
as shown in figure 1, U6 is used as an internal reference gene, and qRT-PCR (quantitative reverse transcription-polymerase chain reaction) is used for detecting the expression level of miR-9820-5p in healthy and antral follicles of pigs, and the result shows that the expression level of miR-9820-5p in healthy and antral follicles of pigs is extremely lower than that of the antral follicles. The function of miR-9820-5p is probably related to promoting follicular atresia.
Example 2
Cell localization of porcine miR-9820-5p
(1) Design of pig miR-9820-5p fluorescent probe
According to the complementary pairing principle, a digoxin labeled probe of miR-9820-5p is designed. The sequence of the probe is shown as SEQ ID NO. 4. This probe was synthesized by Probe Synthesis.
(2) Climbing sheet culture of pig granular cells
The pig ovaries were washed with sterile saline containing gentamicin (80mg/L) and follicular fluid and granulosa cells were removed from clear, lumened follicles of approximately 5mm diameter using a syringe with a 20-gauge needle. The porcine granulocytes were then cultured in DMEM/F12 medium (purchased from Gibco, USA) containing 10% Fetal Bovine Serum (FBS), 100 units/mL penicillin and 100mg/mL streptomycin, and the cells were seeded in a six-well plate with cover slips placed therein under conditions of 37 ℃ and 5% CO 2. And taking out the cover glass after the cells adhere to the wall, and carrying out probe incubation.
(3) Incubation and microscopic observation of pig miR-9820-5p fluorescent probe
Porcine granulosa cells cultured on coverslips were first fixed with 4% paraformaldehyde (containing DEPC) for 20 minutes, washed three times with PBS (pH 7.4) with shaking, and finally digested by addition of proteinase K (20. mu.g/ml) for 5 minutes. The procedures of probe prehybridization, hybridization and washing, and mouse-digoxigenin-labeled peroxidase (anti-DIG-HRP) and DAPI counterstaining nuclei are all performed according to the instructions of the manufacturer of the cell slide fluorescent probe in situ hybridization double-label (TSA) experimental kit (Wuhan Seville Biotech., China). Fluorescence images were acquired using a Nikon upright fluorescence microscope (Nikon DS-U3, Japan). FAM (488) green light excitation wavelength of 465-495nm, emission wavelength of 515-555nm and green light emission; the ultraviolet excitation wavelength is 330-380nm, the emission wavelength is 420nm, and blue light is emitted. The experiment was performed in triplicate.
Results
As shown in FIG. 2, the left image is the location of the green fluorescence labeled miR-9820-5p probe, the middle image is the DAPI stained nucleus, and the right image is the overlay of the left and middle images, which identifies the relative position of miR-9820-5p expression and nucleus; fluorescence in situ hybridization detection of miR-9820-5p in porcine granulosa cells is marked by green fluorescence, and cell nuclei are stained by DAPI (blue), wherein the location of miR-9820-5p is mainly in cytoplasm. The scale bar is 50 μm. The existence of the miR-9820-5p in the porcine granulosa cells and the distribution of the miR-9820-5p in cytoplasm are further confirmed.
The miRNA of the invention has higher distribution in cytoplasm of healthy ovary granular cells of pigs and extremely low expression in cell nucleus.
Example 3
miR-9820-5p promotes apoptosis of pig granular cells
(1) Design of miR-9820-5p specific mimics and inhibiror
The miR-9820-5p sequence is used as a template and is synthesized by a synthesis company. The miR-9820-5p specific mimics sequence pair is respectively SEQ ID NO. 5 and SEQ ID NO. 6; the sequences of miR-9820-5p specificity inhibiror are SEQ ID NO 7 respectively.
(2) Transfection of porcine granulosa cells miR-9820-5p
The porcine granulosa cells were cultured for 36 hours and transfected when the cells reached 50-80% confluence. Transfection was performed using LipofectamineTM3000 transfection reagent and Opti MEM medium (available from Invitrogen) following the instructions. The experiment was performed in triplicate.
(3) Detection of miR-9820-5p specificity mics and inhibiror efficiency
And respectively taking the cDNA of the pig granular cell transfected with miR-9820-5p specific mimics, miR-9820-5p specific inhibiror and NC as templates, carrying out qPCR amplification by using miR-9820-5p specific primers, and comparing the difference of the expression amount of miR-9820-5p in the pig granular cell. The reaction system and the analysis method are shown in 1.4. Biological and technical repeats were performed 3 each.
(4) Detection of apoptosis in porcine granulosa cells
After transfecting the porcine granulosa cells with miR-9820-5p specific mimics and miR-9820-5p specific inhibior respectively for 72 hours, the cells are digested, and apoptosis staining labeling is carried out by using Annexin V-FITC/PI staining kit (purchased from Vazyme Nanjing GmbH) according to the instructions of a manufacturer. The percentage of apoptotic cells was detected by flow cytometry (Beckman Coulter, break, Cal, USA). Data were analyzed using FlowJo v7.6 software (stanford university, california, usa). Biological and technical repeats were performed 3 each.
Results
As shown in figure 3, the expression level of miR-9820-5p in the porcine granular cells transfected with miR-9820-5pmimics is obviously increased (p is less than 0.01) through qRT-PCR detection, and the result shows that miR-9820-5p specific mimics has the effect of up-regulating miR-9820-5p level. The specific mimics disclosed by the invention can effectively increase the corresponding miRNA expression amount in the granular cells after the granular cells are transfected by the liposome.
As shown in figure 4, the expression level of miR-9820-5p in the porcine granular cells transfected with miR-9820-5p inhibitor is remarkably reduced (p is less than 0.01), and the result shows that the miR-9820-5p specific inhibitor has the effect of reducing the level of miR-9820-5 p. The specific inhibitor can effectively reduce the expression quantity of the corresponding miRNA in the granular cells after the granular cells are transfected by the liposome.
As shown in figure 5, the apoptosis rate of the pig granular cells transfected with miR-9820-5p mimics is obviously higher than that of the pig granular cells transfected with NC (p <0.001) through flow cytometry detection, and the result shows that miR-9820-5p can cause the apoptosis of the pig granular cells. The miRNA disclosed by the invention can be used for obviously improving the apoptosis rate of the pig ovarian granule cells after being up-regulated by the mimics.
As shown in figure 6, flow cytometry detection shows that miR-9820-5p inhibitor transfection can reduce apoptosis rate and maintain normal physiological state of porcine granular cells. The miRNA disclosed by the invention can obviously reduce the apoptosis rate of the pig ovarian granule cells after being down-regulated by the inhibitor.
The results show that the function of miR-9820-5p is possibly related to the promotion of granular cell apoptosis and the promotion of follicular atresia. Therefore, the molecular marker is expected to be used as a molecular marker related to sow reproduction, provides a new theoretical basis and a potential molecular target for evaluation of pig reproduction traits and improvement of reproductive capacity, and occurrence and prevention of diseases related to human follicular abnormality.
The technical means disclosed in the invention scheme are not limited to the technical means disclosed in the above embodiments, but also include the technical scheme formed by any combination of the above technical features. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principle of the present invention, and such improvements and modifications are also considered to be within the scope of the present invention.
Sequence listing
<110> Jinling science and technology institute
<120> miRNA molecule related to porcine antral follicular atresia and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
<210> 1
<211> 21
<212> RNA
<213> pig (Sus scrofa)
<400> 1
gaggaggagg gaagaaaggc u 21
<210> 2
<211> 44
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 2
ctcaactggt gtcgtggagt cggcaattca gttgagagcc tttc 44
<210> 3
<211> 19
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gccgaggagg aggagggaa 19
<210> 4
<211> 21
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
agcctttctt ccctcctcct c 21
<210> 5
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
gaggaggagg gaagaaaggc u 21
<210> 6
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
ccuuucuucc cuccuccucu u 21
<210> 7
<211> 21
<212> RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
agccuuucuu cccuccuccu c 21
<210> 8
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
<210> 9
<211> 20
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
<210> 10
<211> 18
<212> DNA/RNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ctcaactggt gtcgtgga 18
Claims (10)
1. A miRNA molecule related to the atresia of the antral follicles of the pigs is miR-9820-5p, and the nucleotide sequence of the miRNA is shown in SEQ ID NO. 1.
2. A reagent for detecting the expression level of miR-9820-5p in a sample is characterized in that the nucleotide sequence of miR-9820-5p is shown in SEQ ID NO 1, and the reagent contains a reverse transcription stem-loop primer for specifically amplifying miR-9820-5p and an upstream primer for fluorescent quantitative PCR detection, or a fluorescent in situ hybridization probe for specifically marking miR-9820-5 p; the sequences of the reverse transcription stem-loop primer and the upstream primer detected by the corresponding fluorescent quantitative PCR are shown as SEQ ID NO.2 and SEQ ID NO.3, and the sequence of the universal downstream primer detected by the fluorescent quantitative PCR matched with the SEQ ID NO.3 is shown as SEQ ID NO. 10; the sequence of the probe is shown as SEQ ID NO. 4.
3. The miRNA molecule related to the atresia of antral follicles of pigs as claimed in claim 1, wherein the specific mimics sequence of miR-9820-5p is shown in SEQ ID NO. 5 and SEQ ID NO. 6.
4. The miRNA molecule related to the atresia of antral follicles in pigs according to claim 1, wherein the specific inhibitor sequence of miR-9820-5p is shown in SEQ ID NO. 7.
5. The use of the miRNA of claim 1 for detecting the expression level of animal cells and tissues.
6. Use of the miRNA of claim 1 for selective assessment, breeding, genetic improvement, and ovarian pathology detection of sows.
7. Use of the reagent according to claim 2 for the detection of expression levels in animal cells and tissues.
8. Use of the agent of claim 2 for the selective assessment, mating, genetic improvement and ovarian pathology detection of sows.
9. Use of the mimics sequence of claim 3 in a treatment for enhancing the miRNA.
10. Use of the inhibitor sequence of claim 4 in a treatment for inhibiting said miRNA.
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| CN113817846A (en) * | 2021-10-27 | 2021-12-21 | 金陵科技学院 | circRNA related to porcine luminal ovarian follicular atresia, siRNA inhibitor thereof and application thereof |
| CN117165696A (en) * | 2023-11-03 | 2023-12-05 | 四川省畜牧科学研究院 | Application of lncRNA related reagent in regulation and control of chicken follicular development |
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| CN112626220A (en) * | 2021-01-18 | 2021-04-09 | 中国农业大学 | Biomarker and application thereof |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113817846A (en) * | 2021-10-27 | 2021-12-21 | 金陵科技学院 | circRNA related to porcine luminal ovarian follicular atresia, siRNA inhibitor thereof and application thereof |
| CN113817846B (en) * | 2021-10-27 | 2023-09-22 | 金陵科技学院 | circRNA related to pig follicular locking, siRNA inhibitor thereof and application thereof |
| CN117165696A (en) * | 2023-11-03 | 2023-12-05 | 四川省畜牧科学研究院 | Application of lncRNA related reagent in regulation and control of chicken follicular development |
| CN117165696B (en) * | 2023-11-03 | 2024-02-06 | 四川省畜牧科学研究院 | Application of lncRNA related reagent in regulation and control of chicken follicular development |
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